Cis-trimethoxy resveratrol induces intrinsic apoptosis via prometaphase arrest and prolonged CDK1 activation pathway in human Jurkat T cells

Chae Eun Kim; Do Youn Jun; Jong-Sik Kim; Young Ho Kim

doi:10.18632/oncotarget.23576

Cis-trimethoxy resveratrol induces intrinsic apoptosis via prometaphase arrest and prolonged CDK1 activation pathway in human Jurkat T cells

Oncotarget. 2017 Dec 22;9(4):4969-4984. doi: 10.18632/oncotarget.23576. eCollection 2018 Jan 12.

Authors

Chae Eun Kim¹, Do Youn Jun^{1

2}, Jong-Sik Kim³, Young Ho Kim¹

Affiliations

¹ Laboratory of Immunobiology, School of Life Science and Biotechnology, College of Natural Sciences, Kyungpook National University, Daegu 41566, Korea.
² Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease Bio-Medical Research Institute, Kyungpook National University Hospital, Daegu 702-201, Korea.
³ Department of Biological Sciences, Andong National University, Andong 36729, Korea.

Abstract

Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G₂/M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G₂/M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIM_EL and BIM_L) phosphorylation irrespective of BCL-2 overexpression. Enforced G₁/S arrest by using a G₁/S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC₅₀ values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.

Keywords: CDK1-dependent mitochondrial apoptosis; cis-trimethoxy resveratrol; mitotic spindle damage; phosphorylation of BCL-2 family proteins; prometaphase arrest.