Xanthohumol Suppresses Endometrial Cancer Cell Proliferation via Promotion of Smurf1-mediated ACLY Ubiquitination and Degradation

Xanthohumol Suppresses Endometrial Cancer Cell Proliferation via Promotion of Smurf1-mediated ACLY Ubiquitination and Degradation | 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 Article Xanthohumol Suppresses Endometrial Cancer Cell Proliferation via Promotion of Smurf1-mediated ACLY Ubiquitination and Degradation Chenyu Hu, Mulin Yang, Junying Xu, Jiazhen Tian, Ting Zhang, Amier Abulizi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4487101/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 ATP citrate lyase (ACLY) is pivotal in de novo fatty acid synthesis. It emerges as a core metabolic enzyme implicated in malignant tumor progression, especially in Endometrial Cancer (EC). The present investigation revealed that Xanthohumol (XN), a naturally prenylated flavonoid, is a novel inactivator of ACLY. XN demonstrates a significant reduction in de novo fatty acid synthesis and concurrent inhibition of cell proliferation in EC. Moreover, XN directly inhibits ACLY enzyme activity and facilitates Smurf1-mediated ACLY ubiquitination and degradation. The research revealed that the knockdown of ACLY reduced fatty acid synthesis, proliferation, and colony formation in EC cells. Conversely, contrasting results were observed upon ACLY overexpression. Additionally, treatment with XN inhibited fatty acid synthesis, cell proliferation, and colony formation, inducing non-apoptotic cell death and G0/G1 cycle arrest by downregulating ACLY expression. The crucial involvement of Smurf1-mediated ACLY ubiquitination in the XN-induced downregulation of ACLY was also highlighted. Notably, the role of the E3 ubiquitin ligase Smurf1 in mediating the ubiquitination of ACLY is reported here for the first time. Furthermore, these findings indicated the potential of ACLY as a prospective drug target for EC. Considering the inhibitory effect of XN on ACLY, it presents encouraging prospects for treating EC. Xanthohumol Endometrial Cancer ATP citrate lyase E3 ligase Smurf1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Endometrial cancer (EC) ranks among the top three prevalent malignant tumors affecting the female reproductive system and stands as the sixth most commonly diagnosed cancer among women globally 1 . The prevalence and mortality rates of EC are elevated in developed countries in comparison to developing ones. Currently, North America demonstrates the highest incidence of EC 2 , 3 . In the past few decades, the overall incidence of EC has increased by 132%, signifying a rise in the prevalence of risk factors. Key risk factors for EC encompass heightened estrogen levels, obesity, advanced age, tamoxifen use, early menstruation, and late menopause 3 , 4 . Early-stage EC can be effectively treated through surgery or a combination of surgery, radiotherapy, and chemotherapy. Chemotherapy, along with comprehensive treatment, is typically utilized to manage advanced and recurrent EC. Currently, the established first-line treatment for advanced or metastatic EC involves the combination of paclitaxel and carboplatin 5 . However, the efficacy of this treatment is constrained, often resulting in poor prognosis, recurrence, and low tolerance. Therefore, there is a need to devise more effective strategies for EC treatment to increase patient survival rates. Obesity is a significant risk factor for EC, and among the 20 most prevalent cancer types, EC is the one strongly associated with obesity. An increase of 5 kg/m 2 in body mass index (BMI) is correlated with a 54% elevated risk of cancer. Women with a BMI exceeding 40 kg/m 2 have a lifetime risk of EC ranging from 10–15% 6,7 . Abnormal lipid metabolism is a crucial metabolic alteration observed in tumors, and several investigations have illustrated its crucial involvement in the development of EC 8 . ATP-citrate lyase (ACLY) is an enzyme in the cytoplasm and nucleus, existing as a homotetramer 9 . It catalyzes the conversion of citric acid to acetyl-CoA, subsequently generating fatty acids when fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) are present 5 . ACLY is a pivotal intermediary connecting glucose metabolism to fatty acid synthesis, serving as an upstream regulator in the fatty acid synthesis pathway. By enabling the de novo synthesis of fatty acids, ACLY facilitates the biosynthesis of cell membranes and is imperative for the formation of tumor cells 10 . Elevated expression or activation of ACLY was observed across various cancer types. Research indicates that interference with ACLY or its knockdown suppresses tumor cell proliferation in both in vitro and in vivo models 11 – 15 . Ubiquitination represents a crucial post-translational modification process in eukaryotes. Three enzyme families regulate this intricate mechanism: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). Ubiquitin is pivotal in regulating proteins, involved in diverse cellular processes, and contributing to the development of diseases, including cancer. The process of ubiquitination is associated with multiple cancer types, including EC 16 . Recent findings indicate the significance of the ubiquitin-proteasome system in the development of EC, which is increasingly recognized as part of the regulatory mechanism 17 , 18 . For instance, in lung cancer, the CUL3-KLHL25 ubiquitin ligase was involved in the ubiquitination and degradation of ACLY, consequently suppressing lipid synthesis. 18 . However, no specific E3 ubiquitin ligase is identified for ACLY that controls energy metabolism, particularly in EC. Natural products have gained popularity recently due to their effectiveness, multi-target capabilities, and safety. These characteristics make them appealing for diverse applications, including anti-tumor endeavors 19 . The exploration and development of natural compounds for the prevention and treatment of cancer have received considerable attention. Xanthohumol (XN), a pentenyl flavonoid extracted from hops, possesses anticancer, antioxidant, antibacterial, and anti-inflammatory activities 20 . Research has demonstrated that XN can impede the growth of various cancer types, such as lung cancer 21 , breast cancer 22 , colon cancer 23 , prostate cancer 24 , liver cancer 25 , and other cancers. This inhibition effect could be achieved by suppressing tumor cell proliferation, invasion, and migration, causing cell cycle arrest and inducing apoptosis. Furthermore, XN was found to synergize with chemotherapy and radiotherapy, reducing chemotherapy resistance and minimizing its associated side effects 26 . However, the anti-tumor properties of XN on EC have not been reported. In this research, the anti-tumor properties of XN in EC cells were investigated in vitro . It was found that XN inhibits the proliferation of EC cells through decreased expression of ACLY, which was associated with Smurf1-mediated ACLY ubiquitination and degradation. Moreover, XN decreases the levels of ACC and FASN by disrupting ACLY function, resulting in reduced lipid synthesis. Results Impacts of XN on EC cell proliferation To assess the impacts of XN on EC cells, varying concentrations of XN (0, 10, 20, and 40 µM) were utilized to treat the EC cells for 24 and 48 hours (Fig. 1 A, 1 B). The impact of XN on the proliferation of EC cells was then assessed utilizing the CCK-8 assay. XN demonstrated a dose- and time-dependent inhibition of EC cell proliferation in comparison to the control group. The colony formation assays and trypan blue assays illustrated that XN substantially inhibited the growth of EC cells and stimulated cell death in a dose-dependent manner in comparison to the control group (Fig. 1 C- 1 F). Moreover, flow cytometry analysis of the cell cycle and apoptotic rate indicated that XN stimulated G0/G1 cell cycle arrest in EC cells (Fig. 1 G, 1 H). However, the apoptosis of EC cells was not substantially altered by XN. In the G1 phase of the cell cycle, Cyclin-dependent kinase 4 (CDK4) and CDK6 interact with Cyclin D1 (CCND1), cyclin D2, and cyclin D3, respectively. Subsequent Western blot analysis demonstrated a substantial reduction in the CDK6 and CCND1 expression relative to the control group (Fig. 1 I, 1 J). These findings suggested that XN exhibits a remarkable anti-tumor impact in EC cells. XN-mediated ACLY suppression resulting in reduced de novo lipid synthesis in EC cells ACLY is a pivotal enzyme in cancer metabolism. To examine whether the anti-tumor efficacy of XN on EC cells is dependent on ACLY downregulation, the AutoDock Vina bioinformatics website was utilized for molecular docking analysis. This analysis aimed to predict the potential interactions between XN and ACLY. The prediction results revealed a favorable binding effect between XN and ACLY (Fig. 2 A). Following XN treatment, a dose-dependent reduction in ACLY protein expression was observed in Ishikawa and HEC-1-A cells (Fig. 2 B, 2 C). Subsequent experiments revealed that XN treatment inhibited ACLY enzyme activity in both Ishikawa and HEC-1-A cells (Fig. 2 D, 2 E). To explore whether XN influenced the stability of ACLY protein, a cycloheximide (CHX)-chase experiment was conducted. It revealed a substantial reduction in the half-life of ACLY in XN-treated EC cells in comparison to control groups (Fig. 2 F, 2 G). In addition, the proteasome inhibitor, MG-132, restored ACLY expression (Fig. 2 H, 2 I). Furthermore, the Western blot method was utilized to illustrate the impact of XN treatment on de novo lipid synthesis. The outcomes revealed that treatment with XN leads to a dose-dependent reduction in the protein expression levels of related fatty acid synthases, including fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) and resulted in a significant decrease in cellular triglyceride content (Fig. 2 B, 2 C, 2 J, 2 K). These outcomes suggest that XN decreases de novo lipid synthesis in EC cells by downregulating ACLY. XN promotes Smurf1-mediated ACLY ubiquitination and degradation In protein metabolism, inducing protein degradation is an effective method to reduce intracellular protein expression. Prior investigations have revealed that XN treatment reduced ACLY expression through protein degradation (Fig. 2 F, 2 G). Subsequently, the ubiquitination analysis demonstrated increased ubiquitination of ACLY after XN treatment in Ishikawa and HEC-1-A cells (Fig. 3 A, 3 B). Additionally, it was observed that XN treatment promoted the formation of Lys 48 (K48)-linked polyubiquitin chains on ACLY protein, indicating proteasome-mediated substrate degradation (Fig. 3 C, 3 D). Furthermore, the bioinformatics-based ubiquitin ligase-substrate interaction programs Ubibrowser were employed to identify potential ACLY target ubiquitin ligase E3. Based on the findings, we hypothesized that Smurf1 might function as a potential E3 ligase regulating ACLY. To confirm the hypothesis, Co-IP analysis was conducted employing an anti-ACLY antibody. The outcomes demonstrated endogenous binding between Smurf1 and ACLY proteins (Fig. 3 F). Moreover, the Co-IP analysis illustrated the formation of a complex between Flag-tagged Smurf1 and GFP-ACLY in HEK-293T cells (Fig. 3 E). These observations corroborate the assertion that Smurf1 effectively interacts with ACLY as a binding partner Moreover, the Co-IP results demonstrated that XN promoted the interaction between ACLY and E3 ligase Smurf1 (Fig. 3 G). Subsequently, an assessment was undertaken to ascertain the influence of Smurf1 on ACLY protein levels. Western blotting analysis revealed that the ectopic overexpression of Smurf1 results in a dose-dependent reduction in ACLY protein levels in EC cells (Fig. 3 H). These findings imply that XN triggers the ubiquitination and degradation of ACLY in a Smurf1-dependent manner. ACLY plays a crucial role in EC cells Growing research highlights the pivotal role of cancer metabolism in the onset and progression of EC 8 . Dysregulation in lipid metabolism emerges as a prominent factor contributing to the development of EC. ACLY exhibits high expression or overactivation across diverse cancer types, including EC. It is critical in facilitating heightened lipid synthesis in cancer cells. To substantiate the influence of ACLY on EC cells, overexpression and knockdown experiments targeting ACLY were conducted in EC cell lines, respectively. Following this, the expression levels of ACC and FASN were evaluated employing qRT-PCR and Western blotting analysis. The findings unveiled notable outcomes that demonstrated a substantial upregulation of mRNA and protein levels for ACC and FASN in EC cells overexpressing ACLY in comparison to the control group (Fig. 4 A- 4 D). A contrasting outcome was noted following the ACLY knockdown. (Fig. 5 A- 5 D). CCK-8 assays, colony formation assays, and trypan blue assays demonstrated overexpressed ACLY-promoted cell viability in Ishikawa and HEC-1-A cells (Fig. 4 E- 4 J). Moreover, elevated expression of cell cycle-related proteins CDK6 and CCND1 was detected (Fig. 4 K, 4 L). Conversely, a contrary result was observed upon ACLY knockdown (Fig. 5 A- 5 L). These observations imply that ACLY is pivotal in the proliferation of EC cells. ACLY reverses the effects of XN-treated on EC cells To further confirm that the suppression of proliferation results from the reduction in ACLY expression induced by XN, EC cells were transfected with an ACLY-overexpressing plasmid subsequent to treatment with XN. The Western blot outcomes revealed considerable upregulation of ACC and FASN expression. This was observed when examining the overexpression-induced increase after transfection with the ACLY-overexpressing plasmid following treatment with XN in comparison to the group treated solely with XN (Fig. 6 A, 6 B). Additionally, transfecting EC cells with an ACLY-overexpressing plasmid after XN treatment substantially reversed the inhibitory effects observed with XN treatment alone on cell proliferation (Fig. 6 C- 6 H). In contrast, the overexpression of ACLY following XN treatment reversed the levels of CDK6 and CCND1 in EC cells in comparison to XN alone (Fig. 6 I, 6 J). These observations substantiated that XN suppresses the proliferation of EC cells by directly targeting ACLY. Discussion EC ranks among the most common tumors affecting the female reproductive system. Its increasing worldwide incidence is closely associated with the rising prevalence of obesity 27 . Dysregulated lipid metabolism is a key metabolic alteration in cancer, with reports indicating that over 30% of genes associated with lipid metabolism demonstrate abnormal expression in EC, including ACLY 28 . This study unveils that by deactivating ACLY, XN impedes de novo fatty acid synthesis and suppresses cell proliferation in EC. XN, a naturally occurring compound derived from hops, exhibited anti-tumor and anti-obesity properties. In vivo research has revealed that XN functions as a ligand for farnesoid X, thereby augmenting glucose and lipid metabolism in KK-A(y) mice 29 . Moreover, XN impedes increased body weight, liver weight, and plasma and hepatic triacylglycerol levels stimulated by a high-fat diet. This effect is achieved through the modulation of hepatic fatty acid metabolism and suppression of intestinal fat absorption in rats 30 . In vitro , XN reduces fatty acid synthesis and cholesterol biosynthesis by suppressing the activation of Sterol regulatory element-binding proteins (SREBPs) 31 . Conversely, the study reveals that XN impedes de novo fatty acid synthesis by inhibiting ACLY activation in EC. XN has undergone extensive investigation as an anticancer agent, with studies reporting its ability to inhibit the proliferation of various cancers. These cancers include gastric cancer 32 , breast cancer 22 , lung cancer 21 , cholangiocarcinoma 33 and colon cancer 23 . Furthermore, our previous experiments revealed that XN impeded cell proliferation by inducing paraptosis in leukemia cells 34 . In the present investigation, substantial anti-tumor activity of XN against EC cells was observed in vitro . XN suppresses the proliferation of EC cells by directly inhibiting the enzymatic activity of ACLY and promoting Smurf1-mediated ubiquitination and degradation of ACLY. Lipids are essential constituents of biological membranes and are vital substrates for energy metabolism. De novo lipid synthesis is critical in tumor progression and represents a target for cancer therapy. ACLY, a key enzyme in de novo lipid synthesis, establishes a connection between glucose metabolism and the lipid synthesis process. 10 , 35 . Increasing research demonstrated the elevated activation or expression of ACLY in multiple tumor tissues, including EC 28 , hepatocellular carcinoma 36 , lung cancer 12 , prostate cancer 37 , bladder cancer 38 , colon cancer 39 , and ovarian cancer 40 . The current investigation has shown that overexpression of ACLY markedly improved the mRNA and protein expression levels of ACC and FASN, crucial components involved in fatty acid synthesis. This overexpression also elevated cell viability, as shown by the trypan blue experiment, and stimulated cell proliferation, as assessed by colony formation and CCK-8 assay. Conversely, the opposite effect was observed when ACLY was downregulated. In this study, by employing the AutoDock Vina bioinformatics platform for molecular docking analysis, a favorable binding interaction between XN and ACLY was predicted. Additionally, it was observed that XN inhibited the enzyme activity of ACLY in EC in a dose-dependent manner. Additional research is necessary to verify whether XN inhibits ACLY enzyme activity by directly binding to ACLY. The ubiquitin-proteasome is a crucial protein degradation pathway in eukaryotic cells 41 . Protein degradation and the regulation of protein stability are primarily governed by K48-linked polyubiquitination. In contrast, k63-linked polyubiquitination predominantly functions in signaling, DNA repair, and the regulation of protein activity 42 – 44 . In the current study, XN was found to augment the ubiquitination and subsequent degradation of ACLY via K48-linked polyubiquitination. Smurf1, identified as an E3 ubiquitin ligase, belongs to the HECT-type ubiquitin ligase classification 45 . Moreover, it was demonstrated that Smurf1 induces K48-linked polyubiquitination of ACLY in EC. However, the specific domains of Smurf1 that are responsible for binding to ACLY warrant further investigation. In conclusion, the anti-tumor impacts of XN in EC were investigated. XN blocked the de novo synthesis of fatty acid and inhibited the proliferation of EC by impairing the enzyme activity and expression of ACLY. Moreover, the study identified that Smurf1 ubiquitinates ACLY, indicating the critical involvement of Smurf1-mediated ACLY ubiquitination in the progression of EC. Collectively, XN emerges as a potent ACLY inactivator with substantial therapeutic promise against EC (Fig. 7 ). Materials and methods Reagents and cell culture XN, with a purity level surpassing 98%, was supplied by Yumen Technology Development Company in Gansu, China. Chemical reagents, comprising DMSO, SDS, Tris base, and NaCl, were procured from Solarbio (China). MG-132 and cycloheximide (CHX) were obtained from MedChemExpress (New Jersey, USA). Biological Industries in Israel was accessed to acquire the DMEM and Fetal Bovine Serum (FBS) for cell culture. The amounts of triglyceride in the cell lysates were measured using a commercial test kit (Jiancheng, Nanjing, China). The human EC cell line Ishikawa was sourced from the Cell Resource Center, Peking Union Medical College (PRCR, Beijing, China). It was maintained in DMEM, having 1% penicillin and streptomycin along with 10% FBS at 37℃ in a humidified atmosphere consisting of 5% CO 2 . Moreover, the human EC cell line HEC-1-A was acquired from the Chinese Academy of Sciences Culture Preservation Committee Cell Bank. It was maintained in McCoy’s 5A medium (Procell, China) supplemented with 1% penicillin and streptomycin as well as 10% FBS at 37℃ in a humidified atmosphere containing 5% CO 2 . Mycoplasma analysis was conducted on all cells within the past three years. Plasmid transfection was carried out utilizing Lipofectamine® 2000 (Thermo Fisher Scientific, USA) following the prescribed procedure from the manufacturer. CCK-8 assay The cells were incubated overnight in 96-well plates at a density of 5×10 3 cells per well. The cells were exposed to varying concentrations of XN (0 µM, 10 µM, 20 µM, and 40 µM) for different time durations (0, 24, and 48 h). Afterward, 10 µL of CCK-8 reagent (Biosharp, China) was introduced to each well. After two hours of incubation at 37℃, a full-wavelength microplate reader (Thermo Fisher Scientific, USA) was utilized to obtain the absorbance values at 450 nm. The cells were seeded into 96-well plates (5×10 3 cells/well) and cultured in a complete medium for 24 hours. Subsequently, cell viability was evaluated every 24 hours, up to 72 hours, following transfection with either the ACLY-overexpression or ACLY-knockdown plasmids. Additionally, the CCK-8 assay was executed as described previously. Colony formation assay and trypan blue assay Both cell lines were cultured into a 6-well plate at a density of 500 cells per well. The plate was gently rotated to ensure even dispersion of the cells. After 24 hours, the EC cells were exposed to XN (0 µM, 10 µM, and 20 µM) and placed in a cell culture incubator for two weeks at 37℃ with 5% CO 2 . Moreover, the cells were rinsed thrice with PBS with subsequent fixation utilizing 4% paraformaldehyde for 30 minutes. Following this, they underwent staining with 0.1% crystal violet for 30 minutes. Subsequently, the stained samples were rinsed with PBS to remove the staining solution. The cells were transfected with the ACLY-overexpression and ACLY-knockdown plasmid. Subsequently, they were cultured in 6-well plates (500 cells/well) for two weeks. The operation was performed as described previously. Trypan blue selectively stains only dead cells, which appear blue under microscopic examination. Cells undergoing exponential growth were inoculated into 6-well plates containing 10% FBS medium and cultured for 24 hours. Following that, the cells were subjected to several treatments. After 24 hours, the cells underwent washing and resuspension in PBS until achieving a cell density of approximately 80–90%. Then, 90 µl of the cell suspension and 10 µl of trypan blue were transferred to an EP tube and gently pipetted to ensure thorough mixing. After incubation for 1 minute, the cells were suspended and added to the blood cell meter to count the blue-stained and unstained cells, which was completed within 3 minutes. Cell cycle and apoptosis analysis EC cells, at a density of 2 × 10 6 cells per well, were seeded onto 6-well plates for cell cycle analysis. Subsequently, they were treated with XN for 24 hours utilizing the Cell Cycle and Apoptosis Analysis Kit (Beyotime, China). For apoptosis analysis, cells were inoculated onto 6-well plates (2 × 10 6 cells/well) and treated with XN for 24 hours. Subsequently, via flow cytometry, apoptosis was detected utilizing the Annexin V/PI staining kit (MULTI SCIENCES, China). Western blotting analysis The cells underwent lysis utilizing an ice-cold RIPA lysis buffer as per the prescribed protocol from the manufacturer. Protein concentration was assessed utilizing the bicinchoninic acid (BCA) assay. Afterward, the proteins underwent separation via SDS-PAGE electrophoresis and electrotransfer separated to the PVDF membrane and were transferred onto PVDF membranes. The 5% BSA Blocking Solution was used for membrane blocking at room temperature for 2 hours and the membranes were treated with the primary antibodies at 4℃ overnight. These antibodies include β-actin (ZSGB-BIO, #TA-09), ACLY (Servicebio, #GB11902-100), ACC (Abways, #CY5575), FASN (Abways, #CY6579), CCND1 (Abways, #CY5404), and CDK6 (Abways, #CY5835). After three washes with TBST buffer, the membranes were subjected to incubation with HRP-conjugated anti-rabbit/mouse antibodies for 2 hours. Subsequently, the membranes were treated with an Enhanced Chemiluminescence Detection reagent and analyzed employing a chemiluminescence analyzer. Immunoprecipitation assays A co-immunoprecipitation (CO-IP) assay was executed utilizing the protein binding IP kit (Absin, #abs955). Initially, cell lysates were treated overnight at 4°C with anti-ACLY (Proteintech, #67166-1-Ig), anti-Smurf1 (Proteintech, #55175-1-AP), anti-Flag (Proteintech, # 66008-4-Ig), anti-GFP (Proteintech, #50430-2-AP) or control IgG (Cell Signaling Technology, #2729). Subsequently, the lysates were exposed to protein A/G-agarose beads at 4°C for 4 hours. The resulting precipitates underwent washing five times in a wash buffer and were subjected to Western blotting analysis for evaluation. RNA extraction and quantitative real-time polymerase chain reaction Total RNA was obtained from cells utilizing TRIzol reagent (Invitrogen, USA), complying with the recommended procedure of the manufacturer. GAPDH was employed as an endogenous control to normalize mRNA. Subsequently, mRNA was reverse transcribed to complementary DNA (cDNA) employing the Revert Aid First-Strand cDNA kit (Thermo Fisher Scientific, USA), respectively, complying with the recommended protocol of the manufacturer. qRT-PCR was executed in triplicate on the Rotor-Gene Q MDx detection system (QIAGEN, Germany) utilizing the SYBR Green PCR kit (QIAGEN, Germany) to examine mRNA. Additionally, relative alterations in the expression levels of mRNA were assessed employing the 2 −ΔΔCT method. The primers utilized for the present investigation are as follows: ACLY forward, 5’-ATCGFGTTCAAGTATGCTCGGG-3’, reverse, 5’-GACCAAGTTTTCCACGACGTT-3’; ACC forward, 5’-TCACACCTGAAGACCTTAAAGCC -3’, reverse, 5’-AGCCCACACTGCTTGTACTG-3’; FASN forward, 5’-ACAGCGGGAATGGGTACT-3’, reverse, 5’-GACTGGTACAACGAGCGGAT-3’. ACLY enzyme activity assay After treating EC cells with XN for 24 hours, ACLY enzyme activity was assessed utilizing a detection reagent (Solarbio, China). Initially, ACLY extractant was employed, followed by centrifugation of the supernatant after ultrasonic fragmentation. The procedure was conducted following the provided instructions. Finally, the absorbance was measured at OD340nm utilizing an enzyme labeling instrument. Statistical analysis The statistical analysis was executed with GraphPad Prism 9. The one-way ANOVA or student’s t-test was employed for assessing the difference between tested groups. A probability value of p < 0.05 reflected statistical significance. The experiment was conducted in triplicate, and quantitative data were presented as mean ± SD. Data availability The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Declarations Author contributions C.H. and M.Y.: Investigation, formal analysis, writing original draft. J.X. and J.T.: Investigation, methodology. T.Z.: Funding acquisition. A.A.: Resources and supervision. X.M. and J.Z.: Conceptualization, project administration, Writing— review and editing, funding acquisition. Funding The work was supported by Shihezi University High-Level Talent Scientific Research Start-up Project (RCSX2018B01), Shihezi University High-Level Talent Scientific Research Project (KX018904), Shihezi University-level colleges independently support scientific research projects (ZZZC202127). Competing interests The authors declare no competing interests. References Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians 71 , 209-249, doi:10.3322/caac.21660 (2021). Matsuo, K. et al. 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ACLY facilitates colon cancer cell metastasis by CTNNB1. J Exp Clin Cancer Res 38 , 401, doi:10.1186/s13046-019-1391-9 (2019). Wei, X. et al. Targeting ACLY Attenuates Tumor Growth and Acquired Cisplatin Resistance in Ovarian Cancer by Inhibiting the PI3K-AKT Pathway and Activating the AMPK-ROS Pathway. Frontiers in oncology 11 , 642229, doi:10.3389/fonc.2021.642229 (2021). Park, J., Cho, J. & Song, E. J. Ubiquitin-proteasome system (UPS) as a target for anticancer treatment. Archives of pharmacal research 43 , 1144-1161, doi:10.1007/s12272-020-01281-8 (2020). Chau, V. et al. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science (New York, N.Y.) 243 , 1576-1583, doi:10.1126/science.2538923 (1989). Komander, D. & Rape, M. The ubiquitin code. Annual review of biochemistry 81 , 203-229, doi:10.1146/annurev-biochem-060310-170328 (2012). Dittmar, G. & Winklhofer, K. F. Linear Ubiquitin Chains: Cellular Functions and Strategies for Detection and Quantification. Frontiers in chemistry 7 , 915, doi:10.3389/fchem.2019.00915 (2019). Shimazu, J., Wei, J. & Karsenty, G. Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148. Cell reports 15 , 27-35, doi:10.1016/j.celrep.2016.03.003 (2016). 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. 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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-4487101","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":311906479,"identity":"9ed776a3-5d0f-4ad4-a4ea-bd2b85914834","order_by":0,"name":"Chenyu Hu","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Chenyu","middleName":"","lastName":"Hu","suffix":""},{"id":311906480,"identity":"a22dbd03-99fb-468d-8d17-16335eeddfc6","order_by":1,"name":"Mulin Yang","email":"","orcid":"","institution":"Nankai University","correspondingAuthor":false,"prefix":"","firstName":"Mulin","middleName":"","lastName":"Yang","suffix":""},{"id":311906481,"identity":"38933d6d-f2ae-4de4-903a-74966e93585c","order_by":2,"name":"Junying Xu","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Junying","middleName":"","lastName":"Xu","suffix":""},{"id":311906482,"identity":"922fba32-a4f8-493a-83e0-aef272cff571","order_by":3,"name":"Jiazhen Tian","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Jiazhen","middleName":"","lastName":"Tian","suffix":""},{"id":311906483,"identity":"6af2a4d0-1f31-4fe3-be5d-71288418f571","order_by":4,"name":"Ting Zhang","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Ting","middleName":"","lastName":"Zhang","suffix":""},{"id":311906484,"identity":"b21f270f-5532-4cc2-8cac-1435cd681004","order_by":5,"name":"Amier Abulizi","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Amier","middleName":"","lastName":"Abulizi","suffix":""},{"id":311906485,"identity":"3d16a56a-e8f3-4989-be2e-6ca796c34124","order_by":6,"name":"Jun Zhang","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zhang","suffix":""},{"id":311906486,"identity":"c1442f61-1d03-443a-8797-b25a8194a2a6","order_by":7,"name":"Xiangquan Mi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDCCA0DM2AAkmJkPPvhQISHHT7wWdrZkwxlnLIwlG4jWws9jJs3ZVpG4gZAWvuO9h1/83GGTx8DMY2zMOE+CcQMD88NHN/BokTxzLs2y90xaMQMzW+Hjwm0SzOYMbMbGOXi0GNzIMTNmbDuc2MDMvNl45jYJNssGHjZpIrT8B2phMJPmnSPBY3CAsBbjx4xtB4BaWIBaGiQkCGqRPHPGjLG3LRmoBRTIxyQMJJsJ+IXveI/xh59tdokN/IeBUVlTV9/P3vzwMT4tQMAmASLtD8D4zPiVg5V8IKxmFIyCUTAKRjQAACkpTHsAHq2WAAAAAElFTkSuQmCC","orcid":"","institution":"Shihezi University","correspondingAuthor":true,"prefix":"","firstName":"Xiangquan","middleName":"","lastName":"Mi","suffix":""}],"badges":[],"createdAt":"2024-05-27 22:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4487101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4487101/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58335524,"identity":"8d672d23-e20f-4219-af9e-8910337ad6ca","added_by":"auto","created_at":"2024-06-14 05:23:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":359641,"visible":true,"origin":"","legend":"\u003cp\u003eXN suppresses the proliferation of EC cells. \u003cstrong\u003e(A, B)\u003c/strong\u003e CCK-8 assay analysis of the effect of XN on Ishikawa \u003cstrong\u003e(A)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(B)\u003c/strong\u003e cells. Cell viability was examined by the CCK-8assay. \u003cstrong\u003e(C, D)\u003c/strong\u003e The effect of XN on colony formation of Ishikawa \u003cstrong\u003e(C)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(D) \u003c/strong\u003ecells. \u003cstrong\u003e(E, F)\u003c/strong\u003e The ratio of dead cells changes after XN treatment on trypan blue assay in Ishikawa \u003cstrong\u003e(E)\u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(F) \u003c/strong\u003ecells. \u003cstrong\u003e(G)\u003c/strong\u003e Ishikawa cells were treated with XN for 24h, followed by flow cytometry analysis with Annexin V/PI staining. Representative data were shown on left and apoptotic rates in right panel. \u003cstrong\u003e(H)\u003c/strong\u003e Ishikawa cells were treated with XN for 24h, followed by flow cytometry analysis with PI staining. \u003cstrong\u003e(I, J)\u003c/strong\u003e Ishikawa \u003cstrong\u003e(I)\u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(J)\u003c/strong\u003e cells were treated with XN for 24h, the whole-cell extract was subjected to WB analysis. ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/b7ed95c1c444ad582668d4f5.png"},{"id":58336557,"identity":"8cdbf720-348c-409e-9fa0-8f3fad7639ea","added_by":"auto","created_at":"2024-06-14 05:31:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2050831,"visible":true,"origin":"","legend":"\u003cp\u003eXN down-regulates the expression of ACLY and induces its degradation in EC cells. (A) The AutoDock software predicts the molecular docking between XN and ACLY, and the dotted line frames the molecular docking area between XN and ACLY. (B, C) XN at concentrations of 0, 10, and 20µM was added to fresh medium and co-cultured with EC cells for 24 h. The protein expression levels of ACLY were measured using Western blot in Ishikawa (B) and HEC-1-A (C) cells. (D, E) XN at concentrations of 0, 10, and 20µM was added to fresh medium and co-cultured with EC cells for 24 h. ATP citrate lyase (ACL) Activity Assay Kit was used to detect ACLY enzyme activity in Ishikawa (D) and HEC-1-A (E) cells. (F, G) XN shortened the half-life of ACLY. Ishikawa (F) and HEC-1-A (G) cells were treated with XN or DMSO control, whole-cell extract was subjected to Western blot analysis. (H, I) MG-132 rescued XN-induced ACLY downregulation. Ishikawa (H) and HEC-1-A (I) cells were treated with XN, followed by MG-132treated for 6h. The whole-cell extract was subjected to Western blot analysis. (J, K) XN at concentrations of 0, 10, and 20µM was added to fresh medium and co-cultured with EC cells for 24 h. Triglyceride Assay Kit was used to detect triglyceride levels in Ishikawa (J) and HEC-1-A (K) cells. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/1e5c17a34b3b8538aceafe26.png"},{"id":58335520,"identity":"c1b3fdc2-01d0-47d8-8808-deaf2aca6608","added_by":"auto","created_at":"2024-06-14 05:23:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":251997,"visible":true,"origin":"","legend":"\u003cp\u003eXN promotes ACLY ubiquitination in a Smurf1-dependent manner.\u003cstrong\u003e (A, B)\u003c/strong\u003e XN promoted ACLY ubiquitination. Ishikawa \u003cstrong\u003e(A)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(B) \u003c/strong\u003ecells were treated with XN and subjected to ubiquitination analysis. \u003cstrong\u003e(C) \u003c/strong\u003eHEK-293T cells were treated with XN and co-expressed GFP-ACLY, His-MYC-Ub for 24h.The polyubiquitylated proteins were detected with anti-Myc antibody. \u003cstrong\u003e(D)\u003c/strong\u003eHEK-293T cells were treated with XN and co-expressed GFP-ACLY, Myc-Ub-K48 for 24h.The polyubiquitylated proteins were detected with anti-MYC antibody. \u003cstrong\u003e(E)\u003c/strong\u003eHEK-293T cells co-expressed Flag-Smurf1 or control vector and GFP-ACLY for 72h were subjected to IP and IB analysis. \u003cstrong\u003e(F)\u003c/strong\u003e Ishikawa cells were subjected to IP analyses with Smurf1antibody or normal mouse IgG. \u003cstrong\u003e(G)\u003c/strong\u003e HEK-293T cells were transfected with Flag-Smurf1 and GFP-ACLY plasmids as indicated, followed by XN treated for 24h, the whole-cell lysate was prepared and subjected to CO-IP and IB analysis.\u003cstrong\u003e (H)\u003c/strong\u003e EC cells were transfected with Flag-Smurf1 for 24h. Cell lysates were subjected to WB analysis.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/08f9d89b82da6ce97440d5be.png"},{"id":58335529,"identity":"b5f5d99e-9c0c-4a4b-be3d-f8562200bd6e","added_by":"auto","created_at":"2024-06-14 05:23:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176673,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACLY overexpression on downstream gene expression level and biological behavior of EC cells.\u003cstrong\u003e (A-D)\u003c/strong\u003e The mRNA and protein expression levels of ACLY, ACC and FASN after ACLY overexpression in Ishikawa \u003cstrong\u003e(A, C)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(B, D)\u003c/strong\u003e cells. \u003cstrong\u003e(E, F)\u003c/strong\u003eThe effect of ACLY overexpression on Ishikawa\u003cstrong\u003e (E)\u003c/strong\u003e and HEC-1-A\u003cstrong\u003e (F)\u003c/strong\u003e cells was used CCK-8 assay. \u003cstrong\u003e(G, H)\u003c/strong\u003e The effect of ACLY overexpression on colony formation in Ishikawa \u003cstrong\u003e(G)\u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(H)\u003c/strong\u003e cells. \u003cstrong\u003e(I, J)\u003c/strong\u003e The effect of ACLY overexpression in Ishikawa \u003cstrong\u003e(I)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(J)\u003c/strong\u003e cells was used trypan blue assay. Cell viability was examined by the CCK-8 assay and trypan blue assay. \u003cstrong\u003e(K, L)\u003c/strong\u003eThe protein expression of CDK6 and CCND1 were assessed following ACLY overexpression in Ishikawa \u003cstrong\u003e(K) \u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(L)\u003c/strong\u003e cells. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/101713700195f47d25a261d3.png"},{"id":58335525,"identity":"96317935-ac1a-455c-b8cf-50a9dd43d65b","added_by":"auto","created_at":"2024-06-14 05:23:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":189020,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACLY knock-down on downstream gene expression level and biological behavior of EC cells.\u003cstrong\u003e (A-D)\u003c/strong\u003e The mRNA and protein expression levels of ACLY, ACC and FASN were assessed following ACLY knock-down in Ishikawa \u003cstrong\u003e(A, C) \u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(B, D)\u003c/strong\u003e cells. \u003cstrong\u003e(E, F)\u003c/strong\u003e The effect of ACLY knock-down on Ishikawa \u003cstrong\u003e(E)\u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(F)\u003c/strong\u003e cells was used CCK-8 assay. \u003cstrong\u003e(G, H)\u003c/strong\u003e The effect of ACLY knock-down on colony formation of Ishikawa \u003cstrong\u003e(G)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(H) \u003c/strong\u003ecells. \u003cstrong\u003e(I, J)\u003c/strong\u003e The effect of ACLY knock-down of Ishikawa \u003cstrong\u003e(I)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(J)\u003c/strong\u003ecells was used trypan blue assay. Cell viability was examined by the CCK-8 assay and trypan blue assay. \u003cstrong\u003e(K, L)\u003c/strong\u003eThe protein expression of CDK6 and CCND1 were assessed following ACLY knock-down in Ishikawa \u003cstrong\u003e(K) \u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(L)\u003c/strong\u003e cells. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/4c14c21f47cbbc80a63cf0fe.png"},{"id":58335527,"identity":"500962fd-1867-4c4d-af11-efd4cf715733","added_by":"auto","created_at":"2024-06-14 05:23:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":225010,"visible":true,"origin":"","legend":"\u003cp\u003eACLY reverses the effects of XN on EC cells. \u003cstrong\u003e(A, B) \u003c/strong\u003eThe protein expression of ACLY, ACC and FASN were detected after treatment of the Ishikawa \u003cstrong\u003e(A)\u003c/strong\u003eand HEC-1-A\u003cstrong\u003e (B)\u003c/strong\u003e cells with the NC, XN, and ACLY+XN, respectively. \u003cstrong\u003e(C, D) \u003c/strong\u003eThe effect of cell proliferation was detected after treatment of the EC cells with the NC, XN, and ACLY+XN on EC cells by used CCK-8 assay. Cell viability was examined by the CCK-8 assay. \u003cstrong\u003e(E, F)\u003c/strong\u003e Colony formation assay of the effect of on Ishikawa \u003cstrong\u003e(E) \u003c/strong\u003eand HEC-1-A \u003cstrong\u003e(F) \u003c/strong\u003ecells. \u003cstrong\u003e(G, H)\u003c/strong\u003eTrypan blue assay was used to detect the ratio of dead cells changes on ACLY overexpression and XN treatment in Ishikawa \u003cstrong\u003e(G)\u003c/strong\u003e and HEC-1-A\u003cstrong\u003e (H)\u003c/strong\u003ecells.\u003cstrong\u003e (I, J)\u003c/strong\u003e Western blot were used to detect the protein expression levels of CDK6 and CCND1 after ACLY overexpression and XN treatment in Ishikawa \u003cstrong\u003e(I)\u003c/strong\u003e and HEC-1-A \u003cstrong\u003e(J)\u003c/strong\u003e cells. *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, #\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ##\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ###\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/15226448080fbf7df61dd378.png"},{"id":58336559,"identity":"281cb4e0-b031-425f-83b8-c15a408715ce","added_by":"auto","created_at":"2024-06-14 05:31:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":99361,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of XN-treatment inhibits EC cell proliferation. XN directly inhibits ACLY enzyme activity, and down-regulates the expression of ACLY via promoting Smurf1-mediated ACLY ubiquitination and degradation.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/266771a1ccef8fa72d50fdcf.png"},{"id":58926092,"identity":"e1be5440-b789-4bb3-9762-7b8aef0e8669","added_by":"auto","created_at":"2024-06-24 08:16:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4046683,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4487101/v1/996418ad-c0cb-43e9-bfb0-03091af466e0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Xanthohumol Suppresses Endometrial Cancer Cell Proliferation via Promotion of Smurf1-mediated ACLY Ubiquitination and Degradation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometrial cancer (EC) ranks among the top three prevalent malignant tumors affecting the female reproductive system and stands as the sixth most commonly diagnosed cancer among women globally \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The prevalence and mortality rates of EC are elevated in developed countries in comparison to developing ones. Currently, North America demonstrates the highest incidence of EC \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In the past few decades, the overall incidence of EC has increased by 132%, signifying a rise in the prevalence of risk factors. Key risk factors for EC encompass heightened estrogen levels, obesity, advanced age, tamoxifen use, early menstruation, and late menopause \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Early-stage EC can be effectively treated through surgery or a combination of surgery, radiotherapy, and chemotherapy. Chemotherapy, along with comprehensive treatment, is typically utilized to manage advanced and recurrent EC. Currently, the established first-line treatment for advanced or metastatic EC involves the combination of paclitaxel and carboplatin \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. However, the efficacy of this treatment is constrained, often resulting in poor prognosis, recurrence, and low tolerance. Therefore, there is a need to devise more effective strategies for EC treatment to increase patient survival rates.\u003c/p\u003e \u003cp\u003eObesity is a significant risk factor for EC, and among the 20 most prevalent cancer types, EC is the one strongly associated with obesity. An increase of 5 kg/m\u003csup\u003e2\u003c/sup\u003e in body mass index (BMI) is correlated with a 54% elevated risk of cancer. Women with a BMI exceeding 40 kg/m\u003csup\u003e2\u003c/sup\u003e have a lifetime risk of EC ranging from 10\u0026ndash;15% \u003csup\u003e6,7\u003c/sup\u003e. Abnormal lipid metabolism is a crucial metabolic alteration observed in tumors, and several investigations have illustrated its crucial involvement in the development of EC \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. ATP-citrate lyase (ACLY) is an enzyme in the cytoplasm and nucleus, existing as a homotetramer \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. It catalyzes the conversion of citric acid to acetyl-CoA, subsequently generating fatty acids when fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) are present \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. ACLY is a pivotal intermediary connecting glucose metabolism to fatty acid synthesis, serving as an upstream regulator in the fatty acid synthesis pathway. By enabling the de novo synthesis of fatty acids, ACLY facilitates the biosynthesis of cell membranes and is imperative for the formation of tumor cells \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Elevated expression or activation of ACLY was observed across various cancer types. Research indicates that interference with ACLY or its knockdown suppresses tumor cell proliferation in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e models \u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUbiquitination represents a crucial post-translational modification process in eukaryotes. Three enzyme families regulate this intricate mechanism: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). Ubiquitin is pivotal in regulating proteins, involved in diverse cellular processes, and contributing to the development of diseases, including cancer. The process of ubiquitination is associated with multiple cancer types, including EC \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recent findings indicate the significance of the ubiquitin-proteasome system in the development of EC, which is increasingly recognized as part of the regulatory mechanism \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. For instance, in lung cancer, the CUL3-KLHL25 ubiquitin ligase was involved in the ubiquitination and degradation of ACLY, consequently suppressing lipid synthesis. \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. However, no specific E3 ubiquitin ligase is identified for ACLY that controls energy metabolism, particularly in EC.\u003c/p\u003e \u003cp\u003eNatural products have gained popularity recently due to their effectiveness, multi-target capabilities, and safety. These characteristics make them appealing for diverse applications, including anti-tumor endeavors \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The exploration and development of natural compounds for the prevention and treatment of cancer have received considerable attention. Xanthohumol (XN), a pentenyl flavonoid extracted from hops, possesses anticancer, antioxidant, antibacterial, and anti-inflammatory activities \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Research has demonstrated that XN can impede the growth of various cancer types, such as lung cancer \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, breast cancer \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, colon cancer \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, prostate cancer \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, liver cancer \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, and other cancers. This inhibition effect could be achieved by suppressing tumor cell proliferation, invasion, and migration, causing cell cycle arrest and inducing apoptosis. Furthermore, XN was found to synergize with chemotherapy and radiotherapy, reducing chemotherapy resistance and minimizing its associated side effects \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the anti-tumor properties of XN on EC have not been reported.\u003c/p\u003e \u003cp\u003eIn this research, the anti-tumor properties of XN in EC cells were investigated \u003cem\u003ein vitro\u003c/em\u003e. It was found that XN inhibits the proliferation of EC cells through decreased expression of ACLY, which was associated with Smurf1-mediated ACLY ubiquitination and degradation. Moreover, XN decreases the levels of ACC and FASN by disrupting ACLY function, resulting in reduced lipid synthesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eImpacts of XN on EC cell proliferation\u003c/h2\u003e \u003cp\u003eTo assess the impacts of XN on EC cells, varying concentrations of XN (0, 10, 20, and 40 \u0026micro;M) were utilized to treat the EC cells for 24 and 48 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The impact of XN on the proliferation of EC cells was then assessed utilizing the CCK-8 assay. XN demonstrated a dose- and time-dependent inhibition of EC cell proliferation in comparison to the control group. The colony formation assays and trypan blue assays illustrated that XN substantially inhibited the growth of EC cells and stimulated cell death in a dose-dependent manner in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, flow cytometry analysis of the cell cycle and apoptotic rate indicated that XN stimulated G0/G1 cell cycle arrest in EC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). However, the apoptosis of EC cells was not substantially altered by XN. In the G1 phase of the cell cycle, Cyclin-dependent kinase 4 (CDK4) and CDK6 interact with Cyclin D1 (CCND1), cyclin D2, and cyclin D3, respectively. Subsequent Western blot analysis demonstrated a substantial reduction in the CDK6 and CCND1 expression relative to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). These findings suggested that XN exhibits a remarkable anti-tumor impact in EC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eXN-mediated ACLY suppression resulting in reduced de novo lipid synthesis in EC cells\u003c/h2\u003e \u003cp\u003eACLY is a pivotal enzyme in cancer metabolism. To examine whether the anti-tumor efficacy of XN on EC cells is dependent on ACLY downregulation, the AutoDock Vina bioinformatics website was utilized for molecular docking analysis. This analysis aimed to predict the potential interactions between XN and ACLY. The prediction results revealed a favorable binding effect between XN and ACLY (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Following XN treatment, a dose-dependent reduction in ACLY protein expression was observed in Ishikawa and HEC-1-A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Subsequent experiments revealed that XN treatment inhibited ACLY enzyme activity in both Ishikawa and HEC-1-A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). To explore whether XN influenced the stability of ACLY protein, a cycloheximide (CHX)-chase experiment was conducted. It revealed a substantial reduction in the half-life of ACLY in XN-treated EC cells in comparison to control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). In addition, the proteasome inhibitor, MG-132, restored ACLY expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Furthermore, the Western blot method was utilized to illustrate the impact of XN treatment on de novo lipid synthesis. The outcomes revealed that treatment with XN leads to a dose-dependent reduction in the protein expression levels of related fatty acid synthases, including fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) and resulted in a significant decrease in cellular triglyceride content (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). These outcomes suggest that XN decreases de novo lipid synthesis in EC cells by downregulating ACLY.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eXN promotes Smurf1-mediated ACLY ubiquitination and degradation\u003c/h2\u003e \u003cp\u003eIn protein metabolism, inducing protein degradation is an effective method to reduce intracellular protein expression. Prior investigations have revealed that XN treatment reduced ACLY expression through protein degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Subsequently, the ubiquitination analysis demonstrated increased ubiquitination of ACLY after XN treatment in Ishikawa and HEC-1-A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Additionally, it was observed that XN treatment promoted the formation of Lys 48 (K48)-linked polyubiquitin chains on ACLY protein, indicating proteasome-mediated substrate degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Furthermore, the bioinformatics-based ubiquitin ligase-substrate interaction programs Ubibrowser were employed to identify potential ACLY target ubiquitin ligase E3. Based on the findings, we hypothesized that Smurf1 might function as a potential E3 ligase regulating ACLY.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm the hypothesis, Co-IP analysis was conducted employing an anti-ACLY antibody. The outcomes demonstrated endogenous binding between Smurf1 and ACLY proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Moreover, the Co-IP analysis illustrated the formation of a complex between Flag-tagged Smurf1 and GFP-ACLY in HEK-293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). These observations corroborate the assertion that Smurf1 effectively interacts with ACLY as a binding partner Moreover, the Co-IP results demonstrated that XN promoted the interaction between ACLY and E3 ligase Smurf1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Subsequently, an assessment was undertaken to ascertain the influence of Smurf1 on ACLY protein levels. Western blotting analysis revealed that the ectopic overexpression of Smurf1 results in a dose-dependent reduction in ACLY protein levels in EC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These findings imply that XN triggers the ubiquitination and degradation of ACLY in a Smurf1-dependent manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eACLY plays a crucial role in EC cells\u003c/h2\u003e \u003cp\u003eGrowing research highlights the pivotal role of cancer metabolism in the onset and progression of EC \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Dysregulation in lipid metabolism emerges as a prominent factor contributing to the development of EC. ACLY exhibits high expression or overactivation across diverse cancer types, including EC. It is critical in facilitating heightened lipid synthesis in cancer cells. To substantiate the influence of ACLY on EC cells, overexpression and knockdown experiments targeting ACLY were conducted in EC cell lines, respectively. Following this, the expression levels of ACC and FASN were evaluated employing qRT-PCR and Western blotting analysis. The findings unveiled notable outcomes that demonstrated a substantial upregulation of mRNA and protein levels for ACC and FASN in EC cells overexpressing ACLY in comparison to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). A contrasting outcome was noted following the ACLY knockdown. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). CCK-8 assays, colony formation assays, and trypan blue assays demonstrated overexpressed ACLY-promoted cell viability in Ishikawa and HEC-1-A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Moreover, elevated expression of cell cycle-related proteins CDK6 and CCND1 was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL). Conversely, a contrary result was observed upon ACLY knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL). These observations imply that ACLY is pivotal in the proliferation of EC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eACLY reverses the effects of XN-treated on EC cells\u003c/h2\u003e \u003cp\u003eTo further confirm that the suppression of proliferation results from the reduction in ACLY expression induced by XN, EC cells were transfected with an ACLY-overexpressing plasmid subsequent to treatment with XN. The Western blot outcomes revealed considerable upregulation of ACC and FASN expression. This was observed when examining the overexpression-induced increase after transfection with the ACLY-overexpressing plasmid following treatment with XN in comparison to the group treated solely with XN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Additionally, transfecting EC cells with an ACLY-overexpressing plasmid after XN treatment substantially reversed the inhibitory effects observed with XN treatment alone on cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). In contrast, the overexpression of ACLY following XN treatment reversed the levels of CDK6 and CCND1 in EC cells in comparison to XN alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). These observations substantiated that XN suppresses the proliferation of EC cells by directly targeting ACLY.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEC ranks among the most common tumors affecting the female reproductive system. Its increasing worldwide incidence is closely associated with the rising prevalence of obesity \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Dysregulated lipid metabolism is a key metabolic alteration in cancer, with reports indicating that over 30% of genes associated with lipid metabolism demonstrate abnormal expression in EC, including ACLY \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This study unveils that by deactivating ACLY, XN impedes de novo fatty acid synthesis and suppresses cell proliferation in EC.\u003c/p\u003e \u003cp\u003eXN, a naturally occurring compound derived from hops, exhibited anti-tumor and anti-obesity properties. \u003cem\u003eIn vivo\u003c/em\u003e research has revealed that XN functions as a ligand for farnesoid X, thereby augmenting glucose and lipid metabolism in KK-A(y) mice \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Moreover, XN impedes increased body weight, liver weight, and plasma and hepatic triacylglycerol levels stimulated by a high-fat diet. This effect is achieved through the modulation of hepatic fatty acid metabolism and suppression of intestinal fat absorption in rats \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vitro\u003c/em\u003e, XN reduces fatty acid synthesis and cholesterol biosynthesis by suppressing the activation of Sterol regulatory element-binding proteins (SREBPs) \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Conversely, the study reveals that XN impedes de novo fatty acid synthesis by inhibiting ACLY activation in EC.\u003c/p\u003e \u003cp\u003eXN has undergone extensive investigation as an anticancer agent, with studies reporting its ability to inhibit the proliferation of various cancers. These cancers include gastric cancer \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, breast cancer \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, lung cancer \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, cholangiocarcinoma \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e and colon cancer \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Furthermore, our previous experiments revealed that XN impeded cell proliferation by inducing paraptosis in leukemia cells \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In the present investigation, substantial anti-tumor activity of XN against EC cells was observed \u003cem\u003ein vitro\u003c/em\u003e. XN suppresses the proliferation of EC cells by directly inhibiting the enzymatic activity of ACLY and promoting Smurf1-mediated ubiquitination and degradation of ACLY.\u003c/p\u003e \u003cp\u003eLipids are essential constituents of biological membranes and are vital substrates for energy metabolism. De novo lipid synthesis is critical in tumor progression and represents a target for cancer therapy. ACLY, a key enzyme in de novo lipid synthesis, establishes a connection between glucose metabolism and the lipid synthesis process. \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Increasing research demonstrated the elevated activation or expression of ACLY in multiple tumor tissues, including EC \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, hepatocellular carcinoma \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, lung cancer \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, prostate cancer \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, bladder cancer \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, colon cancer \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, and ovarian cancer \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The current investigation has shown that overexpression of ACLY markedly improved the mRNA and protein expression levels of ACC and FASN, crucial components involved in fatty acid synthesis. This overexpression also elevated cell viability, as shown by the trypan blue experiment, and stimulated cell proliferation, as assessed by colony formation and CCK-8 assay. Conversely, the opposite effect was observed when ACLY was downregulated.\u003c/p\u003e \u003cp\u003eIn this study, by employing the AutoDock Vina bioinformatics platform for molecular docking analysis, a favorable binding interaction between XN and ACLY was predicted. Additionally, it was observed that XN inhibited the enzyme activity of ACLY in EC in a dose-dependent manner. Additional research is necessary to verify whether XN inhibits ACLY enzyme activity by directly binding to ACLY. The ubiquitin-proteasome is a crucial protein degradation pathway in eukaryotic cells \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Protein degradation and the regulation of protein stability are primarily governed by K48-linked polyubiquitination. In contrast, k63-linked polyubiquitination predominantly functions in signaling, DNA repair, and the regulation of protein activity \u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. In the current study, XN was found to augment the ubiquitination and subsequent degradation of ACLY via K48-linked polyubiquitination. Smurf1, identified as an E3 ubiquitin ligase, belongs to the HECT-type ubiquitin ligase classification \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Moreover, it was demonstrated that Smurf1 induces K48-linked polyubiquitination of ACLY in EC. However, the specific domains of Smurf1 that are responsible for binding to ACLY warrant further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, the anti-tumor impacts of XN in EC were investigated. XN blocked the de novo synthesis of fatty acid and inhibited the proliferation of EC by impairing the enzyme activity and expression of ACLY. Moreover, the study identified that Smurf1 ubiquitinates ACLY, indicating the critical involvement of Smurf1-mediated ACLY ubiquitination in the progression of EC. Collectively, XN emerges as a potent ACLY inactivator with substantial therapeutic promise against EC (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eReagents and cell culture\u003c/h2\u003e \u003cp\u003eXN, with a purity level surpassing 98%, was supplied by Yumen Technology Development Company in Gansu, China. Chemical reagents, comprising DMSO, SDS, Tris base, and NaCl, were procured from Solarbio (China). MG-132 and cycloheximide (CHX) were obtained from MedChemExpress (New Jersey, USA). Biological Industries in Israel was accessed to acquire the DMEM and Fetal Bovine Serum (FBS) for cell culture. The amounts of triglyceride in the cell lysates were measured using a commercial test kit (Jiancheng, Nanjing, China). The human EC cell line Ishikawa was sourced from the Cell Resource Center, Peking Union Medical College (PRCR, Beijing, China). It was maintained in DMEM, having 1% penicillin and streptomycin along with 10% FBS at 37℃ in a humidified atmosphere consisting of 5% CO\u003csub\u003e2\u003c/sub\u003e. Moreover, the human EC cell line HEC-1-A was acquired from the Chinese Academy of Sciences Culture Preservation Committee Cell Bank. It was maintained in McCoy\u0026rsquo;s 5A medium (Procell, China) supplemented with 1% penicillin and streptomycin as well as 10% FBS at 37℃ in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. Mycoplasma analysis was conducted on all cells within the past three years. Plasmid transfection was carried out utilizing Lipofectamine\u0026reg; 2000 (Thermo Fisher Scientific, USA) following the prescribed procedure from the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eThe cells were incubated overnight in 96-well plates at a density of 5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well. The cells were exposed to varying concentrations of XN (0 \u0026micro;M, 10 \u0026micro;M, 20 \u0026micro;M, and 40 \u0026micro;M) for different time durations (0, 24, and 48 h). Afterward, 10 \u0026micro;L of CCK-8 reagent (Biosharp, China) was introduced to each well. After two hours of incubation at 37℃, a full-wavelength microplate reader (Thermo Fisher Scientific, USA) was utilized to obtain the absorbance values at 450 nm.\u003c/p\u003e \u003cp\u003eThe cells were seeded into 96-well plates (5\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well) and cultured in a complete medium for 24 hours. Subsequently, cell viability was evaluated every 24 hours, up to 72 hours, following transfection with either the ACLY-overexpression or ACLY-knockdown plasmids. Additionally, the CCK-8 assay was executed as described previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay and trypan blue assay\u003c/h2\u003e \u003cp\u003eBoth cell lines were cultured into a 6-well plate at a density of 500 cells per well. The plate was gently rotated to ensure even dispersion of the cells. After 24 hours, the EC cells were exposed to XN (0 \u0026micro;M, 10 \u0026micro;M, and 20 \u0026micro;M) and placed in a cell culture incubator for two weeks at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e. Moreover, the cells were rinsed thrice with PBS with subsequent fixation utilizing 4% paraformaldehyde for 30 minutes. Following this, they underwent staining with 0.1% crystal violet for 30 minutes. Subsequently, the stained samples were rinsed with PBS to remove the staining solution.\u003c/p\u003e \u003cp\u003eThe cells were transfected with the ACLY-overexpression and ACLY-knockdown plasmid. Subsequently, they were cultured in 6-well plates (500 cells/well) for two weeks. The operation was performed as described previously.\u003c/p\u003e \u003cp\u003eTrypan blue selectively stains only dead cells, which appear blue under microscopic examination. Cells undergoing exponential growth were inoculated into 6-well plates containing 10% FBS medium and cultured for 24 hours. Following that, the cells were subjected to several treatments. After 24 hours, the cells underwent washing and resuspension in PBS until achieving a cell density of approximately 80\u0026ndash;90%. Then, 90 \u0026micro;l of the cell suspension and 10 \u0026micro;l of trypan blue were transferred to an EP tube and gently pipetted to ensure thorough mixing. After incubation for 1 minute, the cells were suspended and added to the blood cell meter to count the blue-stained and unstained cells, which was completed within 3 minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle and apoptosis analysis\u003c/h2\u003e \u003cp\u003eEC cells, at a density of 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per well, were seeded onto 6-well plates for cell cycle analysis. Subsequently, they were treated with XN for 24 hours utilizing the Cell Cycle and Apoptosis Analysis Kit (Beyotime, China). For apoptosis analysis, cells were inoculated onto 6-well plates (2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well) and treated with XN for 24 hours. Subsequently, via flow cytometry, apoptosis was detected utilizing the Annexin V/PI staining kit (MULTI SCIENCES, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eThe cells underwent lysis utilizing an ice-cold RIPA lysis buffer as per the prescribed protocol from the manufacturer. Protein concentration was assessed utilizing the bicinchoninic acid (BCA) assay. Afterward, the proteins underwent separation via SDS-PAGE electrophoresis and electrotransfer separated to the PVDF membrane and were transferred onto PVDF membranes. The 5% BSA Blocking Solution was used for membrane blocking at room temperature for 2 hours and the membranes were treated with the primary antibodies at 4℃ overnight. These antibodies include β-actin (ZSGB-BIO, #TA-09), ACLY (Servicebio, #GB11902-100), ACC (Abways, #CY5575), FASN (Abways, #CY6579), CCND1 (Abways, #CY5404), and CDK6 (Abways, #CY5835). After three washes with TBST buffer, the membranes were subjected to incubation with HRP-conjugated anti-rabbit/mouse antibodies for 2 hours. Subsequently, the membranes were treated with an Enhanced Chemiluminescence Detection reagent and analyzed employing a chemiluminescence analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation assays\u003c/h2\u003e \u003cp\u003eA co-immunoprecipitation (CO-IP) assay was executed utilizing the protein binding IP kit (Absin, #abs955). Initially, cell lysates were treated overnight at 4\u0026deg;C with anti-ACLY (Proteintech, #67166-1-Ig), anti-Smurf1 (Proteintech, #55175-1-AP), anti-Flag (Proteintech, # 66008-4-Ig), anti-GFP (Proteintech, #50430-2-AP) or control IgG (Cell Signaling Technology, #2729). Subsequently, the lysates were exposed to protein A/G-agarose beads at 4\u0026deg;C for 4 hours. The resulting precipitates underwent washing five times in a wash buffer and were subjected to Western blotting analysis for evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was obtained from cells utilizing TRIzol reagent (Invitrogen, USA), complying with the recommended procedure of the manufacturer. GAPDH was employed as an endogenous control to normalize mRNA. Subsequently, mRNA was reverse transcribed to complementary DNA (cDNA) employing the Revert Aid First-Strand cDNA kit (Thermo Fisher Scientific, USA), respectively, complying with the recommended protocol of the manufacturer. qRT-PCR was executed in triplicate on the Rotor-Gene Q MDx detection system (QIAGEN, Germany) utilizing the SYBR Green PCR kit (QIAGEN, Germany) to examine mRNA. Additionally, relative alterations in the expression levels of mRNA were assessed employing the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The primers utilized for the present investigation are as follows: ACLY forward, 5\u0026rsquo;-ATCGFGTTCAAGTATGCTCGGG-3\u0026rsquo;, reverse, 5\u0026rsquo;-GACCAAGTTTTCCACGACGTT-3\u0026rsquo;; ACC forward, 5\u0026rsquo;-TCACACCTGAAGACCTTAAAGCC\u003c/p\u003e \u003cp\u003e-3\u0026rsquo;, reverse, 5\u0026rsquo;-AGCCCACACTGCTTGTACTG-3\u0026rsquo;; FASN forward, 5\u0026rsquo;-ACAGCGGGAATGGGTACT-3\u0026rsquo;, reverse, 5\u0026rsquo;-GACTGGTACAACGAGCGGAT-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eACLY enzyme activity assay\u003c/h2\u003e \u003cp\u003eAfter treating EC cells with XN for 24 hours, ACLY enzyme activity was assessed utilizing a detection reagent (Solarbio, China). Initially, ACLY extractant was employed, followed by centrifugation of the supernatant after ultrasonic fragmentation. The procedure was conducted following the provided instructions. Finally, the absorbance was measured at OD340nm utilizing an enzyme labeling instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was executed with GraphPad Prism 9. The one-way ANOVA or student\u0026rsquo;s t-test was employed for assessing the difference between tested groups. A probability value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 reflected statistical significance. The experiment was conducted in triplicate, and quantitative data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC.H. and M.Y.: Investigation, formal analysis, writing original draft. J.X. and J.T.: Investigation, methodology. T.Z.: Funding acquisition. A.A.: Resources and supervision. X.M. and J.Z.: Conceptualization, project administration, Writing\u0026mdash; review and editing, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by Shihezi University High-Level Talent Scientific Research Start-up Project (RCSX2018B01), Shihezi University High-Level Talent Scientific Research Project (KX018904), Shihezi University-level colleges independently support scientific research projects (ZZZC202127).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSung, H.\u003cem\u003e et al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. \u003cem\u003eCA: a cancer journal for clinicians\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 209-249, doi:10.3322/caac.21660 (2021).\u003c/li\u003e\n\u003cli\u003eMatsuo, K.\u003cem\u003e et al.\u003c/em\u003e Ovarian conservation for young women with early-stage, low-grade endometrial cancer: a 2-step schema. \u003cem\u003eAmerican journal of obstetrics and gynecology\u003c/em\u003e \u003cstrong\u003e224\u003c/strong\u003e, 574-584, doi:10.1016/j.ajog.2020.12.1213 (2021).\u003c/li\u003e\n\u003cli\u003eGu, B.\u003cem\u003e et al.\u003c/em\u003e Variations in incidence and mortality rates of endometrial cancer at the global, regional, and national levels, 1990-2019. \u003cem\u003eGynecologic oncology\u003c/em\u003e \u003cstrong\u003e161\u003c/strong\u003e, 573-580, doi:10.1016/j.ygyno.2021.01.036 (2021).\u003c/li\u003e\n\u003cli\u003eSun, H.\u003cem\u003e et al.\u003c/em\u003e Expression Profiles of Endometrial Carcinoma by Integrative Analysis of TCGA Data. \u003cem\u003eGynecologic and obstetric investigation\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 30-38, doi:10.1159/000445073 (2017).\u003c/li\u003e\n\u003cli\u003eAbu-Rustum, N.\u003cem\u003e et al.\u003c/em\u003e Uterine Neoplasms, Version 1.2023, NCCN Clinical Practice Guidelines in Oncology. \u003cem\u003eJournal of the National Comprehensive Cancer Network : JNCCN\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 181-209, doi:10.6004/jnccn.2023.0006 (2023).\u003c/li\u003e\n\u003cli\u003eAune, D.\u003cem\u003e et al.\u003c/em\u003e Anthropometric factors and endometrial cancer risk: a systematic review and dose-response meta-analysis of prospective studies. \u003cem\u003eAnnals of oncology : official journal of the European Society for Medical Oncology\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1635-1648, doi:10.1093/annonc/mdv142 (2015).\u003c/li\u003e\n\u003cli\u003eRenehan, A. 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Smurf1 Inhibits Osteoblast Differentiation, Bone Formation, and Glucose Homeostasis through Serine 148. \u003cem\u003eCell reports\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 27-35, doi:10.1016/j.celrep.2016.03.003 (2016).\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":"Xanthohumol, Endometrial Cancer, ATP citrate lyase, E3 ligase Smurf1","lastPublishedDoi":"10.21203/rs.3.rs-4487101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4487101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eATP citrate lyase (ACLY) is pivotal in de novo fatty acid synthesis. It emerges as a core metabolic enzyme implicated in malignant tumor progression, especially in Endometrial Cancer (EC). The present investigation revealed that Xanthohumol (XN), a naturally prenylated flavonoid, is a novel inactivator of ACLY. XN demonstrates a significant reduction in de novo fatty acid synthesis and concurrent inhibition of cell proliferation in EC. Moreover, XN directly inhibits ACLY enzyme activity and facilitates Smurf1-mediated ACLY ubiquitination and degradation. The research revealed that the knockdown of ACLY reduced fatty acid synthesis, proliferation, and colony formation in EC cells. Conversely, contrasting results were observed upon ACLY overexpression. Additionally, treatment with XN inhibited fatty acid synthesis, cell proliferation, and colony formation, inducing non-apoptotic cell death and G0/G1 cycle arrest by downregulating ACLY expression. The crucial involvement of Smurf1-mediated ACLY ubiquitination in the XN-induced downregulation of ACLY was also highlighted. Notably, the role of the E3 ubiquitin ligase Smurf1 in mediating the ubiquitination of ACLY is reported here for the first time. Furthermore, these findings indicated the potential of ACLY as a prospective drug target for EC. Considering the inhibitory effect of XN on ACLY, it presents encouraging prospects for treating EC.\u003c/p\u003e","manuscriptTitle":"Xanthohumol Suppresses Endometrial Cancer Cell Proliferation via Promotion of Smurf1-mediated ACLY Ubiquitination and Degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-14 05:23:12","doi":"10.21203/rs.3.rs-4487101/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":"d559caac-608c-416f-bd1c-0fe7cb448473","owner":[],"postedDate":"June 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T08:08:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-14 05:23:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4487101","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4487101","identity":"rs-4487101","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}