Overexpression of Nrf2 attenuates Carmustine-induced cytotoxicity in U87MG human glioma cells

Abstract

Background: Malignant glioma is one of the most devastating tumors in adults with poor patient prognosis. Notably, glioma often exhibits resistance to conventional chemotherapeutic approaches, complicating patient treatments. However, the molecular mediators involved in tumor chemoresistance remain poorly defined, creating a barrier to the successful management of glioma. In the present study, we hypothesized that the antioxidant transcription factor, Nrf2 (nuclear factor erythroid-derived 2 like 2), attenuates glioma cytotoxicity to Carmustine (BCNU), a widely used chemotherapeutic agent known to modulate cellular oxidative balance.

Methods: To test the hypothesis, we employed human malignant glioma cell line, U87MG and overexpression of Nrf2 in glioma cells was achieved using both pharmacological and genetic approaches.

Results: Notably, induction of Nrf2 was associated with increased expression of heme oxygenase-1 (HO-1), a stress inducible enzyme involved in anti-oxidant defense. In addition, over expression of Nrf2 in U87MG cells significantly attenuated the cytotoxicity of Carmustine as evidenced by both cellular viability assay and flow cytometry analysis. Consistent with this, antioxidants such as glutathione and N-acetyl cysteine significantly reduced Carmustine mediated glioma cytotoxicity.

Conclusions: Taken together, these data strongly implicate an unexplored role of Nrf2 in glioma resistance to Carmustine and raise the possible use of Nrf2 inhibitors as adjunct to Carmustine for the treatment of malignant glioma.

Figure 1

TBHQ and Nrf2 upregulation. U87MG cells were treated with either vehicle or TBHQ for 6 h and the induction of Nrf2 (MW: ~102 kDa) was quantified using (A) western blotting followed by (B) densitometry analysis. The immunocytochemistry analysis followed by (C) confocal imaging further confirmed induction and nuclear translocation of Nrf2 in glioma cells by TBHQ (scale bar = 50 μm). TBHQ treatment of glioma cells also resulted in the induction of HO-1 (MW : ~31 kDa), one of the Nrf2 regulated molecular targets, as evidenced by (D) western blotting followed by (E) densitometry analysis. Densitometry is expressed as the mean ± SEM from three independent trials and data were analyzed using One-way ANOVA followed by Dunnett’s post-hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. vehicle-treated cultures).

Figure 2

TBHQ and glioma cell proliferation. U87MG cells were treated with either vehicle or TBHQ for 24 h and the cellular proliferation was assessed using (A) BrdU incorporation assay and (B) MTT assay as described in Methods. Data are representative of at least three independent trials (n = 3/trial) and are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001 vs. vehicle-treated cultures.

Figure 3

TBHQ and resistance to Carmustine. U87MG cells were treated with either vehicle or TBHQ (30 μM) for 6 h. After respective treatment, the media were removed, cells were replenished with media containing either vehicle or Carmustine at indicated concentrations and incubated for 18 h. The cell viability was measured using (A) MTT reduction assay and (B) flow cytometry analysis. Data from MTT Assay are representative of three independent experiments and are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001 vs. vehicle treated cells. For flow cytometry analysis, the cells after treatments were collected and stained with 7-AAD (y-axis) a marker of cell death and Annexin V (x –axis), a marker of early apoptotic cell death. The percentages of viable cells (C) which are both Annexin V/7-AAD negative are shown. Data are representative of two independent experiments and are expressed as mean ± SEM. *** p < 0.001 vs. Carmustine alone treated cells.

Figure 4

Nrf2 and resistance to Carmustine. U87MG cells were stably transduced with the Precision LentiORF lentiviral particles to accomplish Nrf2 (MW: ~102 kDa) overexpression evidenced by (A) western blotting. The genetic over expression of Nrf2 resulted in increased Nrf2 transcriptional activity as assessed by (B) TransAM Elisa and augmented expression of HO-1 as assessed by western blotting (C). The confocal image (D) illustrates U87MG cells overexpressed with Red Fluoresent protein (RFP) that served as experimental control (scale bar = 200 μm). The U87MG cells stably over expressing either RFP or Nrf2 were subjected to ether vehicle or Carmustine treatment for 18 h and the cellular viability was assessed by (E) MTT reduction assay and the Figure 4D demonstrates the cellular morphology of cells upon Carmustine treatment using bright field microscopy (Scale bar =200 μm). Data are representative of at least three independent trials (n = 3/trial) and are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001 vs. vehicle-treated cultures.

Figure 5

Antioxidants and Carmustine cytotoxicity. U87MG cells were treated with Carmustine (40 μg/ml) for 18 h in the presence of either (A) glutathione (GSH;5 mM) or (B) N-acetyl cysteine (NAC; 5 mM) and cellular viability was quantified using MTT assay. Data are representative of at least three independent trials (n = 3/trial) and are expressed as mean ± SEM. *** p < 0.001 vs. Carmustine alone treated cultures.