Dichloroacetate Induces Apoptosis And Enhances Cytotoxicity Of Adriamycin In Hepatoma Cells Via Elevated Intracellular ROS Levels

Part1Dichloroacetate induces apoptosis in hepatoma cell linesvia elevated intracellular ROS levelsOBJECTIVE: To investigate the effects of dichloroacetate(DCA) onthe glucose metabolism, cell viability in hepatoma cells anddetermine whether DCA induces apoptosis in hepatoma cells.METHODS: A panel of hepatoma cell lines was treated with DCAand analyzed for apoptosis via flow cytometry. Biologicalcorrelates such as glucose uptake, lactate production, ROSproduction, and gene expression were examined to assessapoptotic mechanism.RESULTS: DCA significantly inhibited the glucose uptake andlactate production, and elevated the intracellular ROS levels aswell in hepatoma cells, while sparing normal liver cells. DCA alsoinhibited the cell viability and induced apoptosis in hepatoma cells,while sparing normal liver cell lines. Simultaneous treatment with the thiol antioxidant N-acetylcysteine (NAC) inhibited theelevated ROS levels, as well as protected hepatoma cells from thecytotoxic effects of DCA. Simultaneous treatment withL-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of glutathionesynthesis, sensitized hepatoma cells to the cytotoxic effects of DCA.DCA treatment increased p53up-regulated modulator ofapoptosis (PUMA) transcripts in HCC-LM3cell lines, suggestinginvolvement of a PUMA-mediated mechanism. Western blot assayof caspase-9and caspase-3suggests DCA induces apoptosis inhepatoma cells via a caspase-dependent mitochondrial pathway.CONCLUSION: DCA effectively sensitizes hepatoma cell lines toapoptosis via elevating intracellular ROS levels in hepatoma cells.Part2Dichloroacetate enhances the cytotoxicity ofAdriamycin in hepatoma cell line in vitroOBJECTIVE: To investigate whether dichloroacetate (DCA)combined with Adriamycin (ADM) can enhance cytotoxicity inhuman hepatoma cells.METHODS: Two human hepatoma cell lines HCC-LM3,SMMC-7721, and a LO2normal hepatocyte cell line were treatedwith DCA,ADM alone, or a combination of DCA and ADM. Thecell viability was determined with Cell Counting Kit-8assay, apoptosis was analyzed via flow cytometry after Annexin-V/PIstaining. Intracellular ROS levels were detected usingCM-H2DCFDA.RESULTS: Exposure of hepatoma cells to the combination of DCAand ADM resulted in a significant decrease in cell viability andincrease of apoptotic cells when compared with DCA or ADMalone. Treatment with DCA+ADM also caused significant increaseof ROS levels in hepatoma cells as compared with DCA or ADMalone. Simultaneous treatment with the10mmol/L NAC inhibitedthe elevated ROS levels, as well as protected hepatoma cells fromthe cytotoxic effects of DCA and ADM.1mmol/L BSO sensitizedhepatoma cells to the cytotoxic effects of DCA and ADM.Treatment with DCA and ADM never caused significant increaseof cytotoxicy in normal hepatocyte. Immunoblotting performed formitochondrial respiratory chain complex found that DCAtreatment down-regulated complex III, suggested that DCA mayinduce induce ROS generation through complex III of the ETC.CONCLUSION: Our data suggest that DCA enhances thecytotoxicity of ADM in hepatoma cell line via elevatedintracellular ROS levels. Part3Dichloroacetate enhances the cytotoxicity ofAdriamycin in hepatoma in vivoOBJECTIVE: To investigate whether dichloroacetate (DCA)combined with Adriamycin (ADM) may enhance cytotoxicity innude mouse xenograft models of human hepatoma.METHODS: Nude mice implanted with HCC-LM3hepatoma cellswere divided into four groups as follows:(a) untreated controls;(b)mice treated with ADM alone;(c) mice treated with DCA alone; or(d) mice treated with a combination of DCA+ADM. Treatmentbegan when tumors were either200mm3in volume.RESULTS: The DCA+ADM combination treatment resulted insignificantly slower tumor growth and significantly less tumorweight than the control, DCA, or ADM treatments (P<0.05).CONCLUSION: DCA combined with ADM may enhancecytotoxicity in nude mouse xenograft models of human hepatoma.