Suppression of microtubule dynamics by sulforaphane: A novel mechanistic basis for its antiproliferative action

C64 Sulforaphane (SFN, (R)-1-isothiocyanato-4-(methylsulfinyl)-butane), a naturally-occurring dietary isothiocyanate found in cruciferous vegetables, has been shown to possess multiple anticarcinogenic activities, including cell cycle arrest and induction of apoptosis. However, SFN’s molecular targets and its mechanism(s) of action are not well defined. We previously found that SFN inhibits proliferation of MCF7 breast cancer cells in concert with mitotic arrest by suppressing microtubule dynamic instability, similar to but weaker than the actions of well-known antimitotic anticancer drugs including Paclitaxel and the Vinca alkaloids. In this study, we examined the interaction of SFN with pure mammalian brain tubulin and microtubules in vitro. We have found that SFN inhibits the polymerization of pure and microtubule associated protein (MAP)-rich brain tubulin in a concentration-dependent manner with 50% inhibition occurring at a concentration of 89 uM SFN (pure tubulin) and at 389 uM SFN (MAP-rich tubulin). High concentrations of SFN (≥ 80 uM SFN) resulted in the formation of unproductive tubulin aggregates as observed by electron microscopy. Furthermore, in agreement with our previous findings that SFN stabilizes microtubules in breast cancer cells, we established that it strongly suppresses the plus end dynamic instability behavior of individual microtubules assembled from pure tubulin in vitro as determined by differential interference video microscopy. Specifically, it reduces the growing and the shortening rates and lengths that the microtubules grew and shortened in a concentration-dependent manner, at concentrations substantially below those required to change the mass of assembled polymer. The overall dynamicity of the microtubules was reduced by 78 % at 15 uM SFN, while the time microtubules spent in an attenuated (paused) state significantly increased. We also investigated the effects of SFN on soluble tubulin by fluorescence spectroscopy. We have established that SFN quenches the intrinsic tryptophan fluorescence of tubulin and increases the fluorescence of 8-anilino-1-naphthalenesulfonate (ANS)-tubulin complexes in a concentration-dependent manner, suggesting that the binding of SFN to tubulin induces conformational changes in tubulin. Competitive binding studies indicate that SFN does not appear to bind at the known colchicine or vinblastine drug binding sites. Moreover, SFN significantly reduced the number of cysteine residues accessible to the sulfhydryl-specific reagent DTNB. There were 18 ± 0.2 sulfhydryl residues accessible per tubulin dimer in the absence of SFN and 14.10 ± 0.4 sulfhydryl residues in the presence of 50 uM SFN, providing evidence that the binding of SFN to tubulin induces a conformational change in the tertiary structure of tubulin and that it may interact with cysteine residues. Our combined results suggest that SFN exerts its antiproliferative activity by stabilizing spindle microtubule dynamics by binding to tubulin and/or microtubules and affecting their function in a manner similar to but less potently than that of currently used antimitotic anticancer drugs.