Synergistic induction of apoptosis by the Bcl-2 inhibitor ABT-737 and imatinib mesylate in gastrointestinal stromal tumor cells

Abstract

Background: Although imatinib mesylate has revolutionized the management of patients with gastrointestinal stromal tumor (GIST), resistance and progression almost inevitably develop with long-term monotherapy. To enhance imatinib-induced cytotoxicity and overcome imatinib-resistance in GIST cells, we examined the antitumor effects of the pro-apoptotic Bcl-2/Bcl-x(L) inhibitor ABT-737, alone and in combination with imatinib.

Methods: We treated imatinib-sensitive, GIST-T1 and GIST882, and imatinib-resistant cells with ABT-737 alone and with imatinib. We determined the anti-proliferative and apoptotic effects by cell viability assay, flow cytometric apoptosis and cell cycle analysis, immunoblotting, and nuclear morphology. Synergism was determined by isobologram analysis.

Results: The IC(50) of single-agent ABT-737 at 72 h was 10 μM in imatinib-sensitive GIST-T1 and GIST882 cells, and 1 μM in imatinib-resistant GIST48IM cells. ABT-737 and imatinib combined synergistically in a time- and dose-dependent manner to inhibit the proliferation and induce apoptosis of all GIST cells, as evidenced by cell viability and apoptosis assays, caspase activation, PARP cleavage, and morphologic changes. Isobologram analyses revealed strongly synergistic drug interactions, with combination indices <0.5 for most ABT-737/imatinib combinations. Thus, clinically relevant in vitro concentrations of ABT-737 have single-agent antitumor activity and are synergistic in combination with imatinib.

Conclusion: We provide the first preclinical evidence that Bcl-2/Bcl-x(L) inhibition with ABT-737 synergistically enhances imatinib-induced cytotoxicity via apoptosis, and that direct engagement of apoptotic cell death may be an effective approach to circumvent imatinib-resistance in GIST.

Figure 1

ABT‐737, but not its enantiomer A‐793844, significantly inhibits the viability of GIST cells. (A) GIST‐T1 and GIST882 cells were incubated with 1 μM imatinib or with DMSO vehicle‐control for 24–72 h, and lysates were analyzed by immunoblotting for expression of Bcl‐2, Bcl‐xL, and Mcl‐1. A lysate of untreated A204 sarcoma cells was used as a positive control to demonstrate pro‐survival Bcl‐2 protein expression (rightmost lane). β‐actin was used to demonstrate equal protein loading. (B) GIST‐T1 and GIST882 cells were incubated with increasing concentrations (0, 0.1, 1, 10, 20 μM) of ABT‐737 (blue) or enantiomer A‐793844 (red) as single agents for 24, 48, and 72 h. Inhibition of viability was determined by the MTS cell viability assay with absorbance measured at 490 nm. Symbols represent the mean of duplicate experiments; error bars represent standard deviation (SD). Three asterisks (***) represent Bonferroni's post‐test p‐value<0.0001 versus enantiomer A‐793844 at identical timepoints.

Figure 2

ABT‐737 and imatinib synergistically inhibit the viability of GIST cells. (A) GIST‐T1 and GIST882 cells were treated in a checkerboard fashion with increasing concentrations of imatinib (0, 0.1, 1, 10 μM) and ABT‐737 (0, 0.1, 1, 10, 20 μM) and analyzed at 72 h by MTS cell viability assay with absorbance measured at 490 nm. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by one‐way ANOVA, and three asterisks (***) represent p < 0.0001 versus DMSO control (0 μM ABT‐737 + 0 μM imatinib) by Bonferroni's post‐test comparison. (B) The Combination Indices (CI) corresponding to the Imatinib/ABT‐737 combinations tested in Figure 2A was determined by isobologram analysis (see Materials and methods). A representative normalized isobologram generated for GIST882 cells (Figure 2B, top) and a Fraction affected (Fa)‐CI plot (Figure 2B, bottom), graphically depict the interaction between imatinib and ABT‐737. Similar results for GIST‐T1 cells are available in Supplementary figure 1. Overall results of isobologram (synergy) analyses for all three cell lines are available in the Supplementary table.

Figure 3

ABT‐737 and imatinib induce apoptosis synergistically in imatinib‐sensitive cells. GIST‐T1 and GIST882 cells were treated with imatinib (0, 0.1, 1 μM) and ABT‐737 (0, 0.1, 1, 10 μM) for 48 h at 37 °C and apoptosis was determined by cell cycle analysis (PI‐labeling of sub‐G1 phase cells) and TUNEL assay, using flow cytometry. Apoptosis was quantified in GIST‐T1 and GIST882 cells by (A) sub‐G1 DNA content, and (B) FITC‐positivity. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by one‐way ANOVA, and three asterisks (***) represent p < 0.0001 versus DMSO control by Bonferroni's post‐test. (C) Representative western blots of GIST882 cells treated with ABT‐737 and imatinib as single agents (Figure 3C, left) and in combination Figure 3C, right). Cells were treated for 72 h with vehicle (DMSO) or with increasing concentrations of imatinib and/or ABT‐737, and caspase 3 and PARP cleavage were assessed by immunoblotting. Treatment with Etoposide (10 μM) was used as a positive control for caspase activation. β‐actin was used to demonstrate equal protein loading. Abbreviations: (F), full length; (C), cleaved.

Figure 4

The morphologic features of apoptosis are induced by ABT‐737 in GIST cells. GIST882 cells were treated with imatinib (1 μM) alone, or in combination with ABT‐737 (0.1, 1, 10, 20 μM) for 72 h and apoptotic cell death was evaluated by assessment of nuclear morphology after ethidium bromide/acridine orange (EB/AO) staining. (A) Representative micrographs of GIST882 cells treated with vehicle (DMSO), 1 μM imatinib, 10 μM ABT‐737, or both, demonstrating nuclear fragmentation and condensation in ABT‐737‐treated cells. Original magnification, ×200. Abbreviations: (N), normal nuclei; (A), apoptotic nuclei; Thick arrow, late apoptosis; Thin Arrow, early apoptosis. (B) Quantitative assessment of normal and apoptotic GIST882 cells treated with 1 μM imatinib, alone or with ABT‐737 (0, 0.1, 1, 10, 20 μM).

Figure 5

Combined treatment with ABT‐737 and imatinib induces apoptosis synergistically to overcome imatinib‐resistance in GIST48IM cells. (A) The antiproliferative effect of single‐agent imatinib (left) and single‐agent ABT‐737 (right) in imatinib‐resistant GIST48IM cells was examined after 24, 48 and 72 h of treatment, using the MTS cell viability assay. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by two‐way ANOVA. (B) The effect of combined ABT‐737 (0, 0.1, 1, 10, 20 μM) and imatinib (0, 0.1, 1, 10 μM) on the viability of GIST48IM cells at 72 h. Columns, mean of duplicate experiments; error bars, SD. Results were analyzed by one‐way ANOVA. (C) Normalized isobologram (top) and Fa‐CI plot (bottom) of GIST48IM cells, graphically depicting synergistic, additive, and antagonistic interactions between imatinib and ABT‐737 in this cell line. (D) To determine whether reductions of GIST48IM cell viability were due to apoptosis, nuclear morphology was assessed by EB/AO staining after treatment with ABT‐737 and imatinib for 72 h. Representative micrographs of ethidium bromide/acridine orange‐stained GIST48IM cells. Original magnification, ×200. Abbreviations: (N), normal nuclei; Thick arrow, late apoptosis; (A), apoptotic nuclei; Thin Arrow, early apoptosis. (E) Quantification of normal and apoptotic cells treated with 1 μM imatinib alone, or combined with ABT‐737 (0.1, 1, 10, 20 μM). (F) Immunoblot analysis of the expression of Bcl‐2, Bcl‐xL and Mcl‐1, as well as the cleavage of caspase 3 and PARP, after treatment with DMSO, 1 μM imatinib, 10 μM ABT‐737, or a combination for 72 h. Actin was used to demonstrate equal loading. Abbreviations: (F), full length; (C), cleaved.