Combination of Manumycin A and Mebendazole in Human Breast Cancer Cell Lines
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We evaluated the combination of MA and Mbz in wild-type and HER2 transfected MDA-MB-231 and MCF-7 human breast cancer cell-lines in vitro and in xenografted mouse model. Methods: XTT colorimetric and SRB assays were used to determine cell viability in culture after single and combination treatment. Flow cytometry and western blotting were used to test the role of cell cycle arrest and apoptosis in cytotoxicity of single and combination treatment. We used PI for cell cycle and Annexin-V-FITC for apoptosis. We probed for Cyclins E and B and cleaved PARP. In vivo MDA-MB-231cell pair was used for dorsal subcutaneous xenogratfs in nu/nu Swiss mice. MA and Mbz were administered ip in single and combination treatments. The change in tumor volume was used to assess effectiveness. Results: MA and Mbz were cytotoxic in all four cell-lines at micro-molar levels. Mbz is more effective in MDA-MB-231 cells. MA 1st and Mbz1st showed additional benefit in MDA-MB-231/ErbB2 and MCF-7/Her18 cells, respectively. MA arrested MCF-7cells at G1/S and MDA-MB-231 cells at G2/M phase. No cleaved PARP was detected at 89kDa in all four cell-lines. In vivo, concurrent treatment showed additional benefit in MDA-MB-23/ErbB2. Mbz1st treatment showed additional benefit in male but not female mice with MDA-MB-231 xenografts. Liver histopathology showed necrosic, apoptosic and microangiopathic changes with combination treatment.Discussion: MA and Mbz were cytotoxic in all four cell-lines at micro-molar levels, with Mbz being more effective in MDA-MB-231 cells. Combination therapy showed additional benefit over single agent treatment in HER2 transfected but not wild-type cells. Apoptotic cell death did not play a major role in cytotoxicty. Sequence of drug administration, drug concentration, ratio of MA to Mbz, and targeted cells affect final outcome of combination treatment in vitro. Sequence of drug administration, type of cancer cells, and gender affect treatment outcome in vivo. Liver toxicity was observed with combination treatment. Conclusion: We were able to identify factors affecting MA and Mbz combination outcome. This combination is antagonistic with some exceptions. We are the first to show anticancer activity of Mbz in breast cancer xenografts using a microemulsion formulation.