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Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis

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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 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 shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21Cip1, leading to p38 MAPK activation, and finally, promoting apoptosis.

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Acknowledgments

This work was supported, in part, by Basic Science Research (RBM3301213) and by the Bio & Medical Technology Development Program (No. 2012053532) of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and a Grant from the Korea Research Council of Fundamental Science and Technology (KGM2011211).

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  1. Jeong-Hae Choi

    Present address: Department of Oral Anatomy and Cell Biology, School of Dentistry, Pusan National University, Yangsan, 626-870, Republic of Korea

Authors and Affiliations

  1. Genome Research Center, KRIBB, Daejeon, 305-806, Republic of Korea

    Jiwon Ahn, Misun Won, Jeong-Hae Choi, Yong Sung Kim, Cho-Rock Jung, Dong-Su Im, Mi-Lang Kyun & Kyung-Sook Chung

  2. Functional Genomics, Korea University of Science and Technology, Daejeon, 305-806, Republic of Korea

    Mi-Lang Kyun & Kyung-Sook Chung

  3. College of Pharmacy, Dongguk University, Seoul, 100-715, Republic of Korea

    Kyeong Lee

  4. Department of Food Science and Technology, Chungnam National University, Daejeon, 305-764, Republic of Korea

    Kyung-Bin Song

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Jiwon Ahn and Misun Won contributed equally to this work.

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10495_2013_835_MOESM1_ESM.ppt

Fig. S1 IC50 of shikonin in different human cancer cell lines. Various human cell lines, i.e., WI38 (normal lung), PC3 (prostate cancer), MIA-paca2 (pancreatic cancer), MCF7 (breast cancer), HT29 (colon cancer), Hep3B (liver cancer), HeLa (cervical cancer), HCT116 (colon cancer), A549 (small lung cancer), and MDA-MB-231 (breast cancer), were maintained in RPMI 1640 supplemented with 1.5 g/L sodium bicarbonate, 5 % fetal bovine serum, 100 μg/mL streptomycin, 100 U/mL penicillin, and 2 mM l-glutamine in a humidified incubator containing 5 % CO2. Cells were treated with varying concentrations of shikonin (0.01–100 μM) for 48 h and cell viability was analyzed by the SRB assay. Results are presented as means ± SE of triplicate experiments (P < 0.05). Fig. S2 Cell morphology. Effects of different signaling pathway inhibitors on cell death and survival. After pretreatment for 2 h with 100 μM N-acetyl cysteine (NAC, a ROS inhibitor), 10 μM SB203580 (SB, a p38 inhibitor), 10 μM SP600125 (SP, a JNK inhibitor), or 10 μM LY294002 (LY, an Akt inhibitor), HeLa cells were treated with 2 or 4 μM shikonin for 24 h. Pretreatment of cells with NAC and SB203580 reduced the cytotoxic effect of shikonin. In contrast, pretreatment with LY294002 enhanced the cytotoxic effect of shikonin. Fig. S3 Shikonin generates ROS in HeLa cells. HeLa cells were treated with 2 μM shikonin for 6 h and the amount of generated ROS was determined by ROS assay kit (Cell Biolab, CA, USA). Briefly, cells were incubated with 100 μL of DCFH-DA in complete medium for 30 min at 37 °C to allow cellular incorporation. After, cells were incubated with a new medium containing shikonin with or without 100 μM NAC. ROSdependent DCF fluorescence was monitored at 37 °C with an emission wavelength set at 530 nm and an excitation wavelength set at 480 nm in a fluorescence microplate reader (SpectraMax, Molecular Devices, USA). Hydrogen peroxide was used as positive control for ROS. Each bar represents the mean ± SE of the three independent experiments. Fig. S4 Shikonin induced Akt dephosphorylation at Ser473 and ASK1 phosphorylation at Ser83. HeLa cells were treated with 2 μM shikonin for different times, followed by immunoblot analysis of Akt, phospho-Akt (Ser473), and phospho-ASK1 (Ser83). Supplementary material 1 (PPT 1822 kb)

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Ahn, J., Won, M., Choi, JH. et al. Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21Cip1 downregulation are required for shikonin-induced apoptosis. Apoptosis 18, 870–881 (2013). https://doi.org/10.1007/s10495-013-0835-5

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