The breast cancer BT474 and MCF7 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 medium and Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen Life Technologies). PF4708671 was purchased from Sigma-Aldrich (St. Louis, MO, USA), ABT263 (navitoclax) from Selleckchem (Houston, TX, USA) and Sorafenib from Bayer Healthcare Pharmaceuticals (New Haven, CT, USA).

The BT474 cells were treated with 0, 0.5, 1 and 2 µM ABT263, or 0, 5, 10 and 20 µM PF4708671. The treated cells were stained with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; BD Biosciences, San Jose, CA, USA) for the assessment of apoptotic cell death, as previously described (15). As a positive control, MCF7 cells were treated with 5 µM sorafenib and deprived of glucose for 24 h.

The cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS] supplemented with a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). The protein concentrations were then measured using the Bradford method (16). The cell lysates were separated via SDS-PAGE and transferred to nitrocellulose membranes, followed by immunoblotting with specific primary and horseradish peroxidase-conjugated secondary antibodies. Antibodies specific for cleaved poly(ADP-ribose) polymerase (PARP; rabbit polyclonal; 1:1,000; cat no. 9541), p-S6 at Ser240/244 (rabbit monoclonal; 1:2,000; cat no. 4838), S6K1 (rabbit polyclonal; cat no. 9202; 1:1,000) and survivin (rabbit monoclonal; 1:1,000; cat no. 2808) were obtained from Cell Signaling Technology (Beverly, MA, USA), an antibody for Bcl-xL (mouse monoclonal; 1:1,000; cat no. 610746) was obtained from BD Biosciences (Franklin Lakes, NJ, USA), and an antibody for β-actin (mouse monoclonal; 1:5,000; cat no. A5316) was obtained from Sigma-Aldrich. The immunoreactive bands were visualized using SuperSignal West Pico Chemiluminescent Substrates (Thermo Scientific Pierce, Rockford, IL, USA).

Bcl-2 (cat no. sc-29214), Bcl-xL (cat no. sc-43630) and control (cat no. sc-37007) siRNAs were purchased from Santa Cruz Biotechnology, Inc. (San Jose, CA, USA). S6K1 (cat no. SI00301721) and control siRNAs (cat no. 1022076) were purchased from Qiagen, Inc. (Valencia, CA, USA). Transient 24-h siRNA transfections were performed with Lipofectamine® RNAiMAX reagent, according to the manufacturer's instructions (Invitrogen Life Technologies). The BT474 cells were transiently transfected with the indicated siRNAs for 24 h.

The cell viability was determined by measuring the mitochondrial conversion of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide to a colored product, as previously described (15). Images of cell populations were captured microscopically with an Olympus CKX41 microscope (Olympus Corporation, Tokyo, Japan).

RNA isolation and RT-PCR analysis were conducted as previously described (17). The following primers were used: Survivin, sense, 5′-GGACCACCGCATCTCTAC-3′ and anti-sense, 5′-CAGCCTTCCAGCTCCTTG-3′, with a 156-bp product (15); and β-actin, sense, 5′-GGATTCCTATGTGGGCGACAG-3′ and anti-sense, 5′-CGCTCGGTGAGGATCTTCATG-3′, with a 438-bp product (15).

The effects of two-drug combinations were assessed by isobologram analysis. The cells were treated with different concentrations of ABT263 (0–20 µM) and PF4708671 (0–40 µM). Combinations resulting in 25±1% cytotoxicity were expressed as a percentage of each single drug, alone, producing an equivalent level of cytotoxicity [fractional inhibitory concentrations (FIC)=concentration of each drug in the combination/concentration of each drug alone]. When the sum of the FIC was 1, the combination was additive and the graph was expressed as a straight line; when the sum was <1, the combination was synergistic and the graph demonstrated a concave shape; and when the combination was >1, the combination was antagonistic and the graph demonstrated a convex shape.

Data are expressed as the mean ± standard deviation of three independent experiments. A Student's t-test was performed to analyze differences between groups. All statistical analyses was performed using SigmaPlot software (version 11; Systat Software Inc., San Jose, CA, USA) and P<0.05 was considered to indicate a statistically significant difference.

Initially, the effects of ABT263 on BT474 on breast cancer cell apoptosis were examined. Apoptosis assays were performed using Annexin V/PI staining and western blot analysis of cleaved PARP expression. As indicated in Fig. 1A and B, treatment with ≤2 µM ABT263 did not induce marked apoptotic cell death in the BT474 cells. Furthermore, protein expression levels of myeloid cell leukemia-1 [Mcl-1 (a Bcl-2-related protein)] were upregulated upon treatment with ABT-263, however, the expression levels of Bcl-2 and Bcl-xL did not change (Fig. 1B).

BT474 breast cancer cells are characterized by the overexpression of S6K1 (18). PF4708671 treatment suppressed S6K1 activity in the BT474 cells, as evidenced by the decreased phosphorylation of S6 at Ser240/244 (Fig. 1C). However, PF4708671 (≤20 µM) resulted in no significant cell death (Fig. 1D). These data indicate that ABT263 or PF4708671 treatment alone is not sufficient to cause apoptosis.

The present study examined the effect of combined treatment with ABT263 and PF4708671 on the induction of cell death. Cell viability was reduced by 20% following treatment with 10 µM PF4708671 and by 27% following treatment with 1 µM ABT263. However, combination treatment with the two agents resulted in a significant reduction in cell viability (~60%; P<0.01) (Fig. 2A) and the induction of apoptotic cell death, as examined by PARP cleavage and microscopy images of apoptosis (Fig. 2B and C). Furthermore, isobolographic analysis clarified the synergy between ABT263 and PF4708671 (Fig. 2D). These data indicate that combined treatment with ABT263 and PF4708671 synergistically induces cell death in BT474 breast cancer cells.

To further investigate the effect of the targeting of S6K1 and Bcl-2/Bcl-xL on BT474 cell death, the inhibitory effects of siRNAs targeting S6K1 and Bcl-2/Bcl-xL were investigated. The siRNAs caused marked knockdown of the target genes (Fig. 3A). The combined siRNA knockdown of S6K1 and Bcl-2/Bcl-xL significantly inhibited cell viability and induced PARP cleavage (P<0.001). By contrast, S6K1 or Bcl-2/Bcl-xL siRNA alone did not induce PARP cleavage (Fig. 3B) and exhibited marginal inhibitory effects on cell viability (Fig. 3C). These data indicate that the synergistic targeting of S6K1 and Bcl-2/Bcl-xL may induce BT474 cell death.

The present study invesitgated the mechanisms involved in ABT263- and PF4708671-induced cell death. Combined treatment with ABT263 and PF4708671 appeared to have no effect on the expression of Bcl-2-related proteins, such as Bcl-2, Bcl-xL and Mcl-1. Notably, survivin protein, unlike inhibitor of apoptosis (IAP) family members, such as X-linked IAP (XIAP) and cellular IAP 1 (cIAP1), was significantly decreased in the presence of ABT263 and PF4708671 (Fig. 4A). However, combined treatment with ABT263 and PF4708671 did not modulate survivin mRNA expression levels (Fig. 4B), indicating that the observed changes in survivin expression following ABT263 and PF4708671 exposure did not involve transcriptional regulation.

To further clarify the role of survivin in ABT263 and PF4708671-induced cell death, empty or myc-survivin vectors were transiently transfected into treated BT474 cells prior to receiving combined ABT263 and PF4708671 treatment. The expression of myc-tagged survivin in the BT474 cells significantly suppressed the cell cytotoxicity induced by PF4708671 and ABT263 (P<0.001; Fig. 4C and D), and attenuated the PARP cleavage induced by these inhibitors (Fig. 4E). These data indicate that the cell death induced by PF4708671 and ABT263 is at least in part mediated by the downregulation of survivin protein.