Enhanced Effects Of Bcl-2/mTOR Inhibitor On Epirubicin-based Chemotherapy In HepG2 Cells

PartⅠThe growth inhibition of epirubicin in hepG2 cellsObjective: To explore the appropriate level and incubation concentration of epirubicin that cause the early stress of HepG 2 cells, also known as self-repaired.Methods MTT assay was performed to determine the inhibition of HepG2 cell proliferation and 50% inhibiting concentration (IC50) induced by epirubicin at the different times and levels.Results: The cells growth inhibition was apparently in a time- and dose-dependent manner and the IC50 about epirubicin for incubation 12 hours was 78.14±3.27μg/ml in HepG2 cells.Conclusion: When in the early stress (after incubation of epirubicin) or in a lower incubation level of epirubicin(<40μg/ml), autophagy will be up-regulated. Such a point of time and a drug level can be used as the optimal experiment condition for the next step.PartⅡEnhanced effects on epirubicin-based chemotherapy by blocking bcl-2 pathway in hepG2 cells.Objective: To explore the effects of ABT737 or epirubicin singled or combined on cell proliferation , apoptosis and autophagy in hepG2 cells.Methods: MTT assay was used to determine cytotoxic effects of ABT737 on HEPG2 cells. Apoptosis was assessed using flow cytometry analysis after staining DNA with propidium iodide. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Activation of autophagy was monitored with monodansylcadaverin (MDC) staining, Western blot analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p62, LC3 and Beclin1.Results: After ABT737 treatment, the viability of HEPG2 cells was inhibited and mitochondrial membrane potential collapsed. ABT737 induced apoptosis. Western blot analysis detected inductions in the expression of p53 and Beclin 1 as well as autophagic proteins LC3 and p62 after ABT737 treatment. When combined with epirubicin, ABT737 enhanced the effects of inducing apoptosis of HEPG2 cells, decreased mitochondrial membrane potential and up-regulated the expression of p53, beclin1 and LC3Ⅱ.Conclusion: ABT737 induced death of cancer cells through both apoptotic and autohagic mechanisms, and ABT737 may enhanced the effects of activating autopahgy and inducing apoptosis of Epirubicin.PartⅢEnhanced effects on epirubicin-based chemotherapy by blocking mTOR pathway in hepG2 cells.Objective: To explore the effects of rapamycin or epirubicin singled or combined on cell proliferation , apoptosis and autophagy in hepG2 cells.Methods: MTT assay was used to determine cytotoxic effects of rapamycin on HEPG2 cells. Apoptosis was assessed using flow cytometry analysis after staining DNA with propidium iodide. Mitochondrial membrane potential was measured using the fluorescent probe JC-1. Activation of autophagy was monitored with monodansylcadaverin (MDC) staining, Western blot analysis were used to determine the expression of proteins involved in apoptosis and autophagy including p53, p62, LC3 and Beclin1.Results: After rapamycin treatment, the viability of HEPG2 cells was inhibited and mitochondrial membrane potential collapsed. rapamycin induced apoptosis. Western blot analysis detected inductions in the expression of p53 and Beclin 1 as well as autophagic proteins LC3 and p62 after rapamycin treatment. When combined with epirubicin, rapamycin enhanced the effects of inducing apoptosis of HEPG2 cells, decreased mitochondrial membrane potential and up-regulated the expression of p62 and LC3Ⅱ.Conclusion: Blocking the mTOR pathway can induce death of cancer cells through both apoptotic and autohagic mechanisms, and Rapamycin may enhanced the effects of activating autopahgy and inducing apoptosis of Epirubicin.