Covalent binding to tubulin by isothiocyanates: A mechanism of cell growth arrest.

A95

Isothiocyanates (ITCs) found in cruciferous vegetables, including benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), inhibit carcinogenesis in animal models. They have been shown to decrease cell proliferation by inducing apoptosis and cell cycle arrest in a variety of cell types. The chemical mechanisms by which these compounds induce cell growth arrest or apoptosis are not fully understood. Our recent data showed that binding to intracellular proteins by these electrophiles underlies a mechanism for apoptosis. The 2-D gel electrophoresis using human lung cancer cells treated with the radiolabeled PEITC and SFN revealed that their binding to proteins is selective and tubulin is one of the potential cellular protein targets. The purpose of this study was to examine by treating A549 cells with BITC, PEITC, and SFN whether ITC binding to tubulin underlies a molecular mechanism for cell growth arrest. The growth of cells was significantlysuppressed by all ITCs with different potencies (BITC IC₅₀ = 13.5 µM,PEITC IC₅₀ = 18.3 µM, SFN IC₅₀ = 43.0 µM). When cells were treated with 10 µM of BITC and PEITC, the proportionof cells residing in G₁ and S phaseswere decreased, while the cell population in G₂/M phasewas markedly increased. Immuno-staining studies using phosphor-histone 3 as a mitotic biomarker indicated that cells were arrested in mitosis, not G₂ phase. Interestingly, cells treated with 10 µM of SFN showed only G₁ arrest. However, at 30 µM SFN can also block mitosis. Therefore, the order of potency for arresting cells in mitosis was BITC > PEITC > SFN. The induction of apoptosis measured by caspase-3 activity also followed the same order. The inhibition of tubulin polymerization in an in vitro cell-freesystem and the in vivo microtubule network in treated cells was examined. We found in both systems ITCs disrupted tubulin assembly and this activity, again, was found following the same order. Ellman assay and mass spectrometry study of the ITCs-treated tubulin showed that the ITCs can covalently modify cysteine residues in tubulin with different levels of modification following the order of BITC > PEITC > SFN. Taken together, the modification of tubulin by ITCs is well correlated with the down-stream biological events, including microtubule destabilization, anti-mitosis and apoptosis induction. Thisstudy is the first to demonstrate that ITC binding to tubulin is an important chemical event leading to cell growth arrest.