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

• Published in: Biochemical pharmacology 2015-04, Vol.94 (4), p.257-269
• Main Authors: Lee, Seung Tae, Lee, Ji Young, Han, Cho Rong, Kim, Yoon Hee, Jun, Do Youn, Taub, Dennis, Kim, Young Ho
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Caspase 3 - metabolism; Cdk1 activation; Enzyme Activation; Estradiol - analogs & derivatives; Estradiol - pharmacology; G1 Phase Cell Cycle Checkpoints - drug effects; G2 Phase Cell Cycle Checkpoints - drug effects; Humans; Jurkat Cells; Microtubules - drug effects; Microtubules - metabolism; Mitochondria - drug effects; Mitochondria - physiology; Mitochondrial apoptosis; Mitosis; Mitotic spindle damage; Phosphorylation; Phosphorylation of Bcl-2 family proteins; Prometaphase - drug effects; Prometaphase arrest; Proto-Oncogene Proteins c-bcl-2 - genetics; Proto-Oncogene Proteins c-bcl-2 - metabolism; Spindle Apparatus - drug effects; Spindle Apparatus - physiology
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2015.02.011

The apoptogenic activity of 2-MeO-E2 was attributable to mitotic spindle damage, prometaphase arrest, Cdk1 activation, phosphorylation of Bcl-2, Mcl-1, and Bim, and activation of Bak and mitochondria-dependent caspase cascade. The present study sought to determine the correlation between 2-methoxyestradiol (2-MeO-E2)-induced cell cycle arrest and 2-MeO-E2-induced apoptosis. Exposure of Jurkat T cell clone (JT/Neo) to 2-MeO-E2 (0.5–1.0μM) caused G2/M arrest, Bak activation, Δψm loss, caspase-9 and -3 activation, PARP cleavage, intracellular ROS accumulation, and apoptotic DNA fragmentation, whereas none of these events except for G2/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 Bim phosphorylation were detected irrespective of Bcl-2 overexpression. Concomitant treatment of JT/Neo cells with 2-MeO-E2 and the G1/S blocking agent aphidicolin resulted in G1/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-E2-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-E2-induced mitotic arrest was caused by mitotic spindle network impairment and prometaphase arrest. Whereas 10–20μM 2-MeO-E2 reduced the proportion of intracellular polymeric tubulin to monomeric tubulin, 0.5–5.0μM 2-MeO-E2 increased it. These results demonstrate that the apoptogenic effect of 2-MeO-E2 (0.5–1.0μM) 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.