695 Sulforaphane Sensitizes Colorectal Cancer Cells to Oxaliplatin Induced Cell Growth Inhibition: Key Role of p38 MAPK

Abstract

Background: Microtubule-targeting agents (MTAs) are cancer drugs that act primarily by altering microtubule dynamics and interfering with mitosis. While MTAs are in widespread clinical use, innate and acquired resistance is an ongoing problem. Resistance is thought to stem primarily from drug efflux and expression of alternate tubulin isoforms with MTAresistant microtubule dynamics. We found that the compound T0070907 reduced tumor growth in a murine xenograft model of colon cancer. In human colorectal cancer (CRC) cell lines, T0070907 caused near-total loss of the α and β tubulin proteins, leading to collapse of visible microtubule filaments and eventual cell death. Dramatic tubulin loss is a novel phenotype not reported for any of the standard MTAs. This observation suggests the possible development of MTAs that work by a mechanism less susceptible to escape via expression of alternate tubulin isoforms. As a first step toward determining the molecular mechanisms of tubulin loss, we determined whether T0070907 affects degradation and activity of the microtubule associated proteins that control microtubule polymerization. Methods: The effects of T0070907 on tubulin levels and phosphorylation of the microtubuleassociated protein stathmin/Op18 and its upstream kinase MARK kinase were examined using Western blot in the CRC cell lines HT-29, SW620, and DLD-1. Tubulin protein halflives were measured after addition of the protein synthesis inhibitor cycloheximide. Results: T0070907 caused loss of α and β, but not γ, δ, or e tubulin. The magnitude of the tubulin reduction was much greater than that found when microtubule destabilization leads to tubulin synthesis inhibition. Tubulin half lives decreased from >12h to 6h, suggesting increased degradation. Consistent with this idea, treatments that sequestered tubulin in polymerized microtubules (paclitaxel), leaving the tubulin unavailable for degradation, reduced T0070907-induced tubulin loss. Conversely, the microtubule-destabilizing agents nocodazole and vinblastine increased the rate of tubulin loss. T0070907 did not strongly affect microtubule polymerization In Vitro, suggesting that it does not directly bind to either microtubules or tubulin. However, T0070907 led to increases in MARK kinase activation as well as phosphorylation and inactivation of its downstream target, the microtubulesequestering protein stathmin/Op18. Conclusion: T0070907 promotes tubulin degradation and stathmin inactivation. As knockdown of stathmin in Drosophila leads to a drastic reduction in tubulin levels, we propose that T0070907 reduces tubulin in part by targeting stathmin.