| Summary: | 碩士 === 國立中興大學 === 生物醫學研究所 === 104 === Colorectal cancer is the most commonly diagnosed cancer around the world and the third cause of cancer mortality in Taiwan. Up-regulation of prosurvival BCL-2 family members has been shown to be correlated with early colorectal cancer formation. Obatoclax (a.k.a GX15-070) is a pan-BCL-2 inhibitor. Previous studies have pointed out the antiproliferative effect of obatoclax, but the mechanisms remain unclear. In this study, we used colorectal cancer cell lines HCT116, HT-29 and LoVo as the cellular model to study the antiproliferative action of obatoclax. We found that obatoclax causes cell cycle arrest at the G1 phase and also marked decrease in the clonogenic capacity in dose-dependently. Immunoblotting analyses indicated that a decline of cyclin D1 levels following obatoclax treatment. Importantly, overexpression of cyclin D1 conferred cells resistance to obatoclax-induced G1 arrest and suppression of clonogenicity, indicating that cyclin D1 decrease is a fundamental mechanism for obatoclax to induce antiproliferation. To further understand how obatoclax down-regulates cyclin D1, we found that the levels of the cyclin D1 mRNA and promoter activity were not significantly affected. In contrast, pre-treatment with the proteasome inhibitor MG132 markedly rescued cyclin D1 protein levels in obatoclax-treated cells. Additionally, cycloheximide chase analyses revealed that the half-life of cyclin D1 protein was evidently decreased following obatoclax stimulation, indicating that obatoclax reduces cyclin D1 expression mainly by destabilizing cyclin D1 protein. It is known that a major mechanism of cyclin D1 destabilization is through glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation of Thr286 of cyclin D1 (p-Cyclin D1 (Thr286). However, we found that obatoclax induced ser473 phosphorylation and thus activation of AKT, which then causes inactivation of GSK3β as evidenced by the increase in the levels of p-GSK3β (Ser9), suggesting that GSK3β activity is not required for obatoclax-induced cyclin D1 destabilization. In summary, we herein provide the first evidence that obatoclax induced antiproliferation in human colorectal cancer cells by down-regulating cyclin D through cyclin D1 protein destabilization.
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