The role of Resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer

The role of Resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The role of Resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer Fangfang Chen, Thomas Kolben, Sarah Meister, Bastian Czogalla, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-198498/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2021 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted 4 You are reading this latest preprint version Abstract Objective: Ovarian cancer is the most lethal gynecologic cancer. Resveratrol (RSV) is known to alter metabolism in cancer. It affects the nuclear retinoid-X-receptor (RXR), which implies a modulating effect of RXR to gynecologic cancers. Furthermore, RSV targets Sirtuin1 (Sirt1), a histone deacetylase. Study design: 123 tissue samples of patients with serous or mucinous ovarian cancer were examined for expression of Sirt1 and RXR. Ovarian cell-lines were treated with RSV and consequences on viability and apoptosis were evaluated. The influence of RSV to Sirt1 and RXR expression was analyzed by western blotting. Results: A correlation of nuclear Sirt1 and RXRα expression could be detected (p=0.006). Co-expression of nuclear RXRα and cytoplasmic (p=0.026) or nuclear (p=0.041) Sirt1 was associated with significantly increased overall survival in advanced tumor stages. Viability was decreased in all cell-lines after stimulation with resveratrol, while cell apoptosis was increased. RSV treatment led to significant lower Sirt1 expression in A2780 cells (p=0.025) and significant increased RXR expression in cisA2780 cells (p=0.012). Conclusion: In order to use RSV as medical target, studies could be developed to improve the understanding of drug resistance mechanisms and consequently improve treatment outcome. Sexual & Reproductive Medicine ovarian cancer survival epigenetics Resveratrol Sirtuin1 RXR Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ovarian cancer is the most lethal gynecologic malignancy and the eighth leading cause of cancer-related mortality among women worldwide (1, 2). The overall 5-years survival rates have barely improved over the past few decades remaining at 40-45% for advanced stages and around 80% of patients progressing within 18 months (3, 4). The poor prognosis of ovarian cancer is mainly related to late-stage diagnosis and the rapid development of resistance to current chemotherapy regimens (2, 5) . Resveratrol (RSV), a naturally plant polyphenol originates from grapes and berries, has been proven to alter metabolism in cancer (3) and to regulate tumor microenvironment (6). The regulatory effect of RSV on cancer is complex: besides inhibition of cell growth, RSV is also involved in enhancing chemo-sensitivity and blocking the cancer invasion of cancer cells in vitro (7). In addition, it has been confirmed that RSV improves the efficacy of cisplatin in ovarian cancer (8) . RSV is also able to modulate vitamin-D-receptor (VDR)-signaling and it can induce dimerization of VDR with one of its partners, the nuclear retinoid X receptor (RXR) (9). Three retinoic X receptors are known: RXRα (10) , RXRβ (11) and RXRγ (12). Although the distribution of RXR subtypes is different, their functions are similar: modulating gene expression they control numerous functions by dimerization with other nuclear hormone receptors, contributing thereby to activities of different cell fates (13). As the VDR is known to be involved in gynecologic cancers, the interaction of VDR with the RXR implies that also the RXR may have a modulating effect on gynecological cancers (14). Sirtuin1 (Sirt1) is a NADP-dependent histone deacetylase, which regulates cellular metabolism and cellular pathways (15-20). Its role regarding cancer progression remains controversial as it can act as tumor suppressor or as tumor promotor (21). In ovarian cancer Sirt1 overexpression was correlated with improved overall survival (22). By complex mechanisms (via interaction of NF-kB) Sirt1 can be influenced by RSV: it can be targeted by RSV, which can lead to suppression of tumorigenesis in colorectal carcinoma (23). Recently, RSV was described to activate RXR and stimulate Sirt1 in mammalians (9). The competitive binding of RXR and Sirt1 to PPARα (a peroxisome proliferator-activated receptor) could be due to structural similarity between these proteins. Sirt1 binds to PPARα more strongly than RXRα suggesting that Sirt1 interacts with PPARα directly rather than RXR (24). Regnault et al. noticed that RSV induced Sirt1 and RXR in muscle hypoxia (25). Unfortunately, the effect of RSV on Sirt1 and RXR expression in ovarian cancer cells has not been well documented until now. In the present study, we immunohistochemically examined the expression of RXRα and Sirt1 in mucinous and serous ovarian cancer and analyzed the relationship between RSV, RXRα and Sirt1 in ovarian cancer in vitro. Considering the potential medical role of resveratrol in ovarian cancer, we evaluated the effects of RSV by proliferation and apoptosis experiments. In addition, we investigated the mechanism of RSV resistance to apoptosis in ovarian cancer cell lines. This study aimed to analyze RXRα and Sirt1 as potential therapeutic targets in ovarian cancer. Materials And Methods 1. Material In this study we used ovarian cancer tissue samples of 123 patients who underwent surgery for ovarian cancer from 1990 to 2002 at the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany. Patients who underwent surgery due to serous or mucinous ovarian cancer were included while other histological subtypes were excluded due to low number. The median age was 59 years (range 20–88 years) and median overall survival was 2.67 years. The distribution of clinic-pathological variables can be seen in table 1 . As positive controls for immunohistochemical staining, we utilized palatine tonsil for Sirt1 staining and first trimester placenta for RXR staining, both received from the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University of Munich. Clinical and follow-up data for statistical analyses were provided by the Munich cancer registry and retrieved from medical records. Table 1. Patients’ characteristics. N % Subtype serous 110 89.4 mucinous 13 10.6 Age ≥60 61 49.6 <60 62 50.4 FIGO I/II 29 23.6 III/IV 92 74.8 NA’s 2 01.6 Grading 1/2 76 61.8 3 40 32.5 NA’s 7 05.7 Progression (18 years) No progression Progression NA’s 101 21 1 82.1% 17.1% 0.1% Overall-Survival (18 years) Right censured Died NA’s 38 84 1 30.1% 68.3% 0.1% 2. Ethics Approval All ovarian cancer specimens had been collected for histopathological diagnostics during surgery. They were no longer used for clinical tests. Patients’ data were anonymized and authors were blinded for clinical information during experimental analyses. The study was conducted in consent to the Declaration of Helsinki and was approved by the local ethics committee of the Ludwig-Maximilians University of Munich (reference number 227-09 and 18-392). 3. Immunohistochemistry Paraffin-embedded slides of 3µm were dewaxed in xylol and washed in 100% ethanol. For inhibition of the endogen peroxidases, tissue samples were incubated in 3% methanol/H 2 O 2 and rehydrated in a descending alcohol series. Slides were afterwards heated in a pressure cooker using sodium citrate buffer (pH = 6.0; containing 0.1 M citric acid and 0.1 M sodium citrate in distilled water). After cooling and washing in PBS (phosphate-buffered saline), all slides were incubated with blocking solution to avoid non-specific binding of the primary antibodies. Subsequently, the slides were stained with the primary antibodies anti-Sirt1 and anti-RXRα ( table 2 ) and incubated. After washing, the secondary complexes of the ABC detection kits were applied following the manufacturer’s protocols to detect reactivity. Immunostaining was visualized with the substrate and the chromogen-3, 3′-diaminobenzidine (DAB) for 1 min. For exact staining protocol see table 2 . For the light microscopy analysis, the semi-quantitative immune-reactive score (IRS) is calculated via the multiplication of optical staining intensity (grades: 0 = no, 1 = weak, 2 = moderate and 3 = strong staining) and the percentage range of positive stained cells (0 = no staining, 1 = ≤10% of the cells; 2 = 11–50% of the cells; 3 = 51–80% of the cells and 4 = ≥81% of the cells were stained for the antibody, respectively). Palatine tonsil was used as control for Sirt1 and first trimester placenta was used as control for RXR staining. Table 2. Antibodies and chemicals used for the immunohistochemistry anti-Sirt1 1 anti-RXRα 2 PBS 3 PBS 3 Blocking solution 4 : 20 min Blocking solution 4 : 20 min primary antibody 1 : 1:1000 incubation: 16 h, 4◦C min primary antibody 2 : 1:200 incubation: 16 h, 4◦C ABC detection kid 5 ABC detection kid 5 Chromogen: DAB 6 (1 min) Chromogen: DAB 5 (1 min) 1 anti-Sirt1 rabbit IgG, polyclonal antibody , concentration: 1:1000; Atlas Antibody, Sweden; order number: SHPA006295. 2 anti-RXRα rabbit IgG, polyclonal antibody, concentration: 1:200; PPMX, Japan; order number: pp-k8508-10. 3 HRP-Polymer-Kit (mouse/rabbit); Zytomed Systems, Germany; order number: POLHRP-100. 4 ABC detection kid; Vectastain, USA; order number: AK-6401. 5 Dulbecco’s Phosphate Buffered Saline; Gibco, USA; order number: 14190-094. 4. Cell culture Human ovarian cancer cell lines with different characteristics (A2780, UWB1.289 and cisA2780, see table 3 ) were used in the study. The cell lines were ordered from Gibco (see table 3 ). Table 3. Cell lines A2780 1 UWB1.289 2 cisA2780 3 Cell type Epithelial ovarian cancer cell Epithelial ovarian cancer cell Epithelial ovarian cancer cell Characteristics Mucinous Serous brca1-null Mucinous, carboplatin-resistant Culture medium RPMI 1640 4 + 10% FBS 5 RPMI 1640 4 + 10% FBS 5 RPMI 1640 4 + 10% FBS 5 1/2/3 Gibco . 4 Gibco, USA; Order number: 21875-034. 5 Foetal Bovine Serum, biochrom, Germany; order number S0615 Cell lines were cultured (see table 3 ) and seeded into 96-well plates for MTT and BrdU (see below) and 6-well plates for Western blotting. After 20 h, cell culture medium was replaced with fresh culture medium with resveratrol (50 and 100µM RSV; Sigma, America; order number: R5010-100MG) for the remaining 24 h, which included dimethyl sulfoxide (DMSO; concentration: 0.5%; SERVA, Germany; order number: 20385, 0.5%) as vehicle control. ELISAs Cell viability assay A2780, UWB1.289 and cisA2780 ovarian cancer cells were seeded at the density of 1.5 × 10 4 cells/well in 96-well plates with 200μl medium. After 20 h cells were incubated with 50µM and 100µM of resveratrol for 24 h. Untreated control cells were plated in medium only. To each well, 20μg MTT (Sigma, USA; order number: M-5655) were added for 1.5 h at 37°C in order to show viability. After removing MTT from the plates, 200μL DMSO were added and mixed thoroughly on the shaker for 5 min at room temperature. The optical density was examined at 595 nm using Elx800 universal Microplate Reader. Each experiment was carried out in triplicate. Marker of proliferation: BrdU A2780, UWB1.289 and cisA2780 ovarian cancer cells were cultured at the density of 1.0 × 10 4 cells/well together with various dilutions (50/100µM) of resveratrol in 96-well plates. For the labelling of DNA replication BrdU (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) was added to the culture medium for 2h. The final concentration of BrdU was 10μM. After removal of BrdU by pipette, 200µl/well FixDenat (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) were added and cells were incubated for 30 min at room temperature. Afterwards, FixDenat solution had to be removed thoroughly and 100µl/well anti-BrdU-POD (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) working solution were added. Cells were then incubated for approximately 90 min at room temperature and washed 3 times with PBS. 100µ/well substrate solution BrdU was added and incubation for 20 minutes was performed. To each well 25µl 1M H 2 SO 4 were added and the absorbance of the samples was measured by an ELISA reader at 450nm. Apoptosis assay Apoptosis was evaluated by measuring the level of caspase-cleaved cytokeratin 18 (M30, Roche, Switzerland; order number: 121140322001). The ovarian cell lines A2780, cisA2780 and UWB1.289 were seeded at a density of 1.0 × 10 4 cells/well on 96-well plates in 200μl medium. After 20 h, 50 or 100µM RSV were added and cells were incubated for 24 h. M30 CytoDeath (Roche, Switzerland; order number: 121140322001, dilution 1:1000) was used to detect the apoptotic cells. TUNEL Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. TUNEL staining was performed to assess in situ DNA fragmentation using a commercial kit (FragELTM DNA Fragmentation Detection Kit, Colorimeric- TdT Enzyme , USA; order number: Qia33-1EA) following the manufacturer’s protocol. Western blotting Cell lysates were extracted from A2780, cisA2780 and UWB1.289 cells with radio-immuno-precipitation assay buffer (RIPA, Sigma-Aldrich, St. Luis, USA; order-number: R0278-50ML). For Western blotting, 20µg of cell lysates were first separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 10% casein and then incubated with the primary antibodies for 16 h at room temperature. We used the same antibodies as for immunohistochemistry (see table 2 ). GAPDH was used as a housekeeping gene and mouse monoclonal anti-GAPDH antibody (GeneTex, America; order number: GTX277408) was diluted 1:1000 in 10% CASEIN (Vector, Germany; order number: ZE0925). Afterwards, the membrane was incubated with the goat-anti-rabbit secondary antibody (Vector; bioZol; Germany; order number: VEC-BA-1000, dilution 1:1000) conjugated with alkaline phosphatase, and detected with 5-bromo-4-chloro-3′-indolylphosphate/nitro-blue tetrazolium (BCIP/NBT) -chromogen substrate solution (Vector; bioZol; Germany; order number: Vec-SP-5020). Western blots were scanned and quantified using the GelScan V6.0 1D Analysis Software (SERVA, Electrophoresis GmbH, Heidelberg, Germany). Band intensities of Sirt1 and RXRα were normalized with band intensities of GAPDH. The blots were repeated three times. 5. Statistics SPSS Statistics 25 was used for data collection, processing and analysis. The Wilcoxon test was used for the evaluation of Sirt1, RXR, and GAPDH values between related groups. Spearman’s test was applied to compare the IRS of Sirt1 and RXR staining in the ovarian cancer patients. Survival rates were shown by Kaplan-Meier curves. P-value <0.05 was considered as statistically significant. Results 1. Correlation of RXRα and Sirt1 expression with Clinical and Pathological Data Sirt1 and RXRα expression was analyzed in 123 cases of ovarian cancer (110 serous and 13 mucinous cases) ( table 1 ). Sirt1 expression was distinguished into cytoplasmic and nuclear staining ( table 4 ). Cytoplasmic and nuclear Sirt1 expression was detectable in 115 cases (93.5%). 8 cases (6.5) did not express Sirt1 in the cytoplasm and the nucleus. Median IRS was 4. In the examined subcategories (mucinous, serous, high grade, low grade and different FIGO stages; table 4; figure 1A-D ) the median IRS was also 4 for both, nuclear and cytoplasmic expression. No significant differences regarding histological subtype (p=0.915), FIGO stage (p=0.568) or grading (p=0.076) in cytoplasmic as well as in nuclear expression (histology: p=0.639; FIGO: p=0.408; Grading: p=0.514) were found ( table 4 ). High cytoplasmic Sirt1 expression (IRS≥4) was detectable in 75 cases (61.0%), 48 cases had an IRS<4 (low IRS; 39.0%). Increased nuclear expression with an IRS ≥4 was found in 82 cases (high; 66.7%). 41 cases had a nuclear Sirt1 IRS smaller than 4 (low; 33.3%). A total of 114 cases of ovarian cancer expressed RXRα (see table 4 ) in the nucleus with a median of 2, 4 did not express RXRα at all and 5 cases were not evaluable. Cytoplasmic RXRα staining was not detectable. High RXRα expression (IRS ≥3) was detectable in 44 cases (35.7%) compared to low expression (IRS<3) in 74 cases (60.2%). Analysis of the correlation between RXRα expression and histopathological parameters revealed: median IRS in serous specimens was 2 (SD+/-0.147) compared to a median IRS of 3.5 in mucinous carcinomas (SD +/- 0.499; p=0.424; table 4 ; figure 1 E-G ). Regarding the grading and FIGO, the median IRS was 2 in high grade and low grade cancers (p=0.309) as well as in different FIGO stages (p=0.405). A significant positive correlation between nuclear Sirt1 and RXRα in IRS staining was detected using Spearman’s test (p=0.006; table 5 ). Table 4. Expression profile of RXR α and Sirt1 staining regarding clinical and pathological characteristics Sirt1 cytoplasm Sirt1 nucleus RXRα Nucleus Median (+/- SD) p Median (+/- SD) p Median (+/- SD) p Histology serous mucinous 4 (+/- 1.94) 4 (+/- 3.23) 0.915 4 (+/- 1.81) 4 (+/- 2.39) 0.639 2 (+/- 0.15) 3,5 (+/- 0.50) 0.424 FIGO I/II III/IV 4 (+/- 2.43) 4 (+/- 1.98) 0.568 4 (+/- 1.78) 4 (+/- 1.91) 0.408 2 (+/- 1.48) 2 (+/- 1.55) 0.405 Grading G1/G2 G3 4 (+/- 2.20) 4 (+/- 1.50) 0.076 4 (+/- 1.96) 4 (+/- 1.82) 0.514 2 (+/- 1.394) 2 (+/- 1.773) 0.309 Table 5. Spearmann’s Correlation analysis between SIRT 1 and RXR α . Sirt1nucleus Sirt1cytoplasm RXRα Correlation coefficient -0.259 -0.163 p 0.006 0.085 Significant results (p<0.05) are shown in bold. Table 6. Multivariate analysis Covariate Coefficient (Bi) Exp(B) 95%CI p-value Lower upper Subtype 0.109 1.115 0.642 1.937 0.699 FIGO 1.327 3.771 1.956 7.271 0.000 Grade (I/II vs. III/IV) -0.604 0.547 0.355 0.843 0.006 Age (<60 vs. ≥60 years) 0.359 1.432 0.944 2.170 0.091 Sirt1cytoplasm 0.004 1.004 0.868 1.161 0.959 Sirt1nucleus 0.035 0.965 0.821 1.135 0.670 RXRα nucleus -0.096 0.908 0.908 1.057 0.213 Significant results (p<0.05) are shown in bold. Prognostic impact of Sirt1-cytoplasm (p=0.959), Sirt1-nucleus (p=0.670) and RXRα nucleus (p=0.213) alone was not significant ( table 6 ). As shown in the Kaplan-Meier curve ( figure 2 ), co-expression of Sirt1 and nuclear RXRα were associated with significant longer survival time after diagnosis in advanced tumor stages (FIGO III/IV). This is significant for cytoplasmic Sirt1 expression (p= 0.026; figure 2a ) as well as for nuclear Sirt1 expression (p=0.041; figure 2b ). 2. Correlation of Resveratrol to apoptosis of ovarian cancer Results of the MTT assay showed that viability was decreased in all cell-lines (A2780, cis A2780 and UWB1.289) after stimulation with resveratrol (RSV). This effect was dose-dependent ( figure 3 ). Cell apoptosis, measured via BrdU ELISA, indicated that apoptotic features were obviously improved in 100μM RSV treated cells ( figure 4 , p<0.003), meaning that apoptosis was increased. Furthermore, cell morphology observation showed that apoptotic markers (the brown cytoplasm, marked by M30) were increased in 100μM RSV treated cells ( figure 5; p<0.05). After RSV treatment, the percentage of TUNEL stained cells increased, meaning that apoptosis rate increased ( figure 6; p=0.043). 3. The relationship between Sirt1 and RXRα We tested the involvement of Sirt1 and RXRα in RSV-induced apoptosis in ovarian cancer cell lines. Sirt1 expression was significant lower in A2780 cells treated with resveratrol 100µM (p=0.025; 50µM: p= 0.208; figure 7A ) compared to its control. No significant difference in the Sirt1 expression after RSV-treatment was found in cisA2780 (p=0.327 and 0.069; figure 7B ) and UWB1.289 (p=0.401 and 0.575; figure 7C ). RXRα expression was significantly increased in cisA2780 cells treated with RSV 50µM and RSV 100 µM in comparison with the control on protein level (p =0.012 and 0.017; figure 7E ). In A2780 (p=0.208 and 0.069) and UWB1.289 cells (p=0.093 and 0.069) treated with RSV 50µM or RSV 100µM for 24 h, no significant differences were found compared to their controls ( figure 7D and F ). Expressions were analyzed by western blot ( figure 7G ). Discussion Our report shows that the expression of nuclear RXRα and Sirt1 in advanced ovarian cancer is significantly associated with longer overall survival. Resveratrol could reduce the proliferation and even increase apoptosis of ovarian cancer cells. On protein level, resveratrol (100µM, 24h) upregulated the expression of RXRα in the carboplatin-resistant cell-line cisA2780 and downregulated Sirt1 expression in A2780. Current studies focus on RSV, a naturally plant polyphenol which is able to inhibit cell growth. Furthermore, it was shown to enhance chemo-sensitivity and to stop cancer invasion (7). Additionally, RSV was observed to induce apoptosis in ovarian cancer cells (26, 27). Pizarro et al. reported that 100 µM RSV reduced cell viability and caused apoptosis after 24 h of treatment in neuroblastoma cells. In our experiment, we used the same concentration and treatment. We could confirm that RSV partially blocked cell proliferation and induced apoptosis in all examined ovarian cancer cell lines. The effect of apoptosis seems to be synergistic to cisplatin (28). In addition, recent studies have demonstrated that RSV (200 μM/48 h) promoted an excessive cellular ROS (2-3 times) production which induced cellular death (29). With reference to this study, it may also be of significance that the used concentrations were higher and time of RSV-exposition was longer (200µM-500µM, 48hours) compared to our study design. Sirt1, a member of the Sirtuin-family, is a NADP-dependent histone deacetlyase and has a conserved catalytic core domain. Sirt1 regulates cellular defense and cell fate (15-20). It has been considered to act dualistically either suppressing or promoting cancer, depending on the temporal and special distribution of different Sirt1 upstream and downstream factors (30). As Sirt1 is described to induce chemo-resistance and to be associated with poor prognosis in ovarian cancer (31-34), we intended to analyze treatment with RSV in regard to Sirt1. Exposure to RSV was correlated with decreased Sirt1 expression in mucinous ovarian cancer cell-lines (A2780). Pizarro et al. determined that the decrease of Sirt1 stimulated by RSV is not responsible for apoptosis induction (35). Based on these results, Sirt1 inhibitors could not change cell viability or apoptosis rates (35). Bjorklund et al. showed that the RSV induced potentiation of platinum drugs in ovarian cancer was not correlated to the Sirt1 1 level by using RSV-concentrations up to 40µM (36). Our results contrast these findings. However, in our experiments higher concentrations of RSV were used. Nevertheless, these findings seem not to be transferable to all ovarian cancer cells, as RSV did not decrease Sirt1 expression in carboplatin-resistant cell lines. This finding has to be explored more accurately in further experiments since this patient group still suffers from very poor survival rates. Sirt1 affects many nuclear receptors. Some of them, for example VDR, need the RXR for dimerization (37-39). RXR plays a critical role in mediating ovarian cancer growth suppression (40). Recent studies have demonstrated that RSV can either bind to RXR directly, or modulate RXR dimerization (9). Wang et al. also reported that RARα/RXR synergism prompt apoptosis and dampened cell proliferation (41). Further reports showed that overexpression of RXRα could promote tumor growth by interacting with tumor necrosis factor-alpha-induced phosphoinositide 3-kinase and NF-κB signal transduction pathways (42). In addition, a recent study showed that the "rexinoid apoptosis" involves activation of both iNOS and eNOS by RXR-PPARgamma, resulting in the production of apoptogenic NO, which induced cell apoptosis (43). In the present study, we evaluated RXRα inhibited resveratrol-stimulated apoptosis of ovarian cancer cells. Our results suggest that RXRα could play an important role in the regulation of apoptosis in human ovarian cancer. Increased expression of RXRα and Sirt1 was associated with increased survival rates in advanced stages of ovarian cancer compared. Even though Sirt1 is described to be associated with poor prognosis in ovarian cancer, its interaction with RXRα seem to neutralize this effect. RSV seems to be an excellent candidate for potentiation of platinum treatment and to induce apoptosis in ovarian cancer. Nevertheless, these findings have to be confirmed in a larger number of specimens. Therefore, further investigations focusing on RSV and its role in anticancer effect in combination with platinum is warranted. Conclusion In conclusion, we observed that the combination of nucleus RXRα and Sirt1 expression was correlated with increased overall survival in late stage ovarian cancer. RSV, which induces apoptosis and decreases proliferation in human ovarian cancer cell lines, was associated with decreased expression of Sirt1 in mucinous ovarian cancer and increased expression of RXRα in mucinous, carboplatin resistant ovarian cancer cells. Novel strategies should be developed in order to improve the understanding of drug resistance mechanisms and to improve medical treatment. Undoubtedly, new studies of ovarian cancer for efficient, rapid and effective treatments are required. Abbreviations BrdU Bromodeoxyuridine IRS Immunoreactivity score PPAR α Peroxisome proliferator-activated receptor alpha RSV Resveratrol RXR Retinoid X receptor Sirt1 Sirtuin 1 TUNEL Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling VDR Vitamin D receptor Declarations Author Contributions: FChen: performed the experiments, analyzed and interpreted the data and wrote the manuscript T Kolben: revised the manuscript S Meister: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. B Czogalla: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. T M. Kolben: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. A Hester: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. A Burges: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. F Trillsch: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. E Schmoeckel: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. D Mayr: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. A Mayerhofer: conceived and designed the experiments. S Mahner: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript. U Jeschke: conceived and designed the experiments, revised the manuscript. S Beyer: analyzed and interpreted the data and wrote the manuscript. All authors have read and approved the manuscript. This study is part of the doctoral thesis of Fangfang Chen. Funding: Fangfang Chen was a recipient of a scholarship from China Scholarship council. The sponsors did not participate in the study design, data analysis or the manuscript writing. AM & DM are supported by a grant from the German Israeli Foundation GIF I-1417-201.2/2017. The funders did not have any influence neither on the design of the experiments nor on the results. Compliance with ethical standards: Conflicts of interests T. Kolben: holds stock of Roche, relative employed at Roche. T.M.Kolben: holds stock of Roche, employed at Roche. A. Burges: receives advisory board and honoraria from AstraZeneca, Roche, and Tesaro. F. Trillsch: declares Research support, advisory board, honoraria and travel expenses from AstraZeneca, Clovis, Medac, PharmaMar, Roche, Tesaro. S.Mahner: receives Research support, advisory board, honoraria and travel expenses from AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro¸ All other authors declare that they have no conflict of interest. Ethics approval: All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethics Committee of the Ludwig-Maximilians University of Munich (reference number 227-09 and 18-392). Informed consent: The study was approved by the ethics committee of the Ludwig-Maximilians University Munich (reference number: 048-08; 2008). Patient data were anonymized. Consent to participate: not applicable as all data are anonymized Consent to publish: not applicable as all data are anonymized References Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pineros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53. Mor G, Alvero A. The duplicitous origin of ovarian cancer. Rambam Maimonides Med J. 2013;4(1):e0006. Luvero D, Plotti F, Aloisia A, Montera R, Terranova C, Carlo De Cicco N, et al. Ovarian cancer relapse: From the latest scientific evidence to the best practice. Crit Rev Oncol Hematol. 2019;140:28-38. Allemani CW, H.K.; Carreira, H.; Harewood, R.; Spika, D.; Wang, X.S.; Bannon, F.; Ahn, J.V.; Johnson, C.J.; Bonaventure, A.; et al. Global surveillance of cancer survival 1995–2009: Analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet 2015;385:977–1010. Vaughan S, Coward JI, Bast RC, Jr., Berchuck A, Berek JS, Brenton JD, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer. 2011;11(10):719-25. Han Y, Jo H, Cho JH, Dhanasekaran DN, Song YS. Resveratrol as a Tumor-Suppressive Nutraceutical Modulating Tumor Microenvironment and Malignant Behaviors of Cancer. Int J Mol Sci. 2019;20(4). Mikami S, Ota I, Masui T, Uchiyama T, Okamoto H, Kimura T, et al. Resveratrolinduced REG III expression enhances chemo and radiosensitivity in head and neck cancer in xenograft mice. Oncol Rep. 2019;42(1):436-42. Engelke LH, Hamacher A, Proksch P, Kassack MU. Ellagic Acid and Resveratrol Prevent the Development of Cisplatin Resistance in the Epithelial Ovarian Cancer Cell Line A2780. J Cancer. 2016;7(4):353-63. Dampf Stone A, Batie SF, Sabir MS, Jacobs ET, Lee JH, Whitfield GK, et al. Resveratrol potentiates vitamin D and nuclear receptor signaling. Journal of cellular biochemistry. 2015;116(6):1130-43. Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990;345(6272):224-9. Fitzgibbon J, Gillett GT, Woodward KJ, Boyle JM, Wolfe J, Povey S. Mapping of RXRB to human chromosome 6p21.3. Ann Hum Genet. 1993;57(3):203-9. Almasan A, Mangelsdorf DJ, Ong ES, Wahl GM, Evans RM. Chromosomal localization of the human retinoid X receptors. Genomics. 1994;20(3):397-403. Krezel W, Ruhl R, de Lera AR. Alternative retinoid X receptor (RXR) ligands. Mol Cell Endocrinol. 2019;491:110436. Deuster E, Jeschke U, Ye Y, Mahner S, Czogalla B. Vitamin D and VDR in Gynecological Cancers-A Systematic Review. Int J Mol Sci. 2017;18(11). Qu Y, Zhang J, Wu S, Li B, Liu S, Cheng J. SIRT1 promotes proliferation and inhibits apoptosis of human malignant glioma cell lines. Neurosci Lett. 2012;525(2):168-72. Srisuttee R, Koh SS, Malilas W, Moon J, Cho IR, Jhun BH, et al. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis. Biochem Biophys Res Commun. 2012;429(1-2):45-50. Saunders LR, Verdin E. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene. 2007;26(37):5489-504. Vazquez MJ, Velasco I, Tena-Sempere M. Novel mechanisms for the metabolic control of puberty: implications for pubertal alterations in early-onset obesity and malnutrition. J Endocrinol. 2019. Ren Z, He H, Zuo Z, Xu Z, Wei Z, Deng J. The role of different SIRT1-mediated signaling pathways in toxic injury. Cell Mol Biol Lett. 2019;24:36. Vargas-Ortiz K, Perez-Vazquez V, Macias-Cervantes MH. Exercise and Sirtuins: A Way to Mitochondrial Health in Skeletal Muscle. Int J Mol Sci. 2019;20(11). Lin Z, Fang D. The Roles of SIRT1 in Cancer. Genes & Cancer. 2013;4(3-4):97-104. Jang KY, Kim KS, Hwang SH, Kwon KS, Kim KR, Park HS, et al. Expression and prognostic significance of SIRT1 in ovarian epithelial tumours. Pathology. 2009;41(4):366-71. Buhrmann C, Shayan P, Popper B, Goel A, Shakibaei M. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells. Nutrients. 2016;8(3):145. Oka S, Zhai P, Yamamoto T, Ikeda Y, Byun J, Hsu CP, et al. Peroxisome Proliferator Activated Receptor-alpha Association With Silent Information Regulator 1 Suppresses Cardiac Fatty Acid Metabolism in the Failing Heart. Circ Heart Fail. 2015;8(6):1123-32. Regnault TR, Zhao L, Chiu JS, Gottheil SK, Foran A, Yee SP. Peroxisome Proliferator-Activated Receptor -beta/delta, -gamma Agonists and Resveratrol Modulate Hypoxia Induced Changes in Nuclear Receptor Activators of Muscle Oxidative Metabolism. PPAR Res. 2010;2010:129173. Kim TH, Park JH, Woo JS. Resveratrol induces cell death through ROSdependent downregulation of Notch1/PTEN/Akt signaling in ovarian cancer cells. Mol Med Rep. 2019;19(4):3353-60. Said RS, Mantawy EM, El-Demerdash E. Mechanistic perspective of protective effects of resveratrol against cisplatin-induced ovarian injury in rats: emphasis on anti-inflammatory and anti-apoptotic effects. Naunyn Schmiedebergs Arch Pharmacol. 2019. Hu S, Li X, Xu R, Ye L, Kong H, Zeng X, et al. The synergistic effect of resveratrol in combination with cisplatin on apoptosis via modulating autophagy in A549 cells. Acta Biochim Biophys Sin (Shanghai). 2016;48(6):528-35. Rodriguez-Enriquez S, Pacheco-Velazquez SC, Marin-Hernandez A, Gallardo-Perez JC, Robledo-Cadena DX, Hernandez-Resendiz I, et al. Resveratrol inhibits cancer cell proliferation by impairing oxidative phosphorylation and inducing oxidative stress. Toxicol Appl Pharmacol. 2019;370:65-77. Fang Y, Nicholl MB. Sirtuin 1 in malignant transformation: friend or foe? Cancer Lett. 2011;306(1):10-4. Shuang T, Wang M, Zhou Y, Shi C. Over-expression of Sirt1 contributes to chemoresistance and indicates poor prognosis in serous epithelial ovarian cancer (EOC). Med Oncol. 2015;32(12):260. Liang XJ, Finkel T, Shen DW, Yin JJ, Aszalos A, Gottesman MM. SIRT1 contributes in part to cisplatin resistance in cancer cells by altering mitochondrial metabolism. Mol Cancer Res. 2008;6(9):1499-506. Akhter MZ, Sharawat SK, Kumar V, Kochat V, Equbal Z, Ramakrishnan M, et al. Aggressive serous epithelial ovarian cancer is potentially propagated by EpCAM(+)CD45(+) phenotype. Oncogene. 2018;37(16):2089-103. Asaka R, Miyamoto T, Yamada Y, Ando H, Mvunta DH, Kobara H, et al. Sirtuin 1 promotes the growth and cisplatin resistance of endometrial carcinoma cells: a novel therapeutic target. Laboratory investigation; a journal of technical methods and pathology. 2015;95(12):1363-73. Pizarro JG, Verdaguer E, Ancrenaz V, Junyent F, Sureda F, Pallas M, et al. Resveratrol inhibits proliferation and promotes apoptosis of neuroblastoma cells: role of sirtuin 1. Neurochem Res. 2011;36(2):187-94. Bjorklund M, Roos J, Gogvadze V, Shoshan M. Resveratrol induces SIRT1- and energy-stress-independent inhibition of tumor cell regrowth after low-dose platinum treatment. Cancer Chemother Pharmacol. 2011;68(6):1459-67. Germain P, Staels B, Dacquet C, Spedding M, Laudet V. Overview of nomenclature of nuclear receptors. Pharmacol Rev. 2006;58(4):685-704. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83(6):835-9. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995;83(6):841-50. Wu S, Zhang D, Zhang ZP, Soprano DR, Soprano KJ. Critical role of both retinoid nuclear receptors and retinoid-X-receptors in mediating growth inhibition of ovarian cancer cells by all-trans retinoic acid. Oncogene. 1998;17(22):2839-49. Wang L, DeMarco SS, Peaks MS, Maiorana-Boutilier AL, Chen J, Crouch MJ, et al. RARalpha/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines. Exp Dermatol. 2017;26(11):1004-11. Zhang X, Zhou H, Su Y. Targeting truncated RXRalpha for cancer therapy. Acta Biochim Biophys Sin (Shanghai). 2016;48(1):49-59. Shankaranarayanan P, Rossin A, Khanwalkar H, Alvarez S, Alvarez R, Jacobson A, et al. Growth factor-antagonized rexinoid apoptosis involves permissive PPARgamma/RXR heterodimers to activate the intrinsic death pathway by NO. Cancer Cell. 2009;16(3):220-31. Cite Share Download PDF Status: Published Journal Publication published 06 Dec, 2021 Read the published version in Archives of Gynecology and Obstetrics → Version 1 posted Reviews received at journal 07 Mar, 2021 Reviewers invited by journal 05 Mar, 2021 Editor invited by journal 31 Jan, 2021 First submitted to journal 03 Jan, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-198498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":30239251,"identity":"3613d5c7-9246-4be6-bd0f-6fdb9ea754d0","order_by":0,"name":"Fangfang Chen","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Chen","suffix":""},{"id":30239252,"identity":"45e1269c-d84e-4525-8e4a-98b95df14d89","order_by":1,"name":"Thomas Kolben","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Kolben","suffix":""},{"id":30239253,"identity":"9e151720-9203-4f9b-9368-9920d50c319d","order_by":2,"name":"Sarah Meister","email":"","orcid":"","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Meister","suffix":""},{"id":30239254,"identity":"be08c775-613d-45ea-bdf3-95ec5edc733c","order_by":3,"name":"Bastian Czogalla","email":"","orcid":"https://orcid.org/0000-0001-6589-4736","institution":"University Hospital, LMU Munich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bastian","middleName":"","lastName":"Czogalla","suffix":""},{"id":30239255,"identity":"4d5bf898-2599-4cec-b4e3-47fdd4dc859b","order_by":4,"name":"Theresa M Kolben","email":"","orcid":"","institution":"University Hospital, LMU 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Beyer","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8603-6714","institution":"University Hospital, LMU Munich","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Susanne","middleName":"","lastName":"Beyer","suffix":""}],"badges":[],"createdAt":"2021-02-01 14:25:54","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-198498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-198498/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00404-021-06262-w","type":"published","date":"2021-12-06T08:56:44+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":9850155,"identity":"b1dab010-386c-4996-a7f7-b2f4ec0a21d7","added_by":"auto","created_at":"2021-06-01 18:54:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":181360,"visible":true,"origin":"","legend":"Representative immunohistochemistry images of Sirt1 and RXRα in the same view of ovarian cancer samples. a: Sirt1 expression in serous ovarian cancer on a TMA (tissue micro array) with a 2.5 magnification and an insert at 10x magnification. b: Sirt1 expression in mucinous ovarian cancer on a TMA with a 2.5 magnification and an insert at 10x magnification. C and d: boxplot: Sirt1 expression in the nucleus (c; p=0.639) and in the cytoplasm (d; p=0.915) with a median IRS of 4 in mucinous and serous ovarian carcinoma. e: RXRα expression in serous ovarian cancer with a 10x magnification and an insert at 25x magnification. f: RXRα expression in mucinous ovarian cancer with a 10x magnification and an insert at 25x magnification. g: boxplot: RXRα expression with a median IRS of 2 and 3.5 in mucinous and serous ovarian carcinoma on slides (p=0.424)","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/97e3e2eaf347330972f2614a.jpg"},{"id":9850152,"identity":"dbb4e020-c3cd-4e39-a4a2-95c96cbae29f","added_by":"auto","created_at":"2021-06-01 18:54:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56348,"visible":true,"origin":"","legend":"Overall survival in patients with cytoplasmic Sirt1expression (a; p=0.026) and overall survival in patients with nuclear RXRα expression (b; p=0.041). Statistical significance for all tests was assumed for p \u003c 0.05","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/62f39ac995b83af4a3cfa0de.jpg"},{"id":9849502,"identity":"5b4cc9b3-2e1f-4a91-b570-f77460d9584c","added_by":"auto","created_at":"2021-06-01 18:51:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56043,"visible":true,"origin":"","legend":"Cytotoxity of RSV: ovarian cancer cell-lines were treated with RSV (50µM and 100µM) for 24 hour. The cell viability was determined with MTT assay. a: A2780 (*A2780 control vs. RSV 40µM p=0.0032; **A2780 control vs RSV 60µM p=0.002; *** A2780 control vs. RSV 80µM p=0.0004; ****A2780 control vs. RSV 100µM p\u003c0.0001), b: cisA2780 (*cisA2780 control vs RSV 80µM/RSV 100µM p\u003c0.0001) and C: UWB1.289 ( *UWB1.289 control vs. RSV 20µM p=0.0326; **UWB1.289 control vs. RSV 40µM p=0.0013; ****UWB1.289 control vs. RSV 50/60/80/100µM p\u003c0.0001). The data are presented as the means ± SEM. N=3. *p \u003c 0.05","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/52d08ea2e1b7b44813c6ccf0.jpg"},{"id":9849636,"identity":"90d453f9-8c3e-482c-9918-d40ba357efea","added_by":"auto","created_at":"2021-06-01 18:51:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":41592,"visible":true,"origin":"","legend":"Cells were treated with RSV (50µM and 100 µM) for 24 h and BrdU (final concentration is 10µM) was added. BrdU in corporation was determined by measuring the absorbance at 450nm.a: A2780 (**A2780 control vs. RSV 50µM p=0.0019; ***A2780 control vs. RSV 100µM p=0.003); b: cisA2780 (**cisA2780 control vs. RSV 50/100µM p\u003c0.0001); c: UWB1.289 (***UWB1.289 control vs. RSV 50µM p=0.0007; ***UWB1.289 control vs. RSV 100µM p=0.0003). Representative results are presented as the means ± SEM. (N=3) *p \u003c 0.05","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/3f93178e89d7d36a6b3fa62e.jpg"},{"id":9849648,"identity":"8a0649c1-17c7-4a8d-a331-22fe37e7de1b","added_by":"auto","created_at":"2021-06-01 18:51:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":92398,"visible":true,"origin":"","legend":"The effects of RSV-treatment and M30 identification on A2780 (a-c), cisA2780 (d-f) and UWB1.289 (g-i) cells with 50 µM and 100 µM resveratrol for 24 h. (N=5). Apoptosis rates in dependent of RSV concentration are shown in a boxplot (j). The data are presented as means ± SEM. *p \u003c 0.05","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/faa9ed876b798276748b5754.jpg"},{"id":9849633,"identity":"44259f4b-ae59-44cc-a69f-9c4ce8a15278","added_by":"auto","created_at":"2021-06-01 18:51:46","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":158275,"visible":true,"origin":"","legend":"The apoptosis of A2780 (a-c), cisA2780 (d-f) and UWB1.289 (g-i) were determined by TUNEL assay. All images are at 2.5× magnification with an insert at 10× magnification. Apoptosis rates in dependence of RSV concentration are shown in a boxplot (J).The data are presented as means ± SEM. (N=5) *p= 0.043","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/3b5119f2c3f44cc7e1b0282d.jpg"},{"id":9849499,"identity":"968146ed-0762-414e-96e5-86b7f43b0daf","added_by":"auto","created_at":"2021-06-01 18:51:43","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":88084,"visible":true,"origin":"","legend":"Ovarian cancer cells were treated with resveratrol 50µM and resveratrol 100µM for 24 hours. Expression of Sirt1 in A2780 cells (a), cisA2780 (b) and UWB1.289 (c) cell-lines. RXRα expression in A2780 (d), cisA2780 (e) and UWB1.289 (f) cell-lines after RSV treatment. Finally, expressions were analyzed by western blotting (g). Representative results are presented as the means ± SEM. *p\u003c 0.05","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/5713e8ffd6e25fd52c1bcbaf.jpg"},{"id":16197057,"identity":"48793839-2ed8-488b-b855-7b529e4f218f","added_by":"auto","created_at":"2021-12-06 08:56:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1032703,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-198498/v1/a13dd539-82e4-416c-9037-63a9e3478c4d.pdf"}],"financialInterests":"","formattedTitle":"The role of Resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is the most lethal gynecologic malignancy and the eighth leading cause of cancer-related mortality among women worldwide (1, 2). The overall 5-years survival rates have barely improved over the past few decades remaining at 40-45% for advanced stages and around 80% of patients progressing within 18 months (3, 4). The poor prognosis of ovarian cancer is mainly related to late-stage diagnosis and the rapid development of resistance to current chemotherapy regimens (2, 5) .\u003cbr /\u003e Resveratrol (RSV), a naturally plant polyphenol originates from grapes and berries, has been proven to alter metabolism in cancer (3) and to regulate tumor microenvironment (6). The regulatory effect of RSV on cancer is complex: besides inhibition of cell growth, RSV is also involved in enhancing chemo-sensitivity and blocking the cancer invasion of cancer cells in vitro (7). In addition, it has been confirmed that\u0026nbsp; RSV improves the efficacy of cisplatin in ovarian cancer (8) .\u003cbr /\u003e RSV is also able to modulate vitamin-D-receptor (VDR)-signaling and it can induce dimerization of VDR with one of its partners, the nuclear retinoid X receptor (RXR) (9). Three retinoic X receptors are known: RXR\u0026alpha; (10) , RXR\u0026beta; (11)\u0026nbsp; and RXR\u0026gamma; (12). Although the distribution of RXR subtypes is different, their functions are similar: modulating\u0026nbsp; gene expression they control numerous functions by dimerization with other nuclear hormone receptors, contributing thereby to activities of different cell fates (13). As the VDR is known to be involved in gynecologic cancers, the interaction of VDR with the RXR implies that also the RXR may have a modulating effect on gynecological cancers (14).\u003cbr /\u003e Sirtuin1 (Sirt1) is a NADP-dependent histone deacetylase, which regulates cellular metabolism and cellular pathways (15-20). Its role regarding cancer progression remains controversial as it can act as tumor suppressor or as tumor promotor (21). In ovarian cancer Sirt1 overexpression was correlated with improved overall survival (22). By complex mechanisms (via interaction of NF-kB) Sirt1 can be influenced by RSV: it can be targeted by RSV, which can lead to suppression of tumorigenesis in colorectal carcinoma (23).\u003c/p\u003e\n\u003cp\u003eRecently, RSV was described to activate RXR and stimulate Sirt1 in mammalians (9).\u0026nbsp; The competitive binding of RXR and Sirt1 to PPAR\u0026alpha; (a peroxisome proliferator-activated receptor) could be due to structural similarity between these proteins. Sirt1 binds to PPAR\u0026alpha; more strongly than RXR\u0026alpha; suggesting that Sirt1 interacts with PPAR\u0026alpha; directly rather than RXR (24).\u0026nbsp; Regnault et al. noticed that RSV induced Sirt1 and RXR in muscle hypoxia (25).\u0026nbsp; Unfortunately, the effect of RSV on Sirt1 and RXR expression in ovarian cancer cells has not been well documented until now.\u003c/p\u003e\n\u003cp\u003eIn the present study, we immunohistochemically examined the expression of RXR\u0026alpha; and Sirt1 in mucinous and serous ovarian cancer and analyzed the relationship between RSV, RXR\u0026alpha; and Sirt1 in ovarian cancer in vitro. Considering the potential medical role of resveratrol in ovarian cancer, we evaluated the effects of RSV by proliferation and apoptosis experiments. In addition, we investigated the mechanism of RSV resistance to apoptosis in ovarian cancer cell lines. \u0026nbsp;This study aimed to analyze RXR\u0026alpha; and Sirt1 as potential therapeutic targets in ovarian cancer.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003e1. Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study we used ovarian cancer tissue samples of 123 patients who underwent surgery for ovarian cancer from 1990 to 2002 at the Department of Gynecology and Obstetrics, Ludwig-Maximilians-University of Munich, Germany. Patients who underwent surgery due to serous or mucinous ovarian cancer were included while other histological subtypes were excluded due to low number. The median age was 59 years (range 20\u0026ndash;88 years) and median overall survival was 2.67 years. The distribution of clinic-pathological variables can be seen in\u003cstrong\u003e table 1\u003c/strong\u003e. As positive controls for immunohistochemical staining, we utilized palatine tonsil for Sirt1 staining and first trimester placenta for RXR staining, both received from the Department of Obstetrics and Gynecology of the Ludwig-Maximilians-University of Munich. Clinical and follow-up data for statistical analyses were provided by the Munich cancer registry and retrieved from medical records.\u003c/p\u003e\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003eTable 1. Patients\u0026rsquo; characteristics.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 3.2e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 18.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-image: initial;border-left: none;padding: 0in 5.4pt;height: 18.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1pt solid windowtext;border-right: none;padding: 0in 5.4pt;height: 18.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 14pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eSubtype\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 14pt;vertical-align: top;\"\u003e\u003cspan style=\"font-size: 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70.85pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e92\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e74.8\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eNA\u0026rsquo;s\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e2\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e01.6\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eGrading\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e1/2\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e76\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e61.8\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e3\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e40\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e32.5\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eNA\u0026rsquo;s\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e7\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e05.7\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eProgression (18 years)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eNo progression\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eProgression\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eNA\u0026rsquo;s\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e101\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e21\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e82.1%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e17.1%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e0.1%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 133pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 55.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eOverall-Survival (18 years)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eRight censured\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eDied\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eNA\u0026rsquo;s\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 70.85pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;height: 55.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e38\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e84\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e1\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 120.5pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;height: 55.05pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e30.1%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e68.3%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e0.1%\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif; font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e2. Ethics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll ovarian cancer specimens had been collected for histopathological diagnostics during surgery. They were no longer used for clinical tests. Patients\u0026rsquo; data were anonymized and authors were blinded for clinical information during experimental analyses. The study was conducted in consent to the Declaration of Helsinki and was approved by the local ethics committee of the Ludwig-Maximilians University of Munich (reference number 227-09 and 18-392).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParaffin-embedded slides of 3\u0026micro;m were dewaxed in xylol and washed in 100% ethanol. For inhibition of the endogen peroxidases, tissue samples were incubated in 3% methanol/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and rehydrated in a descending alcohol series. Slides were afterwards heated in a pressure cooker using sodium citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0; containing 0.1\u0026thinsp;M citric acid and 0.1\u0026thinsp;M sodium citrate in distilled water). After cooling and washing in PBS (phosphate-buffered saline), all slides were incubated with blocking solution to avoid non-specific binding of the primary antibodies. Subsequently, the slides were stained with the primary antibodies anti-Sirt1 and anti-RXR\u0026alpha; (\u003cstrong\u003etable 2\u003c/strong\u003e) and incubated. After washing, the secondary complexes of the ABC detection kits were applied following the manufacturer\u0026rsquo;s protocols to detect reactivity. Immunostaining was visualized with the substrate and the chromogen-3, 3\u0026prime;-diaminobenzidine (DAB) for 1\u0026thinsp;min. For exact staining protocol see \u003cstrong\u003etable 2\u003c/strong\u003e.\u003cbr /\u003e For the light microscopy analysis, the semi-quantitative immune-reactive score (IRS) is calculated via the multiplication of optical staining intensity (grades: 0\u0026thinsp;=\u0026thinsp;no, 1\u0026thinsp;=\u0026thinsp;weak, 2\u0026thinsp;=\u0026thinsp;moderate and 3\u0026thinsp;=\u0026thinsp;strong staining) and the percentage range of positive stained cells (0\u0026thinsp;=\u0026thinsp;no staining, 1\u0026thinsp;=\u0026thinsp;\u0026le;10% of the cells; 2\u0026thinsp;=\u0026thinsp;11\u0026ndash;50% of the cells; 3\u0026thinsp;=\u0026thinsp;51\u0026ndash;80% of the cells and 4\u0026thinsp;=\u0026thinsp;\u0026ge;81% of the cells were stained for the antibody, respectively). Palatine tonsil was used as control for Sirt1 and first trimester placenta was used as control for RXR staining.\u003c/p\u003e\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003eTable 2. Antibodies and chemicals used for the immunohistochemistry\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.6e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eanti-Sirt1\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003eanti-RXR\u0026alpha;\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003ePBS\u003csup\u003e3\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003ePBS\u003csup\u003e3\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eBlocking solution\u003csup\u003e4\u003c/sup\u003e: 20 min\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eBlocking solution\u003csup\u003e4\u003c/sup\u003e: 20 min\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eprimary antibody\u003csup\u003e1\u003c/sup\u003e: 1:1000\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eincubation: 16 h, 4◦C min\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eprimary antibody\u003csup\u003e2\u003c/sup\u003e: 1:200\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eincubation: 16 h, 4◦C\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eABC detection kid \u003csup\u003e5\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eABC detection kid \u003csup\u003e5\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eChromogen: DAB \u003csup\u003e6\u003c/sup\u003e (1 min)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 230.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003eChromogen: DAB \u003csup\u003e5\u003c/sup\u003e (1 min)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003csup\u003e1\u003c/sup\u003e anti-Sirt1 rabbit IgG, polyclonal antibody\u003cspan style=\"color:black;\"\u003e, concentration:\u0026nbsp;\u003c/span\u003e1:1000; Atlas Antibody, Sweden;\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003eorder number: SHPA006295. \u003cbr\u003e \u003csup\u003e\u003cspan style=\"color:black;\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"color:black;\"\u003e\u0026nbsp;\u003c/span\u003eanti-RXR\u0026alpha; rabbit IgG, polyclonal antibody, concentration: 1:200; PPMX, Japan; order number: pp-k8508-10. \u003cbr\u003e \u003csup\u003e3\u003c/sup\u003e HRP-Polymer-Kit (mouse/rabbit); Zytomed Systems, Germany; order number: POLHRP-100.\u003cbr\u003e \u003csup\u003e4\u003c/sup\u003e ABC detection kid; Vectastain, USA; order number: AK-6401.\u003cbr\u003e \u003csup\u003e5\u003c/sup\u003e Dulbecco\u0026rsquo;s Phosphate Buffered Saline; Gibco, USA; order number: 14190-094.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e4. Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman ovarian cancer cell lines with different characteristics (A2780, UWB1.289 and cisA2780, see \u003cstrong\u003etable 3\u003c/strong\u003e) were used in the study. The cell lines were ordered from Gibco (see \u003cstrong\u003etable 3\u003c/strong\u003e).\u003c/P\u003e\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; color: black; font-family: Calibri, sans-serif;\"\u003eTable 3. Cell lines\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.6e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003eA2780 \u003csup\u003e1\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003eUWB1.289 \u003csup\u003e2\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003ecisA2780 \u003csup\u003e3\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003eCell type\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eEpithelial ovarian cancer cell\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eEpithelial ovarian cancer cell\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eEpithelial ovarian cancer cell\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003eCharacteristics\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eMucinous\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eSerous brca1-null\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eMucinous, carboplatin-resistant\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cstrong\u003e\u003cspan style=\"color: black;\"\u003eCulture medium\u003c/span\u003e\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eRPMI 1640\u003csup\u003e4\u003c/sup\u003e + 10% FBS \u003csup\u003e5\u003c/sup\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eRPMI 1640\u003csup\u003e4\u003c/sup\u003e + 10% FBS \u003csup\u003e5\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 115.15pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"color: black;\"\u003eRPMI 1640\u003csup\u003e4\u003c/sup\u003e + 10% FBS \u003csup\u003e5\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003csup\u003e\u003cspan style=\"color: black;\"\u003e1/2/3\u0026nbsp;\u003c/span\u003e\u003c/sup\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan style=\"font-size: 15px; color: black; font-family: Calibri, sans-serif;\"\u003eGibco\u003csup\u003e. 4\u003c/sup\u003e Gibco, USA; Order number: 21875-034. \u003csup\u003e5\u003c/sup\u003e Foetal Bovine Serum, biochrom, Germany; order number S0615 \u0026nbsp;\u003c/span\u003e\u003c/p\u003e\u003cp\u003eCell lines were cultured (see \u003cstrong\u003etable 3\u003c/strong\u003e) and seeded into 96-well plates for MTT and BrdU (see below) and 6-well plates for Western blotting. After 20\u0026thinsp;h, cell culture medium was replaced with fresh culture medium with resveratrol (50 and 100\u0026micro;M RSV; Sigma, America; order number: R5010-100MG) for the remaining 24\u0026thinsp;h, which included dimethyl sulfoxide (DMSO; concentration: 0.5%; SERVA, Germany; order number: 20385, 0.5%) as vehicle control.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eELISAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA2780, UWB1.289 and cisA2780 ovarian cancer cells were seeded at the density of 1.5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plates with 200\u0026mu;l medium. After 20 h cells were incubated with 50\u0026micro;M and 100\u0026micro;M of resveratrol for 24\u0026thinsp;h. Untreated control cells were plated in medium only. To each well, 20\u0026mu;g MTT (Sigma, USA; order number: M-5655) were added for 1.5\u0026thinsp;h at 37\u0026deg;C in order to show viability. After removing MTT from the plates, 200\u0026mu;L DMSO were added and mixed thoroughly on the shaker for 5\u0026thinsp;min at room temperature. The optical density was examined at 595\u0026thinsp;nm using Elx800 universal Microplate Reader. Each experiment was carried out in triplicate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMarker of proliferation: BrdU\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA2780, UWB1.289 and cisA2780 ovarian cancer cells were cultured at the density of 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells/well together with various dilutions (50/100\u0026micro;M) of resveratrol in 96-well plates. For the labelling of DNA replication BrdU (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) was added to the culture medium for 2h. The final concentration of BrdU was 10\u0026mu;M. After removal of BrdU by pipette, 200\u0026micro;l/well FixDenat (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) were added and cells were incubated for 30 min at room temperature. Afterwards, FixDenat solution had to be removed thoroughly and 100\u0026micro;l/well anti-BrdU-POD (Bromodeoxyuridine; Roche, Switzerland; order number: 11647229001) working solution were added. Cells were then incubated for approximately 90 min at room temperature and washed 3 times with PBS. 100\u0026micro;/well substrate solution BrdU was added and incubation for 20 minutes was performed. To each well 25\u0026micro;l 1M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were added and the absorbance of the samples was measured by an ELISA reader at 450nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptosis assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptosis was evaluated by measuring the level of caspase-cleaved cytokeratin 18 (M30, Roche, Switzerland; order number: 121140322001). The ovarian cell lines A2780, cisA2780 and UWB1.289 were seeded at a density of 1.0\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e4\u003c/sup\u003e cells/well on 96-well plates in 200\u0026mu;l medium. After 20 h, 50 or 100\u0026micro;M RSV were added and cells were incubated for 24 h. M30 CytoDeath (Roche, Switzerland; order number: 121140322001, dilution 1:1000) was used to detect the apoptotic cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTerminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay has been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis. TUNEL staining was performed to assess in situ DNA fragmentation using a commercial kit (FragELTM DNA Fragmentation Detection Kit, Colorimeric- TdT Enzyme , USA; order number: Qia33-1EA) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell lysates were extracted from A2780, cisA2780 and UWB1.289 cells with radio-immuno-precipitation assay buffer (RIPA, Sigma-Aldrich, St. Luis, USA; order-number: R0278-50ML). For Western blotting, 20\u0026micro;g of cell lysates were first separated in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 10% casein and then incubated with the primary antibodies for 16\u0026thinsp;h at room temperature. We used the same antibodies as for immunohistochemistry (see \u003cstrong\u003etable 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eGAPDH was used as a housekeeping gene and mouse monoclonal anti-GAPDH antibody (GeneTex, America; order number: GTX277408) was diluted 1:1000 in 10% CASEIN (Vector, Germany; order number: ZE0925). Afterwards, the membrane was incubated with the goat-anti-rabbit secondary antibody (Vector; bioZol; Germany; order number: VEC-BA-1000, dilution 1:1000) conjugated with alkaline phosphatase, and detected with 5-bromo-4-chloro-3\u0026prime;-indolylphosphate/nitro-blue tetrazolium (BCIP/NBT) -chromogen substrate solution (Vector; bioZol; Germany; order number: Vec-SP-5020). Western blots were scanned and quantified using the GelScan V6.0 1D Analysis Software (SERVA, Electrophoresis GmbH, Heidelberg, Germany). Band intensities of Sirt1 and RXR\u0026alpha; were normalized with band intensities of GAPDH. The blots were repeated three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5.\u0026nbsp;Statistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS Statistics 25 was used for data collection, processing and analysis. The Wilcoxon test was used for the evaluation of Sirt1, RXR, and GAPDH values between related groups.\u0026nbsp; Spearman\u0026rsquo;s test was applied to compare the IRS of Sirt1 and RXR staining in the ovarian cancer patients. Survival rates were shown by Kaplan-Meier curves. P-value \u0026lt;0.05 was considered as statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. Correlation of RXR\u0026alpha; and Sirt1 expression with Clinical and Pathological Data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSirt1 and RXR\u0026alpha; expression was analyzed in 123 cases of ovarian cancer (110 serous and 13 mucinous cases) (\u003cstrong\u003etable 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eSirt1 expression was distinguished into cytoplasmic and nuclear staining (\u003cstrong\u003etable 4\u003c/strong\u003e). Cytoplasmic and nuclear Sirt1 expression was detectable in 115 cases (93.5%). 8 cases (6.5) did not express Sirt1 in the cytoplasm and the nucleus. Median IRS was 4. In the examined subcategories (mucinous, serous, high grade, low grade and different FIGO stages; \u003cstrong\u003etable 4; figure 1A-D\u003c/strong\u003e) the median IRS was also 4 for both, nuclear and cytoplasmic expression. No significant differences regarding histological subtype (p=0.915), FIGO stage (p=0.568) or grading (p=0.076) in cytoplasmic as well as in nuclear expression (histology: p=0.639; FIGO: p=0.408; Grading: p=0.514) were found (\u003cstrong\u003etable 4\u003c/strong\u003e). High cytoplasmic Sirt1 expression (IRS\u0026ge;4) was detectable in 75 cases (61.0%), 48 cases had an IRS\u0026lt;4 (low IRS; 39.0%). Increased nuclear expression with an IRS \u0026ge;4 was found in 82 cases (high; 66.7%). 41 cases had a nuclear Sirt1 IRS smaller than 4 (low; 33.3%).\u003c/p\u003e\n\u003cp\u003eA total of 114 cases of ovarian cancer expressed RXR\u0026alpha; (see \u003cstrong\u003etable 4\u003c/strong\u003e) in the nucleus with a median of 2, 4 did not express RXR\u0026alpha; at all and 5 cases were not evaluable. Cytoplasmic RXR\u0026alpha; staining was not detectable. High RXR\u0026alpha; expression (IRS \u0026ge;3) was detectable in 44 cases (35.7%) compared to low expression (IRS\u0026lt;3) in 74 cases (60.2%).\u0026nbsp; Analysis of the correlation between RXR\u0026alpha; expression and histopathological parameters revealed:\u0026nbsp; median IRS in serous specimens was 2 (SD+/-0.147) compared to a median IRS of 3.5 in mucinous carcinomas (SD +/- 0.499; p=0.424; \u003cstrong\u003etable 4\u003c/strong\u003e; \u003cstrong\u003efigure 1 E-G\u003c/strong\u003e). Regarding the grading and FIGO, the median IRS was 2 in high grade and low grade cancers (p=0.309) as well as in different FIGO stages (p=0.405).\u003c/p\u003e\n\u003cp\u003eA significant positive correlation between nuclear Sirt1 and RXR\u0026alpha; in IRS staining was detected using Spearman\u0026rsquo;s test (p=0.006; \u003cstrong\u003etable 5\u003c/strong\u003e).\u003c/p\u003e\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003eTable 4. Expression profile of RXR\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026alpha;\u003cstrong\u003e\u0026nbsp;and Sirt1 staining regarding clinical and pathological characteristics\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.6e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 99.2pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1 cytoplasm\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 85.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1 nucleus\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 35.45pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 127.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eRXR\u0026alpha; Nucleus\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eMedian (+/- SD)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 56.7pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eMedian (+/- SD)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 42.55pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eMedian (+/- SD)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eHistology\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eserous\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003emucinous\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.94)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 3.23)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 56.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.915\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.81)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 2.39)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 42.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.639\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2 (+/- 0.15)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e3,5 (+/- 0.50)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.424\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eFIGO\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eI/II\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eIII/IV\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 2.43)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.98)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 56.7pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.568\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.78)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.91)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 42.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.408\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2 (+/- 1.48)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2 (+/- 1.55)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.405\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 76.3pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eGrading\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eG1/G2\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eG3\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 2.20)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.50)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 56.7pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.076\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.96)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e4 (+/- 1.82)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 42.55pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.514\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77.95pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2 (+/-\u0026nbsp;1.394)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2 (+/- 1.773)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.309\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eTable 5. Spearmann\u0026rsquo;s Correlation analysis between SIRT 1 and RXR\u003c/strong\u003e\u0026alpha;\u003cstrong\u003e.\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"width: 4.6e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153.5pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1nucleus\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1cytoplasm\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153.5pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eRXR\u0026alpha;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153.5pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eCorrelation coefficient\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: none;border-bottom: none;border-left: none;border-image: initial;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e-0.259\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e-0.163\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 153.5pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003ep\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e0.006\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 153.55pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.085\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003eSignificant results (p\u0026lt;0.05) are shown in bold.\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eTable 6. Multivariate analysis\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: none;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eCovariate\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: none;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eCoefficient (Bi)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: none;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eExp(B)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 113.4pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;95%CI\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.15pt;border-top: 1pt solid windowtext;border-left: none;border-bottom: none;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-right: none;border-bottom: none;border-left: none;border-image: initial;border-top: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003ep-value\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eLower\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1.5pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eupper\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1.5pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSubtype\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.109\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.115\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.642\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.937\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.699\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eFIGO\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.327\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e3.771\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.956\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e7.271\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e0.000\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eGrade (I/II vs. III/IV)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e-0.604\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.547\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.355\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.843\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003e0.006\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eAge (\u0026lt;60 vs. \u0026ge;60 years)\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.359\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.432\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.944\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e2.170\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.091\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1cytoplasm\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.004\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.868\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.161\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.959\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eSirt1nucleus\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.035\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.965\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.821\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.135\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.670\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 125.9pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cstrong\u003eRXR\u0026alpha; nucleus\u003c/strong\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85.05pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e-0.096\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.908\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.908\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 63.75pt;border-top: none;border-left: none;border-bottom: 1pt solid windowtext;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e1.057\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 58.3pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e0.213\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003ctd style=\"border:none;\"\u003e\u003cspan style=\"font-family: Calibri, sans-serif;\"\u003e\u003cspan style=\"font-size: 15px;\"\u003e\u003cbr\u003e\u003c/span\u003e\u003c/span\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size: 15px; font-family: Calibri, sans-serif;\"\u003eSignificant results (p\u0026lt;0.05) are shown in bold.\u003c/span\u003e\u003c/p\u003e\u003cp\u003ePrognostic impact of Sirt1-cytoplasm (p=0.959), Sirt1-nucleus (p=0.670) and RXR\u0026alpha; nucleus (p=0.213) alone was not significant (\u003cstrong\u003etable 6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAs shown in the Kaplan-Meier curve (\u003cstrong\u003efigure 2\u003c/strong\u003e), co-expression of Sirt1 and nuclear RXR\u0026alpha; were associated with significant longer survival time after diagnosis in advanced tumor stages (FIGO III/IV). This is significant for cytoplasmic Sirt1 expression (p= 0.026; \u003cstrong\u003efigure 2a\u003c/strong\u003e) as well as for nuclear Sirt1 expression (p=0.041; \u003cstrong\u003efigure 2b\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.\u0026nbsp;Correlation of Resveratrol to apoptosis of ovarian cancer\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResults of the MTT assay showed that viability was decreased in all cell-lines (A2780, cis A2780 and UWB1.289) after stimulation with resveratrol (RSV). This effect was dose-dependent (\u003cstrong\u003efigure 3\u003c/strong\u003e). Cell apoptosis, measured via BrdU ELISA, indicated that apoptotic features were obviously improved in 100\u0026mu;M RSV treated cells (\u003cstrong\u003efigure 4\u003c/strong\u003e, p\u0026lt;0.003), meaning that apoptosis was increased. Furthermore, cell morphology observation showed that apoptotic markers (the brown cytoplasm, marked by M30) were increased in 100\u0026mu;M RSV treated cells (\u003cstrong\u003efigure 5; \u003c/strong\u003ep\u0026lt;0.05). After RSV treatment, the percentage of TUNEL stained cells increased, meaning that apoptosis rate increased (\u003cstrong\u003efigure 6; \u003c/strong\u003ep=0.043).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. The relationship between Sirt1 and RXR\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe tested the involvement of Sirt1 and RXR\u0026alpha; in RSV-induced apoptosis in ovarian cancer cell lines. Sirt1 expression was significant lower in A2780 cells treated with resveratrol 100\u0026micro;M (p=0.025; 50\u0026micro;M: p= 0.208; \u003cstrong\u003efigure 7A\u003c/strong\u003e) compared to its control. No significant difference in the Sirt1 expression after RSV-treatment was found in cisA2780 (p=0.327 and 0.069; \u003cstrong\u003efigure 7B\u003c/strong\u003e) and UWB1.289 (p=0.401 and 0.575; \u003cstrong\u003efigure 7C\u003c/strong\u003e). RXR\u0026alpha; expression was significantly increased in cisA2780 cells treated with RSV 50\u0026micro;M and RSV 100 \u0026micro;M in comparison with the control on protein level (p =0.012 and 0.017; \u003cstrong\u003efigure 7E\u003c/strong\u003e). \u0026nbsp;In A2780 (p=0.208 and 0.069) and UWB1.289 cells (p=0.093 and 0.069) treated with RSV 50\u0026micro;M or RSV 100\u0026micro;M for 24 h, no significant differences were found compared to their controls (\u003cstrong\u003efigure 7D and F\u003c/strong\u003e). Expressions were analyzed by western blot (\u003cstrong\u003efigure 7G\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur report shows that the expression of nuclear RXR\u0026alpha; and Sirt1 in advanced ovarian cancer is significantly associated with longer overall survival. Resveratrol could reduce the proliferation and even increase apoptosis of ovarian cancer cells. On protein level, resveratrol (100\u0026micro;M, 24h) upregulated the expression of RXR\u0026alpha; in the carboplatin-resistant cell-line cisA2780 and downregulated Sirt1 expression in A2780.\u003c/p\u003e\n\u003cp\u003eCurrent studies focus on RSV, a naturally plant polyphenol which is able to inhibit cell growth. \u0026nbsp;Furthermore, it was shown to enhance chemo-sensitivity and to stop cancer invasion (7). Additionally, RSV was observed to induce apoptosis in ovarian cancer cells (26, 27). Pizarro et al.\u0026nbsp; reported that 100 \u0026micro;M RSV reduced cell viability and caused apoptosis after 24 h of treatment in neuroblastoma cells. In our experiment, we used the same concentration and treatment. We could confirm that RSV partially blocked cell proliferation and induced apoptosis in all examined ovarian cancer cell lines. The effect of apoptosis seems to be synergistic to cisplatin (28). In addition, recent studies have demonstrated that RSV (200 \u0026mu;M/48 h) promoted an excessive cellular ROS (2-3 times) production which induced cellular death (29). With reference to this study, it may also be of significance that the used concentrations were higher and time of RSV-exposition was longer (200\u0026micro;M-500\u0026micro;M, 48hours) compared to our study design.\u003c/p\u003e\n\u003cp\u003eSirt1, a member of the Sirtuin-family, is a NADP-dependent histone deacetlyase and has a conserved catalytic core domain. Sirt1 regulates cellular defense and cell fate (15-20). It has been\u0026nbsp; considered to act dualistically either suppressing or promoting cancer, depending on the temporal and special distribution of different Sirt1 upstream and downstream factors (30). As Sirt1 is described to induce chemo-resistance and to be associated with poor prognosis in ovarian cancer (31-34), we intended to analyze treatment with RSV in regard to Sirt1. Exposure to RSV was correlated with decreased Sirt1 expression in mucinous ovarian cancer cell-lines (A2780). Pizarro et al. determined that the decrease of Sirt1 stimulated by RSV is not responsible for apoptosis induction (35). Based on these results, Sirt1 inhibitors could not change cell viability or apoptosis rates (35). Bjorklund et al. showed that the RSV induced potentiation of platinum drugs in ovarian cancer was not correlated to the Sirt1 1 level by using RSV-concentrations up to 40\u0026micro;M (36). Our results contrast these findings. However, in our experiments higher concentrations of RSV were used. Nevertheless, these findings seem not to be transferable to all ovarian cancer cells, as RSV did not decrease Sirt1 expression in carboplatin-resistant cell lines. This finding has to be explored more accurately in further experiments since this patient group still suffers from very poor survival rates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSirt1 affects many nuclear receptors. Some of them, for example VDR, need the RXR for dimerization (37-39). RXR plays a critical role in mediating ovarian cancer growth suppression (40). Recent studies have demonstrated that RSV can either bind to RXR directly, or modulate RXR dimerization (9). Wang et al. also reported that RAR\u0026alpha;/RXR synergism prompt apoptosis and dampened cell proliferation (41). Further reports showed that overexpression of RXR\u0026alpha; could promote tumor growth by interacting with tumor necrosis factor-alpha-induced phosphoinositide 3-kinase and NF-\u0026kappa;B signal transduction pathways (42). In addition, a recent study showed that the \"rexinoid apoptosis\" involves activation of both iNOS and eNOS by RXR-PPARgamma, resulting in the production of apoptogenic NO, which induced cell apoptosis (43). In the present study, we evaluated RXR\u0026alpha; inhibited resveratrol-stimulated apoptosis of ovarian cancer cells. Our results suggest that RXR\u0026alpha; could play an important role in the regulation of apoptosis in human ovarian cancer.\u003c/p\u003e\n\u003cp\u003eIncreased expression of RXR\u0026alpha; and Sirt1 was associated with increased survival rates in advanced stages of ovarian cancer compared. Even though Sirt1 is described to be associated with poor prognosis in ovarian cancer, its interaction with RXR\u0026alpha; seem to neutralize this effect.\u003c/p\u003e\n\u003cp\u003eRSV seems to be an excellent candidate for potentiation of platinum treatment and to induce apoptosis in ovarian cancer. Nevertheless, these findings have to be confirmed in a larger number of specimens. Therefore, further investigations focusing on RSV and its role in anticancer effect in combination with platinum is warranted.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we observed that the combination of nucleus RXR\u0026alpha; and Sirt1 expression was correlated with increased overall survival in late stage ovarian cancer. RSV, which induces apoptosis and decreases proliferation in human ovarian cancer cell lines, was associated with decreased expression of Sirt1 in mucinous ovarian cancer and increased expression of RXR\u0026alpha; in mucinous, carboplatin resistant ovarian cancer cells. Novel strategies should be developed in order to improve the understanding of drug resistance mechanisms and to improve medical treatment. Undoubtedly, new studies of ovarian cancer for efficient, rapid and effective treatments are required.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eBrdU\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eBromodeoxyuridine\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eIRS\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eImmunoreactivity score\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003ePPAR\u003c/strong\u003e\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003ePeroxisome proliferator-activated receptor alpha\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eRSV\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eResveratrol\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eRXR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eRetinoid X receptor\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eSirt1\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eSirtuin 1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eTerminal deoxynucleotidyl transferase dUTP Nick-End Labeling\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"102\"\u003e\n\u003cp\u003e\u003cstrong\u003eVDR\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"512\"\u003e\n\u003cp\u003eVitamin D receptor\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFChen: performed the experiments, analyzed and interpreted the data and wrote the manuscript\u003c/p\u003e\n\u003cp\u003eT Kolben: revised the manuscript\u003c/p\u003e\n\u003cp\u003eS Meister: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eB Czogalla: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eT M. Kolben: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eA Hester: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eA Burges: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eF Trillsch: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eE Schmoeckel: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eD Mayr: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eA Mayerhofer: conceived and designed the experiments.\u003c/p\u003e\n\u003cp\u003eS Mahner: interpreted the results and read and approved the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eU Jeschke: conceived and designed the experiments, revised the manuscript.\u003c/p\u003e\n\u003cp\u003eS Beyer: analyzed and interpreted the data and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003eThis study is part of the doctoral thesis of Fangfang Chen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Fangfang Chen was a recipient of a scholarship from China Scholarship council. The sponsors did not participate in the study design, data analysis or the manuscript writing. AM \u0026amp; DM are supported by a grant from the German Israeli Foundation GIF I-1417-201.2/2017. The funders did not have any influence neither on the design of the experiments nor on the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. Kolben: holds stock of Roche, relative employed at Roche. T.M.Kolben: holds stock of Roche, employed at Roche. A. Burges: receives advisory board and honoraria from AstraZeneca, Roche, and Tesaro. F. Trillsch: declares Research support, advisory board, honoraria and travel expenses from AstraZeneca, Clovis, Medac, PharmaMar, Roche, Tesaro. S.Mahner: receives Research support, advisory board, honoraria and travel expenses from AstraZeneca, Clovis, Eisai, GlaxoSmithKline, Medac, MSD, Novartis, Olympus, PharmaMar, Roche, Sensor Kinesis, Teva, Tesaro\u0026cedil; All other authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study was approved by the Bioethics Committee of the Ludwig-Maximilians University of Munich (reference number 227-09 and 18-392).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent:\u003c/strong\u003e The study was approved by the ethics committee of the Ludwig-Maximilians University Munich (reference number: 048-08; 2008). Patient data were anonymized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e not applicable as all data are anonymized\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish:\u003c/strong\u003e not applicable as all data are anonymized\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFerlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pineros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-53.\u003c/li\u003e\n\u003cli\u003eMor G, Alvero A. The duplicitous origin of ovarian cancer. Rambam Maimonides Med J. 2013;4(1):e0006.\u003c/li\u003e\n\u003cli\u003eLuvero D, Plotti F, Aloisia A, Montera R, Terranova C, Carlo De Cicco N, et al. Ovarian cancer relapse: From the latest scientific evidence to the best practice. Crit Rev Oncol Hematol. 2019;140:28-38.\u003c/li\u003e\n\u003cli\u003eAllemani CW, H.K.; Carreira, H.; Harewood, R.; Spika, D.; Wang, X.S.; Bannon, F.; Ahn, J.V.; Johnson, C.J.; Bonaventure, A.; et al. Global surveillance of cancer survival 1995\u0026ndash;2009: Analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet 2015;385:977\u0026ndash;1010.\u003c/li\u003e\n\u003cli\u003eVaughan S, Coward JI, Bast RC, Jr., Berchuck A, Berek JS, Brenton JD, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer. 2011;11(10):719-25.\u003c/li\u003e\n\u003cli\u003eHan Y, Jo H, Cho JH, Dhanasekaran DN, Song YS. Resveratrol as a Tumor-Suppressive Nutraceutical Modulating Tumor Microenvironment and Malignant Behaviors of Cancer. Int J Mol Sci. 2019;20(4).\u003c/li\u003e\n\u003cli\u003eMikami S, Ota I, Masui T, Uchiyama T, Okamoto H, Kimura T, et al. Resveratrolinduced REG III expression enhances chemo and radiosensitivity in head and neck cancer in xenograft mice. Oncol Rep. 2019;42(1):436-42.\u003c/li\u003e\n\u003cli\u003eEngelke LH, Hamacher A, Proksch P, Kassack MU. Ellagic Acid and Resveratrol Prevent the Development of Cisplatin Resistance in the Epithelial Ovarian Cancer Cell Line A2780. J Cancer. 2016;7(4):353-63.\u003c/li\u003e\n\u003cli\u003eDampf Stone A, Batie SF, Sabir MS, Jacobs ET, Lee JH, Whitfield GK, et al. Resveratrol potentiates vitamin D and nuclear receptor signaling. Journal of cellular biochemistry. 2015;116(6):1130-43.\u003c/li\u003e\n\u003cli\u003eMangelsdorf DJ, Ong ES, Dyck JA, Evans RM. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990;345(6272):224-9.\u003c/li\u003e\n\u003cli\u003eFitzgibbon J, Gillett GT, Woodward KJ, Boyle JM, Wolfe J, Povey S. Mapping of RXRB to human chromosome 6p21.3. Ann Hum Genet. 1993;57(3):203-9.\u003c/li\u003e\n\u003cli\u003eAlmasan A, Mangelsdorf DJ, Ong ES, Wahl GM, Evans RM. Chromosomal localization of the human retinoid X receptors. Genomics. 1994;20(3):397-403.\u003c/li\u003e\n\u003cli\u003eKrezel W, Ruhl R, de Lera AR. Alternative retinoid X receptor (RXR) ligands. Mol Cell Endocrinol. 2019;491:110436.\u003c/li\u003e\n\u003cli\u003eDeuster E, Jeschke U, Ye Y, Mahner S, Czogalla B. Vitamin D and VDR in Gynecological Cancers-A Systematic Review. Int J Mol Sci. 2017;18(11).\u003c/li\u003e\n\u003cli\u003eQu Y, Zhang J, Wu S, Li B, Liu S, Cheng J. SIRT1 promotes proliferation and inhibits apoptosis of human malignant glioma cell lines. Neurosci Lett. 2012;525(2):168-72.\u003c/li\u003e\n\u003cli\u003eSrisuttee R, Koh SS, Malilas W, Moon J, Cho IR, Jhun BH, et al. SIRT1 sensitizes hepatocellular carcinoma cells expressing hepatitis B virus X protein to oxidative stress-induced apoptosis. Biochem Biophys Res Commun. 2012;429(1-2):45-50.\u003c/li\u003e\n\u003cli\u003eSaunders LR, Verdin E. Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene. 2007;26(37):5489-504.\u003c/li\u003e\n\u003cli\u003eVazquez MJ, Velasco I, Tena-Sempere M. Novel mechanisms for the metabolic control of puberty: implications for pubertal alterations in early-onset obesity and malnutrition. J Endocrinol. 2019.\u003c/li\u003e\n\u003cli\u003eRen Z, He H, Zuo Z, Xu Z, Wei Z, Deng J. The role of different SIRT1-mediated signaling pathways in toxic injury. Cell Mol Biol Lett. 2019;24:36.\u003c/li\u003e\n\u003cli\u003eVargas-Ortiz K, Perez-Vazquez V, Macias-Cervantes MH. Exercise and Sirtuins: A Way to Mitochondrial Health in Skeletal Muscle. Int J Mol Sci. 2019;20(11).\u003c/li\u003e\n\u003cli\u003eLin Z, Fang D. The Roles of SIRT1 in Cancer. Genes \u0026amp; Cancer. 2013;4(3-4):97-104.\u003c/li\u003e\n\u003cli\u003eJang KY, Kim KS, Hwang SH, Kwon KS, Kim KR, Park HS, et al. Expression and prognostic significance of SIRT1 in ovarian epithelial tumours. Pathology. 2009;41(4):366-71.\u003c/li\u003e\n\u003cli\u003eBuhrmann C, Shayan P, Popper B, Goel A, Shakibaei M. Sirt1 Is Required for Resveratrol-Mediated Chemopreventive Effects in Colorectal Cancer Cells. Nutrients. 2016;8(3):145.\u003c/li\u003e\n\u003cli\u003eOka S, Zhai P, Yamamoto T, Ikeda Y, Byun J, Hsu CP, et al. Peroxisome Proliferator Activated Receptor-alpha Association With Silent Information Regulator 1 Suppresses Cardiac Fatty Acid Metabolism in the Failing Heart. Circ Heart Fail. 2015;8(6):1123-32.\u003c/li\u003e\n\u003cli\u003eRegnault TR, Zhao L, Chiu JS, Gottheil SK, Foran A, Yee SP. Peroxisome Proliferator-Activated Receptor -beta/delta, -gamma Agonists and Resveratrol Modulate Hypoxia Induced Changes in Nuclear Receptor Activators of Muscle Oxidative Metabolism. PPAR Res. 2010;2010:129173.\u003c/li\u003e\n\u003cli\u003eKim TH, Park JH, Woo JS. Resveratrol induces cell death through ROSdependent downregulation of Notch1/PTEN/Akt signaling in ovarian cancer cells. Mol Med Rep. 2019;19(4):3353-60.\u003c/li\u003e\n\u003cli\u003eSaid RS, Mantawy EM, El-Demerdash E. Mechanistic perspective of protective effects of resveratrol against cisplatin-induced ovarian injury in rats: emphasis on anti-inflammatory and anti-apoptotic effects. Naunyn Schmiedebergs Arch Pharmacol. 2019.\u003c/li\u003e\n\u003cli\u003eHu S, Li X, Xu R, Ye L, Kong H, Zeng X, et al. The synergistic effect of resveratrol in combination with cisplatin on apoptosis via modulating autophagy in A549 cells. Acta Biochim Biophys Sin (Shanghai). 2016;48(6):528-35.\u003c/li\u003e\n\u003cli\u003eRodriguez-Enriquez S, Pacheco-Velazquez SC, Marin-Hernandez A, Gallardo-Perez JC, Robledo-Cadena DX, Hernandez-Resendiz I, et al. Resveratrol inhibits cancer cell proliferation by impairing oxidative phosphorylation and inducing oxidative stress. Toxicol Appl Pharmacol. 2019;370:65-77.\u003c/li\u003e\n\u003cli\u003eFang Y, Nicholl MB. Sirtuin 1 in malignant transformation: friend or foe? Cancer Lett. 2011;306(1):10-4.\u003c/li\u003e\n\u003cli\u003eShuang T, Wang M, Zhou Y, Shi C. Over-expression of Sirt1 contributes to chemoresistance and indicates poor prognosis in serous epithelial ovarian cancer (EOC). Med Oncol. 2015;32(12):260.\u003c/li\u003e\n\u003cli\u003eLiang XJ, Finkel T, Shen DW, Yin JJ, Aszalos A, Gottesman MM. SIRT1 contributes in part to cisplatin resistance in cancer cells by altering mitochondrial metabolism. Mol Cancer Res. 2008;6(9):1499-506.\u003c/li\u003e\n\u003cli\u003eAkhter MZ, Sharawat SK, Kumar V, Kochat V, Equbal Z, Ramakrishnan M, et al. Aggressive serous epithelial ovarian cancer is potentially propagated by EpCAM(+)CD45(+) phenotype. Oncogene. 2018;37(16):2089-103.\u003c/li\u003e\n\u003cli\u003eAsaka R, Miyamoto T, Yamada Y, Ando H, Mvunta DH, Kobara H, et al. Sirtuin 1 promotes the growth and cisplatin resistance of endometrial carcinoma cells: a novel therapeutic target. Laboratory investigation; a journal of technical methods and pathology. 2015;95(12):1363-73.\u003c/li\u003e\n\u003cli\u003ePizarro JG, Verdaguer E, Ancrenaz V, Junyent F, Sureda F, Pallas M, et al. Resveratrol inhibits proliferation and promotes apoptosis of neuroblastoma cells: role of sirtuin 1. Neurochem Res. 2011;36(2):187-94.\u003c/li\u003e\n\u003cli\u003eBjorklund M, Roos J, Gogvadze V, Shoshan M. Resveratrol induces SIRT1- and energy-stress-independent inhibition of tumor cell regrowth after low-dose platinum treatment. Cancer Chemother Pharmacol. 2011;68(6):1459-67.\u003c/li\u003e\n\u003cli\u003eGermain P, Staels B, Dacquet C, Spedding M, Laudet V. Overview of nomenclature of nuclear receptors. Pharmacol Rev. 2006;58(4):685-704.\u003c/li\u003e\n\u003cli\u003eMangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83(6):835-9.\u003c/li\u003e\n\u003cli\u003eMangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995;83(6):841-50.\u003c/li\u003e\n\u003cli\u003eWu S, Zhang D, Zhang ZP, Soprano DR, Soprano KJ. Critical role of both retinoid nuclear receptors and retinoid-X-receptors in mediating growth inhibition of ovarian cancer cells by all-trans retinoic acid. Oncogene. 1998;17(22):2839-49.\u003c/li\u003e\n\u003cli\u003eWang L, DeMarco SS, Peaks MS, Maiorana-Boutilier AL, Chen J, Crouch MJ, et al. RARalpha/RXR synergism potentiates retinoid responsiveness in cutaneous T-cell lymphoma cell lines. Exp Dermatol. 2017;26(11):1004-11.\u003c/li\u003e\n\u003cli\u003eZhang X, Zhou H, Su Y. Targeting truncated RXRalpha for cancer therapy. Acta Biochim Biophys Sin (Shanghai). 2016;48(1):49-59.\u003c/li\u003e\n\u003cli\u003eShankaranarayanan P, Rossin A, Khanwalkar H, Alvarez S, Alvarez R, Jacobson A, et al. Growth factor-antagonized rexinoid apoptosis involves permissive PPARgamma/RXR heterodimers to activate the intrinsic death pathway by NO. Cancer Cell. 2009;16(3):220-31.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ovarian cancer, survival, epigenetics, Resveratrol, Sirtuin1, RXR","lastPublishedDoi":"10.21203/rs.3.rs-198498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-198498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eOvarian cancer is the most lethal gynecologic cancer. Resveratrol (RSV) is known to alter metabolism in cancer. It affects the nuclear retinoid-X-receptor (RXR), which implies a modulating effect of RXR to gynecologic cancers. Furthermore, RSV targets Sirtuin1 (Sirt1), a histone deacetylase. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eStudy design:\u003c/strong\u003e 123 tissue samples of patients with serous or mucinous ovarian cancer were examined for expression of Sirt1 and RXR. Ovarian cell-lines were treated with RSV and consequences on viability and apoptosis were evaluated. The influence of RSV to Sirt1 and RXR expression was analyzed by western blotting. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eA correlation of nuclear Sirt1 and RXRα expression could be detected (p=0.006). Co-expression of nuclear RXRα and cytoplasmic (p=0.026) or nuclear (p=0.041) Sirt1 was associated with significantly increased overall survival in advanced tumor stages. Viability was decreased in all cell-lines after stimulation with resveratrol, while cell apoptosis was increased. RSV treatment led to significant lower Sirt1 expression in A2780 cells (p=0.025) and significant increased RXR expression in cisA2780 cells (p=0.012). \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e In order to use RSV as medical target, studies could be developed to improve the understanding of drug resistance mechanisms and consequently improve treatment outcome.\u003c/p\u003e","manuscriptTitle":"The role of Resveratrol, Sirtuin1 and RXRα as prognostic markers in ovarian cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-06-01 18:51:31","doi":"10.21203/rs.3.rs-198498/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-03-08T00:00:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-03-06T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Archives of Gynecology and Obstetrics","date":"2021-02-01T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Archives of Gynecology and Obstetrics","date":"2021-01-03T05:53:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"archives-of-gynecology-and-obstetrics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"arch","sideBox":"Learn more about [Archives of Gynecology and Obstetrics](https://www.springer.com/journal/404)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/arch/default.aspx","title":"Archives of Gynecology and Obstetrics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d29d1eba-fe18-46f0-9660-bd23e77e928c","owner":[],"postedDate":"June 1st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":4723489,"name":"Sexual \u0026 Reproductive Medicine"}],"tags":[],"updatedAt":"2021-12-06T08:56:44+00:00","versionOfRecord":{"articleIdentity":"rs-198498","link":"https://doi.org/10.1007/s00404-021-06262-w","journal":{"identity":"archives-of-gynecology-and-obstetrics","isVorOnly":false,"title":"Archives of Gynecology and Obstetrics"},"publishedOn":"2021-12-06 08:56:44","publishedOnDateReadable":"December 6th, 2021"},"versionCreatedAt":"2021-06-01 18:51:31","video":"","vorDoi":"10.1007/s00404-021-06262-w","vorDoiUrl":"https://doi.org/10.1007/s00404-021-06262-w","workflowStages":[]},"version":"v1","identity":"rs-198498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-198498","identity":"rs-198498","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}