Study On The Effect Of Metformin Combined With Glucose Metabolism Inhibition On Proliferation And Apoptosis In Liver Cancer Cells

Primary liver cancer is one of the most common malignant tumors.It is the second leading cause of cancer death in the world.It has high degree of malignancy,poor treatment effect and high mortality.The five years survival rate of Liver cancer patients is less than 15%,lacking of effective treatment.Therefore,exploring the specific mechanism of its carcinogenesis and development process,which aims at prevention and diagnosis and treatment,is of great significance to the prevention and treatment of liver cancer.In the process of tumor cell development,a large amount of glucose is needed,even in the absence of oxygen;the malignant tumor cells are more likely to obtain the energy by anaerobic glycolysis,which is the famous Warburg effect.Therefore,glucose is the energy source of most malignant tumors.At present,low sugar therapy has been used as a new method of tumor treatment.Targeting energy metabolism is a new direction of anticancer therapy.Metformin is a commonly used hypoglycemic agent.In recent years,the study found that metformin has a certain anti-tumor effect,by reducing the risk of diabetes patients to develop malignant tumors,and by enhancing the efficacy of drugs.Studies have reported that metformin can reduce hepatic gluconeogenesis and blood glucose release,limit the buffer function of the liver,and increase serum glucose levels when intake of nutrients decreased;on theother hand,metformin has been shown to act possibly through direct inhibition of glucose induced by short-term lack of mitochondrial energy to supplement respiratory complexes I consume,and hinder the growth of cancer cells.2-deoxy-D-glucose,a glucose analogue,is able to block glucose metabolism and reduce the ability of tumor cells to supply energy.It has been reported that2-deoxy-D-glucose can increase oxidative stress,inhibit glycosylation,and induce autophagy.2-deoxy-D-glucose also can effectively delay cell growth and promote apoptosis of tumor cells.Although 2-deoxy-D-glucose itself has been limited in the treatment of many types of cancer,it can be combined with other therapeutic drugs or radiation therapy with synergistic antitumor effects.Autophagy is a process dependent on lysosomal pathway for degradation of cytoplasm and organelles.It is highly conserved in evolution and it plays an important role in cell death,and cell senescence,and physiological,and pathological processes,and metabolism.What's more,autophagy is a key mechanism to promote cell survival.PI3K/AKT/m TOR signaling pathway can regulate autophagy.Phosphorylation of AKT is an important part of this pathway,which plays an important role in the survival and apoptosis of tumor cells.In this study,we investigated the survival of hepatocellular carcinoma cells by metformin combined with glucose deficiency.Then we explored the survival and apoptosis of hepatoma cells by replacing glucose deficiency with 2-deoxy-D-glucose and combining with metformin,and further explored the mechanism of apoptosis,in order to provide a new target for the direction of prevention and treatment of HCC patients.Chapter one Effect of metformin on proliferation and apoptosis of hepatocellular carcinoma cells under glucose deprivationObjective: To explore the effect and mechanism of metformin on proliferation and apoptosis of hepatocellular carcinoma cells under glucose deprivation.Methods: 1.Hep G2,Hep3 B,HCCM,Huh7 and PLC/5 cells were cultured in the full medium,serum-free medium and sugar-free medium.The proliferation rate of HCC cells was observed by Wst-1 assay.2.The apoptosis rate of 48 h treatment was detected by flow cytometry in full medium,glutamine and glucose-free mudium,glutamine-supplemented medium,glucose-supplemented medium or both.3.Metformin was added into the culture medium.There were four groups,the Ctrl group,Metformin group,glucose deprivation group and metformin plus glucose deprivation group.Huh7 and Hep G2 were cultured respectively and the morphology change of cell were observed after 24 h and48h,and cell survival by flow cytometry.5 Western blotting to analyze the protein expressions of Caspase 3,PARP,Mcl-1,LC3 and p-AKT of Huh7 cells.Results: 1.HCC cells were cultured in glucose deprivation,full medium and serum-free medium.The survival rate decreased gradually with the increase of time.The changes of HCCM and Hep G2 cells were most obvious.2.The sub G1 apoptosis rate of Hep G2 cells cultured in glucose-double free and glutamine-double free medium,or supplemented with glutamine(Gln)were(94±0.50)% and(74.80±1.92)%,and compared with the sub G1 apoptosis rate of Hep G2 cells cultured in full medium(4.40±0.21)%,supplemented with glucose(11.00±0.21)% and both(7.20±0.29)%.The difference was statistically significant(P<0.05);While the sub G1 apoptosis rate of Hep3 B,Huh7 and PLC/5 cells were no significant differences(P>0.05).3.Microscopy observation showed that combined treatment of Met and glucose deprivation caused less viable adherent cells of Huh7 after 48 h,but more floating dead cells.4.The death rate of Hep G2 cells in Met and glucose deprivation was(52.21±0.29)%,when compared with glucose free group(12.45±0.71)%,metformin group(11.53±0.25)% and full medium group(7.72±0.42)%.The difference was statistically significant(P<0.05).The death rate of Huh7 cells in Met and glucose deprivation was(74.97±0.21)%,when compared with glucose free group(32.69±0.89)%,metformin group(11.98±0.29)% and full medium group(7.74±0.23)%.The difference was statistically significant(P<0.05);The death rate of Huh7 cells in glucose free medium was(32.69±0.89)%,when compared with metformin group(11.98±0.29)% and full medium group(7.74±0.23)%.The difference was statistically significant(P<0.05).5.Western blotting indicated that the Metformin plus glucose free group had Caspase-3 activation and the phosphorylation of AKT,PARP cleavage,reduction of the expression of Mcl-1 and the autophagy related protein LC3 B.Conclusion: 1.Glucose deprivation can inhibit the proliferation of hepatocellular carcinoma Hep G2 and HCCM cells,promote cell apoptosis.2.The Hep G2 and HCCM cells are more sensitive to glucose deprivation,but Hep3 B,Huh7 and PLC/5 cells are tolerant.3.Metformin could promote the apoptosis of Huh7 cells in glucose deprivation.4.Metformin can further promote the apoptosis of Huh7 cells in glucose deprivation,which may be related to AKT phosphorylation and autophagy.Chapter two Effect of Metformin combined with 2-deoxy-D-glucose on proliferation and apoptosis in liver cancer cellsObjective: To investigate the effect and mechanism of metformin combined with glucose antagonist 2-deoxy-D-glucose on proliferation and apoptosis in hepatocellular carcinoma cells.Methods: 1.Wst-1 assay was used to observe the survival of HCC cells after48 htreatment with different concentrations of metformin and 2-deoxy-D-glucose.2.Wst-1 reagent was used to determine the anti-proliferation effects after treatments with Metformin,2-deoxy-D-glucose alone or combined in Hep G2 and Hep3B cells after 48 h.3.Microscopy was used to observe cell morphological changes after treatments with Met,2DG alone or combined in Hep G2 and Hep3 B cell.4.The cell sub G1 apoptosis was observed by flow cytometry after treatment of different drugs;5.Western blotting analyzed the protein expressions of Caspase 3,PARP,Mcl-1 in Hep G2 cell.Results: 1.The proliferation rate of Hep G2,HCCM,Hep3 B and PLC/5 cells decreased with the increased concentrations of Metformin and2-deoxy-D-glucose.2.The survival rate of Hep G2 cells in the combination group was(22.48±0.51)%,when compared with the full medium group(100.00±5.05)%,Metformin group(80.68±5.10)% and 2-deoxy-D-glucose group(72.56±4.34)%,respectively.The differences were statistical significant(P<0.05).The survival rate of Hep3 B cells in the combination group was(29.16±1.34)%,when compared with the full medium group(100.00±1.23)%,Metformin group(59.58±1.92)% and 2-deoxy-D-glucose group(33.87±1.95)%,respectively.The difference was statistically significant(P<0.05).3.Microscopy observation showed that combined treatment of Metformin and 2-deoxy-D-glucose caused less viable adherent cells of Hep G2,but more floating dead cells.While the combination group also caused a decrease in the density of Hep3 B cells,but did not significantly increase the shedding of cells.4.The apoptosis of Hep G2 cells in combination group was(39.63±0.21)%,when compared with the full medium group(7.12±0.14)%,Metformin group(12.56±0.35)% and 2-deoxy-D-glucose group(15.16±1.93)%,respectively.The differences were statistically significant(P<0.05).The apoptosis of Hep3 B cells in combination group was(12.58±1.03)%,when compared with the full medium group(2.82±0.51)% and Metformin treatment group(8.98±0.86)%,respectively.The differences were statistically significant(P<0.05).The combination group compared with the treatment group(12.40±1.78)%.The difference was not statistically significant(P>0.05).5.Western blotting method demonstrated that the combined treatment induced evident Caspase-3 activation and PARP cleavages,and decreased expression of Mcl-1.Conclusion: 1.The combination of Metformin and 2-deoxy-D-glucose can effectively inhibit liver cancer cell proliferation of Hep G2,Hep3 B and induce apoptosis in Hep G2 cells.2.The mechanism may be involved with Caspase-3activation,PARP cleavage and reduction of Mcl-1 protein.