| Hepatocellular carcinoma(HCC)is the fifth most common malignant tumor,third in mortality worldwide.Despite multimodal therapy,including surgery,chemotherapy,and radiotherapy,the curative effects on HCC patients are not good as anticipated.Mammalian target of rapamycin(mTOR)is a class of non-conserved evolutionary protein kinase,and is involved in a variety of physiological and pathological processes.The mTOR signaling pathway is a promising target in the light of its frequent dysregulation in hepatocellular carcinoma and its central role in regulating cell proliferation,migration,survival and angiogenesis.Aberrant mTOR signaling was detected in up to 48%of hepatocellular carcinoma,and a correlation between poor outcome and mTOR signaling activation has been shown.Celasorus orbiculatus is a member of the family Celastraceae and the genus Celastrus.Celastrus and several of its defined constituents possess anti-cancer,anti-inflammatory and anti-oxidant properties.The preliminary results of our study suggested that COE could inhibit the activity of mTOR signaling pathway,but the underlying molecular mechanism has not yet to be revealed completely.This study explored the effects of COE on the proliferation and apoptosis in the HepG2/mTOR-cells,which may shed new light on clinical treatment for cancer characterized by mTOR activation.This study was divided into four parts.Part ?Construction of HepG2/mTOR-cell lineObjective:To construct the human hepatoma HepG2 cells with low mTOR expression.Methods:The GV248-mTOR-RNAi-EGFP lentivirus vector was constructed by our laboratory using molecular biological technology.The recombinant plasmid of Lentiviral vector GV248 carrying mTOR RNAi(NM 004958)gene.The mTOR-RNAi viruses infected HepG2 cells,after 24 hours of cell culture,the cells were observed under an inverted fluorescence microscope and the mTOR expression level was detected by western blotting and RT-qPCRResults:After 24 hours of transfection,the cells was observed under inverted microscope.The outline of the wild-type cells was clear,the structure was spindle shape,the cell wall was smooth and no protrusion,the refractive index was good,and the nucleus was homogeneous.The morphology of HepG2/mTOR" cells was not significantly different from that of wild-type cells,but some cells were long and thin.The green fluorescence was strongly expressed in the cells.Compared with the negative control,the expression of mTOR RNA and protein in HepG2/mTOR-cells were significantly decreased(P<0.05).Conclusion:The HepG2 cell line with mTOR knockdown was successfully constructed.Part ? Effects of Celastrus Orbiculatus Extracts on HepG2/mTOR-CellsObjective:To study the effects of Celastrus Orbiculatus Extracts(COE)on the proliferation,apoptosis,invasion and migration in HepG2/mTOR-cells.Methods:The logarithmic growth phase human hepatocellular carcinoma HepG2/mTOR-cells were devided into COE groups(20?40?80?160?320 mg/L),positive control group(Cisplatin,DDP,2mg/L).Cell viability was determined using MTT assays at 24,48 and 72 hours,respectively.A transmission electron microscope(CM 100,Philips,the Netherlands)was used to observe ultrathin sections stained with uranyl acetate and lead citrate.The effects of COE on the apoptosis were analyzed by using flow cytometry.The transwell migration assay was used to observe cell migration.Results:COE inhibited the proliferation significantly in a concentration-dependent manner(P<0.01)in HepG2/mTOR-cells.COE significantly induced the apoptosis of HepG2/mTOR"cells(P<0.01)and the apoptotic bodies can be observed under transmission electronic microscopy.COE inhibited the invasion and migration of HepG2/mTOR-cells in a concentration-dependent manner(P<0.05).Conclusion:COE effectively inhibited the proliferation,invasion and migration,and promoted the apoptosis of HepG2/mTOR-cells.Part ? The Molecular Mechanisms of COE Induce the Apoptosis of HepG2/mTOR-cellsObjective:To characterize the molecular mechanism underlying the antineoplastic activity of COE induced the apoptosis in HepG2/mTOR-cells.Methods:Cell viability was determined using MTT assays.According to the IC50 value(140 mg/L),set the concentrations of COE in the subsequent experiment was to alleviate the cytotoxicity.The HepG2/mTOR-cells were divided into 5 groups:negative control(untreatment),COE treatment groups(40,80,120 mg/L),positive control(Cisplatin,DDP,2 mg/L).Wild-type HepG2 cells were used as blank control.After 24 hours of the treatment,the protein expression levels of mTOR signal pathways were determined by western blotting.Results:COE decreased the expression of Bcl-2 and Bcl-xL.Meanwhile,the expression of Bax and Caspase-3 were increased in a concentration-dependent manner.Compared to the untreated control group,the protein levels of mTOR and its downstream proteins,such as 4EBP1,p-4EBP1,P70S6k and p-P70S6k,reduced significantly in a concentration-dependent manner(P<0.05).Conclusion:COE induced the apoptosis through Bcl-2 family signaling pathway and PI3K/AKT signaling pathway in HepG2/mTOR" cells.Part? COE Attenuated Growth of Solid Hepatic Carcinoma in Nude MiceObjective:To study the anticancer effect of Celastrus Orbiculatus Extracts(COE)in vivo.Methods:HepG2/mTOR-cells(2×106 cell/mice)were subcutaneously injected into the right flank of nude mice.36 female nude mice were randomly assigned to 6 groups according to body weight(6 mice per group)as follows:solvent vehicle control,Banmao Capsule treated group(BM,195 mg/kg),Tegafur,Gimeracil and Oteracil Potassium Capsules treated group(TJA,10 mg/kg),and different dosages of COE(10,20,40 mg/kg).Tumor growth was monitored and immunohistochemical staining was used to examine the expression of apoptosis-related proteins in tumor tissues.Results:COE significantly inhibited tumor growth in nude mice.Moreover,the results showed that COE could down-regulate the expression of Bcl-2 and Bcl-xL,while up-regulating the levels of Bax and caspase-3(P<0.05).Conclusion:COE was a potential chemotherapeutic drug in HCC treatments via targeting mTOR signal pathway. |
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