Crocin, a carotenoid, suppresses spindle microtubule dynamics and activates the mitotic checkpoint by binding to tubulin

Abstract

Crocin, a constituent of the saffron spice, exhibits promising antitumor activity in animal models and also inhibits the proliferation of several types of cancer cells in culture. Recently, we have shown that crocin binds to purified tubulin at the vinblastine site, depolymerizes microtubules and induces a mitotic block in cultured cells. Here, we extend our previous suggestion and explore the cellular effects of crocin to further understand its mechanism of action. In a kinetic study, we observed that the crocin-induced depolymerization of microtubules preceded both DNA damage and reactive oxygen species generation indicating that depolymerizing microtubules is the primary action of crocin. Crocin also inhibited the growth of cold-depolymerized microtubules in HeLa cells indicating that it can inhibit microtubule dynamics. Using fluorescence recovery after photobleaching, crocin was found to suppress the spindle microtubule dynamics in live HeLa cells. Further, crocin treatment resulted in activation of spindle assembly checkpoint proteins, BubR1 and Mad2. Similar to other microtubule-targeting agents, crocin also perturbed the localization of end-binding protein EB1 from the growing microtubule ends and enhanced the acetylation of remaining microtubules. Further, crocin was found to bind to purified tubulin with a dissociation constant of 12 ± 1.5 μM. The results suggested that crocin exerted its antiproliferative effect primarily by inhibiting the assembly and dynamics of microtubules. Importantly, the combination of crocin with known anticancer agents like combretastatin A-4, cisplatin, doxorubicin or sorafenib, exerted a strong synergistic cytotoxic effect in HeLa cells indicating that crocin may enhance the effectiveness of other anticancer agents.