The MCF-7 human breast carcinoma cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% FCS and insulin, and penicillin and streptomycin (Life Technologies, Gaithersburg, MD, USA). Stably transfected MCF-7 cells with full-length HER2 cDNA (MCF-7/HER2–18) or control vector (MCF-7/neo) have been previously described (9). Tam/MCF-7 cells were grown in the presence of 4-OH Tam (10⁻⁶ M) (20). The cells obtained from ATCC were immediately expanded and frozen, and restarted every 3–4 months from a frozen vial of the same batch of cells and no additional authentication was done on these cells. 4-OH Tam was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The anti-glyceraldehyde-3-phosphate dehydrogenase (G3PDH) rabbit polyclonal was purchased from Trevigen (Gaithersburg, MD, USA). Monoclonal antibodies of PARP and Bid were purchased from BD Biosciences (San Diego, CA, USA). Caspase-9 antibodies were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). Caspase-3 inhibitor Z-DEVD-FMK, Caspase-8 inhibitor Z-IETD-FMK, Caspase-9 inhibitor Z-LEHD-FMK, and pan-caspase inhibitor Z-VAD-FMK were purchased from R&D System (Minneapolis, MN, USA). Unless otherwise specified, any other chemicals were obtained from Sigma Chemical Co. at highest suitable purities.

Briefly, 5×10⁴ cells were added in 96-well tissue culture plates. After 24 h, the cells were treated with TRAIL (10 ng/ml), cisplatin (10 μg/ml), or a combination of TRAIL plus cisplatin for another 24 or 48 h. Then, 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (1 mg/ml) was added into each sample and incubated for 3 h under 5% CO₂ and 37°C. The cell viability was measured by MTT, which was converted by succinate dehydrogenase in the mitochondria of viable cells to yield a purple formazan dye. The formazan dye was dissolved in dimethyl sulfoxide (DMSO) and measured by absorption at a wavelength of 550 nm using Benchmark^® microplate reader from Bio-Rad (Hercules, CA, USA).

Apoptosis was assessed using the Cell Death Detection ELISAplus kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions. This kit quantitatively detects cytosolic histone-associated DNA fragments. Briefly, the cells were treated with cisplatin (10 μg/ml) and TRAIL (10 ng/ml) or a combination of cisplatin (10 μg/ml) plus TRAIL (10 ng/ml) for 16 h of treatment. Apoptosis was quantified by ELISA and normalized to values measured in untreated cells. Data were presented as the mean ± SEM of triplicate determination.

The cells were grown in 6-well plates, to near confluence in the presence or absence of various treatments. The cells were lysed and western blotting was performed as previously described (5) using a standard protocol. Briefly, the cell extracts were obtained by lysing the cells in RIPA buffer (20 mM Hepes, 100 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 1 mM Na₃VO₄, 1 mM EGTA, 50 mM NaF, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1X protease inhibitor mixture). The samples containing 100 μg of total protein were electrophoresed on 8% or 15% SDS-polyacrylamide gels and transferred on to PVDF membranes by electroblotting. The membranes were probed with antibodies as indicated, followed by HRP-conjugated mouse or rabbit secondary antibodies (Amersham Amersham Biosciences, Piscataway, NJ, USA). Anti-G3PDH was used for loading controls.

Data are presented as mean ± SEM, and statistically analyzed using the unpaired Student’s t-test. Differences were considered statistically significant when P<0.05.

To develop new strategies to eliminate anti-estrogen-resistant and -sensitive breast cells, we examined the effect of cisplatin on Tam/MCF-7 and Tam/T47D cells. Cell survival is shown in Fig. 1. Treatment of the cells with Cisplatin induced a significant decrease in cell survival in the Tam/MCF-7 and Tam/T47D cells. The efficacy of cisplatin was dose-dependent; however, a significant risk of nephrotoxicity frequently hindered the use of higher doses to maximize the antitumor effects of cisplatin (21). Our data showed that 10 μg/ml of cisplatin exhibited optimum inhibition in MCF-7- and T47D-derived anti-estrogen-resistant cells (Fig. 1). In addition, the results showed that cisplatin was more effective in killing anti-estrogen-resistant cells when compared to sensitive cells in the MCF-7 cell line. However, in the T47D-derived cell lines Tam-resistant and -sensitive cells were killed (Fig. 1). The reasons for this discrepancy in ER-positive breast cell lines remains to be determined.

We evaluated whether a combination of cisplatin plus TRAIL enhanced killing in anti-estrogen-resistant cells. Fig. 2 shows that cisplatin or TRAIL treatment decreased cell survival in Tam/MCF-7 (acquired resistance to Tam), HER2/MCF-7 (de novo resistance) and Tam/T47D (acquired resistance to Tam) cells to varying degrees. However, cisplatin plus TRAIL had a maximum effect on anti-estrogen-resistant cells by 48 h. A similar treatment of CRL8799 normal breast cells resulted in a moderate increase in cell death.

Most chemotherapy agents and irradiation trigger apoptosis through the cell-intrinsic pathway, as an indirect consequence of cell damage. The intrinsic pathway usually requires functional p53. However, inactivation of p53, either directly through mutation(s) or indirectly through p53 modulation through the MDM2 protein, is common in many human tumors. The extrinsic pathway induces apoptosis in response to the engagement of death receptors by their ligands such as TRAIL. This pathway stimulates the apoptotic caspase mechanism independent of p53 (22). The effect of cisplatin, TRAIL, and cisplatin plus TRAIL treatment on apoptosis in Tam/MCF-7 and Tam/T47D cells are shown in Fig. 3. Although treatment with cisplatin or TRAIL increased apoptosis to varying degrees, a combination of cisplatin plus TRAIL had a maximum effect on apoptosis in Tam/MCF-7 (Fig. 3A), and Tam/T47D (Fig. 3B) cells (p<0.05). These data suggested that cisplatin plus TRAIL increased apoptosis in anti-estrogen-resistant cells compared with the untreated controls.

Caspases have been previously shown to play an important role in TRAIL-induced apoptosis (23). It was previously shown that 75% of human breast tumors lack caspase-3 transcripts, whereas other caspases, such as caspase-8 and -9 were shown to be normal (24). MCF-7 cells lack caspase-3 expression as a result of a functional deletion mutation in the caspase-3 gene, while the expression of other caspase-8 and -9 remains normal. We used MCF-7 and T47D anti-estrogen cells to determine the possible mechanism by which cisplatin plus TRAIL increase cell death. The results from the western blot analyses revealed an increased caspase-9 activation in Tam/MCF-7 and Her-2/MCF-7 anti-estrogen-resistant cells compared to -sensitive cells and this led to increased PARP and Bid cleavage (Fig. 4A) and apoptosis (Fig. 3A). By contrast, T47D and Tam/T47D (express caspase-3) showed a higher activation of caspase-3 and -9 in resistant and sensitive cells in the presence of cisplatin plus TRAIL, leading to increased PARP and Bid cleavage (Fig. 4B).

To determine whether caspase activation is essential for cisplatin plus TRAIL-induced cell death, we blocked caspase-3, -8, -9 or pan-caspase using relatively specific caspase inhibitor(s). The effect of caspase inhibitors on cisplatin plus TRAIL treatment in Tam/MCF-7 HER2/MCF-7 and Tam/T47D cells are shown in Fig. 5. However, the addition of caspase inhibitors alone to anti-estrogen-resistant cells had a minimal or no effect on these cells. These data suggested that cisplatin plus TRAIL-induced apoptosis is predominantly mediated through the activation of caspases in Tam-resistant cells.