Effects Of Celastrus Orbiculatus Extracts On The HepG2 Cells With The Overexpression Of VEGF-A

Hepatocellular carcinoma is one of the most common malignancies in the world,seriously endangering human health.Only in 2012,there are 782,000 new cases of diagnosis of liver cancer,which is from World Cancer Research Foundation statistics in 2015.Traditional treatments including surgical and radiotherapy and chemotherapy have a certain limitation.The metastasis is also great injury to the body.Many Chinese herbal medicines can inhibit the proliferation of hepatic carcinoma cells,angiogenesis and metastasis.There are more than 30 factors related to tumor angiogenesis.Vascular endothelial growth factor(VEGF)and its receptor(VEGFR)play vital roles,they can specifically promote vascular-related cell division,proliferation and migration.VEGF family includes VEGFA,VEGFB,VEGFC,VEGFD,VEGFE and placental growth factor,among which VEGFA is the most studied and widely used.Celastrus orbiculatus Thunb.,also named guo shan feng,etc.We found that Corbiculatu ethylacetate extract(COE)have the anti-tumor activity in vitro and in vivo including inhibit tumor invasion,metastasis,angiogenesis.The effects of Celastrus orbiculatus Thunb.extract on the VEGF-A in hepatocellular carcinoma cells has not been reported.Therefore,we constructed the human hepatic carcinoma HepG2 cells with the overexpression of VEGF-A.And we explored the effects of COE on the proliferation and apoptosis in HepG2/VEGF-A+ Cells.The results showed that we successfully constructed the recombinant VEGF-A-EGFP cloning vectors,and established the stable HepG2 cell line with the overexpression of VEGF-A.The Celastrus orbiculatu extract can inhibit the proliferation and the apoptosis of HepG2/VEGF-A+ cells.COE inhibit the expression level of VEGF-A in a dose-dependent manner.And the mechanism may be related to MAPK signaling pathway.The study will be described in two parts.Part ? Establishment the stable HepG2 cell line with the overexpression of VEGF-AObjective:To construct the human hepatic carcinoma HepG2 cells with the overexpression of VEGF-A.Methods:The GV287-VEGFA-EGFP recombinant vector was constructed by using PCR technology and transfected into 293-T cells.The fluorescence intensity of the cells was observed by fluorescence microscopy.And the expression of the recombinant protein was detected by Western Blot.Then,the VEGFA-EGFP recombinant plasmids were integrated into the DNA of HepG2 cells using virus infection technology.After 72 h of infection,the expression level of VEGF-A was detected by the fluorescence intensity and Western Blot.Results:The characteristic band at 48 KDa was foud,and its size was consistent with the VEGFA-EGFP fusion protein.And the protein expression of VEGF-A in transfected HepG2 cells was significantly enhanced.Conclusion:We successfully constructed the recombinant VEGF-A-EGFP cloning vectors,and established the stable HepG2 cell line with the overexpression of VEGF-A.Part ? Effects of Celastrus orbiculatu extract on the proliferation and apoptosis in HepG2/VEGF-A+ cellsObjective:To study the effects of Celastrus orbiculatu extract on the proliferation and apoptosis in HepG2/VEGF-A+ Cells.Methods:The human hepatic carcinoma HepG2/VEGF-A+ cells were divided into the following groups:untreated control,vehicle control,positive control(RG3 50?mol/L,bevacizumab 80mg/mL),and COE-treated groups.The HepG2/VEGF-A+ cells were treated with C.orbiculatu extract in different concentrations(10,20,40,80?g/mL)for 24 h.The cell proliferation was detected by MTT assay.The expression level of VEGF-A and the MAPK signaling pathway related proteins were detected by Western Blot,respectively.Results:COE suppress the growth of HepG2/VEGF-A+ cells in a dose-dependent manner.The proliferation of the cells was began to inhibited When the COE at a concentration of 40 ?g/mL(P<0.01).Compared with wild-type control group,the expression level of VEGF-A was significantly increased in HepG2/VEGF-A+ cells.The western blotting assays indicated that COE inhibit the expression level of VEGF-A in a dose-dependent manner.And the expression of ERK1/2,p-ERK1/2,p38MAPK and p-p38MAPK were significantly decreased in a dose-dependent manner(P<0.05).Conclusion:The Celastrus orbiculatu extract can inhibit the proliferation and the apoptosis of HepG2/VEGF-A+ cells.COE inhibit the expression level of VEGF-A in a dose-dependent manner.And the mechanism may be related to MAPK signaling pathway.