Cinobufotalin Inhibits Hepatocellular Carcinoma Epithelial-Mesenchymal Transition By Down-regulation ?-catenin

Objective:Hepatocellular carcinoma(HCC)is one of the most mortality cancers in the world,and it is a high risk cancer in China.The postoperative recurrence rate and metastasis rate of HCC are very high,which will lead to poor prognosis of patients.Epithelial-mesenchymal transition(EMT)plays a key role in the metastasis of HCC,which is closely related to the invasion,intrahepatic metastasis and low survival rate of HCC.Therefore,it is important to inhibit EMT of HCC.Cinobufotalin is one of the most components from Bufonis Venenum,which has shown a variety of anti-tumor activity.In this study,we aim to investigate whether cinobufotalin can inhibit EMT and metastasis of HCC,and then clarify the molecular mechanism.Methods:The effect of cinobufotalin on the proliferation of Hep G2 and SMMC-7721 cells was determined by CCK-8 assay.Morphological change of cells was observed by the inverted biological microscope.Western blotting assay was used to detect the expression of EMT-related proteins(such as E-cadherin,N-cadherin,Snail,Vimentin,Slug and ZEB1)and migration-related proteins(such as ?-catenin,DKK1 and MMP7)in Hep G2 and SMMC-7721 cells after treated with cinobufotalin.Wound healing,transwell migration and transwell invasion assay were used to investigate the effect of cinobufotalin on the migration and invasion ability of HCC.The effects of cinobufotalin on the expression of ?-catenin mRNA and ?-catenin cytoplasm and nuclear protein in HCC were detected by Real-time PCR assay and western blotting assay.?-catenin overexpressed and knockdown Hep G2 and SMMC-7721 cells were established using transient transfection method.And these cell models were used to investigate the role of ?-catenin in cinobufotalin-mediated EMT inhibition.Furthermore,the lung metastasis model of BALB/c mice was established to examine the effect of cinobufotalin on H22 cells EMT and metastases in vivo.Immunohistochemistry was conducted to determine the expression of EMT-related protein in lung tumors of mice.Results:1.The result of CCK-8 assay showed that cinobufotalin could inhibit cell proliferation of Hep G2 and SMMC-7721 cells in time-and dose-dependent manner.2.After treatment with cinobufotalin for 24 h,the cell morphology of Hep G2 and SMMC-7721 cells have changed from a spindle-mesenchymal like shape to an oval-epithelial like shape.3.Western blotting result showed that cinobufotalin significantly regulates the expression of EMT-related proteins in Hep G2 and SMMC-7721 cells,such as the expression of epeithelial marker(E-cadherin)was up-regulated and the expression levels of mesenchymal markers(Vimentin,ZEB1,Slug,Snai and N-cadherin)were down-regulated.4.The results of wound healing,transwell migration and invasion assays displayed that cinobufotalin attenuated the migration and invasion of hepatocellular carcinoma cells.5.The results of Real-time PCR and western blotting assay showed that cinobufotalin significantly reduces the mRNA and protein expression of ?-catenin,and then inhibits the nucleus expression of ?-catenin.6.The experiments successfully used the transient transfection method to build ?-catenin overepressed cell lines.Western blotting assay indicated that overexpression of ?-catenin promotes EMT of hepatocellular carcinoma cell lines,but cinobufotalin reverses the regulation of overexpression of ?-catenin on EMT-related proteins.The results of wound healing,transwell migration and invasion assays displayed that cinobufotalin significantly reversed the induction of overexpression of ?-catenin on the migration and invasion of hepatocellular carcinoma cells.7.The experiments successfully used the transient transfection method to build ?-catenin knockdown cell lines.Then the experiment results showed that knockdown of ?-catenin inhibits EMT activity,migration and invasion in Hep G2 and SMMC-7721 cells and cinobufotalin further enhance this inhibition.8.Animal experiments showed that cinobufotalin significantly inhibits the formation and metastasis of lung metastases in BALB/c mice.9.The result of immunohistochemistry showed that cinobufotalin inhibited tumor EMT at the animal level.Conclusions:Cinobufotalin inhibits EMT of HCC by down-regulating ?-catenin,and then prevent the migration and invasion of HCC.