Synergistic Effect of Supercritical CO Extract of Mango Ginger (Curcuma amada- 2Roxb.) With Glycolytic Inhibitors in Human Glioblastoma Cells In Vitro

Abstract

Background: Cancer cells, unlike normal cells, principally use aerobic glycolysis with reduced mitochondrial oxidative phosphorylation for glucose metabolism, a phenomenon referred to as the Warburg effect. Glioblastoma, one of the most aggressive, lethal and incurable human tumors with a survival rate of 12-15 months in patients undergoing standard of care treatment involving surgery, chemotherapy and radiation therapy, has been shown to have a preferential metabolism of glucose
through aerobic glycolysis. The cytotoxic effects of a previously described supercritical CO extract of 2 mango ginger (Curcuma amada-CA) with two glycolytic inhibitors [2-deoxy-D-glucose (2-DG) and sodium oxamate (SO)] was investigated in the U-87MG human glioblastoma cell line. Methods: Cytotoxicity assay was performed with increasing concentrations of CA, 2-DG and SO as single agents and in combinations in U-87MG glioblastoma cells by the MTT assay. The cytotoxicity data was analyzed using CompuSyn software to determine the synergism/additive effect/antagonism between drugs. The effect of CA and glycolytic inhibitors on ATP and lactate synthesis was analyzed to establish the inhibitory effects of individual drugs as well as their combinations on glycolysis pathway. The modulatory effect of CA, 2-DG and SO as single agents or combinations on mRNA and protein expression of apoptotic and metastasis genes were also analyzed by RT-PCR and western blot hybridization, respectively. Results: The hexokinase inhibitor 2-DG and the lactate dehydrogenase-A inhibitor SO, both inhibiting the glycolysis pathway, showed synergistic cytotoxic effects with CA in the glioblastoma cell line with combination index values of <1 in the CompuSyn analysis. CA inhibits cellular ATP synthesis in a dosedependent manner and it has better inhibition profile than 2-DG and SO. CA inhibits cellular lactate synthesis significantly better than 2-DG and SO at low concentrations, and CA+2-DG combination appears to be better than single agents at low doses for lactate inhibition in glioblastoma cells. Gene expression analysis by RT-PCR and western blot hybridization showed that CA, 2-DG and SO as well as their combinations up regulate the ratio of Bax/Bcl-2, p21, TIMP1 and caspase-3 expression and down regulate mutant p53 and MMP2 expression that may increase apoptosis and inhibit cell proliferation as well as metastasis of tumor cells. Conclusion: The combination of CA with glycolytic inhibitors like 2-DG and SO is beneficial for inhibition of growth, proliferation and migration of glioblastoma cells. These in vitro results support the rationale for conducting in vivo studies combining CA with 2-DG and SO in human glioblastomas