Withaferin A (Synonyms: NSC 101088, NSC 273757)

Cell experiment:

Ovarian epithelial cancer cell line A2780 is maintained in RPMI1640 medium supplemented with insulin (5 μg/mL), penicillin/streptomycin (100 IU/mL and 100 μg/mL respectively) and 10% fetal bovine serum (FBS) from Hyclone. Withaferin A, cisplatin (CIS) and other reagents are prepared in DMSO. Cisplatin is prepared fresh each time[3].

Animal experiment:

Mice[2]Vimentin homozygous-deficient mice (Vim−/−) and mice that are vimentin-heterozygous deficient (Vim+/−) in the 129/Svev background are used in the assay. In brief, mice between 4 and 6 wk of age are anesthetized by intraperiteoneal (i.p.) injection of ketamine and xylazine. Corneas are topically anesthetized by application of proparacain eye drop, and 1 μL drop of dilute 0.15 M sodium hydroxide is applied for 1 min. The cornea is immediately washed extensively in saline solution, and corneal and limbal epithelium gently removed by scraping. The cornea is topically treated with atropine eye drop and covered with tobramycin and erythromycin antibiotic eye ointment. Withaferin A or 12-D WS (2 mg/kg solubilized in DMSO) or vehicle (DMSO) is injected i.p. in respective drug or control groups of mice after their recovery from corneal injury, and subsequently every day thereafter for a period of 10 days. Mice are humanely killed and eyes enucleated. The anterior segment half of eyes are dissected and corneal buttons are prepared. Corneal tissues are fixed in 100% acetone for 20 min, washed in PBS for 1 hr, and blocked for 18 hr in 1% BSA-PBS at 4°C. Cornea whole-mount staining is performed by incubating tissues in FITC-conjugated rat anti-mouse CD31 antibody (1:333 dilution in 1% BSA-PBS) for 12 hr, washed away for 24 hr at 4°C in 1% BSA-PBS, and affixed to glass slides with a coverslip. Fluorescent staining is visualized on microscope, and quantified by importing digital images to NIH ImageJ[2].

References:

[1]. Kaileh M, et al. Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem. 2007 Feb 16;282(7):4253-64. Epub 2006 Dec 6.
[2]. Bargagna-Mohan P, et al. The tumor inhibitor and antiangiogenic agent withaferin A targets the intermediate filament protein vimentin. Chem Biol. 2007 Jun;14(6):623-34.
[3]. Kakar SS, et al. Withaferin A (WFA) inhibits tumor growth and metastasis by targeting ovarian cancer stem cells. Oncotarget. 2017 Aug 10;8(43):74494-74505.