title: Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of β-tubulin.
abstract: 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 and withanolide A). 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 bipolar 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 withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys(303) of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of 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.
authors.affiliation: From the Departments of Pharmacology and Chemical Biology and.
authors.forename: Marie L
authors.initials: ML
authors.lastname: Antony
authors.forename: Joomin
authors.initials: J
authors.lastname: Lee
authors.forename: Eun-Ryeong
authors.initials: ER
authors.lastname: Hahm
authors.forename: Su-Hyeong
authors.initials: SH
authors.lastname: Kim
authors.forename: Adam I
authors.initials: AI
authors.lastname: Marcus
authors.forename: Vandana
authors.initials: V
authors.lastname: Kumari
authors.forename: Xinhua
authors.initials: X
authors.lastname: Ji
authors.forename: Zhen
authors.initials: Z
authors.lastname: Yang
authors.forename: Courtney L
authors.initials: CL
authors.lastname: Vowell
authors.forename: Peter
authors.initials: P
authors.lastname: Wipf
authors.forename: Guy T
authors.initials: GT
authors.lastname: Uechi
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Yates
authors.forename: Guillermo
authors.initials: G
authors.lastname: Romero
authors.forename: Saumendra N
authors.initials: SN
authors.lastname: Sarkar
authors.forename: Shivendra V
authors.initials: SV
authors.lastname: Singh
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D015536:Down-Regulation / Q000187:drug effects* / Q000235:genetics; D005260:Female; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D051379:Mice; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*; D054358:Withanolides / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D014404:Tubulin; D054358:Withanolides; C009684:withaferin A
keywords: Breast Cancer; Cancer Chemoprevention; Cell Cycle; Natural Products; Tubulin; Withaferin A