Effect of Rapamycin Treatment in Human Seminoma TCam-2 Cells through Inhibition of G1-S Transition

Effect of Rapamycin Treatment in Human Seminoma TCam-2 Cells through Inhibition of G1-S Transition | 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 Effect of Rapamycin Treatment in Human Seminoma TCam-2 Cells through Inhibition of G1-S Transition Tugce Onel, Cihan S. Erdogan, Basak Aru, Ecem Yildirim, Gulderen Yanikkaya Demirel, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2083681/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Mammalian Target of Rapamycin (mTOR) is an important serine/threonine kinase that plays a critical role in several processes including cell cycle, protein synthesis and energy metabolism. Due to its multiple roles and general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited. Therefore, our aim was to investigate the expression of mTOR signaling pathway proteins in the TCam-2 cell line after rapamycin treatment. Methods: TCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin) for 48 h and 72 h. mTOR, p-mTOR, P70S6K, p-P70S6K, proliferating cell nuclear antigen (PCNA) and Caspase-3 expression levels were analyzed by western blot. Apotosis and cell cycle were analyzed by flow cytometry. Results: After 48 hours of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p<0.05). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (***p<0.001, ****p<0.0001). After 72 hours of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500 and 1000 nM rapamycin-treated groups (p<0.05). Moreover, P70S6K expression decreased in all treatment groups (****p<0.0001). Caspase-3 expression were similar in all groups. While PCNA expression tended to decrease at 48 h in a dose-dependent manner, this decrease was not significant. We detected decreased PCNA expression at 1000 nM rapamycin at 72 h (p<0.05). The rate of apoptosis increased especially at 1000 nM rapamycin at 72 hours (***p<0.001). On the other hand, according to the results of the cell cycle experiment, G1 phase arrest was detected at all rapamycin doses at 48 and 72 hours (***p<0.001). Conclusion: our study indicated that 1000 nM rapamycin may inhibit TCam-2 seminoma cells growth by halting cell proliferation through inhibition of G1-S transition. Therefore we believe that the findings obtained will contribute to the development of new treatment approaches for seminoma patients in the future and in the process of restoring testicular functions and preserving fertility. mTOR Rapamycin TCam-2 cell line Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Testicular cancer (TC) is the most common solid organ malignancy in young men and the leading cause of cancer-related deaths in this population [ 1 ]. TC usually occurs during the reproductive period and can cause infertility [ 2 ]. Testicular tumors can be broadly classified germ cell tumors, sex cord-stromal tumors, mixed germ cell/sex cord-stromal tumors [ 1 ]. Although testicular cancer can arise from any cell type found in the testes, more than 95% of testicular cancers are germ cell tumors. The remaining 5% are sex cord-gonadal stromal tumors orginate from Leydig or Sertoli cells. Among germ cell tumors, testicular seminoma is a germ cell tumor of the testis and the most common testicular tumor, accounting for approximately 45% of all primary testicular tumors [ 3 ]. The TCam-2 cell line is the first seminoma-derived cell line that retains most of the characteristic features of seminoma [ 4 ]. TCam-2 cells express typical marker genes for primordial germ cells and germ cell neoplasia in situ (GCNIS) ( SOX17 , PRAME , cKIT , TFAP2C , PRDM1 / BLIMP1 ) and show typical GCNIS/seminoma morphology (large round cells with a large nucleus and clear cytoplasm) [ 5 – 7 ]. Mammalian target of rapamycin (mTOR) is an important serine/threonine kinase [ 8 ] that plays critical roles in cell cycle, protein synthesis, energy metabolism, cell proliferation, growth, differentiation, and apoptosis [ 9 – 13 ]. mTOR is a downstream factor of PI3K/Akt and positively regulates tumor progression [ 14 ]. mTORC1 is one of the two major mTOR complexes and the main downstream effector of PI3K/PTEN/AKT signaling in response to growth factors which phosphorylates ribosomal protein S6 kinase (S6K1) to increase protein synthesis [ 15 ]. Rapamycin is a natural product and non-ATP competitive inhibitor of mTORC1; it exert its’ effects via binding to cytosolic FK binding protein (FKBP12) with high affinity, to interact with mTORC1 and via interfering with mTOR activity or complex assembly [ 16 ]. Previosly, we have reported mTOR signal pathway immunoreactivity in testicular seminoma, phosphorylated serine 2448 (p-mTOR), P70S6K and p-P70S6K and hypothesized that mTOR signaling pathway may contribute to the development or progression of testicular cancer [ 3 ]. Due to its diverse roles and it’s general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited [ 3 , 17 – 20 ]. Herein, we analyzed the effect of mTOR inhibition in seminoma-like TCam-2 cells with regard to cell proliferation, migration, cell cycle, and apoptosis. Materials And Methods Cell Culture and Reagents The human TCam-2 cell line was derived in 1992 from a testicular tumor sample of a pure classical seminoma [ 21 ]. TCam-2 (obtained from Prof. Dr. Hubert Schorle, Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany) was grown in RPMI 1640 medium (Gibco, #11875093) supplemented with 10% FBS (fetal bovine serum) (Gibco, #16000044), 1% penicillin/streptomycin (Gibco, #15140122) and 200 mM glutamine (Gibco, #25030081). Cells were grown at 37°C and under 5% CO2 humidified atmosphere. Rapamycin (Glentham Life Sciences, #GA7417) was dissolved in DMSO (Sigma, #D8418) at a stock concentration of 10 mM. Experimental groups were: control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin for 48 h and 72 h [ 22 ]. Western blotting Western blot was performed as described previously [ 23 ] . In summary, a total of 4 × 10 5 cells/well were seeded on six-well plates and let attached overnight. T otal protein from the TCam-2 cells were extracted using RIPA lysis buffer (SantaCruz, #sc-24948) supplemented with protease (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78446) and phosphatase inhibitor cocktails (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78420). After the total protein concentration was determined, 20 μg of total lysate was loaded into each lane, separated electrophoretically using NuPAGE 4-12% Bis-Tris gel (Invitrogen™, #NP0321BOX), and then electroblotted onto PVDF membrane (Thermo Scientific, #88585). Later, membrane was blocked in TBS-T buffer (0.1% Tween-20 in Tris-buffered saline) with 5% nonfat dry milk (Sigma, #70166) for one hour. The membrane was then incubated with appropriate primary antibodies anti-mTOR (1:1000 dilution; Cell Signaling, #2983), anti-p-mTOR (1:1000 dilution; Cell Signaling, #2971), anti-P70S6K (1:1000 dilution; Cell Signaling, #9202), anti-p-P70S6K (1:1000 dilution; Cell Signaling, #701064), anti-PCNA (1:1000 dilution; Cell Signaling, #2586) and anti-Caspase-3 (1:1000 dilution; Cell Signaling, #9662) overnight at 4°C. After incubation with primary antibody, membrane was washed with TBS-T for three times for 10 min each, incubated with HRP-conjugated secondary antibody (1:5000 dilution, Cell Signaling, #7074) for one hour at room temperature and subsequently washed with TBS-T. Phospho-specific antibodies were always used for the first round of probing. Membrane was strippped with western blot stripping buffer (Takara Bio, #T7135A). Immunobolt images were acquired and bands were quantified by using Image Lab Software (BioRad, #1708265). All experiments were repeated at least three times. β-Actin was used as an internal control and ratio of expression level of individual protein to that of β-Actin from the same samples was used to determine the final expression levels each protein. Wound-Healing Scratch Assay A total of 5 × 10 5 cells/well were seeded on six-well plates and let attached overnight. After overnight attachment, a straight line was created by scratching with a 100 μL pipette tip. Then the medium was removed, fresh medium with respective drug concentrations were added and images were obtained for t=0 h. The migration/healing was determined for 48 and 72 hours by obtaining microscopy images of the same area (Axiovert 135; Carl Zeiss Microscopy). Migration/healing was determined as previously described by using Fiji [ 24 ] and the percent healing against the control group was determined. Assesment of Apoptosis Viability, apoptosis and necrosis were evaluated by Annexin V/Propidium Iodide (PI) staining. For this purpose, cells were seeded into six-well plates as 5 × 10 5 cell per well and incubated for overnight for attachment. Cells were then treated with rapamycin at respective doses for 48 and 72 hours. For staining, cells were detached by trypsinization, washed once with Dulbecco’s phosphate buffered saline solution (DPBS, Thermo Fisher Scientific, #14190144) and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 5 μL Annexin V-FITC reagent (Biolegend, #640906) and 1 µL PI solution (Thermo Fisher Scientific, #P3566, diluted to 250 μg/mL in DPBS) by incubating for 10 minutes under dark conditions at room temperature. Cells were read with DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed by CytExpert software [ 25 ]. The experiment was done as triplicates and 2.5 × 10 4 cells per analysis were acquired at medium flow rate. Cell cycle Assessment DNA content analysis was performed by using Cell Cycle Kit (Beckman Coulter Inc., #C03551). Cells were seeded as 5 × 10 5 cell/six-well plate and incubated overnight for attachment. After incubating with rapamycin at respective doses for 48 and 72 hours, cells were detached by tyripsinization, washed once with DPBS, fixed with ice cold 70% ethanol by adding dropwise followed by incubation at 4°C for an hour. Tubes were stored at -20 °C overnight, and ethanol was removed by centrifuging cells at 400 x g for 5 minutes. Pellet was suspended in 500 µ L cell cycle kit reagent, and tubes were incubated at room temperature under dark for 30 minutes. DNA content was analyzed by DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed with ModFit LT 5.0 software (Verify Software House) [ 26 ]. The experiment was done as triplicates and 5 × 10 4 cells per test were acquired at medium flow rate. Analyses were performed Statistical Analysis The statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. A p value lower than 0.05 was considered statistically significant. Results Dose-dependent inhibition of mTORC1 in TCam-2 Cells by Rapamycin Rapamycin exerts its biological activity by inhibiting the mTOR, which is a key regulator of cell growth and survival in many cell types [ 27 ]. In our study, TCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin) for 48 h and 72 h [ 22 ]. After 48 hours of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p<0.05; Figure 1a and b; Table 1). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (**p<0.01, ***p<0.001, ****p<0.0001; Figure 1a and c; Table 1). After 72 hours of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500 and 1000 nM rapamycin-treated groups (p<0.05; Figure 1a and d; Table 1). Moreover, P70S6K activity decreased at all treatment groups (****p<0.0001; Figure 1a and e; Table 1). Table 1 Main outcome measures for cell cycle distribution of TCam-2 cell line at 48 and 72 h following exposure to 0 - 1000 nM rapamycin doses. Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. p values are shown statistically. P values G0/G1 S G2/M 48 h 0 vs. 4 0.0065 0.184 0.9999 0 vs. 20 0.0001 0.0012 0.1845 0 vs. 100 0.0005 0.0313 0.9974 0 vs. 500 0.0008 0.1589 0.8954 0 vs. 1000 0.0002 0.0092 0.8518 72 h 0 vs. 4 0.0001 0.0415 0.9955 0 vs. 20 0.0002 0.0121 0.8967 0 vs. 100 0.0001 0.001 0.1951 0 vs. 500 0.0001 0.0004 0.2086 0 vs. 1000 0.0011 0.0006 0.0276 Rapamycin does not significantly alter Caspase-3 and PCNA protein levels in TCam-2 Cells In order to check apoptosis and proliferation in TCam-2 cells after administration of specific doses of rapamycin, we investigated Caspase-3 and PCNA protein expression. Caspase-3 expression did not change at either of the doses nor the time points (Figure 2a, c and e). While levels of PCNA proteins seemed to decrease in the at 48 h in a dose dependent manner, this decrease was not significant (Figure 2a and b). On the other hand, PCNA expression decreased significantly at 1000 nM rapamycin at 72 h (p<0.05; Figure 2a and d; Table 1). Wound-Healing Scratch Assay in TCam-2 Cell Line In order to evalutae TCam-2 cell migration after rapamycin treatment, wound-healing scratch assay were used. 4 nM rapamycin treatment significantly inhibited cell migration after 48 h (p<0.05; Figure 3a). On the other hand, rapamycin at doses of 20 and 1000 nM significantly decreased cell migration at 72 h (**p<0.01; Figure 3b; Table 1). No significant changes were detected at other doses for 48 h and 72 h (Figure 3a). mTOR inhibition by using rapamycin does not promote apoptosis in the TCam-2 Cells The apoptotic effect of rapamycin mediated mTOR inhibition was evaluated by Annexin V/PI staining on 48 and 72 hours. Comparisons between the control and treatment groups revealed no significant change in viability, early and late apoptosis and necrosis rates at 48 hours (p>0.05) (Figure 4a, b, c, d and i). On the other hand, rapamycin treatment increased late apoptosis rates at all concentrations tested significantly (p<0.05 for 4, 20, 100 and 500 nM; p0.05; Figure 4e, f, h and i) mTOR inhibition leads to G1-phase arrest in TCam-2 cells in a dose dependent manner It was previously reported that inhibition of mTOR by physiological cell stressors or pharmacological agents decrease proliferation mainly by leading to cell cycle arrest at G1 phase [ 28 ]. It should be noted that inhibition of mTOR during interphase would be responsible for arrest in G1, and therefore the interphase expression patterns of p-mTOR, and p-P70S6K (Figure 1a-c) should not be ignored. Consistent with the literature, our results revealed that rapamycin at 1000 nM promoted G1 phase arrest in TCam-2 cells in both 48 and 72 hours significantly compared to the control group (p<0.001 and p<0.01, respectively) whereas 4, 20,100 and 500 nM showed a lower rate of rapamycin treatment percentage of cells (approximately 25%) were in G1 (**p<0.01, ***p<0.001, ****p<0.0001; Figure 5a, b and c; Table 1). Discussion And Conclusion The mTOR is frequently deregulated in cancer and activating somatic mutations of mTOR have recently been identified in various types of human cancer and so the mTOR is therapeutically targeted [29]. mTOR inhibitors have been commonly used as immunosuppressants, and are currently approved for the treatment of human malignancies [30]. In this study, we aimed to investigate the effect of inhibition of the mTORC1 signaling pathway in TCam-2 seminoma cells. This study demonstrates that treatment with 1000 nM rapamycin can inhibit proliferation of TCam-2 seminoma cells by downregulating PCNA through mTOR inhibition. In a previous study, analyzed whether mTORC1 and MAPK signaling pathway activities were differentially active in both Germ Cell Tumors (GCT) classes in seminoma and non-seminoma germ cell tumors. In this study, it was shown that the mTORC1 signaling pathway is activated in non-seminoma germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma), but not in seminomas. Seminomas have been shown to negatively regulate mTORC1 activity with high REDD1 levels to maintain an undifferentiated state. On the other hand, members of the EGF and FGF receptor families have been shown to be more highly expressed in NSGCTs and stimulate the signaling of the EGF and FGF2 ligands mTORC1 and MAPK. This suggests that mTORC1 activation in NSGCTs contributes to cell proliferation [20]. We investigated the activity of mTORC1 in the TCam-2 cell line and propose to show for the first time that the mTOR signaling pathway acts as an oncogene in the TCam-2 seminoma cell line. This is evidenced by its dramatic downregulation in seminomas based on western blot experiments, as well as its positive regulation of cell proliferation and migration. mTOR functions as a key control protein that integrates signals from a variety of environmental factors, and mTOR signalling proteins are essential for cell growth, migration, survival, and development. To date, we know that mTOR signaling proteins play a role in cell adhesion in the testis and are involved in the regulation of the blood–testis barrier [31]. mTOR signaling proteins express in the testis of adult mice, and mTOR pathway proteins may play a role in proliferation and stimulation of meiotic initiation of spermatogonial stem cells [32]. Treatment with mTOR inhibitors, sirolimus and everolimus, had destructive effects on the adult mouse testis and impairs gonadal function [33]. Therefore, we suggested that investigating the role of mTOR signalling inhibition in the TCam-2 cell line might be important to understand underlying mechanism of male reproductive cancers. Rapamycin is widely used in the clinic as an antiproliferative drug and immunosuppressant, moreover, its analogs have been shown to retard tumor proliferation in clinical trials[34]. Currently, more studies are focusing on the effect of rapamycin in tumor therapy [35]. . As an mTOR inhibitor, rapamycin has been reported to block cell cycle progression from G1 to S phase by inhibiting p70 ribosomal protein S6 kinase (p70S6K) [36, 37]. As a threonine kinase, mTOR phosphorylates S6K1 and 4E-BP1 and promotes transcription of key mRNAs associated with cell cycle progression from G1 to S phase [37]. Through its interaction with p70S6K, it is associated with cell growth, proliferation and differentiation by regulating ribosome biogenesis, protein synthesis, cell cycle progression and metabolism [38]. mTOR inhibition via rapamycin was reported to inhibit proliferation and migration of hemangiomas and reduce growth of vascular tumors [39]. Consistent with this finding, we showed that the activity of the downstream effector molecule mTOR, P70S6K was significantly reduced upon rapamycin treatment at all concentrations tested in TCam-2 seminoma cells, indicating mTOR inhibiton decreases cell cycle progression and in turn, proliferation. Moreover, in our study, rapamycin induced mTOR inhibition led to G1 phase-arrest during cell cycle progression, which is also in line with reports showing that mTOR is involved in cell cycle progression in G1 [40, 41]. PCNA is a nuclear protein expressed in proliferating cells, is required for the maintenance of cell proliferation, and is used as a marker of cell proliferation [42]. AKT/mTOR pathway have shown to inhibit skin cancer proliferation by downregulating PCNA [43]. In prostate cancer, agents such as vicenin-2 reduce the expression of PCNA and cyclin-D1 by inhibiting the EGFR/Akt/mTOR pathway [44]. The expression levels of p-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K were also significantly downregulated in hemangioma tissues and cells compared with the control group, and the expressions of Ki67 and PCNA were significantly decreased [45]. In another study, syndecan binding protein was reported to inhibit apoptosis of gastric cancer cells and promote the growthof gastric cancer by inducing PCNA expression and inactivating the PI3K/AKT/mTOR pathway [46]. In our study, we showed that rapamycin decreases PCNA expression in a dose dependent manner on 72 h, though a significant decrease was only observed upon treatment with 1000 nM. Yet, according to cell cycle analysis, rapamycin promoted G1-phase arrest in all concentrations tested compared to the control group, indicating other possible mechanisms involving in cell cyce arrest rather than PCNA should be clarified. In short, Inhibition of mTOR by rapamycin prevents cells growth, thus division and proliferation. Therefore, we suggest that rapamycin, an mTOR inhibitor, can potentially be used as a chemotherapeutic agent in human seminoma cells. We hope that the knowledge gained from our study will contribute to the development of new treatment approaches for seminoma patients and to the restoration of testicular functions and preservation of fertility in the future. Declarations Author information Affiliations Department of Histology and Embryology, Yeditepe University, School of Medicine, 34755, Istanbul, Turkey. Ecem Yildirim, Tugce Onel, Aylin Yaba. Department of Physiology Yeditepe, University, School of Medicine, 34755, Istanbul, Turkey. Cihan Suleyman Department of Immunology, Yeditepe University, School of Medicine, 34755, Istanbul, Turkey. Basak Aru, Gulderen Yanikkaya Demirel Competing Interests The authors declare no competing interests. Corresponding author Correspondence to Aylin Yaba. Acknowledgement We would like to thank Prof. Dr. Hubert Schorle (Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany) for providing TCam-2 cells. Authorship contribution statement Conceptualization: TO, CSE and AY; Data curation: TO, CSE, BA, EY; Formal analysis: TO, CSE, BA, EY, AY; Investigation: TO, CSE, BA, EY, AY; Methodology: TO, CSE, BA, EY, AY; Software: TO, CSE, BA, EY, AY; Supervision: AY; Validation: TO, CSE; Visualization: TO, CSE; Roles/Writing- original draft: TO, CSE, BA, EY, GYD, AY; Writing- review & editing: TO, CSE, BA, EY, GYD, AY. References Ye H and Ulbright TM (2012) Difficult differential diagnoses in testicular pathology. Arch Pathol Lab Med 136:435-46. doi: 10.5858/arpa.2011-0475-RA Jacobs LA and Vaughn DJ (2012) Hypogonadism and infertility in testicular cancer survivors. J Natl Compr Canc Netw 10:558-63. doi: 10.6004/jnccn.2012.0053 Yaba A, Bozkurt ER and Demir N (2016) mTOR expression in human testicular seminoma. 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Bioscience Reports 38. doi: 10.1042/BSR20181822 Shafer A, Zhou C, Gehrig PA, Boggess JF and Bae-Jump VL (2010) Rapamycin potentiates the effects of paclitaxel in endometrial cancer cells through inhibition of cell proliferation and induction of apoptosis. Int J Cancer 126:1144-54. doi: 10.1002/ijc.24837 Marqués L, Núñez-Córdoba JM, Aguado L, Pretel M, Boixeda P, Nagore E, Baselga E and Redondo P (2015) Topical rapamycin combined with pulsed dye laser in the treatment of capillary vascular malformations in Sturge-Weber syndrome: phase II, randomized, double-blind, intraindividual placebo-controlled clinical trial. J Am Acad Dermatol 72:151-8.e1. doi: 10.1016/j.jaad.2014.10.011 Shin S, Wolgamott L, Yu Y, Blenis J and Yoon SO (2011) Glycogen synthase kinase (GSK)-3 promotes p70 ribosomal protein S6 kinase (p70S6K) activity and cell proliferation. Proc Natl Acad Sci U S A 108:E1204-13. doi: 10.1073/pnas.1110195108 Medici D and Olsen BR (2012) Rapamycin inhibits proliferation of hemangioma endothelial cells by reducing HIF-1-dependent expression of VEGF. PLoS One 7:e42913. doi: 10.1371/journal.pone.0042913 Song J, Salek-Ardakani S, So T and Croft M (2007) The kinases aurora B and mTOR regulate the G1-S cell cycle progression of T lymphocytes. Nat Immunol 8:64-73. doi: 10.1038/ni1413 Ponticelli C (2004) The pleiotropic effects of mTor inhibitors. J Nephrol 17:762-8. Strzalka W and Ziemienowicz A (2011) Proliferating cell nuclear antigen (PCNA): a key factor in DNA replication and cell cycle regulation. Annals of botany 107:1127-1140. doi: 10.1093/aob/mcq243 Amornphimoltham P, Leelahavanichkul K, Molinolo A, Patel V and Gutkind JS (2008) Inhibition of Mammalian target of rapamycin by rapamycin causes the regression of carcinogen-induced skin tumor lesions. Clinical cancer research : an official journal of the American Association for Cancer Research 14:8094-8101. doi: 10.1158/1078-0432.CCR-08-0703 Singhal SS, Jain D, Singhal P, Awasthi S, Singhal J and Horne D (2017) Targeting the mercapturic acid pathway and vicenin-2 for prevention of prostate cancer. Biochimica et biophysica acta. Reviews on cancer 1868:167-175. doi: 10.1016/j.bbcan.2017.03.009 Wang T, Zhang FL, Zhao Y, Guo DD and Yang R (2021) [Effects of miR-125b-5p on the proliferation and apoptosis of human hemangioma endothelial cells HemES and its mechanism]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 37:247-253. doi: 10.12047/j.cjap.6068.2021.021 Qian B, Yao Z, Yang Y, Li N and Wang Q (2021) Downregulation of SDCBP inhibits cell proliferation and induces apoptosis by regulating PI3K/AKT/mTOR pathway in gastric carcinoma. Biotechnol Appl Biochem. doi: 10.1002/bab.2103 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-2083681","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":138052569,"identity":"a312f6b6-6e86-4054-8a12-aa1a4d0f08d7","order_by":0,"name":"Tugce Onel","email":"","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tugce","middleName":"","lastName":"Onel","suffix":""},{"id":138052570,"identity":"177979c4-90b5-4ad6-a413-aeca8874bd59","order_by":1,"name":"Cihan S. Erdogan","email":"","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Cihan","middleName":"S.","lastName":"Erdogan","suffix":""},{"id":138052571,"identity":"55be352b-9a2c-4999-baac-5320a0a08117","order_by":2,"name":"Basak Aru","email":"","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Basak","middleName":"","lastName":"Aru","suffix":""},{"id":138052572,"identity":"a5fed0aa-4910-465f-8dd8-d10f5e0783e7","order_by":3,"name":"Ecem Yildirim","email":"","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ecem","middleName":"","lastName":"Yildirim","suffix":""},{"id":138052573,"identity":"4cacbde0-2cb4-48a8-95e0-e170b72fc8b3","order_by":4,"name":"Gulderen Yanikkaya Demirel","email":"","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gulderen","middleName":"Yanikkaya","lastName":"Demirel","suffix":""},{"id":138052574,"identity":"c8e87751-1b9a-41e6-a26f-bf1b77eea36f","order_by":5,"name":"Aylin Yaba","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6klEQVRIiWNgGAWjYFAC5oYDQDKBgb0HzOXhI6yFEaqF5wwDA5DFw0aMFgawFokcsBYGglr4ZyQ2Hi5ss8vjn/n24OOPOXYybAzMDx/dwKNF4kZiw+GZbcnFErfzkg0ObksGOozN2DgHnzUgLbxtzIkNt3PMJA5uYwZq4WGTxqdFHqKlPnH+zTMgLfWEtRhAtBxO3HCDB6TlMGEthmceNhzmOXe82PBMjrHB2W3HediYCfhF7njy4c88ZdV5csfPGD6o3FZtz8/e/PAxXu8LJACjBiUumPEpBwH+A0DiDyFVo2AUjIJRMKIBAAadTv3aMtOQAAAAAElFTkSuQmCC","orcid":"","institution":"Yeditepe University Faculty of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Aylin","middleName":"","lastName":"Yaba","suffix":""}],"badges":[],"createdAt":"2022-09-20 07:44:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2083681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2083681/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26847602,"identity":"f77faa82-b7e1-4cd8-b677-987261944ecf","added_by":"auto","created_at":"2022-09-22 20:44:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":662466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative immunoblot image for p-mTOR, mTOR, p-P70S6K, P70S6K at 0 - 1000 nM rapamycin doses at 48 and 72 hours. \u003cstrong\u003e(b) \u003c/strong\u003emTOR and \u003cstrong\u003e(c) \u003c/strong\u003eP70S6K activities at 48 hour. \u003cstrong\u003e(d) \u003c/strong\u003emTOR and \u003cstrong\u003e(e) \u003c/strong\u003eP70S6K activities at 72 hour. Statistical analysis is shown in Table 1.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/bdf436506c755af6d91d7b44.png"},{"id":26848913,"identity":"dbd6d6f9-2295-4e5e-867c-5745ab82152f","added_by":"auto","created_at":"2022-09-22 20:49:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":458290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative immunoblot image for PCNA and Caspase-3 at 0 - 1000 nM rapamycin doses at 48 and 72 hours. \u003cstrong\u003e(b) \u003c/strong\u003ePCNA and \u003cstrong\u003e(c) \u003c/strong\u003eCaspase-3 activities at 48 hour. \u003cstrong\u003e(d) \u003c/strong\u003ePCNA and \u003cstrong\u003e(e) \u003c/strong\u003eCaspase-3 activities at 72 hour. Statistical analysis is shown in Table 1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/b66f1c79392bed01177cd78a.png"},{"id":26847606,"identity":"bc543406-d08e-4a0c-bba0-d40a6be87750","added_by":"auto","created_at":"2022-09-22 20:44:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":34509,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative in the Wound-Healing Scratch for migration at 0 - 1000 nM rapamycin doses at 48 hours. Rapamycin treatment for 48 h significantly decreased migration in all cells, with a greater effect on dose of 4 nM compared to the control (0 nM). \u003cstrong\u003e(b) \u003c/strong\u003eat 72 hours. Rapamycin treatment for 72 h significantly decreased migration in all cells, with a greater effect on dose of 20 and 1000 nM compared to the control (0 nM). Statistical analysis is shown in Table 1.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/9ce49b6ccc846ad0e1fb4f34.png"},{"id":26847605,"identity":"bf00d985-cfa6-42a1-af3c-b23cabff6abd","added_by":"auto","created_at":"2022-09-22 20:44:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":233452,"visible":true,"origin":"","legend":"\u003cp\u003eTCam-2 cells promotes apoptosis without altering necrosis significantly when treated with 1000 nM rapamycin at 72 hours\u003cstrong\u003e. (a,b,c,d)\u003c/strong\u003e Bar graphs indicating viability, early apoptosis, late apoptosis and necrosis. TCam-2 cells was found ineffective in initiating the this pathway at 0 - 1000 nM rapamycin doses at 48 hours. \u003cstrong\u003e(e,f,h)\u003c/strong\u003e Bar graphs indicating viability, early apoptosis and necrosis. TCam-2 cells was found ineffective in initiating the this pathway at 0, 4, 20, 100, 500 and 1000 nM rapamycin doses at 72 hours. \u003cstrong\u003e(g)\u003c/strong\u003e Rapamycin treatment for 72 h significantly increased late apoptosis in all cells, with a greater effect on higher doses (4, 20, 100, 500 and 1000 nM) compared to the control (0 nM). \u003cstrong\u003e(i)\u003c/strong\u003e Representative flow cytometry quadrants. Statistical analysis is shown in Table 1.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/03ef8bb69d28a1fd585bba87.png"},{"id":26847604,"identity":"fa3b6888-7f64-483e-8eb8-e1c12aaf1be2","added_by":"auto","created_at":"2022-09-22 20:44:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142063,"visible":true,"origin":"","legend":"\u003cp\u003eRapamycin induces the G0/G1phase accumulation. Cell cycle distribution of TCam-2 cell line at \u003cstrong\u003e(a)\u003c/strong\u003e 48 h, \u003cstrong\u003e(b)\u003c/strong\u003e 72 h following exposure to 0 - 1000 nM rapamycin doses. \u003cstrong\u003e(c)\u003c/strong\u003eRepresentative flow cytometry quadrants. Statistical analysis is shown in Table 1.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/e89726f8ddf4f5a548d7c96c.png"},{"id":26848918,"identity":"2e8038d1-3c5e-4e65-8c98-744f1e627968","added_by":"auto","created_at":"2022-09-22 20:49:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1107414,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2083681/v1/fdc27838-b64e-4aab-a086-52ce3e8a0d2b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of Rapamycin Treatment in Human Seminoma TCam-2 Cells through Inhibition of G1-S Transition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTesticular cancer (TC) is the most common solid organ malignancy in young men and the leading cause of cancer-related deaths in this population [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. TC usually occurs during the reproductive period and can cause infertility [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Testicular tumors can be broadly classified germ cell tumors, sex cord-stromal tumors, mixed germ cell/sex cord-stromal tumors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although testicular cancer can arise from any cell type found in the testes, more than 95% of testicular cancers are germ cell tumors. The remaining 5% are sex cord-gonadal stromal tumors orginate from Leydig or Sertoli cells. Among germ cell tumors, testicular seminoma is a germ cell tumor of the testis and the most common testicular tumor, accounting for approximately 45% of all primary testicular tumors [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe TCam-2 cell line is the first seminoma-derived cell line that retains most of the characteristic features of seminoma [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. TCam-2 cells express typical marker genes for primordial germ cells and germ cell neoplasia in situ (GCNIS) (\u003cem\u003eSOX17\u003c/em\u003e, \u003cem\u003ePRAME\u003c/em\u003e, \u003cem\u003ecKIT\u003c/em\u003e, \u003cem\u003eTFAP2C\u003c/em\u003e, \u003cem\u003ePRDM1\u003c/em\u003e/\u003cem\u003eBLIMP1\u003c/em\u003e) and show typical GCNIS/seminoma morphology (large round cells with a large nucleus and clear cytoplasm) [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMammalian target of rapamycin (mTOR) is an important serine/threonine kinase [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] that plays critical roles in cell cycle, protein synthesis, energy metabolism, cell proliferation, growth, differentiation, and apoptosis [\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. mTOR is a downstream factor of PI3K/Akt and positively regulates tumor progression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. mTORC1 is one of the two major mTOR complexes and the main downstream effector of PI3K/PTEN/AKT signaling in response to growth factors which phosphorylates ribosomal protein S6 kinase (S6K1) to increase protein synthesis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Rapamycin is a natural product and non-ATP competitive inhibitor of mTORC1; it exert its\u0026rsquo; effects via binding to cytosolic FK binding protein (FKBP12) with high affinity, to interact with mTORC1 and via interfering with mTOR activity or complex assembly [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Previosly, we have reported mTOR signal pathway immunoreactivity in testicular seminoma, phosphorylated serine 2448 (p-mTOR), P70S6K and p-P70S6K and hypothesized that mTOR signaling pathway may contribute to the development or progression of testicular cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to its diverse roles and it\u0026rsquo;s general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Herein, we analyzed the effect of mTOR inhibition in seminoma-like TCam-2 cells with regard to cell proliferation, migration, cell cycle, and apoptosis.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Culture and Reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human TCam-2 cell line was derived in 1992 from a testicular tumor sample of a pure classical seminoma\u0026nbsp;[\u003ca href=\"#_ENREF_21\" title=\"Mizuno, 1993 #222\"\u003e21\u003c/a\u003e].\u0026nbsp;TCam-2 (obtained from Prof. Dr. Hubert Schorle, Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany) was grown in RPMI 1640 medium (Gibco, #11875093) supplemented with 10% FBS (fetal bovine serum) (Gibco, #16000044), 1% penicillin/streptomycin (Gibco, #15140122) and 200 mM glutamine (Gibco, #25030081). Cells were grown at 37\u0026deg;C and under 5% CO2 humidified atmosphere. Rapamycin (Glentham Life Sciences, #GA7417) was dissolved in DMSO (Sigma, #D8418) at a stock concentration of 10 mM.\u0026nbsp;Experimental groups were: control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin for 48 h and 72 h\u0026nbsp;[\u003ca href=\"#_ENREF_22\" title=\"Yu, 2011 #994\"\u003e22\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWestern blot\u003c/em\u003e was\u0026nbsp;\u003cem\u003eperformed\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eas described\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003epreviously\u0026nbsp;\u003c/em\u003e\u003cem\u003e[\u003c/em\u003e\u003ca href=\"#_ENREF_23\" title=\"Yaba, 2012 #372\"\u003e\u003cem\u003e23\u003c/em\u003e\u003c/a\u003e\u003cem\u003e]\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e\u0026nbsp;In summary, a total of\u0026nbsp;\u003c/em\u003e4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well were seeded on six-well plates and let attached overnight.\u0026nbsp;\u003cem\u003eT\u003c/em\u003eotal protein from the TCam-2 cells were extracted using RIPA lysis buffer (SantaCruz, #sc-24948) supplemented with protease (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78446) and phosphatase inhibitor cocktails\u0026nbsp;(Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78420). After the total protein concentration was determined, 20 \u0026mu;g of total lysate was loaded into each lane, separated electrophoretically using NuPAGE 4-12% Bis-Tris gel (Invitrogen\u0026trade;, #NP0321BOX), and then electroblotted onto PVDF membrane (Thermo Scientific, #88585). Later, membrane was blocked in TBS-T buffer (0.1% Tween-20 in Tris-buffered saline) with 5% nonfat dry milk (Sigma, #70166) for one hour. The membrane was then incubated with appropriate primary antibodies anti-mTOR (1:1000 dilution; Cell Signaling, #2983), anti-p-mTOR (1:1000 dilution; Cell Signaling, #2971), anti-P70S6K (1:1000 dilution; Cell Signaling, #9202), anti-p-P70S6K (1:1000 dilution; Cell Signaling, #701064), anti-PCNA (1:1000 dilution; Cell Signaling, #2586) and anti-Caspase-3 (1:1000 dilution; Cell Signaling, #9662) overnight at 4\u0026deg;C. After incubation with primary antibody, membrane was washed with TBS-T for three times for 10 min each, incubated with HRP-conjugated secondary antibody (1:5000 dilution, Cell Signaling, #7074) for one hour at room temperature and subsequently washed with TBS-T. Phospho-specific antibodies were always used for the first round of probing. Membrane was strippped with western blot stripping buffer (Takara Bio, #T7135A). Immunobolt images were acquired and bands were quantified by using Image Lab Software (BioRad, #1708265).\u0026nbsp;All experiments were repeated at least three times.\u0026nbsp;\u0026beta;-Actin\u0026nbsp;was used as an internal control and ratio of expression level of individual protein to that of\u0026nbsp;\u0026beta;-Actin\u0026nbsp;from the same samples was used to determine the final expression levels each protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound-Healing Scratch Assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well were seeded on six-well plates and let attached overnight. After overnight attachment, a straight line was created by scratching with a 100\u0026nbsp;\u0026mu;L pipette tip. Then the medium was removed, fresh medium with respective drug concentrations were added and images were obtained for t=0 h. The migration/healing was determined for 48 and 72 hours by obtaining microscopy images of the same area (Axiovert 135; Carl Zeiss Microscopy). Migration/healing was determined as previously described by using Fiji\u0026nbsp;[\u003ca href=\"#_ENREF_24\" title=\"Treloar, 2013 #552\"\u003e24\u003c/a\u003e]\u0026nbsp;and the percent healing against the control group was determined.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssesment of Apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViability, apoptosis and necrosis were evaluated by Annexin V/Propidium Iodide (PI) staining. For this purpose, cells were seeded into six-well plates as 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cell per well and incubated for overnight for attachment. Cells were then treated with rapamycin at respective doses for 48 and 72 hours. For staining, cells were detached by trypsinization, washed once with Dulbecco\u0026rsquo;s phosphate buffered saline solution (DPBS, Thermo Fisher Scientific, #14190144) and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 5\u0026nbsp;\u0026mu;L Annexin V-FITC reagent (Biolegend, #640906) and 1 \u0026micro;L PI solution (Thermo Fisher Scientific, #P3566, diluted to 250\u0026nbsp;\u0026mu;g/mL in DPBS) by incubating for 10 minutes under dark conditions at room temperature. Cells were read with DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed by CytExpert software\u0026nbsp;[\u003ca href=\"#_ENREF_25\" title=\"Aru, 2020 #850\"\u003e25\u003c/a\u003e]. The experiment was done as triplicates and 2.5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per analysis were acquired at medium flow rate.\u0026nbsp;\u003c/p\u003e\n\u003ch3\u003eCell cycle Assessment\u003c/h3\u003e\n\u003cp\u003eDNA content analysis was performed by using Cell Cycle Kit (Beckman Coulter Inc., #C03551). Cells were seeded as 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cell/six-well plate and incubated overnight for attachment. After incubating with rapamycin at respective doses for 48 and 72 hours, cells were detached by tyripsinization, washed once with DPBS, fixed with ice cold 70% ethanol by adding dropwise followed by incubation at 4\u0026deg;C for an hour. Tubes were stored at -20 \u0026deg;C overnight, and ethanol was removed by centrifuging cells at 400 x g for 5 minutes. Pellet was suspended in 500\u0026nbsp;\u003cstrong\u003e\u0026micro;\u003c/strong\u003eL cell cycle kit reagent, and tubes were incubated at room temperature under dark for 30 minutes. DNA content was analyzed by DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed with ModFit LT 5.0 software (Verify Software House)\u0026nbsp;[\u003ca href=\"#_ENREF_26\" title=\"Aru, 2019 #851\"\u003e26\u003c/a\u003e]. The experiment was done as triplicates and 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per test were acquired at medium flow rate. Analyses were performed\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s multiple comparison tests. A p value lower than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDose-dependent inhibition of mTORC1 in TCam-2 Cells by Rapamycin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRapamycin exerts its biological activity by inhibiting the mTOR, which is a key regulator of cell growth and survival in many cell types\u0026nbsp;[\u003ca href=\"#_ENREF_27\" title=\"Zhao, 2008 #993\"\u003e27\u003c/a\u003e]. In our study,\u0026nbsp;TCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin)\u0026nbsp;for 48 h and 72 h\u0026nbsp;[\u003ca href=\"#_ENREF_22\" title=\"Yu, 2011 #994\"\u003e22\u003c/a\u003e]. \u0026nbsp;After 48 hours of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p\u0026lt;0.05; Figure 1a and b; Table 1). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (**p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001; Figure 1a and c; Table 1). After 72 hours of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500 and 1000 nM rapamycin-treated groups (p\u0026lt;0.05; Figure 1a and d; Table 1). Moreover, P70S6K activity decreased at all treatment groups (****p\u0026lt;0.0001; Figure 1a and e; Table 1).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMain outcome measures for cell cycle distribution of TCam-2 cell line at 48 and 72 h following exposure to 0 - 1000 nM rapamycin doses. Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s multiple comparison tests. p values are shown statistically.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable align=\"left\" border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003eP values\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003cstrong\u003eG0/G1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;S \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; G2/M\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;48 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0065\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.184\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0012\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.1845\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0005\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0313\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.9974\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;500\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0008\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.1589\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.8954\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;1000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0092\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.8518\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; 72 h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0415\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.9955\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;20\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0121\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.8967\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;100\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.1951\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;500\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0004\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0.2086\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e0 vs.\u003cstrong\u003e\u0026nbsp;1000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0011\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0006\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0276\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eRapamycin does not significantly alter Caspase-3 and PCNA protein levels in TCam-2 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to check apoptosis and proliferation in TCam-2 cells after administration of specific doses of rapamycin, we investigated Caspase-3 and PCNA protein expression. \u0026nbsp;Caspase-3 expression did not change at either of the doses nor the time points (Figure 2a, c and e). While levels of PCNA proteins seemed to decrease in the at 48 h in a dose dependent manner, this decrease was not significant (Figure 2a and b). On the other hand, PCNA expression decreased significantly at 1000 nM rapamycin at 72 h (p\u0026lt;0.05; Figure 2a and d; Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound-Healing Scratch Assay in TCam-2 Cell Line\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evalutae TCam-2 cell migration after rapamycin treatment, wound-healing scratch assay were used. 4 nM rapamycin treatment significantly inhibited cell migration after 48 h (p\u0026lt;0.05; Figure 3a). On the other hand, rapamycin at doses of 20 and 1000 nM significantly decreased cell migration at 72 h (**p\u0026lt;0.01; Figure 3b; Table 1). No significant changes were detected at other doses for 48 h and 72 h (Figure 3a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emTOR inhibition by using rapamycin does not promote apoptosis in the TCam-2 Cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptotic effect of rapamycin mediated mTOR inhibition was evaluated by Annexin V/PI staining on 48 and 72 hours. Comparisons between the control and treatment groups revealed no significant change in viability, early and late apoptosis and necrosis rates at 48 hours (p\u0026gt;0.05) (Figure 4a, b, c, d and i).\u0026nbsp;On the other hand, rapamycin treatment increased late apoptosis rates at all concentrations tested significantly (p\u0026lt;0.05 for 4, 20, 100 and 500 nM; p\u0026lt;0.001 for 1000 nM) at 72 hours\u0026nbsp;(Figure 4g and i; Table 1)\u0026nbsp;thoughno significant changes in viability, early apoptosis and necrosis rates between doses for this timepoint was observed (p\u0026gt;0.05; Figure 4e, f, h and i)\u003c/p\u003e\n\u003ch3\u003emTOR inhibition leads to G1-phase arrest in TCam-2 cells in a dose dependent manner\u003c/h3\u003e\n\u003cp\u003eIt was previously reported that inhibition of\u0026nbsp;mTOR\u0026nbsp;by physiological cell stressors or pharmacological agents decrease proliferation\u0026nbsp;mainly by leading to cell cycle arrest at G1 phase\u0026nbsp;[\u003ca href=\"#_ENREF_28\" title=\"Tian, 2019 #627\"\u003e28\u003c/a\u003e]. It should be noted that inhibition of mTOR during \u003cem\u003einterphase\u003c/em\u003e would be responsible for arrest in G1, and therefore the interphase expression patterns of p-mTOR, and p-P70S6K (Figure 1a-c) should not be ignored. Consistent with the literature, our results revealed that rapamycin at 1000 nM promoted G1 phase arrest in TCam-2 cells in both 48 and 72 hours significantly compared to the control group (p\u0026lt;0.001 and p\u0026lt;0.01, respectively) whereas 4, 20,100 and 500 nM showed a lower rate of rapamycin treatment percentage of cells (approximately 25%) were in G1 (**p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001; Figure 5a, b and c; Table 1).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion And Conclusion","content":"\u003cp\u003eThe mTOR is frequently deregulated in cancer and activating somatic mutations of \u003cem\u003emTOR\u003c/em\u003e\u003cem\u003e \u003c/em\u003ehave recently been identified in various types of human cancer and so the mTOR is therapeutically targeted [29]. mTOR inhibitors have been commonly used as immunosuppressants, and are currently approved for the treatment of human malignancies [30]. In this study, we aimed to investigate the effect of inhibition of the mTORC1 signaling pathway in TCam-2 seminoma cells. This study demonstrates that treatment with 1000 nM rapamycin can inhibit proliferation of TCam-2 seminoma cells by downregulating PCNA through mTOR inhibition. In a previous study, analyzed whether mTORC1 and MAPK signaling pathway activities were differentially active in both Germ Cell Tumors (GCT) classes in seminoma and non-seminoma germ cell tumors. In this study, it was shown that the mTORC1 signaling pathway is activated in non-seminoma germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma), but not in seminomas. Seminomas have been shown to negatively regulate mTORC1 activity with high REDD1 levels to maintain an undifferentiated state. On the other hand, members of the EGF and FGF receptor families have been shown to be more highly expressed in NSGCTs and stimulate the signaling of the EGF and FGF2 ligands mTORC1 and MAPK. This suggests that mTORC1 activation in NSGCTs contributes to cell proliferation [20]. We investigated the activity of mTORC1 in the TCam-2 cell line and propose to show for the first time that the mTOR signaling pathway acts as an oncogene in the TCam-2 seminoma cell line. This is evidenced by its dramatic downregulation in seminomas based on western blot experiments, as well as its positive regulation of cell proliferation and migration.\u003c/p\u003e\n\n\u003cp\u003emTOR functions as a key control protein that integrates signals from a variety of environmental factors, and mTOR signalling proteins are essential for cell growth, migration, survival, and development. To date, we know that mTOR signaling proteins play a role in cell adhesion in the testis and are involved in the regulation of the blood\u0026ndash;testis barrier [31]. mTOR signaling proteins express in the testis of adult mice, and mTOR pathway proteins may play a role in proliferation and stimulation of meiotic initiation of spermatogonial stem cells [32]. Treatment with mTOR inhibitors, sirolimus and everolimus, had destructive effects on the adult mouse testis and impairs gonadal function [33]. Therefore, we suggested that investigating the role of mTOR signalling inhibition in the TCam-2 cell line might be important to understand underlying mechanism of male reproductive cancers.\u003c/p\u003e\n\n\u003cp\u003eRapamycin is widely used in the clinic as an antiproliferative drug and immunosuppressant, moreover, its analogs have been shown to retard tumor proliferation in clinical trials[34]. Currently, more studies are focusing on the effect of rapamycin in tumor therapy [35]. . As an mTOR inhibitor, rapamycin has been reported to block cell cycle progression from G1 to S phase by inhibiting p70 ribosomal protein S6 kinase (p70S6K) [36, 37]. As a threonine kinase, mTOR phosphorylates S6K1 and 4E-BP1 and promotes transcription of key mRNAs associated with cell cycle progression from G1 to S phase [37]. Through its interaction with p70S6K, it is associated with cell growth, proliferation and differentiation by regulating ribosome biogenesis, protein synthesis, cell cycle progression and metabolism [38]. mTOR inhibition via rapamycin was reported to inhibit proliferation and migration of hemangiomas and reduce growth of vascular tumors [39]. Consistent with this finding, we showed that the activity of the downstream effector molecule mTOR, P70S6K was significantly reduced upon rapamycin treatment at all concentrations tested in TCam-2 seminoma cells, indicating mTOR inhibiton decreases cell cycle progression and in turn, proliferation. Moreover, in our study, rapamycin induced mTOR inhibition led to G1 phase-arrest during cell cycle progression, which is also in line with reports showing that mTOR is involved in cell cycle progression in G1 [40, 41].\u003c/p\u003e\n\n\u003cp\u003ePCNA is a nuclear protein expressed in proliferating cells, is required for the maintenance of cell proliferation, and is used as a marker of cell proliferation [42]. AKT/mTOR pathway have shown to inhibit skin cancer proliferation by downregulating PCNA [43]. In prostate cancer, agents such as vicenin-2 reduce the expression of PCNA and cyclin-D1 by inhibiting the EGFR/Akt/mTOR pathway [44]. The expression levels of p-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K were also significantly downregulated in hemangioma tissues and cells compared with the control group, and the expressions of Ki67 and PCNA were significantly decreased [45]. In another study, syndecan binding protein was reported to inhibit apoptosis of gastric cancer cells and promote the growthof gastric cancer by inducing PCNA expression and inactivating the PI3K/AKT/mTOR pathway [46]. In our study, we showed that rapamycin decreases PCNA expression in a dose dependent manner on 72 h, though a significant decrease was only observed upon treatment with 1000 nM. Yet, according to cell cycle analysis, rapamycin promoted G1-phase arrest in all concentrations tested compared to the control group, indicating other possible mechanisms involving in cell cyce arrest rather than PCNA should be clarified. In short, Inhibition of mTOR by rapamycin prevents cells growth, thus division and proliferation. Therefore, we suggest that rapamycin, an mTOR inhibitor, can potentially be used as a chemotherapeutic agent in human seminoma cells.\u003c/p\u003e\n\n\u003cp\u003eWe hope that the knowledge gained from our study will contribute to the development of new treatment approaches for seminoma patients and to the restoration of testicular functions and preservation of fertility in the future.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e \u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003eAffiliations\u003c/h3\u003e\n\u003cp\u003eDepartment of Histology and Embryology, Yeditepe University, School of Medicine, 34755, Istanbul, Turkey.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEcem Yildirim, Tugce Onel, Aylin Yaba.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Physiology Yeditepe, University, School of Medicine, 34755, Istanbul, Turkey.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCihan Suleyman\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDepartment of Immunology, Yeditepe University, School of Medicine, 34755, Istanbul, Turkey.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBasak Aru, Gulderen Yanikkaya Demirel\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003eCompeting Interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch3\u003eCorresponding author\u003c/h3\u003e\n\u003cp\u003eCorrespondence to Aylin Yaba.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eProf. Dr. Hubert Schorle (Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany) for providing TCam-2 cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: TO, CSE and AY; Data curation: TO, CSE, BA, EY; Formal analysis: TO, CSE, BA, EY, AY; Investigation: TO, CSE, BA, EY, AY; Methodology: TO, CSE, BA, EY, AY; Software: TO, CSE, BA, EY, AY; Supervision: AY; Validation: TO, CSE; Visualization: TO, CSE; Roles/Writing- original draft: TO, CSE, BA, EY, GYD, AY; Writing- review \u0026amp; editing: TO, CSE, BA, EY, GYD, AY.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYe H and Ulbright TM (2012) Difficult differential diagnoses in testicular pathology. 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Biotechnol Appl Biochem. doi: 10.1002/bab.2103\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mTOR, Rapamycin, TCam-2 cell line","lastPublishedDoi":"10.21203/rs.3.rs-2083681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2083681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction:\u003c/strong\u003e Mammalian Target of Rapamycin (mTOR) is an important serine/threonine kinase that plays a critical role in several processes including cell cycle, protein synthesis and energy metabolism. Due to its multiple roles and general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited. Therefore, our aim was to investigate the expression of mTOR signaling pathway proteins in the TCam-2 cell line after rapamycin treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eTCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin) for 48 h and 72 h. mTOR, p-mTOR, P70S6K, p-P70S6K, proliferating cell nuclear antigen (PCNA) and Caspase-3 expression levels were analyzed by western blot. Apotosis and cell cycle were analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e After 48 hours of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p\u0026lt;0.05). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (***p\u0026lt;0.001, ****p\u0026lt;0.0001). After 72 hours of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500 and 1000 nM rapamycin-treated groups (p\u0026lt;0.05). Moreover, P70S6K expression decreased in all treatment groups (****p\u0026lt;0.0001). Caspase-3 expression were similar in all groups. While PCNA expression tended to decrease at 48 h in a dose-dependent manner, this decrease was not significant. We detected decreased PCNA expression at 1000 nM rapamycin at 72 h (p\u0026lt;0.05). The rate of apoptosis increased especially at 1000 nM rapamycin at 72 hours (***p\u0026lt;0.001). On the other hand, according to the results of the cell cycle experiment, G1 phase arrest was detected at all rapamycin doses at 48 and 72 hours (***p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e our study indicated that 1000 nM rapamycin may inhibit TCam-2 seminoma cells growth by halting cell proliferation through inhibition of G1-S transition. Therefore we believe that the findings obtained will contribute to the development of new treatment approaches for seminoma patients in the future and in the process of restoring testicular functions and preserving fertility.\u003c/p\u003e","manuscriptTitle":"Effect of Rapamycin Treatment in Human Seminoma TCam-2 Cells through Inhibition of G1-S Transition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-22 20:44:49","doi":"10.21203/rs.3.rs-2083681/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"074e075a-27ca-4e37-a4d3-69484afe0ede","owner":[],"postedDate":"September 22nd, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-22T20:44:51+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-22 20:44:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2083681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2083681","identity":"rs-2083681","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}