Dependency of 2-methoxyestradiol-induced mitochondrial apoptosis on mitotic spindle network impairment and prometaphase arrest in human Jurkat T cells

作者：Lee Seung Tae; Lee Ji Young; Han Cho Rong; Kim Yoon Hee; Jun Do Youn; Taub Dennis; Kim Young Ho

来源：Biochemical Pharmacology, 2015, 94(4): 257-269.

DOI：10.1016/j.bcp.2015.02.011

• Mitotic spindle damage
• Prometaphase arrest
• Cdk1 activation
• Phosphorylation of Bcl-2 family proteins
• Mitochondrial apoptosis

The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E-2)-induced cell cycle arrest and 2-MeO-E-2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5-1.0 mu M) caused G(2)/M arrest, Bak activation, Delta psi m loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G(2)/M arrest were induced in Jurkat T cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, Cdk1 phosphorylation at Thr-161 and dephosphorylation at Tyr-15, up-regulation of cyclin B1 expression, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation at Ser-159/Thr-163, and Bins phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E-2 and the G(1)/S blocking agent aphidicolin resulted in G(1)/S arrest and abrogation of all apoptotic events, including Cdk1 activation, phosphorylation of Bcl-2, Mcl-1 and Bim, and ROS accumulation. The 2-MeO-E-2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor, but not by an Aurora A kinase (AURKA), Aurora B kinase (AURKB), JNK, or p38 MAPK inhibitor. Immunofluorescence microscopic analysis revealed that 2-MeO-E-2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10-20 mu M 2-MeO-E-2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5-5.0 mu M 2-MeO-E-2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E-2 (0.5-1.0 mu M) was attributable to mitotic spindle defect-mediated prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade.

• 出版日期2015-4-15