Molecular Mechanisms Of The Celastrus Orbiculatus Extracts Induce The Apoptosis By Targeting MTOR In Human Hepatocellular Carcinoma HepG2 Cells

Celastrus orbiculatus(Celastraceae),has been used as a folk medicine against many diseases in China,including arthritis and other inflammation.Our research has showed that Celastrus Orbiculatus extracts(COE)had antitumor activity include inhibiting the proliferation and inducing the apoptosis in tumor cells.But the molecular mechanisms underlying had not been cleared.Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world.The incidence and mortality rate of HCC is increasing in recent years.Despite multimodal therapy,including surgery,chemotherapy,and radiotherapy,the curative effect on HCC patients is not as good as anticipated and the prognosis remains poor.Therefore,searching for the anticancer drugs with high efficiency,and low toxicity is very important.Mammalian target of rapamycin(mTOR),as a key signaling pathway,involved in a variety of physiological and pathological processes(such as proliferation,migration,etc).Its dysfunction is closely related to the occurrence and the development of tumor.To investigate the molecular mechanisms underlying the effects of COE on the proliferation and the apoptosis in hepatocellular carcinoma cells,HepG2 cells with high expression of mTOR(HepG2/mTOR+)were constructed,and then treated with different concentrations of COE.The results showed that COE inhibited the proliferation and induced the apoptosis in HepG2/mTOR+ cells in a concentration-dependent manner.Its molecular mechanism may be related to mTOR signaling pathways.The study will be described in four parts.Part ?Construction of the HepG2/mTOR+ cell line Objective:To construct the human hepatocellular carcinoma HepG2 cells with mTOR overexpression.Methods:The recombinant plasmid of GV238-mTOR was transfected into HepG2 cells by using molecular biology technique.After 24 hours of the transfection,the morphology of the cells was observed under inverted microscope.The mTOR protein expression level was detected by western blotting in HepG2 cells.Results:After 24 hours of the transfection,the cellular morphology of the wild type cell was clear,the refractive index was good,and the nucleus was homogeneous.Most of the morphology of HepG2/mTOR+ cells were not significantly different,but some cells became rounded.Compared with the control,the results of western blot showed that the expression level of mTOR protein was significantly increased in HepG2/mTOR+ cells.Conclusion:The HepG2/mTOR+ cell line has been constructed successfully.Part ?Effects of C.Orbiculatus extracts on HepG2/mTOR+ cells Objective:To study the effects of C.Orbiculatus extracts(COE)on the proliferation,apoptosis,invasion and migration in HepG2/mTOR+ cells.Methods:The logarithmic HepG2/mTOR+ cells were treated with different concentrations of COE(20,40,80,160 and 320mg/L).The proliferation of HepG2/mTOR+ cells were detected using MTT assay.The morphological changes of the cells were observed by inverted microscope and transmission electron microscopy.The effects of COE on the apoptosis in the HepG2/mTOR+ cells were detected by flow cytometry.The migration was observed using trans-well migration assay in HepG2/mTOR+ cells.Results:COE inhibited the proliferation,invasion and migration of HepG2/mTOR+ cells.Meanwhile,COE induced the apoptosis of HepG2/mTOR+ cells in a concentration-dependent manner.Conclusion:COE inhibited the proliferation,invasion and migration in HepG2/mTOR+ cells.Meanwhile,COE induced the apoptosis in HepG2/mTOR+ cells.Part IIIMolecular mechanisms of COE induce the apoptosis in HepG2/mTOR+ cellsObjective:To study the molecular mechanism underlying COE induce the apoptosis in HepG2/mTOR+ cells.Methods:The human hepatocellular carcinoma HepG2/mTOR+ cells were treated with different concentrations of COE(20,40,80 mg/L),and 2 mg/L DDP,respectively.After 24 hours of the treatment,the protein expression levels were detected by western blotting.Results:COE inhibited the expression of mTOR protein in a concentration-dependent manner.The expression of Bcl-2 and Bcl-2L12 were decreased,at the same time,the expression of Bax,Caspase-3,4E-BP1 and P70 proteins were increased in a concentration-dependent manner.Conclusion:The molecular mechanisms underlying COE induce the apoptosis of HepG2/mTOR+cells may be related to Bcl-2 family and mTOR signaling pathway.Part ?The synergistic effects of COE and rapamycinObjective:To study whether there are synergistic effects between COE and mTOR inhibitor Rapamycin(RAPA).Methods:The logarithmic HepG2/mTOR+ cells were treated with COE(80 mg/L),RAPA(100 nmol/L),COE+RAPA,respectively.The effects of drugs on the apoptosis were assayed by flow cytometry in HepG2/mTOR+ cells.The protein expression levels were detected by western blotting.Results:Compared with the control,COE or RAPA group could induce the apoptosis,inhibit the expression of apoptosis-related protein,COE and RAPA combination therapy showed synergistic effect.Conclusion:COE could further promote tumor cell apoptosis when mTOR signaling pathway was blocked.