Dietary Isothiocyanate-induced Apoptosis via Thiol Modification of DNA Topoisomerase IIα

Ren-Kuo Lin,Nai Zhou,Yi Lisa Lyu,Yuan-Chin Tsai,Chang-Hsien Lu,John Kerrigan,Yu-Tsung Chen,Ziqiang Guan,Tao-Shih Hsieh,Leroy F Liu

doi:10.1074/jbc.m111.258137

Abstract

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.

Highlights

Many dietary components have been shown to possess cancer preventive activities [1]
Our results suggest that thiol modification of Top2␣ may contribute to apoptosis induction in transformed cells by ITCs
ITCs can stimulate the formation of Top2␣-DNA covalent adducts in vitro through a thiol modification mechanism. These results suggest that thiol modification of Top2␣ and, the formation of Top2␣-DNA covalent adducts may contribute to apoptosis induction in transformed cells by ITCs

Summary

Chemicals and Reagents

BITC, PEITC, SFN, glutathione (GSH), selenomethionine, selenocysteine, VP-16 (etoposide), DSF, sodium selenite, and thiazolyl blue tetrazolium bromide were purchased from Sigma. Anti-␣-tubulin antibodies were purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. The anti-Top2␤ antibodies were purchased from Santa Cruz Biotechnology. The anti-Top2␣ antibodies were gift from Dr Jaulang Hwang (IMB, Acdemia Sinica, Taiwan). DMEM, and RPMI 1640, were purchased from Invitrogen. All other tissue culture media and reagents were purchased from Invitrogen. The reaction mixture (20 ␮l total volume) containing 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, 30 ␮g/ml bovine serum albumin, 1 mM ATP, 10 ng of 3Ј-end 32P-labeled YEpG DNA, 10 ng of purified human Top2␣, and the compound of interest were incubated at 37 °C for 30 min. Agarose gel, 0.5ϫ Tris-Phosphate-EDTA buffer (45 mM TrisPhosphate, pH 8.0, 1 mM EDTA), 2.5 V/cm.

Apoptosis Assay

Gene Silencing by siRNAs

BITC PEITC

RESULTS

DISCUSSION