Covalent Binding to Tubulin by Isothiocyanates: A MECHANISM OF CELL GROWTH ARREST AND APOPTOSIS

Lixin Mi,Zhen Xiao,Brian L Hood,Sivanesan Dakshanamurthy,Xiantao Wang,Sudha Govind,Thomas P Conrads,Timothy D Veenstra,Fung-Lung Chung

doi:10.1074/jbc.m802330200

Abstract

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl-ITC (BITC), phenethyl-ITC (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models and induce apoptosis and cell cycle arrest in various cell types. The biochemical mechanisms of cell growth inhibition by ITCs are not fully understood. Our recent study showed that ITC binding to intracellular proteins may be an important initiating event for the induction of apoptosis. However, the specific protein target(s) and molecular mechanisms were not identified. In this study, two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC. We examined whether binding to tubulin by ITCs could lead to cell growth arrest. The proliferation of A549 cells was significantly reduced by ITCs, with relative activities of BITC > PEITC > SFN. All three ITCs also induced mitotic arrest and apoptosis with the same order of activity. We found that ITCs disrupted microtubule polymerization in vitro and in vivo with the same order of potency. Mass spectrometry demonstrated that cysteines in tubulin were covalently modified by ITCs. Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN. Together, these results support the notion that tubulin is a target of ITCs and that ITC-tubulin interaction can lead to downstream growth inhibition. This is the first study directly linking tubulin-ITC adduct formation to cell growth inhibition.

Highlights

IntroductionEllman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN
Two-dimensional gel electrophoresis of human lung cancer A549 cells treated with radiolabeled PEITC and SFN revealed that tubulin may be a major in vivo binding target for ITC
BITC, PEITC, and SFN Inhibited Cell Proliferation—Incubation of A549 cells with BITC, PEITC, and SFN for 24 h at a concentration range of 1–100 ␮M caused a dose-dependent inhibition of cell proliferation (Fig. 1B)

Summary

Introduction

Ellman assay results indicated that the modification levels follow the same order, BITC > PEITC > SFN Together, these results support the notion that tubulin is a target of ITCs and that ITCtubulin interaction can lead to downstream growth inhibition. The time course of this protein binding correlated well with the inhibition of proliferation and the induction of apoptosis, providing preliminary evidence suggesting that modification of cellular proteins via direct covalent binding to ITCs may be an early event for apoptosis induction These results prompted us to identify which specific proteins are ITC binding targets in vivo, to determine how these proteins desorption/ionization-time-of-flight/time-of-flight; NMPEA, N-methyl phenethylamine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PI, propidium iodide; RPLC, reversed-phase liquid chromatography; MS/MS, tandem mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; HDAC, histone deacetylase. The studies described here are important because they define the chemical basis of ITC-induced cell growth inhibition, they identify structural information that will enable the rational design of new, more specific and more potent ITC-related compounds for cancer prevention and treatment studies

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Results

Conclusion