Research Paper Volume 12, Issue 17 pp 17582—17600

Figure 2. Metformin increased cisplatin sensitivity of HepG2/DDP through down-regulation of Nrf2-dependent transcription. (A) Metformin sensitized HepG2/DDP cells to cisplatin. HepG2/DDP and HepG2 cells were treated with metformin at various concentrations and cell survival was measured by Cell Titer-Glo. Relative percentage of survival after 10 ug/mL cisplatin treatment for 24 hours was plotted. Difference between cisplatin-treated HepG2/DDP and HepG2 was tested by student’s t-test (** P<0.001). (B) Data from (A) were normalized to cisplatin non-treated controls and shown in bar plot. Metformin’s effect on HepG2/DDP and HepG2 was tested by student’s t-test (** P<0.001). (C) Metformin treatment did not affect Nrf2 protein levels. HepG2/DDP cells were treated with or without 1mM metformin for 24 hours and total cell lysates were subjected to Western blotting. Culture #1 and #2 were different cell clones. Representative images of were shown. Quantification of N= 4 biological repeats were shown in bar graph. (D) Metformin repressed Nrf2 target genes expression. HepG2/DDP cells were treated with or without 1mM metformin for 24 hours and mRNA was isolated and reversed transcribed. RT-qPCR were carried out with HO-1 and GCLM specific primers. For each gene, data of cisplatin-treated samples were normalized to that of non-treated controls. Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (E) Metformin and Nrf2(siRNA) had no additive effect on increasing cisplatin toxicity. HepG2/DDP and HepG2 cells were transfected with Nrf2-specific siRNAs for 24 hours then treated metformin for 24 hours. 10ug/ml cisplatin were added for another 24 hours and relative cell viability was measured with Cell Titer-Glo. Data from 2 independent experiments were normalized to the average of non-treated controls. Significance was tested by student’s t-test (ns, not significant, * P<0.01, ** P<0.001). (F) Nrf2 activation prevented metformin from increasing cisplatin toxicity. HepG2/DDP cells were treated as in (E) except KEAP1-specific siRNA was transfected. Data from 4 independent experiments were normalized to the average of metformin non-treated control. Significance was tested by student’s t-test (ns, not significant, ** P<0.001).