Emodin Regulates The Migration And Invasion Of Epithelial Ovarian Cancer Cells Via ILK Pathway And The Underlying Mechanism

Background:Epithelial ovarian cancer(EOC)is the most lethal gynecologic malignancy.The lack of effective early detection markers,coupled with the vague,nonspecific symptoms of EOC,often results in the late diagnosis of the disease with widespread metastases.Mortality of EOC is directly related to the prevalence of metastatic diseases,however,the underlying molecular mechanism of metastases are still not fully elucidated.In recent studies,a crucial link has been discovered between the acquisition of metastatic traits and the epithelial-mesenchymal transition(EMT)in ovarian cancer.Integrin-linked kinase(ILK)is a serine/threonine protein kinase,which was initially discovered through its interactions with the β1 and β3 integrin subunits.ILK has a fundamental role in the regulation of cell survival,proliferation and migration by mediating integrin signaling in diverse cell types.Once activated,ILK directly phosphorylates several key signaling molecules,including protein kinase B/Akt(PKB/Akt)and glycogen synthase kinase 3β(GSK3β),to affect cell survival,cycle,adhesion and extracellular matrix(ECM)modification.The expression of ILK is often up-regulated in various human malignancies and correlated with advanced tumor stage and grade.Moreover,ILK has been reported to promote cancer cell migration and invasion by inducing EMT process.Emodin(1,3,8-trihydroxy-6-methylanthraquinone),an anthraquinone,isolated from rhizomes of Rhubarb,aloes and other plants is known to have anti-proliferative,anti-tumorigenic,anti-metastatic and anti-angiogenic effects on various cancers.Recent studies have shown that emodin repressed the EMT of cells through WNT/β-catenin pathway in several cancer cells.Meanwhile,previous studies demonstrate that emodin inhibited the migration and invasion abilities of human endometrial stromal cells by facilitating the mesenchymal-epithelial transition(MET)through targeting ILK.Objective:This study aimed to investigate whether emodin could inhibit EOC cells invasion and migration by suppressing EMT and then to explore the underlying mechanism in vitro and in vivo.Methods:1.The cell counting kit-8(CCK-8)was performed to detect the effect of emodin on the proliferation of EOC cells.SK-OV-3 and A2780 cells were treated with a range of concentrations of emodin for up to 24 hours,48 hours and 72 hours.2.Transwell migration and invasion assays were used to explore the migratory and invasive abilities of A2780 and SK-OV-3 cells.3.Western blot analysis and immunohistochemical(IHC)was conducted to evaluate the expression of epithelial factors(E-cadherin and Claudin),mesenchymal markers(N-cadherin and Vimentin)and ILK pathway associated factors in EOC cells and tumors.4.ILK and Slug were knockdown by small interfering RNA(siRNA)-ILK and siRNA-Slug,respectively.ILK was exogenously expressed by transfecting with ILK lentiviral vector(pLVX-C1-ILK)in EOC cells.Results:1.The effect of emodin on the proliferation,migration,and invasion of EOC cells.Emodin inhibited the proliferation of SK-OV-3 and A2780 cells in a dose-and time-dependent fashion.The migration and invasion abilities of A2780 and SK-OV-3 cells were significantly decreased after treated with emodin for 48 hours.2.The effect of emodin on the expression of EMT related factors and Slug in EOC cells.The Western Blot showed that emodin up-regulated the expression of epithelial markers(E-cadherin and Claudin)while down-regulated the expression of mesenchymal markers(N-cadherin and Vimentin)and transcription factor(Slug)in a dose-dependent fashion.After transfection of siRNA-Slug,Slug and N-cadherin were both down-regulated in A2780 and SK-OV-3 cells while E-cadherin was up-regulated,which were intensified by emodin.The migration and invasion abilities of A2780 and SK-OV-3 cells were lowest in Slug knockdown cells followed by treating with emodin.3.The effect of emodin on ILK signaling pathway related molecules in EOC cells.Dose-dependent decrease of ILK,p-GSK-3β,β-catenin and Slug expression were observed after treated with emodin for 48 hours in A2780 and SK-OV-3 cells.4.The verification of emodin repressed EMT by targeting ILK in EOC cells.The expression of ILK,p-GSK-3β,β-catenin and Slug were down-regulated after transfection of siRNA-ILK,which was further strengthened by emodin treatment.Meanwhile,the levels of E-cadherin was increased while the levels of N-cadherin,was decreased,which can also be intensified by additional emodin.5.The verification of emodin inhibited the EMT of EOC cells by ILK/GSK-3βsignaling pathway.SB216763,an inhibitor of GSK-3β,could up-regulate the expression of p-GSK-3β,β-catenin and Slug except for ILK.Meanwhile the level of E-cadherin was down-regulated while the level of N-cadherin was up-regulated in A2780 and SK-OV-3 cells treated with SB216763.So,SB216763 could reverse the effects of emodin except for ILK expression.6.The effect of emodin on the migration,and invasion of A2780/pLVX-ILK and SK-OV-3/pLVX-ILK cells.The migration and invasion abilities of A2780 and SK-OV-3 cells lines were effectively increased by transfection of pLVX-ILK,which can be abrogated by following treatment with emodin(20μM).After transfection of pLVX-ILK,the expression of E-cadherin was decreased while the levels of Vimentin,MMP-9 and Slug were increased in A2780 and SK-OV-3 cells,which can be counteracted by emodin.7.The effects of emodin on the growth rate of tumors in vivo.Tumors grew more rapidly in pLVX-ILK group compared with the control group(pLVX-Con),which can be significantly inhibited by emodin.8.The effects of emodin on EMT-related factors in tumors in vivo.The expression of E-cadherin was down-regulated,while the expression of ILK,Slug,MMP-9 and Vimentin were up-regulated in pLVX-ILK group.The contrary results were detected in emodin treated groups.9.The effects of emodin on cardiac,liver and renal function of nude mice.Hematologic toxicity test results showed emodin could hardly affect the function indices of liver,kidney and cardiac.Conclusion:1.Emodin could suppress the proliferation,invasion and migration capabilities of A2780 and SK-OV-3 cells.2.Emodin inhibited the migration and invasion abilities of EOC cells by suppressing the EMT through ILK/GSK-3β/Slug signaling pathway.3.Emodin suppressed the growth rate and metastasis of EOC tumors in vivo.4.Emodin was not found to have target toxicity on hepatocytes,nephrocytes and cardiomyocytes.