title: Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21(Cip1) downregulation are required for shikonin-induced apoptosis.
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 p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is 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 p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.
authors.affiliation: Genome Research Center, KRIBB, Daejeon 305-806, Republic of Korea.
authors.forename: Jiwon
authors.initials: J
authors.lastname: Ahn
authors.forename: Misun
authors.initials: M
authors.lastname: Won
authors.forename: Jeong-Hae
authors.initials: JH
authors.lastname: Choi
authors.forename: Yong Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Cho-Rock
authors.initials: CR
authors.lastname: Jung
authors.forename: Dong-Su
authors.initials: DS
authors.lastname: Im
authors.forename: Mi-Lang
authors.initials: ML
authors.lastname: Kyun
authors.forename: Kyeong
authors.initials: K
authors.lastname: Lee
authors.forename: Kyung-Bin
authors.initials: KB
authors.lastname: Song
authors.forename: Kyung-Sook
authors.initials: KS
authors.lastname: Chung
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics* / Q000378:metabolism; D009285:Naphthoquinones / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics* / Q000378:metabolism
chemical_list: D050759:Cyclin-Dependent Kinase Inhibitor p21; D004365:Drugs, Chinese Herbal; D009285:Naphthoquinones; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C016101:shikonin; D051057:Proto-Oncogene Proteins c-akt; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human