Redox-modulating agents target NOX2-dependent IKKε oncogenic kinase expression and proliferation in human breast cancer cell lines

Abstract

Oxidative stress is considered a causative factor in carcinogenesis, but also in the development of resistance to current chemotherapies. The appropriate usage of redox-modulating compounds is limited by the lack of knowledge of their impact on specific molecular pathways. Increased levels of the IKKε kinase, as a result of gene amplification or aberrant expression, are observed in a substantial number of breast carcinomas. IKKε not only plays a key role in cell transformation and invasiveness, but also in the development of resistance to tamoxifen. Here, we studied the effect of in vitro treatment with the redox-modulating triphenylmethane dyes, Gentian Violet and Brilliant Green, and nitroxide Tempol on IKKε expression and cell proliferation in the human breast cancer epithelial cell lines exhibiting amplification of IKKε, MCF-7 and ZR75.1. We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide anion levels and inhibit IKKε expression and cell viability. Treatment with Gentian Violet and Brilliant Green was associated with a reduced cyclin D1 expression and activation of caspase 3 and/or 7. Tempol decreased cyclin D1 expression in both cell lines, while activation of caspase 7 was only observed in MCF-7 cells. Silencing of the superoxide-generating NOX2 NADPH oxidase expressed in breast cancer cells resulted in the significant reduction of IKKε expression. Taken together, our results suggest that redox-modulating compounds targeting NOX2 could present a particular therapeutic interest in combination therapy against breast carcinomas exhibiting IKKε amplification.

Keywords: Breast cancer; Cell growth; IKKε; NADPH oxidase; Tempol; Triphenylmethane dyes.

Graphical abstract

Fig. 1

IKKε expression levels in MCF-7 compared to ZR75.1 cells. (A) Total RNA were extracted from MCF-7 and ZR75.1 cells, subjected to reverse transcription and IKKε mRNA expression was quantified by RT-qPCR and expressed as fold over MCF-7 level after normalization to actin expression. (B) WCE extracts were prepared and resolved by SDS-PAGE. Immunoblot analyses were performed using anti-IKKε and anti-TBK1 antibodies. Anti-actin antibodies were used to control equal loading. Data are representative of two experiments.

Fig. 2

Reduction of intracellular superoxide levels in cells treated with tempol, Gentian Violet or Brilliant Green. In (A), MCF-7 cells were left untreated or treated with Tempol (T, 5 mM), Gentian Violet (GV, 5 µM) or Brilliant Green (BG, 5 µM). In (B), ZR75.1 cells were left untreated or treated with Tempol (3 mM), Gentian Violet (5 µM) or Brilliant Green (5 µM). Intracellular superoxide levels were monitored using staining with the fluorescent dye DHE. Fluorescence (ex: 488 nm/em: 575 nm) was acquired by flow cytometry and the median of fluorescence was quantified. Values are means±SEM from n≥3. Statistical comparison (treated cells vs untreated cells) was performed using a t-test.

Fig. 3

Treatment with Gentian Violet, Brilliant Green or Tempol decreases oncogenic IKKε kinase expression levels. MCF-7 (A–C) and ZR75.1 (B–F) were left untreated or treated with Tempol (T), Gentian Violet (GV) or Brilliant Green (BG) at the indicated concentrations for 24 h (A, C, D and F) or the indicated times (B and E). WCE were resolved by SDS-PAGE and analyzed by immunoblot using anti-IKKε and anti-TBK1 antibodies. Equal loading was monitored using anti-actin antibodies. Representative immunoblots of at least three experiments are shown. In (C) and (F), IKKε levels were quantified by densitometric analysis using the ImageJ software. Quantification data are expressed as mean±SEM from n≥3. Statistical comparison was performed using a t-test.

Fig. 4

Tempol, Gentian Violet and Brillant Green treatment impair cyclin D1 expression. MCF-7 (A) and ZR75.1 (B) were left untreated or treated for 24 h with Tempol (T), Gentian Violet (GV) or Brilliant Green (BG) at the indicated concentrations. WCE were resolved by SDS-PAGE and analyzed by immunoblot using anti-cyclin D1 and anti-actin antibodies. Experiments were performed three times. Representative immunoblots are shown.

Fig. 5

Tempol, Gentian Violet and Brilliant Green significantly decrease viability of MCF-7 and ZR75.1 cells. MCF-7 (A and B) and ZR75.1 (C and D) cells were left untreated or treated with Tempol (T), Gentian Violet (GV) or Brilliant Green (BG). In A and C, inhibitors were used at the indicated concentrations and cell viability was determined at various times by the crystal violet assay. In (B) and (D), cell viability was monitored after treatment with increasing concentrations of either inhibitor. Non-linear regression curve fit (log[inhibitor] vs normalized response) were obtained using GraphPad Prism software. Plotted data correspond to the mean±SD (n≥3).

Fig. 6

Cell-type specific activation of caspases by Tempol, Gentian Violet and Brillant Green. MCF-7 (A) and ZR75.1 (B) were treated with the indicated concentration of Tempol (T), Gentian Violet (GV) or Brilliant Green (BG) for 24 h. WCE were analyzed by immunoblot for cleaved-caspase 3 (19 and 17 kDa), cleaved-caspase 7 (20 kDa) and PARP (Full-length 116 kDa and cleaved 89 kDa). Equal loading was monitored using anti-actin antibodies. Representative immunoblots of three different experiments are shown.

Fig. 7

NOX2-dependent regulation of IKKε expression in MCF-7 cells. Total RNA from MCF-7 or ZR75.1 cells (A) or from 10 distinct primary breast tumors (B) was extracted, subjected to reverse transcription and specific expression of NOX1-5, DUOX1-2 and actin was evaluated by RT-PCR. Specific positive controls (Ctrl⁺) were used for each gene as detailed in the methods section. In (C), control (CTRL), NOX2-, NOX5- or IKKε-specific RNAi were transfected into MCF-7 cells. Efficiency of NOX2 and NOX5 silencing was controlled by RT-qPCR (left panels). IKKε and TBK1 protein expression were analyzed by immunoblot (IB) using specific antibodies. Actin was used as loading control. IKKε levels were quantified by densitometric analysis using the ImageJ software. Quantification data are expressed as mean±SEM from n≥3. A t-test was performed to compare specific RNAi-transfected to CTRL RNAi-transfected cells. (D) Cell viability was monitored at the indicated times post-RNAi transfection and expressed as % of the corresponding siCTRL-transfected condition at each time point.