Xiao Dong, Zeng Yan, Singh Shivendra V
Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Mol Carcinog. 2009 Nov;48(11):1018-29. doi: 10.1002/mc.20553.
Growth suppressive effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, against cultured human cancer cells correlates with checkpoint kinase 1 (Chk1)-mediated mitotic arrest, but the fate of the cells arrested in mitosis remains elusive. Using LNCaP and HCT-116 human cancer cells as a model, we now demonstrate that the Chk1-mediated mitotic arrest resulting from DATS exposure leads to apoptosis. The DATS exposure resulted in G(2) phase and mitotic arrest in both LNCaP and HCT-116 cell lines. The G(2) arrest was accompanied by downregulation of cyclin-dependent kinase 1 (Cdk1), cell division cycle (Cdc) 25B, and Cdc25C leading to Tyr15 phosphorylation of Cdk1 (inactivation). The DATS-mediated mitotic arrest correlated with inactivation of anaphase-promoting complex/cyclosome as evidenced by accumulation of its substrates cyclinB1 and securin. The DATS treatment increased activating phosphorylation of Chk1 (Ser317) and transient transfection with Chk1-targeted siRNA conferred significant protection against DATS-induced mitotic arrest in both cell lines. The Chk1 protein knockdown also afforded partial yet statistically significant protection against apoptotic DNA fragmentation and caspase-3 activation resulting from DATS exposure in both LNCaP and HCT-116 cells. Even though DATS treatment resulted in stabilization and Ser15 phosphorylation of p53, the knockdown of p53 protein failed to rescue DATS-induced mitotic arrest. In conclusion, the results of the present study indicate that Chk1 dependence of DATS-induced mitotic arrest in human cancer cells is not influenced by the p53 status and cells arrested in mitosis upon DATS exposure are driven to apoptotic DNA fragmentation.
二烯丙基三硫化物（DATS）是大蒜中一种很有前景的癌症化学预防成分，其对培养的人癌细胞的生长抑制作用与检查点激酶1（Chk1）介导的有丝分裂停滞相关，但停滞在有丝分裂期的细胞命运仍不清楚。以LNCaP和HCT - 116人癌细胞为模型，我们现在证明，DATS暴露导致的Chk1介导的有丝分裂停滞会导致细胞凋亡。DATS暴露导致LNCaP和HCT - 116细胞系均出现G2期和有丝分裂停滞。G2期停滞伴随着细胞周期蛋白依赖性激酶1（Cdk1）、细胞分裂周期（Cdc）25B和Cdc25C的下调，导致Cdk1的Tyr15磷酸化（失活）。DATS介导的有丝分裂停滞与后期促进复合物/环体的失活相关，这可通过其底物细胞周期蛋白B1和securin的积累得到证明。DATS处理增加了Chk1（Ser317）的激活磷酸化，并且用靶向Chk1的小干扰RNA进行瞬时转染可在两种细胞系中显著保护细胞免受DATS诱导的有丝分裂停滞。Chk1蛋白敲低也为LNCaP和HCT - 116细胞中DATS暴露导致的凋亡性DNA片段化和半胱天冬酶-3激活提供了部分但具有统计学意义的保护。尽管DATS处理导致p53稳定并使其Ser15磷酸化，但p53蛋白的敲低未能挽救DATS诱导得有丝分裂停滞。总之，本研究结果表明，人癌细胞中DATS诱导的有丝分裂停滞对Chk1的依赖性不受p53状态影响，并且DATS暴露后停滞在有丝分裂期的细胞会发生凋亡性DNA片段化。
