Research Paper Volume 12, Issue 17 pp 17582—17600

Figure 1. Nrf2 activation contributes to cisplatin-resistance of HepG2/DDP cells. (A) Nrf2 protein levels were increased in hepatocellular carcinoma tissues from patients undergone cisplatin treatment. Sections of paraffin-embedded hepatocellular carcinoma tissues were stained with a Nrf2-specific antibody in immunochemistry (IHC) experiments. Representative images were shown. Scale bars are 20 μm. (B) Quantifying Nrf2 expression in tumor tissues from 16 patients (8 undergone cisplatin treatment). Scores were obtained by pathologists according to hospital protocols. Statistical analysis by student’s t-test showed significant difference (*, P<0.05). (C) Nrf2 protein levels were elevated in cisplatin-resistant HepG2/DDP cells. Cell lysates of HepG2 and cisplatin-resistant HepG2/DDP from 2 different cultures were subjected to Western blot analysis with Nrf2 and Tubulin antibodies separately. Shown are samples from two different cultures. Representative images of were shown. Quantification of N= 2 biological repeats were shown in bar graph. (D) Nrf2 target gene expression was upregulated in HepG2/DDP cells. mRNA was isolated from HepG2 and HepG2/DDP and the relative mRNA levels of Nrf2 target genes (NQO1, GCLC, GCLM and HO-1) was compared by RT-qPCR. Relative expression of each gene as compared to that in HepG2 cells in fold change. Significance was tested by student’s t-test (** P<0.001, *** P<0.0001). (E, F) Nrf2 knockdown repressed target genes expression in HepG2 and HepG2/DDP cells. Nrf2 was knocked down by transfecting cells with siRNA pools specific to Nrf2 gene for 48 hours. mRNA was isolated and RT-qPCR was conducted with specific primers for both Nrf2 gene and its target genes (HO-1 and GCLM). For each gene, data were normalized to non-transfected controls (Ctrl). Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (G) Nrf2 knockdown sensitized HepG2/DDP cells to cisplatin. HepG2/DDP and HepG2 cells were transfected with siRNAs specific to Nrf2 gene for 48 hours and cells were treated with cisplatin at indicated concentrations for 24 hours. Cell survival was measured with Cell Titer-Glo reagent. Data from 3 independent experiments was normalized to the average of non-treated controls. Significance was tested by student’s t-test (* P<0.05, ** P<0.001). (H) Survival fold change by Nrf2 knockdown. Data in (G) were used to calculate the fold change caused by Nrf2 siRNA knockdown at each cisplatin concentration. Significance was tested by student’s t-test (* P<0.05, ns, not significant).