Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Downregulation and Covalent Binding at Cysteine-303 of {beta}-Tubulin [Signal Transduction]

December 2nd, 2013 by Antony, M. L., Lee, J., Hahm, E.-R., Kim, S.-H., Marcus, A. I., Kumari, V., Ji, X., Yang, Z., Vowell, C. L., Wipf, P., Uechi, G. T., Yates, N. A., Romero, G., Sarkar, S. N., Singh, S. V.

Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally-occurring C6,C7-epoxy analogs of WA [withanone (WE) and withanolide A (WLA)]. A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bi-polar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in WE or WLA, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA at cysteine-303 of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket was located on the surface of the β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.
  • Posted in Journal of Biological Chemistry, Publications
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