The Novel Synergetic Combination Of Chemotherapy Based On Metformin Induced Selective Anti-proliferation Effects Against Hepatoma Cells

Part I Metformin synergetic with doxorubicin induced anti-proliferation effect on human hepatoma cellsObjective: To study whether metformin can enhance the cytotoxicity of classical chemotherapy agent,doxorubicin towards human hepatoma cells,and providing scientific proof of such combination protocols.Methods: Human hepatoma cells Hep G2 and SMMC7721,human normal liver cells L-02 were exposed to different dosage of medications for 24 hrs as a mimic of chemotherapy in vitro.SRB assay and Brd U ELISA were usage to determine the cell viability after designed time episodes.Alkaline single-cell gel electrophoresis was introduced to determine the DNA damage level.Western blot was used to determine the expression level of poly ADP-ribose polymerase 1(PARP1).Other measurement was including the intracellular ATP,reactive oxygen species and NAD(H).Results: Metformin treatment alone showed the mild selective cytotoxicity between hepatoma and normal liver cells.The inhibitory concentration 50%(IC50)was significantly different among three tested cell lines(p<0.05),i.e.27.53±2.91 m M for L-02 cells,25.94±1.76 m M for SMMC7721 cells and 21.10±1.08 m M for Hep G2 cells,respectively.Besides,metformin could enhance the cytotoxicity of classical chemotherapy agent doxorubicin in human hepatoma cell lines(Hep G2 and SMMC7721).Metformin and doxorubicin combined treatment significantly inhibited hepatoma cell proliferation,while inducing relatively modest impacts on normal liver cells(p<0.05).The underlying mechanisms may attribute to higher expression level of poly ADP-ribose polymerase 1(PARP1),leading to decreased intracellular NAD+/NADH ratio(p<0.05).Targeted inhibiting PARP1 and adding NAD+ biosynthesis intermediate nicotinamide mononucleotide restored the NAD+ level and impaired the anti-proliferation of metformin and doxorubicin combined treatment,while the NAD+/NADH ratio further reduced in the presence of specific inhibitor of nicotinamide phosphoribosyl transferase,enhancing the cellular anti-proliferation that metformin and doxorubicin had towards hepatoma cells.Conclusions: Metformin synergizing with doxorubicin could be a promising chemotherapy in treating hepatocellular carcinoma.The combination protocol may lead to increased ROS and PARP1 levels,reduced intracellular NAD+/NADH ratio and ATP content,which caused the uncoupled process of NAD+ metabolism and redox balance.Part II Metformin synergetic with ascorbate induced anti-proliferation effect on human hepatoma cellsObjective: To study whether metformin combined with ascorbate could be a novel protocol in chemotherapy practice,and to explore the underlying mechanisms in this research context.Methods: Several human neoplasm and normal cell lines were exposed to different dosage of medications for 24 hrs as a mimic of chemotherapy in vitro.SRB assay and colonial formation assay were usage to determine the cell viability after designed time episodes.The contents of intracellular hydrogen peroxide were determined as a reflection of hydrogen peroxide production during the mentioned medications;The enzymatic activity of catalase and clearance of hydrogen peroxide were also determined as represented progress of hydrogen peroxide clearance.The transcriptive effect of antioxidant elements within the nuclear was determined by dual luciferase reporter assay.Western blot was introduced to detect the expression level of Nrf2(both cytosol and nuclear),FOXO3a(phosphorylation and not).Results: Ascorbate treatment,in our study context,i.e.under the pharmacological dosage ascorbate,induced mild selective cytotoxicity between neoplastic cells and normal cells.Besides,metformin could synergize the cytotoxicity of pharmacological ascorbate inducing more damage towards human neoplastic cells(p<0.05).Metformin and pharmacological dosage ascorbate combined treatment significantly increasing intracellular hydrogen peroxide levels in hepatoma cells,while relatively reduced activity of catalase was found during this progress.Metformin and pharmacological dosage ascorbate co-treatment also decreased the glucose uptake capacity and G6 PDH of hepatoma cells and human pancreatic cancer cells.The lower luciferase and Nrf2 expression level was found,along with lower expressing levels of phosphorylated FOXO3 a.Conclusions: Metformin synergizing with ascorbate could be a promising novel chemotherapy in treating various cancer types which never reported by other research groups,may guide the further application research in the near future.The combination protocol may lead to increased hydrogen peroxide and inhibiting the intrinsic hydrogen peroxide clearance pathway in cancer cells.