| Background: Ovarian cancer is one of the most common gynecological malignancies,with the highest mortality rate after cervical cancer and endometrial cancer.The high mortality rate of ovarian cancer is mainly due to the fact that the ovary is located deeply in the pelvic cavity,the early symptoms are not easy to be detected in time,the development of the disease is very hidden,at the same time,lacking effective early screening methods.70% of ovarian cancer patients are already advanced when they visit the doctor,accompanied by pelvic and abdominal metastasis,so ovarian cancer is known as the "silent killer".90% of ovarian cancer is ovarian epithelial cancer,and the main treatment is surgery combined with chemotherapy based on platinum and/or paclitaxel.However,the 5-year overall survival rate of ovarian cancer is only 39%,which is a serious threat to women’s health,because most patients are diagnosed in the advanced stage with metastasis caused missing the best surgical opportunity,and chemotherapy resistance is common.Epidemiological and clinical studies have shown that the psychological stress events caused by the disease,anxiety and lack of social support,promoting the progression of tumors.Stress stimulation activates sympathetic nervous system(SNS)and hypothalamic-pituitary-adrenal axis(HPA),leading to the increase of stress hormones(adrenaline and glucocorticoid,etc.)in vivo.Previous studies have shown that epinephrine can directly promote the proliferation,survival(anti-apoptosis),migration,invasion and metastasis of various tumors,such as prostate cancer,rectal cancer and ovarian cancer.Glucocorticoid(GC)is not only significantly increased in plasma during stress,but also widely used in clinical practice as anti-inflammatory(treatment of rheumatoid arthritis,asthma,inflammatory bowel disease,etc.)and immunosuppressant(treatment of systemic lupus erythematosus,etc.).GC can also be used as an auxiliary drug for tumor chemotherapy.To reduce side effects caused by chemotherapy(nausea,vomiting and allergic reactions,etc.).The role of GC is mediated by Glucocorticoid receptor(GR),which can activate GR and bind GR into the nucleus with Glucocorticoid response element(GRE)in the promoter of target gene in the form of dimer.To regulate gene expression;GR also indirectly regulates gene expression by interacting with other transcription factors,such as NF-κB and AP-1.The effect of GC on solid tumors is very complex.Previous studies have shown that GC can inhibit the immune system of the body and reduce the immune surveillance function of the body,thus leading to immune escape of tumor cells and promoting tumor progression.GC has a powerful anti-inflammatory effect and can reduce the inflammatory microenvironment of tumors and inhibit the proliferation and progression of tumors by inhibiting the production of cytokines and inflammatory mediators.Hidalgo et al.found that GR transcriptional activity of fibroblasts in tumor microenvironment significantly increased,which promoted the proliferation,invasion and migration of tumor cells.In addition,GC can promote cell survival under adverse microenvironment(e.g.,hypoxia and low serum)and enhance the resistance of various tumor cells(e.g.,ovarian,pancreatic,breast and bladder cancer)to chemotherapy drugs.GC can also act directly on tumor cells,for example,GC can directly inhibit the proliferation of various tumor cells(such as ovarian cancer,prostate cancer,lung cancer,breast cancer and kidney cancer,etc.),but GC has been reported to promote and inhibit the migration,invasion and metastasis of tumor cells.GC has been shown to inhibit the progression and metastasis of pancreatic,glioma,and bladder cancers in a combined immune deficiency(SCID)mouse model of human tumor cell xenotransplantation.However,in the nude mouse breast cancer metastasis model of human tumor xenotransplantation,the glucocorticoid receptors(GR)in the metastatic lesions were significantly activated.Further construction of stable knockdown GR breast cancer cell line was found in experiments.GC relies on GR to activate receptor tyrosine kinase-like orphan receptor 1(ROR1)to promote lung metastasis of breast cancer.In addition to this pathway,other studies have found that GC can promote lung metastasis of breast cancer by activating the PI3K/SGK1/CTGF pathway,and in the metastatic tumor model,the number of lung metastasis lesions in nude mice treated with GC combined with paclitaxel significantly increased compared with that treated with paclitaxel alone,indicating that GC promotes chemotherapy resistance and metastasis of breast cancer treated with paclitaxel.Before chemotherapy,the protein levels of SGK1 and MKP1 in cancer cells of breast cancer patients treated with GC were significantly increased.After the expression of SGK1 and MKP1 was interfered,the effect of GC on promoting chemotherapy resistance to paclitaxel was significantly weakened.Wnt/β-catenin signaling pathway regulates important physiological processes such as cell differentiation,cell motility and cell polarity,and abnormal activation of Wnt/β-catenin signaling pathway can promote tumor metastasis,cancer cell survival and chemotherapy resistance.The classical Wnt signaling pathway is a highly conserved complex cascade reaction.In mature cells,the Wnt pathway is closed,and most of theβ-catenin in the cytoplasm binds to E-cadherin on the cell membrane.A small portion ofβ-catenin at Ser33,Ser37,and Thr 41 are phosphorylated by glycogen synthase kinase 3β(GSK3β)in the GSK3β/APC/Axin complex and subsequently ubiquitinated and degraded by proteasome.When Wnt ligand or other cell surface receptors bind to Frizzled,the Wnt signaling pathway is activated and stabilizes β-catenin expression by inhibiting the phosphorylation of β-catenin by GSK3β/APC/Axin complex.β-catenin accumulated in the cytoplasm is transferred to the nucleus,where it forms complexes with members of the t-cytokine/lymphoid enhancer factor binding factor(TCF/LEF)family in the nucleus to regulate the expression of downstream target genes.Enrichment analysis of transcriptional sequencing results in TCGA ovarian cancer database and nude mouse ovarian cancer metastasis model suggested that the progression and metastasis of ovarian cancer were positively correlated with the activation of β-catenin signaling pathway.After β-catenin knockdown,the migration ability of ovarian cancer SKOV-3 cells in vitro and in vivo was significantly reduced.These results suggest that β-catenin activation promotes the metastasis of ovarian cancer.It has also been found that the activity and expression level of E-cadherin and β-catenin are critical in the initiation of EMT in ovarian cancer cells,which is considered to be the initial step of metastasis.Therefore,it is speculated that β-catenin may influence the metastasis of ovarian cancer through EMT.In addition,studies have shown that in rectal cancer cells,the expression of β-catenin protein in the nucleus significantly increased,and was positively correlated with the m RNA level of MMP-7.MMP-7 expression was not found in the cells lacking β-catenin accumulation,suggesting that MMP-7 is the downstream target gene of β-catenin.β-catenin is not only closely related to tumor metastasis,but also found that interfering the expression of β-catenin in breast cancer cells can increase the expression of Caspase3/9 protein and thus enhance the sensitivity of tumor cells to cisplatin.The pathophysiology laboratory of our university has confirmed that GC can promote the invasion and metastasis of Ovarian Cancer cells and chemotherapy resistance,but the specific mechanism of action is not very clear.On this basis,this study further explored the regulation and mechanism of GC on the expression and localization of β-catenin,and whether β-catenin is involved in GC to promote the migration,invasion,metastasis and chemotherapy resistance of ovarian cancer cells.Methods: First,in vitro experiments were carried out in ovarian cancer SKOV-3and HO-8910 celllines.100 n M Dex(synthetic GC)was used to treat the cells.In order to remove the effect of hormones in the serum on the cells,we used a culture medium containing 10% hormone-depleted serum.The m RNA levels of β-catenin,Wnt4 and Wnt5a were detected by RT-QPCR.The protein expression and protein phosphorylation level were detected by Western Blot.Transwell chamber was used to detect the vertical migration ability of cells.Transwell invasion chamber was used to detect the invasive ability of cells.Inhibitors inhibit the ability of the gene of interest.CCK-8 was used to detect cell proliferation ability.The cellular localization of β-catenin was detected by immunofluorescence.In vivo experiments,puromycin screening method was used to screen ovarian cancer cells that stably interfered with β-catenin.4-week-old BABL/c female nude mice were injected intraperitoneally β-catenin-sh RNA tumor cells with puromycin to screen.Adding corticosterone(glucocorticoid in mice)to drinking water to increase corticosterone levels in the plasma of nude mice.Elisa measures corticosterone levels of plasma.HE staining was used to determine the pathological nature of the metastases.The expression and distribution of β-catenin in the ovarian tissue of tumor-bearing nude mice were detected by immunohistochemistry.Results:Part I: The role of β-catenin in glucocorticoid-induced ovarian cancer cell migration,invasion and metastasis1.Synthetic GC-dexamethasone(Dex)could increase the expression and nuclear localization of β-catenin protein in vitro and in vivo.2.β-catenin inhibitor PNU74654 could completely block the promoting effect of GC on ovarian cancer cell’s migration and invasion.3.In a metastatic tumor model prepared by intraperitoneal injection of ovarian cancer cells in BABL/c nude mice,interfering the expression of β-catenin blocked the effect of GC on ovarian cancer cells to promote liver metastasis in vivo;4.the β-catenin inhibitor PNU74654 could partially block the GC-induced chemoresistance in ovarian cancer cells.Part II: Mechanism of glucocorticoid up-regulating β-catenin to promote ovarian cancer cell metastasis1.GC can up-regulate the protein expression of matrix metallopeptidase MMP-3and MMP-7,but did not affect the protein expression of MMP2 and MMP9.2.GC up-regulates the protein expression of mesenchymal cell markers Vimentin and N-cadherin and reduces the expression of epithelial cell marker E-cadherin protein expression.3.after PNU74654 inhibited the β-catenin signaling pathway,the up-regulation of N-cadherin,Vinmentin,MMP-3 and MMP-7 proteins and the down-regulation of E-cadherin protein by GC were inhibited.Part III: Mechanism of glucocorticoid up-regulating β-catenin in ovarian cancer cells1.In ovarian cancer cells,GC can down-regulate the phosphorylation level of serines 33 and 37 of β-catenin and up-regulate the level of serine 9 of GSK3β.2.GC did not affect the m RNA and protein expressions of Wnt pathway ligands Wnt4 and Wnt5 a.3.After inhibiting the AKT signaling pathway,the inhibition of GSK3β activity by GC was completely blocked(the phosphorylation level of GSK3β 9-position serine was reduced),and the protein expression of β-catenin was reduced.Conclusion: Activation of β-catenin signaling pathway mediates the effect of glucocorticoids on promoting the invasion and metastasis of ovarian cancer cells.Glucocorticoids can promote the occurrence of EMT and the increase of MMP-3and MMP-7 protein expressions through β-catenin signaling pathway.In ovarian cancer cells,GC inhibits the activity of GSK3β by activating the AKT signaling pathway,reducing the phosphorylation of β-catenin at serine 33 and 37,inhibiting its proteasomal degradation,and then increasing the β-catenin protein level. |
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