| Objective: Chronic stress has been shown to facilitate progression of epithelial ovarian cancer(EOC),however,the neuro-endocranial mechanism participating in this process still remains unclear.This study is designed to investigate whether and how chronic restraint stress(CRS)promotes abdominal implantation metastasis of EOC cells in animal models and in vitro.In addition,whether melatonin inhibits CRS-mediated prometastatic effects on EOC cells.Methods: In this study,a tumor-bearing animal model was established to evaluate the pro-tumoral effects of CRS and the protective effects of melatonin on ovarian cancer cells in vivo.ELISA was conducted to measure the content of corticosterone,norepinephrine(NE)and melatonin(MLT)in tumor tissues or serum of mouse models.Immunofluorescence technique and confocal laser scanning microscope were used to evaluate the effects of CRS and MLT on expression of epithelial-mesenchymal transition-inducing transcriptional factors(EMT-TFs)in tumor tissues obtained from animal models.CCK-8 was used to evaluate the pro-or anti-proliferation of NE,β-Catenin agonist SKL2001,β-Catenin inhibitor KYA1797,AKT inhibitor AKTi VIII and MLT on epithelial ovarian cancer cell lines SK-OV-3 and HO-8910 pm.Transwell assays were conducted to examine the motility of cancer cells.Western blotting and immunocytochemistry were used to test the influence of NE on AKT/β-Catenin/SLUG axis in ovarian cancer cells in vitro.A human phosphor-kinase array was used to explore whether NE regulates phosphor-kinases in SK-OV-3 cells.Informatic analysis was conducted to further analyze how NE regulates these phosphor-kinases,including Protein-Protein Interaction analysis,Gene Ontology analysis,pathway analysis,and protein domain analysis.Furthermore,ovarian tumor tissue array was used to analyze the relationship between NE content or β-catenin expression with clinicopathologic significance.Results: 1)CRS facilitated abdominal cavity implantation metastasis of SK-OV-3 cells in animal models.2)CRS promoted expression of EMTTFs in tumor tissues obtained from animal models,including TWIST,SLUG,SNAIL and β-Catenin.3)CRS promoted co-expression of β-Catenin and SLUG in tumor tissues.4)β-Catenin agonist SKL2001 promoted expression of SLUG and exerted pro-migrating and pro-invading effects on ovarian cancer cells in vitro.5)CRS increased NE content in tumor tissues and mouse serum and promoted co-expression of NE and β-Catenin in tumor tissues.6)NE facilitated expression of β-Catenin and SLUG and promoted migration and invasion of ovarian cancer cells in vitro.7)NE significantly promoted phosphorylation levels of various kinds of phosphor kinases in SK-OV-3 cells.8)NE activated β-Catenin / SLUG axis and promoted migration and invasion of ovarian cancer cells through phosphorylating AKT.9)MLT suppressed CRS-mediated abdominal cavity implantation metastasis of SK-OV-3 cells in animal models and inhibited expression of EMT-TFs in tumor tissues.10)MLT inhibited NE/AKT/β-Catenin/SLUG axis and suppressed migration and invasion of ovarian cancer cells in vitro.11)Results from ovarian tumor tissue array revelaed that higher NE content and greater β-Catenin expression were positively related to malignancy and higher FIGO stage of ovarian serous cancer.NE content and β-Catenin expression were more in tumor tissues than paracancerous tissues.Furthermore,NE expression was significantly related to β-Catenin expression in both benign and malignant ovarian tumors.But there was no significance of NE content and β-catenin expression among various subtypes of stage I-II ovarian cancer.Conclusion: NE participated in CRS-mediated abdominal cavity implantation metastasis of ovarian cancer cells which could be reversed by MLT through suppressing AKT/β-Catenin/SLUG axis in vivo and in vitro.These findings suggest a novel mechanism for CRS-mediated ovarian cancer metastasis and MLT has a potential therapeutic target against ovarian cancer. |
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