OBATOCLAX and ABT-737 induce ER stress responses in human melanoma cells that limit induction of apoptosis.
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ABSTRACT:
SUBMITTER: Wroblewski D
PROVIDER: S-EPMC3868604 | biostudies-literature | 2013
REPOSITORIES: biostudies-literature
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Project description:BackgroundAlthough 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.MethodsWe 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.ResultsThe IC(50) of single-agent ABT-737 at 72 h was 10 ?M in imatinib-sensitive GIST-T1 and GIST882 cells, and 1 ?M 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.ConclusionWe 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.
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Project description:BackgroundMalignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance.Methodology/principal findingsHere we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity.Conclusions/significanceOur findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.
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Project description:In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE)-based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose) polymerase (PARP) and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER) stress with increased GRP78 and phosphorylated eIF2? protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA) enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination.
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