| Objective:ABT-737 is a small-molecule chemical that mimics the direct binding to the hydrophobic groove in Bcl-2, Bcl-xL, and Bcl-w, and consequently prevents them from sequestering proapoptotic BH3-only proteins such as t-Bid, Bad, and Bim. The Bcl-2 antagonist ABT-737 targets Bcl-2/Bcl-xL but not Mcl-1, which may confer resistance to this agent in various cancers with high level of Mcl-1. In this study, we plan to explore the potential anti-tumor efficacy by combing ABT-737 with chemotherapeutic agents against human cancer cells.Methods:(1) MTT method was used to detect cytotoxicity of chemotherapeutic agents and/or ABT-737 on human cancer cells. (2) Analysis of apoptosis by propidium iodide staining or Annexin V/PI staining. (3) Determination of mitochondrial membrane depolarization by JC-1 staining. (4) The expression of protein by western blotting. (5) Analysis of Bax conformational change by flow cytometry. (6) Analysis of mRNA by Real-Time reverse transcription. (7) Immunoprecipitation to detect the interaction between protein to protein. (8) Gene silencing by small interfering RNA. (9) Gene amplification by plasmid. (10) Xenografted athymic mice model.Results:(1) Anti-tumor property of gemcitabine and ABT-737 in vitro and in vivo:We demonstrated that the combination of gemcitabine and ABT-737 exhibited synergistic cytotoxicity and induced significant apoptosis in multiple cancer types. The enhanced apoptosis induced by gemcitabine plus ABT-737 was accompanied by mitochondrial depolarization, caspases-3 activation and PARP cleavage in the 95-D and 5637 cells. Importantly, in ABT-737-resistant cancer cells, the interaction between USP9X and Mcl-1, increased by ABT-737 treatment, could be disrupted by gemcitabine, thus enhanced the ubiquitination and the subsequent degradation of Mcl-1 protein, and ultimately resulted in the synergism of these two drugs. Moreover, the increased anti-cancer efficacy of ABT-737 combined with gemcitabine was further validated in a human lung cancer 95-D xenograft nude mice model and confirmed in clinical patient sample.(2) Anti-tumor property of GDC-0941 and ABT-737 in vitro and in vivo:we demonstrated that GDC-0941 potently sensitized breast cancer cells to ABT-737 in vitro and in vivo. ABT-737 exhibited limited cytotoxicity in breast cancer cells; however, when combined with GDC-0941, it displayed strong synergistic anti-tumor effect. The combination of GDC-0941 and ABT-737 caused enhanced apoptosis, accompanied with the greater extent of mitochondrial depolarization and the activation of caspase cascades. In addition, GDC-0941 could promote proteasome-mediated degradation of Mcl-1 protein, thus enhanced the anticancer efficacy of ABT-737. Furthermore, GDC-0941 combined with ABT-737 synergistically inhibited tumor growth on MDA-MB-231 xenograft nude mice models.(3) Anti-tumor property of As2O3 and ABT-737 in vitro:it is demonstrated that As2O3 potently sensitized leukemia cells to ABT-737 in vitro. Furthermore, ABT-737 combined with As2O3 could synergistically induce apoptosis in leukemia cells, and the combination of ABT-737 and As2O3 can synergistically decrease the mitochondrial membrane potential. Additionally, ABT-737 combined with As2O3 could regulate Bax/Bak balance and reduce levels of Caspase-8, Bid, Caspase-3, XIAP protein, etc.Conclusion:(1) Gemcitabine and ABT-737 in combination exhibited substantial synergistic anti-tumor efficacy against human cancer cells both in vitro and in vivo, which attributed to caspase-dependent apoptosis via disrupting the interaction between USP9X and Mcl-1 proteins.(2) GDC-0941 acted in concert with ABT-737 to exert synergistic anticancer efficacy against breast cancer cells in vitro and in vivo, which attributed to the downregulation of Mcl-1 protein.(3) ABT-737 combined with As2O3 could synergistically suppress the proliferation of cancer cells by inducing apoptosis in cancer cells.
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