Figure 4.

TG02 decreases intracellular ATP levels and suppresses glycolysis as a single agent and in combination with TMZ. A–C, TG02 and TMZ inhibit glycolysis. A, ECAR measurements of U251 cells that were treated with TG02, TMZ, and TG02/TMZ. B, Basal glycolysis (a) and maximal capacity of glycolysis (b) were calculated. C, TG02/TMZ downregulates key enzymes in glycolytic pathway. Western blots of HK2, PKM2, and LDHA in GSC923 and GSC827 cells. D, Intracelluar ATP levels were decreased in TG02 and TG02/TMZ-treated GSC923 cells. ATP levels were determined following TG02, TMZ, and TG02/TMZ treatment. E, Schematic illustrating the mechanisms of the synergism between TG02 and TMZ. TG02 decreased phosphorylation of CDK9 and inhibited RNA Pol II–mediated transcriptional regulation of antiapoptotic proteins, including Mcl-1 and Survivin (also known as BIRC5 gene), leading to release of Cyt c, which induced mitochondrial-mediated apoptosis in GBM cells (shown in red). On the other hand, TG02 also caused disruption of the mitochondrial respiration complexes and inhibition of the glycolysis, resulting in ATP depletion. TMZ induced DNA damage, effectively reducing the level of nicotinamide adenine dinucleotide (NAD⁺) through PARP activity, and thus suppressed glycolysis to reduce energy production in the cells (shown in blue). The combination of TG02/TMZ treatment downregulated protein expressions of HK2, PKM2, and LDHA to decrease ATP production. The synergism between TG02 and TMZ is partially due to suppressed glycolysis and inhibited ATP generation (shown in purple). As a consequence of disruption of ATP production by combination of TG02/TMZ, the cells undergo apoptosis and necrosis. EC, elongation complex. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.