Research Paper Volume 12, Issue 17 pp 17582—17600

Figure 5. Metformin regulated Nrf2-dependent transcription and cisplatin sensitivity through glucose metabolism and glycolysis in HepG2/DDP cells. (A, B) Metformin and glucose had no additive effect on Nrf2-dependent transcription. HepG2/DDP cells in normal RPMI (2g/L) were shifted to normal or high glucose (8g/L) medium supplemented with or without 1mM metformin for 24 hours. Total mRNA was extracted and relative expression of indicated gene was quantified by RT-qPCR. Data from 2 independent experiments (3 replicates) were normalized to non-treated controls and statistically analyzed by student’s t-test (ns, not significant, *P<0.05, **P<0.001). (C) Metformin and glucose had no additive effect on cisplatin toxicity. HepG2/DDP cells cultured and treated with glucose and metformin as in (B). After cisplatin (10ug/ml) treatment for another 24 hours, relative cell viability was measured by Cell Titer-Glo. Data from 2 experiments with 3 replicates were plotted and analyzed by student’s t-test (** P<0.001, ns, not significant). (D–E) Inhibition of glycolysis with 2-DG (20mM) attenuated Metformin’s effect on repressing Nrf2-dependent target gene expression. HepG2/DDP cells under normal glucose condition were treated with or without 1mM metformin and 20mM 2-DG for 24 hours. Relative expression of indicated genes was quantified by RT-qPCR for 2 independent times (3 replicates each time). Right panels are normalized data to compare the effect of 2-DG. Student’s t-test was used to test statistical significance (*P<0.05, **P<0.001, ***P<0.0001). (F) Glycolysis inhibition by 2-DG attenuated metformin’s effect on increasing cisplatin toxicity. HepG2/DDP cells were cultured and treated with metformin as in (E) then cisplatin (10ug/ml) was added for 24 hours. Relative cell viability was measured by Cell Titer-Glo. Data from 2 experiments of 3 replicates were plotted and analyzed by student’s t-test (*P<0.05, ***P<0.0001, ns, not significant).