ABT-737 ameliorates docetaxel resistance in triple negative breast cancer cell line

Abstract

Purpose: This study aimed to validate the synergistic effect of ABT-737 on docetaxel using MDA-MB-231, a triple negative breast cancer (TNBC) cell line overexpressing B-cell lymphoma-2 (Bcl-2).

Methods: Western blot analysis was performed to assess expression levels of Bcl-2 family proteins and caspase-related molecules. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution was determined by flow cytometry analysis. Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) was used for pretreatment to assess the role of caspases.

Results: Cell viability of MDA-MB-231 after combination treatment with ABT-737 and docetaxel was significantly lower than that after docetaxel or ABT-737 monotherapy based on MTT assay (both P < 0.001), with a combination index of 0.41. The proportion of sub-G1 population after combination treatment was significantly higher than that after docetaxel or ABT-737 monotherapy (P = 0.001, P = 0.003, respectively). Pretreatment with z-VAD-fmk completely restored cell viability of MDA-MB-231 from apoptotic cell death induced by combination therapy (P = 0.001). Although pro-caspase-8 or Bid did not show significant change in expression level, pro-casepase-9 showed significantly decreased expression after combination treatment. Cleaved caspase-3 showed increased expression while poly (ADP-ribose) polymerase cleavage was induced after combination treatment. However, hypoxia-inducible factor 1-alpha and aldehyde dehydrogenase 1 totally lost their expression after combination treatment.

Conclusion: Combination of ABT-737 with docetaxel elicits synergistic therapeutic effect on MDA-MB-231, a TNBC cell line overexpressing Bcl-2, mainly by activating the intrinsic pathway of apoptosis. Therefore, adjunct of ABT-737 to docetaxel might be a new therapeutic option to overcome docetaxel resistance of TNBCs overexpressing Bcl-2.

Keywords: ABT-737; Bcl-2; Docetaxel; Drug resistance; Triple negative breast neoplasms.

Fig. 1. Western blot analysis for ER, HER2, Bcl-2, Bcl-xL, and Bcl-w using 6 breast cancer cell lines and MTT assay using MDA-MB-231 and MDA-MB-468 after docetaxel or ABT-737 monotherapy. (A) After lysing harvested cells (1 × 10⁶ cells per each cell line), equal amount of cell lysate extracted from 6 breast cancer cell lines using radioimmunoprecipitation assay buffer was subjected to Western blot analysis using specific antibodies. Signal intensity was normalized to that of β-actin as loading control. MTT assay was performed using MDA-MB-231 and MDA-MB-468 (5 × 10³ cells per each cell line) after treatment with indicated concentration of docetaxel (B) and ABT-737 (C) for 72 hours. Cell viability (%) was expressed as a percentage of viable cell proportion for treated sample compared to that of untreated control. Values correspond to mean ± standard deviation of three independent experiments. Black squares indicate MDA-MB-231. White squares indicate MDA-MB-231. Statistical significance was assessed by unpaired Student t-test. ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; Bcl-2, B-cell lymphoma-2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NS, not significant. ^*P < 0.05. ^**P < 0.01. ^****P < 0.0001.

Fig. 2. Cytotoxicity evaluation of docetaxel, ABT-737, and combination treatment using MDA-MB-231. (A) MDA-MB-231 cells (5 × 10³) were treated with 10 nM docetaxel and/or 1 µM ABT-737 for 48 hours. Cellular morphology was observed by phase contrast microscopy (×100). White arrow indicates a suspended shrunk cell. Black arrow points to an attached surviving cell. (B) MDA-MB-231 cells (5 × 10³) were treated with 20 nM docetaxel and/or 1 µM ABT-737 for 72 hours. Cell viability was assessed by MTT assay. Absorbance was measured at wavelength of 570 nm. Values correspond to mean ± standard deviation of three independent experiments. Statistical significance was assessed by unpaired Student t-test. NS, not significant. ^***P < 0.001.

Fig. 3. Cell cycle analysis. (A) MDA-MB-231 cells (1 × 10⁵) were treated with 10 nM docetaxel and/or 1 µM ABT-737 for indicated time periods and stained with 50 µg/mL of propidium iodide including 100 µg/mL of RNase A for 30 minutes at 37℃. Top, middle, and bottom rows were treated with ABT-737, docetaxel, and both drugs, respectively. Graphs depict data of flow cytometry results after treatment for 72 hours according to cell cycle (B) and treatment agents (C). Values correspond to mean ± standard deviation of 3 independent experiments. Statistical significance was assessed by unpaired Student t-test. NS, not significant. ^*P < 0.05. ^**P < 0.01.

Fig. 4. MTT assay and cell cycle analysis after docetaxel, ABT-737, and combination treatment using MDA-MB-231 with or without pretreatment of z-VAD-fmk. Twenty µM of z-VAD-fmk was pretreated for 1 hour before 10 nM docetaxel, 1 µM ABT-737, or combination treatment for 48 hours. (A) Cell viability after treatment was determined by MTT assay. Statistical significance was assessed by unpaired Student t-test. Values correspond to mean ± standard deviation of five independent experiments. (B) After treatment with docetaxel, ABT-737, or combination therapy, MDA-MB-231 cells (1 × 10⁵) were stained with 50 µg/mL of propidium iodide with (bottom) or without (top) pretreatment of 20 µM z-VAD-fmk. (C) Percentage of each phase is shown as 100% stacked bar chart. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NS, not significant. ^**P < 0.01.

Fig. 5. Expression of apoptosis related molecules by Western blot. MDA-MB-231 cells were treated with 10 nM docetaxel, 1 µM ABT-737, or combination of the two drugs for 24 hours and 48 hours. After lysing harvested cells (1 × 10⁶), 40 µg of protein from each cell lysate was loaded and blotted with specific antibodies, including caspase-8, caspase-9, cleaved caspase-3, Bid, and PARP antibodies. Expression levels of ALDH1 and HIF-1α were also tested under the same condition. β-actin was used as loading control. PARP, poly (ADP-ribose) polymerase; ALDH1, aldehyde dehydrogenase 1; HIF, hypoxia-inducible factor.