The Impact of NRF2 Inhibition on Drug-Induced Colon Cancer Cell Death and p53 Activity: A Pilot Study

Abstract

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) protein is the master regulator of oxidative stress, which is at the basis of various chronic diseases including cancer. Hyperactivation of NRF2 in already established cancers can promote cell proliferation and resistance to therapies, such as in colorectal cancer (CRC), one of the most lethal and prevalent malignancies in industrialized countries with limited patient overall survival due to its escape mechanisms in both chemo- and targeted therapies. In this study, we generated stable NRF2 knockout colon cancer cells (NRF2-Cas9) to investigate the cell response to chemotherapeutic drugs with regard to p53 oncosuppressor, whose inhibition we previously showed to correlate with NRF2 pathway activation. Here, we found that NRF2 activation by sulforaphane (SFN) reduced cisplatin (CDDP)-induced cell death only in NRF2-proficient cells (NRF2-ctr) compared to NRF2-Cas9 cells. Mechanistically, we found that NRF2 activation protected NRF2-ctr cells from the drug-induced DNA damage and the apoptotic function of the unfolded protein response (UPR), in correlation with reduction of p53 activity, effects that were not observed in NRF2-Cas9 cells. Finally, we found that ZnCl₂ supplementation rescued the cisplatin cytotoxic effects, as it impaired NRF2 activation, restoring p53 activity. These findings highlight NRF2's key role in neutralizing the cytotoxic effects of chemotherapeutic drugs in correlation with reduced DNA damage and p53 activity. They also suggest that NRF2 inhibition could be a useful strategy for efficient anticancer chemotherapy and support the use of ZnCl₂ to inhibit NRF2 pathway in combination therapies.

Keywords: CHOP; DNA damage; NRF2; TP53; ZnCl2 supplementation; apoptosis; colorectal carcinoma; sulforaphane; unfolded protein response (UPR).

Figure 1

Sulforaphane (SFN) induces NRF2 protein levels and reduces drug-induced cell death. (a) HCT116 and RKO cells were exposed to increasing doses of SFN for 24 h, and cell viability was assessed by XTT assay. The histograms represent the mean plus S.D. from three independent experiments. (b) Cells treated as in (a) were analyzed by Western blot for NRF2 expression levels. Actin was used as protein loading control. The ratio of NRF2 levels vs. β-actin, following densitometric analysis using ImageJ software, is shown. (c) In the upper panel, HCT116 and RKO cell viability was measured by XTT assay at 492 nM and cell death was measured by Trypan blue staining after treatment with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The results are expressed as cell death percentage ± S.D. In the lower panels, the expression levels of PARP cleavage (cl.) were assessed by Western blot. Actin was used as protein loading control and the ratio of cl.PARP vs. β-actin, is reported. (d) Cell viability, as measured by Trypan blue staining, of HCT116-p53^−/− cells treated as in (c) The results are expressed as cell death percentage ± S.D. * p ≤ 0.01.

Figure 2

NRF2 is involved in SFN-induced inhibition of CDDP cytotoxicity. (a) NRF2-proficient (NRF2-ctr) and NRF2-KO (NRF2-Cas9) cells were treated with SFN (2 µM) for 8 h and NRF2 and HO-1 protein levels analyzed by Western blot. Actin was used as protein loading control. Densitometric analysis of NRF2/β-actin and HO-1/β-actin is reported in the right panels. * p ≤ 0.01. (b) NRF2-ctr and NRF2-Cas9 cell proliferation (left panel) was measured by XTT assay and cell viability (right panel) was measured by Trypan blue staining after treatment with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The results are expressed as cell death percentage ± S.D. * p ≤ 0.01.

Figure 3

NRF2 inhibition restores CDDP-induced p53 activity impaired by SFN. (a) NRF2-ctr and NRF2-Cas9 cells were treated with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h before assessing the phospho (p) Ser46, p53 and HO-1 levels by Western blot. Actin was used as protein-loading control. Densitometric analysis of pSer46/p53, p53/β-actin and HO-1/β-actin is reported in the right panels. * p ≤ 0.01. (b) Total mRNA was extracted from NRF2-ctr and NRF2-Cas9 cells untreated or treated as in (a). The indicated gene expression was assayed by semiquantitative RT-PCR. The histograms represent the mean plus S.D. of three independent experiments. Densitometric analysis using ImageJ software was applied to calculate the gene/28S ratio. * p ≤ 0.01.

Figure 4

NRF2 activation impairs the CDDP-induced DNA damage. NRF2-ctr and NRF2-Cas9 cells were treated with CDDP (5 µg/mL) alone or in combination with SFN (2 µM) for 24 h. The indicated proteins’ expression was analyzed by Western blot and the densitometric analyses reported in the right panels with S.D. Actin was used as protein loading control. * p ≤ 0.01.

Figure 5

ZnCl₂ supplementation enhances DNA damage and rescues p53 activity, inhibited by SFN. (a) HCT116 and RKO cells were treated with CDDP (5 µg/mL) alone or in combination with SFN (2 µM) and ZnCl₂ (100 μM). The expression level of the indicated proteins was analyzed by Western blot. Actin was used as protein loading control. Densitometric analyses was performed and reported as histogram in the right panels, plus S.D. * p ≤ 0.01. (b) RT-PCR analysis of total mRNA extracted from HCT116 cells treated as in (a). Densitometric analysis using ImageJ software was applied to calculate the PUMA/28S ratio. (c) HCT116 and RKO cell viability was measured by Trypan blue staining after treatment with cisplatin (CDDP) (5 µg/mL) alone or in combination with SFN (2 µM) and ZnCl₂ (100 μM) for 24 h. The results are expressed as cell death percentage ± S.D. * p ≤ 0.01.

Figure 6

Proposed model for NRF2 role in cancer cell chemosensitivity and p53 activity. Hyperactivation of NRF2 (by SFN) counteracts the CDDP-induced DNA damage, impairing the p53 activity and reducing cell death; ZnCl₂ supplementation counteracts the effect of SFN/NRF2 rescuing the DNA damage, p53 activity, and cell death, induced by CDDP.