Celastrus Orbiculatus Extracts Inhibit Epithelial Mesenchymal Transition In Human Hepatocellular Carcinoma HepG2 Cells Under Hypoxia Microenvironment

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.It ranks as the third incidence of malignant tumors and the fifth cause for cancer death worldwide.Besides surgical resection,systemic chemotherapy,intervention and local radiotherapy are the efficient methods for raising five-year survival rate to the paitients with HCC.High mortality of liver cancer is closely related to tumor invasion and metastasis.Epithelial mesenchymal transition(EMT),the process ofcells losing their epithelial phenotype and acquiring a migratorymesenchymal phenotype,has been accepted to play animportant role in cancer metastasis in several human malignancies.Hypoxia is a common feature of all solid tumors.Increasing evidences suggest that the hypoxic microenvironment is one of the reasonsof the drug resistance to chemotherapy and radiotherapy.Hypoxic alsopromote the early tumor metastasis.Hypoxia inducing factor 1α(HIF-1α)is activated under the hypoxic microenvironment,and then it is reacted with downstream target genes of hypoxia in the promoter region of the components,participate in the transfer process of cascade,increase the tumor cells ability of EMT.Thus,the inhibition of hypoxic microenvironmenthas been considered as one of useful targets of anti-tumor drugs.Celastrus orbicuiatus Thunb.(Nansheteng,celastrus),has been used as a folk medicine in China for the treatment of many diseases,including promoting blood circulation to remove blood stasis,relieve swelling and pain.We have found that Celastrus orbiculatus extracts(COE)could anti-tumor in vivo and in vitro,including inhibition of tumor cell proliferation,invasion,metastasis and promotion of apoptosis of tumor cells under the condition of oxygen.But its effect on tumor cells under hypoxic microenvironment is not clear.In this study,we investigated the effects of COE on CoCl2-induced EMT in vitro.And then we explored the molecular mechanisms underlying the function.Therefore,the present findings suggest that COE might have potential therapeutic effect against hepatocellular carcinoma.The findings will be described in three parts as follows.Part ⅠEffects of the CoCl2-induced hypoxic microenvironment on EMT(epithelial mesenchymal transition)in human hepatocellular carcinoma HepG2 cellsObjective:To set up the hypoxic microenvironment by using CoCl2 in vitro,and investigate whether the CoCl2 affect the process of EMT in human hepatocellular carcinoma HepG2 cells.Methods:Human hepatocellular carcinoma HepG2cells were added with different concentrations(50,100,200,400,800μmol/L)of CoCl2 for 24 h.MTT method was used to detect the cell proliferation.The morphological changes were determined by optical microscopy after CoCl2-induced for 24h.The protein expression levels of HIF-la,E-cadherin,N-cadherin,and Vimentin were analyzed using western blotting and Immunofluorescence,respectively.Results:After treating with different concentration of CoCl2 for 24 h,the viability of HepG2 cells is inhibited in a concentration-dependent manner.Therefore,then non-cytotoxic concentrations(100μmol/L)of CoCl2 was chosen for further experiments.After treating with CoCl2(100 μmol/L)for 24 h,the fusiform shapesof the cells were changed.And the cells link weredisappeared.The expression of E-cadherin was down-regulated(P<0.01),whereas HIF-1α,N-cadherin and Vimentin were up-regulated(P<0.01).Conclusion:The hypoxic microenvironment can be induced successfully by using 100μmol/L CoCl2 inhuman hepatocellular carcinoma HepG2 cells.Part ⅡThe effects of COE on EMT(epithelial mesenchymal transition)in CoCl2-induced HepG2 cellsObjective:To investigate the effects of COE onproliferation,apoptosis,invasion,migration and EMT inCoCl2-induced HepG2cells.Methods:The HepG2 cells were pre-treated with 100μmol/L,the they were devided into different groups,including COE groups(160,200,240μg/mL),positive control group(DDP 2μg/mL).Apoptosis cells were detected by using Flow Cytometry assay.The mitochondrial transmembrane potential was analyzed with the JC-1 staining assay.The wound healing ability was detected through the wound Healing assay.The cell migration was observed by transwell migration assay.The expression ofE-cadherin,N-cadherin,and Vimentin were analyzed using western blotting and Immunofluorescence,respectively.Results:COE inhibits the viability of HepG2 cells in a concentration-dependent manner under the hypoxic microenvironment.JC-1 staining assay shows a decrease of red fluorescence and increase of green fluorescence.The invasionandmigration ofhuman hepatocellular carcinoma HepG2 cells were inhibited.The expression of E-cadherin was up-regulated(P<0.01),whereas the expression of N-cadherin and Vimentin were down-regulated(P<0.01).Conclusion:COE effectively inhibit the proliferation,apoptosis,invasion and migration,EMT in human hepatocellular carcinoma cell line HepG2 cells.Part Ⅲ The molecular mechanisms underlying the COE inhibitthe EMT(epithelial mesenchymal transition)in the CoCl2-induced HepG2 cellsObjective:To investigate the molecular mechanisms of the COE inhibit the EMT in the CoC12-induced human hepatocellular carcinoma HepG2 cells.Methods:After HepG2 cells were treated with different dose(160,200,240 μg/mL)of COE,the protein of HIF-1α、Twistl was detected by western blot and Immunofluorescence,respectively.To further investigate the effects of HIF-1α on cell invasion,migration and EMT in CoCl2-induced HepG2 cells,the cells were pretreated with YC-1(100μmol/L),and then incubated with COE(200μg/mL)for 24h.The expression of E-cadherin,N-cadherin,and Vimentin were analyzed using western blotting and Immunofluorescence,respectively.Results:It is suggested that the protein expression of HIF-la and Twistl were decreased in a dose-dependent manner after COE treatment(P<0.01).In addition,when HIF-1α/Twist1 signaling pathway was blocked by using YC-1,a potential anticancer agent which reduces the protein stability ofHIF-la,western blot analysis showed that the protein expression level of HIF-1α,Twistl were decreased.In addition,YC-1 and COE could further increase the E-cadherin protein level,but decreased N-cadherin and Vimentin protein levels.Conclusion:The effects of COE on inhibiting tumor cells metastasis and EMT,may be closely related to Hif-la/Twistl signaling pathway.