MCF-7, T47-D and MDA-MB-231 human breast cancer cells [American Type Culture Collection (ATCC) Manassas, VA, USA] were maintained in Dulbeccos modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS), in a 5% CO₂ incubator at 37°C. RES and CIS were obtained from Sigma Chemical Company (St. Louis, MO, USA). RES stock solution was solubilized in absolute ethanol and diluted in DMEM. CIS stock solution was solubilized in dimethyl sulfoxide (DMSO) and diluted in culture medium.

Cells were seeded at a density of 2×10⁵ cells/dish in p60 cell culture dishes 24 h before the assay. The cells were treated with different concentrations of RES (0–250 µM in 0.3% ethanol) and/or CIS and cultured for 24, 48 and 72 h. At the end of each treatment period, the cells were incubated in MTT (0.5 mg/ml in DMEM) at 37°C for 30 min. The medium was removed, and the formazan dye crystals were solubilized with 500 µl of acid isopropanol. Absorbance was measured by a colorimetric assay at 540 nm wavelength (Bio-Rad Laboratories, Hercules, CA, USA). The growth percentage was calculated using the initial number of control cells as 100% at 0 h. The IC₅₀ values for RES and CIS were calculated using the GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA).

Total RNA was extracted using TRIzol (Invitrogen) as described elsewhere and purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's protocol. RNA was recovered in 30 µl of nuclease-free water and either used immediately or stored at −80°C until further analysis.

cDNA synthesis was performed with a First Strand kit as previously described (21). Each sample was tested in triplicate, and relative gene expression levels were calculated using the mRNA ratios relative to the β2-microglobulin house-keeping. The primer sequences were designed using Primer Express Software (Table I). SYBR-Green reaction was conducted using a QuantiTect™ SYBR-Green PCR Reagents kit (Qiagen) following the manufacturer's recommendations. Before performing the RT-qPCR, a reaction optimization was performed for each gene-specific pair of primers to confirm the specificity of the amplification signal. Changes in fluorescence were recorded as the temperature was increased from 65–95°C at a rate of 0.2°C/sec to obtain a DNA melting curve.

The data were analysed using the equation described by Livak and Schmittgen (22). Briefly, we used the average ΔCq from RES-untreated MCF-7 or MCF-7R cells as the calibrator for each gene tested to obtain the amount of target = 2^−ΔΔCq. Validation of the method was performed as previously reported (21). Data are presented as the mean ± standard deviation (SD). Statistical evaluation of significant differences was performed using Student's t-test. Differences of P<0.05 were considered statistically significant.

MCF-7 and MCF-7R cells were treated for 48 h with the proper vehicle, RES and/or CIS. Briefly, the cells were lysed with RIPA lysis buffer, and 30 µg protein was loaded on an SDS-10% polyacrylamide gel. The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA), blocked with 5% (w/v) non-fat milk and washed with Tris-buffered saline-Tween solution (TBST). The membrane was probed overnight at 4°C with a specific primary antibody [anti-Rad51, 1:1,000; rabbit polyclonal; cat. no. SC-8349; Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti-H2AX (pSer139), 1:1,000; rabbit polyclonal; cat. no. H5912; Sigma-Aldrich Co. LLC)]. The blots were developed using chemiluminescent detection reagents (Immobilon™ Western; Millipore). After stripping, the blots were re-probed with anti-α-actin (1:200; mouse monoclonal; prepared in the laboratory of PhD José M. Hernandez Hernandez, Department of Cell Biology, Cinvestav-IPN, Mexico City, Mexico) or anti-β-actin (1:20,000; mouse monoclonal; cat. no. A3854; Sigma-Aldrich, Co. LLC).

Data were evaluated in triplicate against the untreated control cells and collected from three independent experiments. The RT-qPCR results were evaluated by the Student's paired t-test. Two-tailed P-values <0.05 were considered statistically significant. Data from CIS and RES treatments were graphed and analysed by GraphPad Prism Software 5.0 using a two-way ANOVA, with post hoc Tukey HSD. P<0.005 was considered statistically significant. The data are presented as the mean ± standard deviation.

In a previous study, we observed that RES decreased the level of HR proteins in MCF-7 breast cancer cells, suggesting that this polyphenol may enhance the efficacy of DNA damage agents (20). We investigated the antiproliferative effect of RES combined with CIS in MCF-7, T47-D and MDA-MB-231 cells using MTT assays. First, we determined the IC₅₀ of RES and CIS on breast cancer cell lines (MCF-7, T47-D and MDA-MB-231) using the MTT assay. The cells were treated for 48 h with different concentrations of RES (Fig. 1A) or CIS (Fig. 1B). At 10 µM RES, the cell viability of MCF-7 and T47-D (both oestrogen receptor-positive cells) increased. By contrast, the viability of MDA-MB-231 (oestrogen receptor-negative cells) decreased at this concentration (Fig. 1A). At higher concentrations of RES, the decrease in viability was similar for all the cell lines tested, with MCF-7 presenting the highest IC₅₀ value (101.1 µM) and T47-D cell line presenting the lowest (78.19 µM) (Fig. 1A). In Fig. 1B, we can observe a dose-dependent reduction in cell viability for the MCF-7, T47-D and MDA-MB-231 cell lines treated with CIS (P<0.005). T47-D cells presented the lowest IC₅₀ (7.27 µM), and MDA-MB-231 cells were the most resistant to CIS (IC₅₀ = 21.48 µM CIS). We then investigated the antiproliferative effect of RES combined with CIS in MCF-7, T47-D and MDA-MB-231 cells. As shown in Fig. 1C, in all the cell lines, the IC₅₀ values for CIS decreased significantly after 48 h of 50 µM RES treatment (P<0.005). In the T47-D cell line, the IC₅₀ obtained for CIS decreased >50% (from 7.3 to 3.0 µM) in the presence of 50 µM RES, and the MCF-7 and MDA-MB-231 cell lines also showed a marked reduction (from 11.9 to 6.2 µM and from 21.3 to 16.9 µM, respectively). The IC₅₀ value for RES and CIS was calculated as indicated in Materials and methods.

It has been reported that many of the anticancer properties of RES are dependent on p53, so although we observed that RES increased the effectiveness of CIS in the three different cell lines, we focused on a scenario where the p53 protein was present in order to understand the mechanisms of action of RES. For this reason, experiments were subsequently focused on the MCF-7 cell line. Since the main action of the reported molecular mechanisms of CIS activity is to cause DNA damage, and HR is the pathway related to the DNA damage response to CIS, one interesting possibility for RES activity would be to interfere with the expression of the canonical HR system components. We examined the effects of RES on the mRNA level of the canonical HR initiation complex components (Nbs-1, Mre-11 and Rad-50) in the MCF-7 cells, using RT-qPCR assays.

Notably, lower doses of RES near the IC₂₅ value (30 and 50 µM) induced an increase in Nbs-1, Mre-11 and Rad-50 mRNA levels at 48 h. However, these values decreased at RES doses near the IC₅₀ concentration (100 µM) and 150–250 µM (Fig. 2) as previously reported (20). These results suggested that at doses similar or greater than the IC₅₀ value, RES appears to reduce the mRNA level of the HR initiation complex components. Consequently, 100 µM of RES was used to sensitize MCF-7 cells to CIS treatment. Thus, RES may contribute to decreased CIS IC₅₀ in MCF-7 cells by negatively regulating HR initiation complex components.

As we observed that 100 µM RES decreases the expression of HR genes, we analysed whether 100 µM RES concentration increased sensibility to CIS in MCF-7 cells. We observed a significant decrease in cell viability when we treated MCF-7 cells with 50 µM RES combined with 10 µM of CIS (P<0.001). In addition, when the MCF-7 cells were treated with 100 µM RES in combination with CIS, the cell viability was significantly decreased after treatment with 2 µM of cisplatin (P<0.001). The IC₅₀ values for CIS decreased ~8-fold (11.91 vs. 1.48 µM) in cells treated for 48 h with the combination of 100 µM RES (Fig. 3) (P<0.001). These data suggested that a large decrease in the IC₅₀ value may be associated with the ability of 100 µM RES to reduce the HR initiation complex mRNA components.

Rad51 is the central recombinase involved in the HR repair of DNA DSBs (23) and overexpression of Rad51 has been detected in various cancer cell types (24–27). We investigated whether RES induced changes in Rad51 protein expression in MCF-7 cells. The cells were treated for 48 h with either vehicle, 50, 100, 150 or 250 µM RES, and cellular extracts were evaluated for the presence of Rad51 protein (Fig. 4A). Western blot analysis revealed that Rad51 expression was decreased in the presence (48 h) of 100 µM or higher RES concentrations. Then, we explored whether the reduced level of Rad51 was related to unrepaired damaged DNA by measuring the levels of phosphorylated H2AX [Ser¹³⁹] since γ-H2AX formation is used as a marker for DNA damage (notably DNA DSB) (28). The cells were treated for 48 h with vehicle, 20 µM CIS or 20 µM CIS plus 100 µM RES. As shown in Fig. 4A, the MCF-7 cells expressed basal levels of Rad51, and as expected this level was further induced by CIS treatment. Of note, however, this induction was blocked by 100 µM RES (Fig. 4B). Induced levels of γ-H2AX after 48 h of CIS treatment were barely detectable (Fig. 4B, lanes 1 and 2). This finding suggested that with high levels and activity of Rad51, DNA damage is rapidly repaired, and γ-H2AX is no longer needed. However, γ-H2AX is significantly present in cells treated with 20 µM CIS plus 100 µM RES (Fig. 4B, lane 3), suggesting that the reduction of HR activity (due to decreased Rad51, Nbs-1, Mre-11 and Rad50) may affect DNA repair, and high levels of γ-H2AX may be evidence of a defective DNA repair. Therefore, these results strongly indicated that RES suppressed the repair of DNA damage caused by CIS in MCF-7 cells.

To demonstrate the contribution of RES to enhance CIS sensitivity in CIS-resistant cells, we generated MCF-7 cells resistant to CIS treatment (MCF-7R) by continuous exposure to low concentrations of CIS (10 µM CIS-IC₄₀). After selection, the IC₅₀ value of these MCF-7R cells increased ~3-fold (from 11.91 to 34.66 µM) (Fig. 5). To investigate whether RES has an impact on cellular sensitivity towards CIS in MCF-7R, the IC₅₀ was determined. According to our previous results with MCF-7 cells (Fig. 3), 100 µM RES was also able to reduce the viability of the MCF-7R cells. We observed a significant decrease in the viability of MCF-7R cells after 2–15 µM of CIS treatment (P<0.001). The CIS IC₅₀ decreased to one-sixth of the original MCF-7R IC₅₀ value when the cells were treated with a combination of 100 µM of RES (from 34.66 to 5.22 µM, Fig. 5). These findings suggested that RES may be a potent adjuvant to recover CIS sensitivity in CIS resistance.

Given that Rad51 is the central recombinase involved in HR (29), we also examined whether RES decreases the Rad51 expression levels and maintains γ-H2AX levels in MCF-7R cells as we previously observed in MCF-7 non-resistant cells. We then examined the levels of Rad51 mRNA in MCF-7R cells with or without RES treatment. The RT-qPCR assay demonstrated that Rad51 mRNA is overexpressed in MCF-7R (Fig. 6A), but successfully reduced by RES treatment at 100 µM. These results suggested that as with MCF-7 cells, a partial inhibition of DNA repair mechanisms by RES may contribute to CIS sensitivity in MCF-7R cells. To induce DNA damage, we treated MCF-7R cells with a high dose of CIS (20 µM), and observed an increase in Rad51 protein levels and γ-H2AX levels, indicating the presence of DNA damage (Fig. 6B). By contrast, 100 µM of RES did not increase the Rad51 levels (Fig. 6B). In the MCF-7R cells treated with 20 µM CIS plus 100 µM RES we observed that the induction of Rad51 expression by CIS DNA damage was partially inhibited by treatment with RES, indicating that RES was able to suppress Rad51 overexpression in MCF-7R cells (Fig. 6B, upper right panel). We also examined whether RES would promote sustained γ-H2AX after CIS induced DNA damage by affecting Rad51. Although MCF-7R cells were created under CIS conditions, at this time, they had no basal γ-H2AX signal (Fig. 6B, lower panel). As opposed to that identified in Fig. 4B, at the same time (48 h), CIS damage was able to promote significant levels of γ-H2AX highlighting the different nature of the resistant cells compared to the normal cells (Fig. 6B). RES alone had no effect on the level of γ-H2AX of MCF-7R, but γ-H2AX was still present in MCF-7R cells treated with 20 µM CIS plus 100 µM RES (Fig. 6B, lower right panel), further confirming that RES may partially inhibit the repair of DNA damage caused by CIS, even in chemoresistant MCF-7R cells.