Enhanced Anticancer Effect of the Combination of Cisplatin and TRAIL in Triple-Negative Breast Tumor Cells Free

Breast cancer continues to be a major health problem worldwide despite recent advances in its diagnosis and treatment. Based on gene expression profiles breast cancer can be the classification of into 5 main groups: (i) luminal A, (ii) luminal B (both are ER+), (iii) basal-like, (iv) HER2, and (v) normal breast-like tumors (1). At present, most treatment in breast cancer is targeted to either to ER or HER-2. Triple-negative breast cancer (TNBC) is characterized by the absence of 3 receptors, viz., estrogen receptor (ER), progesterone receptor (PR), and HER2, hence the term triple-negative. In approximately 60% of cases, these tumor cells express the receptor for epidermal growth factor (EGFR; 2–4) and may also contain mutations in p53 gene (5–7). The disease is diagnosed more frequently in younger and premenopausal women (6, 8–11). It was previously shown that mutations in BRCA1 gene leads to error-prone DNA repair resulting in genomic instability and predisposition to carcinogenesis. In TNBC patient's loss of ER, PR, and HER-2 prevents targeted therapy. In general TNBC tumors are refractory to commonly used chemotherapeutic agents, as a result it leads to relatively poor prognosis (1, 12). Some reports have shown that BRCA1-associated TNBC tumors are sensitive to cisplatin chemotherapy (13). The use of platinum complexes for the breast cancer therapy is an emerging new treatment modality. Platinum drugs including cisplatin and carboplatin are widely used as anticancer drugs. Cisplatin is currently used to treat tumors of the head, neck, lungs, and ovarian cancer (14–16). However, therapeutic efficiency of cisplatin in TNBC is moderate. Therefore, it is important to identify new treatment modalities for TNBC patients.

Tumor necrosis-factor–related apoptosis-inducing ligand (TRAIL, a member of tumor necrosis factor family of death-receptor ligands) is reported to have a potential use in cancer therapy because of its ability to selectively kill cancer cells without affecting normal cells (17). TRAIL is shown to bind with death receptor 4 (DR4) and death receptor 5 (DR5) leading to the formation of death-inducing signaling complex (DISC) and Fas-associated protein with death domain (FADD). In turn, DISC and FADD recruits caspase-8 (or caspase-10) that plays an important role in inducing apoptosis either by direct activation of downstream effector caspases (caspase-3, caspase-6, and caspase-7) or by cleaving apoptotic molecules (Bcl-2 and Bcl-xL) resulting in further activation of caspase-9 complex (18). Studies in animals have also shown that TRAIL regresses cancer xenografts without affecting normal tissues (19).

In this study, we evaluated the molecular mechanism by which cisplatin sensitizes TNBC cell lines to TRAIL-induced apoptosis. Data show that cisplatin and TRAIL downregulates antiapoptotic proteins, survivin, Bcl-2, Bcl-xL and induces activation of caspase-3,-8, and -9 and increases apoptosis. However, in normal breast cell lines combination of cisplatin/TRAIL had a minimal effect on antiapoptotic proteins and shows a marginal increase in apoptosis in comparison with TNBC cells.

The human breast carcinoma cell line CRL8799, CRL2335, and MDA-MB-468 cells were obtained from the American Type Culture Collection (ATCC) and maintained in according to ATCC recommended culture media. All cells obtained from ATCC were immediately expanded and frozen down such that all cell lines could be restarted every 3 to 4 months from a frozen vial of the same batch of cells and no additional authentication was done in our laboratory. DR4/TRAILR1 monoclonal antibody and DR5/TRAILR2 polyclonal antibody was purchased from Imgenex. Monoclonal antibodies of PARP, Bid, actin, caspase-8 were purchased from BD Biosciences. Caspase-3, and caspase-9 antibodies and survivin small interfering RNA (siRNA) were purchased from Cell Signaling Technology. Anti-Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) rabbit polyclonal was obtained from Trevigen. TRAIL and caspase-3 inhibitors were purchased from R&D systems. Cell Death Detection ELISA^PLUS was purchased from Roche. Reagents for protein concentration analysis and protein gel electrophoresis were obtained from Bio-Rad p63 siRNA was obtained from Santa Cruz biotechnology. EGFR and random siRNA were obtained from Millipore. All other chemicals, unless otherwise specified, were obtained from Sigma in the highest suitable purities.

In brief, 5 × 10⁴ cells were added in 96-well tissue culture plates. After 24 hours, cells were treated with TRAIL (10 ng/mL), cisplatin (10 ug/mL), or combination of TRAIL plus cisplatin for another 24 hours. Following treatments, 100 μL of MTT (1 mg/mL) was added into each sample and incubated for 3 hours under 5% CO2 and 37°C. The cell viability was measured by MTT, which is converted by succinate dehydrogenase in 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.

Cells were grown in 6-well plates, to near confluence in the presence or absence of various treatments. Cells were lysed and Western blotting was carried out as described previously (20) using a standard protocol. In brief, cell extracts were obtained by lysing the cells in RIPA buffer (20 mmol/L Hepes, 100 mmol/L NaCl, 0.1% SDS, 1% Nonidet P-40, 1% deoxycholate, 1 mmol/L Na₃VO₄, 1mmol/L EGTA, 50 mmol/L NaF, 10% glycerol, 1mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture). Samples containing 100 μg of total protein were electrophoresed on 10% or 15% SDS-polyacrylamide gels and transferred on to PVDF membrane by electroblotting. Membranes were probed with antibodies as indicated, followed by HRP-conjugated mouse or rabbit secondary antibodies (Amersham). Anti-G3PDH was used for loading controls.

Cells were plated in 6-well tissue culture plates at a density of 3 × 10⁵/well in medium containing 10% FBS. After 24 hours, cells were transfected with 100 pmol of siRNA's from EGFR, and/or p63, or survivin or random siRNA with scrambled sequence was used as control. Lipofectamine transfection reagent was used to transfect sequence according to the manufacturer's instructions. After 48 hours of transfection, cells were treated with or without TRAIL for additional 24 hours. Cells were then harvested for Western blot analysis.

Cells were treated with cisplatin, TRAIL, or combination of cisplatin plus TRAIL for 16 hours. Cells were harvested and stained with FITC Annexin V and propidium iodide (PI) to identify early apoptotic cells. Apoptosis was determined using FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer's instructions.

Crystal violet stain binds to the nuclei of the cells and gives a violet color (an OD₅₉₅ reading) that is proportional to surviving cell. In brief, 5 × 10⁵ cells were plated in 12-well tissue culture plates. After 24 hours, cells were treated with TRAIL, cisplatin, or combination of TRAIL plus cisplatin for another 24 hours. Following treatments, the medium was removed and cells were washed with PBS, fixed, and stained with 0.2% crystal violet in 10% phosphate-buffered formaldehyde for 30 seconds. Excess crystal violet solution was removed and cells were washed 3 times with PBS. The pictures were taken after the plates completely dried.

We used 3 cell lines, 2 TNBC cell lines, CRL2335 and MDA-MB-468 cells, and an immortalized normal breast cell line CRL8799 to determine the effect of cisplatin, TRAIL, or combination of cisplatin plus TRAIL on cell death. Western blot analysis indicated the absence of ER, PR, and HER-2 amplification in both CRL2335 and MDA-MB-468 TNBC cells and thus confirmed the triple-negative status of the cells and the cell lines overexpress EGFR (Fig. 1A). HER-2 expressing T47D cells lysate was used as positive control to determine ER, PR, and HER2 immunoreactivity of antibodies. MTT data indicate that cisplatin or TRAIL treatment induced approximately 10%–30% cell death in TNBC cell lines. However, combination of cisplatin plus TRAIL enhanced cell death to approximately 60%–70% in TNBC cells (Fig. 1B). In contrast, cisplatin plus TRAIL treatment of normal CRL8799 cells had minimal (∼10%–15%) effect on cell death (Fig. 1B). The FITC Annexin V and PI staining indicated that the proportion of cells in apoptosis in CRL2335 and MDA-MB-468 TNBC cells treated with cisplatin plus TRAIL was significantly higher (∼79% and ∼54%, respectively) as compared with those treated with cisplatin or TRAIL alone. Similar treatment of normal CRL8789 cells resulted in a moderate increase in apoptotic cells (∼13%; Fig. 1C). These observations were further confirmed in cells stained with crystal violet (Fig. 1D). Thus, taken together, the results suggested that cisplatin plus TRAIL treatment significantly enhanced cell death in both CRL-2335 and MDA-MB-468 TNBC cell lines whereas it had a minimal or moderate effect on normal CRL8799 cells.

Gobson and colleagues (21, 22) reported that EGFR played a dual role in apoptosis: increased expression gave resistance, whereas decreased expression promoted TRAIL-induced apoptosis. Leong and colleagues (23) observed that the p53 family member ΔNp63α isoform promoted the survival of primary breast cancer cells by binding to TAp73 and thus inhibiting its proapoptotic activity. The effect of cisplatin, TRAIL, or cisplatin plus TRAIL on EGFR protein and ΔNp63 was tested in CRL2335 and MDA-MB-468 TNBC cells as well as in CRL8799 cells in order to understand the mechanism involved in cell death. The data in Fig. 2 indicated that in normal CRL8799 cells treatment with cisplatin, TRAIL, or combination of cisplatin plus TRAIL had a minimal effect on both EGFR and p63 proteins compared with untreated control cells. In comparison, CRL2335 TNBC cells expressed both EGFR and p63 proteins, 24-hour treatment with cisplatin resulted in a significant reduction whereas similar treatment with TRAIL had a minimal effect on these proteins. In contrast, cisplatin plus TRAIL treatment resulted in a significant suppression of both proteins. On the other hand, MDA-MB-468 TNBC cells expressed higher level of EGFR and negligible level of p63 protein. Treatments with cisplatin, TRAIL, and cisplatin plus TRAIL for 24 hours resulted in a significant reduction in the expression of EGFR. Taken together, these data suggested that cisplatin plus TRAIL treatment had a significant effect in reducing EGFR and/or p63 protein levels in TNBC cells, whereas similar treatment had a minimal effect in CRL8799 normal breast cancer cells.

The expression of EGFR and p63 protein in CRL-2335 TNBC cells were knocked down using EGFR and/or p63 siRNA to determine whether inhibition of these proteins is essential for TRAIL-induced apoptosis. The results presented in Fig. 3A and B suggested that the inhibition of both proteins by siRNA did not increase cell death; the percentages of surviving cells were not significantly different than in control cells treated with random siRNA (Fig. 3B). Similar results were obtained in MDA-MB-468 TNBC cells when EGFR expression was knocked down using EGFR siRNA (which expressed EGFR but negligible levels of p63 protein as shown in Fig. 2) and treated with TRAIL. There was no increased cell death and the percentage of surviving cells were not significantly different than in control cells treated with random siRNA (Fig. 3D). These results indicated that inhibition of EGFR and/or p63 may not be sufficient to increase TRAIL-induced apoptosis in TNBC cells. Perhaps, combined treatment with cisplatin may be affecting other death proteins to enhance TRAIL-induced apoptosis.

Caspases are reported to play an important role in TRAIL-induced apoptosis (24). Therefore, the effect of cisplatin, TRAIL, and cisplatin plus TRAIL treatment on activation of caspases -8, -3, and -9, Bid and cleavage of PARP was investigated in CRL8799 normal cells and CRL2335 and MDA-MB-468 TNBC cells. The data in Fig. 4 indicated that the activities of caspases -8, -3, and -9 were increased in TNBC cells by cisplatin treatment leading to enhanced Bid and PARP cleavage. However, the maximum effect was observed when the same cells were treated with cisplatin plus TRAIL. In contrast, similar treatments in CRL8799 normal cells had a minimal effect on activity of caspases -8, -3, and -9, Bid and PARP cleavage.

The effect of cisplatin, TRAIL, cisplatin plus TRAIL treatment was also examined on antiapoptotic protein survivin (25), Bcl-xL and Bcl-2 in CRL8799 normal cells and CRL2335, MDA-MB-468 TNBC cells. The results in Fig. 5A showed maximum inhibition of survivin as well as Bcl-xl and Bcl-2 in TNBC cells treated with cisplatin plus TRAIL compared with cisplatin or TRAIL alone. These observations provided support to the data presented in Fig. 1B where CRL2335 and MDA-MB-468 TNBC cells treated with cisplatin plus TRAIL showed significantly decreased percentage of surviving cells. It is interesting to note that similar treatments had no effect or, at best, a minimal effect in CRL8799 normal cells.

Previous reports have suggested that survivin significantly reduces apoptosis by inhibiting caspases (26–28). If downregulation of survivin sensitizes CRL-2335 TNBC cells to TRAIL-induced apoptosis were examined by inhibiting survivin by siRNA in the presence of TRAIL. Indeed, CRL2335 TNBC cells treated with survivin siRNA and TRAIL exhibited higher activation of caspases -3 and -8 (Fig. 5B) with a concomitant and significant increase TRAIL-induced cell death (Fig. 5C) in comparison with the cells treated with random siRNA. These results provide support that survivin plays an important role in TRAIL-induced apoptosis in TNBC cells. It is interesting to note that random siRNA treatment had no effect on cell survival in CRL8799 normal cells.

The inhibition of CRL2335 TNBC cell xenografts in mice treated with cisplatin plus TRAIL was assessed in vivo in mice. The results in Fig. 6 indicated that there was a gradual regression of tumor xenografts as indicated by reduction in tumor size/volume in mice treated with cisplatin plus TRAIL as compared with control mice that were injected with vehicle. Also, the mice tolerated cisplatin plus TRAIL treatment with no signs of toxicity or weight loss during the observation period (data not shown). These findings suggest that cisplatin plus TRAIL therapy enhanced antitumor effect in vivo and, these observations were consistent with results obtained in vitro.

TNBC is the most aggressive and difficult to treat form of cancer. TNBC patients do not benefit from hormonal or herceptin-based therapies due to loss of target receptors such as ER, PR, and HER-2. The results from the present investigations clearly have shown that CRL2335 and MDA-MB-468 TNBC cells with cisplatin plus TRAIL exhibited significantly enhanced apoptosis (∼60%–70% cell death) as compared with untreated cells. Also, similar treatment had a minimal effect on CRL8799 normal breast cancer cells (∼10%–15% cell death; Fig. 1). In addition, the results from the current study also provided evidence that cisplatin exerted its influence on TRAIL-induced TNBC cell death by simultaneous activation of caspases and inhibition of antiapoptotic protein survivin. Furthermore, suppression of survivin seems to have played a major role in TRAIL-induced cell death in TNBC cells. These observations gain support from Altier (25) report that increased survivin expression enhanced tumor resistance to various apoptotic stimuli, primarily through caspase-dependent mechanism and antagonizing Survivin-induced apoptosis in tumor cells. Bcl-2 and Bcl-xl reside within the mitochondrial membrane where they act by inhibiting adaptor molecules needed for the activation of effector caspases.

The results from this study have suggested that treatment with cisplatin alone induced approximately 20%–30% and approximately 8%–12% cell death in CRL2335 and MDB-MB-468 TNBC cells, which express high and low levels of p63, respectively (Fig. 1, 2). These observations are consistent with recently published clinical (pathological assessment) data where cisplatin treatment was shown to have a significantly higher rate of remission in p63-positive tumors as compared with p63-negative tumors (29). Furthermore, Leong and colleagues (23) have reported that cisplatin treatment of breast cancer cells, which express both p63 and p73 activated c-Abl-dependent phosphorylation of p73 and dissociation of p63/p73 protein complex leading to p73-dependent transcription of proapoptotic Bcl-2 family members and thus, significantly enhanced apoptosis. These observations suggested that cisplatin in very effective in p63-positive TNBC patients. However, only a small percent of tumors are p63 positive in TNBC patients (30). The results from the present investigation have shown that the combination of cisplatin plus TRAIL is effective in both p63-positive and p63-negative TNBC cells.

Approximately 60% of TNBC cells show expression of EGFR and, and drugs that target EGFR could be beneficial to treat TNBC patients. Although Carey and colleagues (31) reported that cetuximan, a chimeric monoclonal antibody whichtargets EGFR, elicited little or no response from patients with advanced TNBC, the results from phase II clinical trial using cetuximab+carboplatin suggested favorable response (18%) and overall therapeutic gain (27%) in 102 patients with advanced TNBC (32). The observations from previous phase II clinical trials have also indicated an overall response rate of approximately 32% when unselected breast cancer patients were treated with cisplatin plus carboplatin (33–35). In more recent studies, platinum-based chemotherapy increased the response rate in TNBC tumors, with a trend toward improved survival in TNBC patients (36). A number of phase III clinical trials are in progress in which advanced TNBC patients were randomized and treated first with carboplatin or docetaxel, with crossover on progression (37). A nonrandomized phase II study using cisplatin to treat TNBC patients is also under way in the USA (38).

In conclusion, the results from the present investigations suggested that treatment of CRL2335 and MDA-MB-468 TNBC cells with cisplatin can enhance TRAIL-induced apoptosis by activating caspases along with simultaneous reduction of antiapoptotic proteins, such as survivin, Bcl-xL, and Bcl-2 expression. Comparatively, similar treatment of immortalized CRL8799 normal breast cells had a minimal effect. Individually, cisplatin and TRIAL are approved by FDA to treat other forms of cancer. As TNBC patients do not benefit from hormonal or herceptin-based therapies as well as other such as surgery, chemo- and radiation-therapy, cisplatin plus TRAIL treatment is suggested as a potential new cancer treatment strategy in TNBC patients.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.