Catalase down-regulation in cancer cells exposed to arsenic trioxide is involved in their increased sensitivity to a pro-oxidant treatment

Human breast cancer cell line MCF-7 was purchased from ATCC (Manassas, VA, United States). Cells were maintained in DMEM medium supplemented with 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) from Gibco (Grand Island, NY, USA). Human mammary epithelial cells 250MK were provided by Dr. M. Stampfer and J. Garbe (Lawrence Berkeley National Laboratory, Berkeley, California, USA). They were maintained in a M87A + CT + X medium and used between passages 8–10 [22]. The cultures were maintained at a density of about 50 × 10³ cells/cm². All cultures were maintained at 37 °C in 95% air/5% CO₂ with 100% humidity.

Cancer cell cultures were treated with sodium ascorbate and menadione sodium bisulfite (Asc/Men) in a ratio of 100/1 (ascorbate in mM/menadione in µM), arsenic trioxide (5–10 μM), hydrogen peroxide (0–1 mM), or 5 mM of 3-amino-1,2,4-triazole (ATA). All reagents were purchased from Sigma-Aldrich (St Louis, MO).

Breast cancer cell line MCF-7 CAT 3 overexpressing catalase was established from wild-type MCF-7 cell line, which was purchased from ECACC (Salisbury, United Kingdom). Plasmid construct pZeoSV2(1) containing human catalase cDNA was a kind gift from Professor A. Cederbaum (Mount Sinai Hospital, New York, USA) [23]. MCF-7 cells were transfected (50% confluence) for 24 h with 1 µg of plasmid using FuGENE 6 reagent transfection (Promega, Madison, WI, USA). The selection of successful transfected cells was obtained by supplementing the culture media with 400 µg/ml of zeocin (InvivoGen, San Diego, CA, USA) for 3 weeks and changing medium every 3–4 days. Then cell clones were obtained and characterized. Only the clone number 3 (MCF-7 CAT 3) was selected for this study.

A Resox cell line was established from wild-type human breast cancer MCF-7 cell line, which was purchased from ATCC, as previously reported [18]. Briefly, oxidative stress-resistant Resox cells were made by exposing MCF-7 cells chronically to increasing concentrations of ascorbate/menadione (Asc/Men) during 6 months. To avoid the development of islets of resistance, which could arise from cooperation between cells, the cells were trypsinized approximately every 2 weeks and seeded into new flasks. After selection, the cell line was stabilized in drug-free medium for 1 month.

Dharmafect reagent 1 was used for transfecting siRNA against catalase (ON-TARGET plus SMART pool siRNA) in all cell lines, according to the protocols provided by Dharmacon (Lafayette, CO, USA). Transfections were performed on cells at 50% confluence for 24 h, with a 0.1 μM siRNA solution. All experiments were performed 48 h after transfection. siRNA-mediated catalase knockdown experiments were compared to those displayed by transfecting a siRNA control (scrambled sequence) under the same experimental conditions described above.

The effects of Asc/Men, arsenic trioxide or hydrogen peroxide on cell metabolic status were assessed by following the reduction of MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) to blue formazan [24]. Blue formazan crystals were solubilized with DMSO and the coloured solution was subsequently read at 550 nm. Results are expressed as % of MTT reduction compared to untreated control conditions.

At the indicated times, cells were washed twice with ice-cold PBS and then resuspended in RIPA lysis buffer supplemented with 1% Protease Inhibitor Cocktail (Sigma-Aldrich) and 3% Phosphatase Inhibitor Cocktail (Calbiochem, Merck KGaA, Darmstadt, Germany). Electrophoresis of proteins (20 µg), electroblotting and further procedures were done as reported elsewhere [17]. Protein bands were detected by chemiluminescence using the ECL detection kit (Pierce, ThermoScientific, Rockford, IL, USA). When appropriate, bands obtained from Western blot analysis were quantified using ImageJ software (http://​rsb.​info.​nih.​gov/​ij/​).

Plasmid pGL3-Basic containing the human catalase gene promoter − 1518/+ 16 was kindly provided by Professor M. Nenoi (National Institute of Radiological Sciences, Chiba, Japan) and all constructions strategies were previously described [25]. The transcription start (+ 1) is located 73 bp upstream of the initiation codon [26]. The purified fragment containing human catalase promoter was excised from pGL3 pCAT plasmid by KpnI and BglII restriction enzymes (Fermentas, Rockford, IL, USA), and ligated into pGL4.15 plasmid (Promega, Madison, WI, USA). For stable transfection, DNA transfection was carried out by using the Xtremegene HP reagent (Roche Applied Science Diagnostics, Mannheim, Germany). Stable transfectants and clones (selected by limiting dilution in a 96-well plate) were selected by supplementing the culture media with 100 µg/ml of Hygromycin B (Invivogen, SanDiego, CA, USA) [17]. Luciferase activities were measured in accordance with manufacturer’s instructions (Luciferase Assay System, Promega, Madison, WI, USA) using a Victor™X2 luminometer (Perkin Elmer, Waltham, MA, USA). Normalized luciferase activity corresponds to measured light units that were normalized to protein concentrations using BCA protein kit (ThermoScientific, Rockford, IL, USA).

Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. RNA was reverse-transcribed using Primer Script RT reagent Kit with gDNA Eraser (Takara BIO INC, Kusatsu, Shiga, Japan). Real-time PCR was performed using the SYBR Premix Ex Taq RNAse H + kit (Takara), and analyzed using the Bio-Rad detection system (Bio-Rad, Hercules, CA, USA). The samples were first incubated 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. The results were calculated (formula: 2-(Ct catalase-Ct EF1)) and matched to the control samples. The primers were produced by Sangon Biotech (Shanghai, China): catalase (sense primer 5′-ccagaagaaagcggtcaagaa-3′; antisense primer 5′-gagatccggactgcacaaag-3′), EF1 (sense primer 5′-cttcactgctcaggtgat-3′; antisense primer 5′-gccgtgtggcaatccaat-3′).

For ROS detection, cells were incubated at indicated time with 10 μM CM-H2DCFDA (Molecular Probes, Rockford, IL, USA) for 20 min. The cells were harvested, washed twice with PBS, and analyzed by flow cytometer (Gallios; Beckman Coulter, Brea, CA, USA).

All experiments were performed at least 3 times and groups were compared by ANOVA test using GraphPad Prism software (San Diego, CA, USA). Two-way ANOVA test was used to analyze the dose–response curves.

Preliminary experiments performed in our laboratory using mixtures of Asc/Men with various chemotherapeutic drugs (cisplatin, 5-fluorouracil, doxorubicin, paclitaxel and etoposide) showed that the combination of ATO + Asc/Men was the most effective treatment to overcome this resistance (data not shown). Therefore, we investigated the effect of ATO + Asc/Men on the following mammary cancer cells: MCF-7, CAT3 and Resox cells. Figure 1a–d shows that ATO (5 and 10 µM) enhanced the cytotoxic effects of sub-lethal doses Asc/Men in MCF-7. Such synergistic effect was also observed but to a lesser extent in catalase-overexpressing MCF-7 cells (CAT3 and Resox cells). In addition, the effect of Asc/Men in combination with 10 μM ATO was more pronounced in Resox cells compared to CAT3 cells (Fig. 1b, d).

The key role of catalase in the acquisition of cell resistance against a pro-oxidant treatment was studied by applying a double experimental approach: genetic invalidation via siRNA against catalase and pharmacological inhibition using aminotroazole (ATA), a well-known inhibitor of catalase activity [27]. Genetic invalidation of catalase in MCF-7 and Resox cells reaches 85–95% efficacy of protein expression decrease (Fig. 2a). In MCF-7 cells no changes in sensitivity to increasing concentrations of H₂O₂ or Asc/Men in a ratio of 100/1 were observed (Fig. 2b, c). On the other hand, an increased sensitivity to higher doses of H₂O₂ and Asc/Men was observed upon catalase knockdown by siRNA in Resox cells (Fig. 2d, e). Regarding the effect by ATA, a much more increase of sensitivity was observed in both cells against H₂O₂ exposure (Fig. 2f–h) while a less marked effect was seen in cells incubated with Asc/Men (Fig. 2g–i). Resox cells appear then as more sensitive most likely due to a higher catalase expression at basal levels as compared to parental MCF-7 cells.

Given the pro-oxidant effect exerted by ATO (Fig. 1), the potentiation of its cytotoxicity when associated with Asc/Men, reinforces the validity of a pro-oxidant approach to remove cancer cells. The question about the underlying ATO mechanism thus explaining the enhanced cytotoxicity was therefore raised.

Since catalase is a key antioxidant enzyme and ATO enhances the sensitivity of catalase-overexpressing cells to Asc/Men (Fig. 1a–d), we further investigated the regulation of the expression of catalase by ATO.

We observed that catalase is down-regulated in MCF-7 cells when compared to normal mammary epithelial 250MK cells but its expression is highly enhanced in oxidative stress-resistant Resox cells (Fig. 3a, b). Interestingly, ATO significantly decreases the amount of catalase in Resox cells (Fig. 3c, d) and increases ROS levels (Fig. 3e), likely explaining the enhanced cytotoxicity previously observed (Fig. 1a, b).

Because previous results suggested that alteration of catalase gene transcription is the main regulator of catalase expression in cancer cells, we first explored the hypothesis that ATO decreased the transcriptional activity of catalase gene promoter.

Figure 3f, g shows that ATO represses, in a dose-dependent manner, mRNA transcription and transcriptional activation of human catalase gene in both MCF-7 and Resox mammary cancer cell lines. Such repression of transcriptional activity may also explain the differences observed in CAT3 and Resox cells (shown in Fig. 1). Indeed, the difference between them is that Resox cells, after chronic exposure to Asc/Men, acquired a higher transcriptional activity of the endogenous catalase promoter, whereas catalase expression in CAT3 cells is the result of a plasmid transfection controlled by SV40 promoter. Thus, in addition to catalase activity, this may explain why CAT3 cells are less sensitive to ATO when its concentration was increased from 5 to 10 µM (Fig. 1c, d). However, CAT3 cells showed an increased sensitivity to the association ATO + Asc/Men compared to the drugs alone. We thus cannot exclude other targets of ATO in these breast cancer cells to explain our findings.