Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21 Cip1 downregulation are requir

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ORIGINAL PAPER

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis Jiwon Ahn • Misun Won • Jeong-Hae Choi • Yong Sung Kim • Cho-Rock Jung • Dong-Su Im • Mi-Lang Kyun • Kyeong Lee • Kyung-Bin Song • Kyung-Sook Chung

Published online: 2 April 2013 Ó Springer Science+Business Media New York 2013

Abstract Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21Cip1. In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is

Jiwon Ahn and Misun Won contributed equally to this work.

Electronic supplementary material The online version of this article (doi:10.1007/s10495-013-0835-5) contains supplementary material, which is available to authorized users. J. Ahn  M. Won  J.-H. Choi  Y. S. Kim  C.-R. Jung  D.-S. Im  M.-L. Kyun  K.-S. Chung (&) Genome Research Center, KRIBB, Daejeon 305-806, Republic of Korea e-mail: [email protected] Present Address: J.-H. Choi Department of Oral Anatomy and Cell Biology, School of Dentistry, Pusan National University, Yangsan 626-870, Republic of Korea M.-L. Kyun  K.-S. Chung Functional Genomics, Korea University of Science and Technology, Daejeon 305-806, Republic of Korea

associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21Cip1 was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21Cip1 have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21Cip1 facilitated shikonin-induced apoptosis, implying that p21Cip1 delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21Cip1 to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21Cip1 actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonininduced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21Cip1, leading to p38 MAPK activation, and finally, promoting apoptosis. Keywords Shik