Resistance index is used to evaluate the drug resistance of drug-resistant cancer cells to chemotherapy drugs. In order to verify SW1990/Gem and Panc-1/Gem cells were gemcitabine-resistant, MTT assay was performed, and resistance index was calculated. As shown in Fig. 1A-B and Table 2, the IC50 values of gemcitabine in SW1990/Gem and Panc-1/Gem cells increased dramatically compared with SW1990 and Panc-1 cells. RI in SW1990/Gem and Panc-1/Gem cells were 57.04 and 37.75 respectively, indicating that the SW1990/Gem and Panc-1/Gem cells were gemcitabine-resistant. Moreover, we found that Nrf2, NQO1, HO-1, GCLC, ABCC1 and ABCC5 protein levels were markedly increased in SW1990/Gem and Panc-1/Gem cells (Fig. 1C–D and Supplementary Fig. S1A–B). In addition, Nrf2 target genes encoding antioxidant enzymes, enzymes of glutathione synthesis and conjugation, drug metabolizing enzymes and transporters, metabolic enzymes were also highly expressed in SW1990/Gem and Panc-1/Gem cells (Fig. 1E–F). We also investigated whether SW1990/Gem and Panc-1/Gem cells were resistant to other anticancer agents including etoposide, paclitaxel, cisplatin, 5-FU, ara-C and doxorubicin. As shown in Table 2, the IC50 values of etoposide, paclitaxel, cisplatin, 5-FU, ara-C and doxorubicin in SW1990/Gem and Panc-1/Gem cells markedly increased compared with their parental cells. The RI of etoposide, paclitaxel, cisplatin, 5-FU, ara-C and doxorubicin were 5.32, 8.41, 3.19, 5.56, 11.67 and 6.4 in SW1990/Gem cells, and the RI of etoposide, paclitaxel, cisplatin, 5-FU, ara-C and doxorubicin were 4.28, 6.58, 2.42, 4.49, 9.65 and 5.06 in Panc-1/Gem cells, respectively. These results demonstrated that gemcitabine-resistant pancreatic cancer cell lines also exhibited chemoresistance to other anticancer agents.
Fig. 1 Nrf2 signaling was upregulated in SW1990/Gem and Panc-1/Gem cells. (A–B) The cytotoxicity of gemcitabine to SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (C–D) Western blot was used to detect the protein level of Nrf2, NQO1, HO-1, and GCLC in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. (E–F) The expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. The colors of the heatmap reflect log2-expression levels of Nrf2 target genes in SW1990, SW1990/Gem, Panc-1 and Panc-1/Gem cells. Data were expressed as mean ± SD, and the results were representative of three independent experiments. Significant differences were indicated as ***P < 0.001 vs. SW1990 or Panc-1 cells.
Table 2 Determination of IC50 of different anticancer drugs.
Group SW1990 SW1990/Gem RI Panc-1 Panc-1/Gem RI
Gemcitabine (μM) 9.33 ± 0.9 532.15 ± 26.13 57.04 7.85 ± 0.56 296.34 ± 22.17 37.75
Etoposide (μM) 117.46 ± 14.02 624.52 ± 44.89 5.32 102.7 ± 9.35 439.24 ± 37.58 4.28
Paclitaxel (nM) 35.43 ± 4.34 298.15 ± 37.68 8.41 32.67 ± 2.97 214.82 ± 16.45 6.58
Cisplatin (μM) 58.74 ± 4.82 187.23 ± 21.09 3.19 65.38 ± 7.74 158.39 ± 13.29 2.42
5-FU (μM) 33.05 ± 2.85 183.68 ± 8.4 5.56 39.14 ± 1.99 175.74 ± 11.55 4.49
Ara-C (μM) 1.38 ± 0.23 16.11 ± 1.43 11.67 1.21 ± 0.09 11.68 ± 0.7 9.65
Doxorubicin (μM) 4.35 ± 0.59 27.85 ± 1.89 6.4 4.66 ± 0.29 23.59 ± 1.64 5.06
IC50 values of different anticancer drugs were detected by using MTT assay. Data were expressed as mean ± SD of three independent experiments.