Shikonin time-dependently induced necrosis or apoptosis in gastric cancer cells via generation of reactive oxygen species Mu-Jang Lee a,1 , Shao-Hsuan Kao b,1 , Jing-En Hunag b , Gwo-Tarng Sheu c , Chi-Wei Yeh b , You-Cheng Hseu d , Chau-Jong Wang b,e,⇑ , Li-Sung Hsu b,e,⇑ a Cardiovascular Center, Antai Tian-Sheng Memorial Hospital, Pingtung 92843, Taiwan b Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 40201, Taiwan c Institute of Medicine, Chung Shan Medical University, Taichung 40201, Taiwan d Department of Cosmeceutics, College of Pharmacy, China Medical University, Taichung 40402, Taiwan e Clinical Laboratory, Chung Shan Medical University Hospital, Taichung 40201, Taiwan article info Article history: Received 11 November 2013 Received in revised form 2 January 2014 Accepted 13 January 2014 Available online 23 January 2014 Keywords: Apoptosis Gastric cancer Necrosis Shikonin abstract The effects of shikonin on gastric cancer cells were investigated in this study. Exposure to shikonin reduced the viability of gastric cancer cells in a time- and dose-dependent manner. However, apoptosis was not observed in gastric cancer cell treatment with different concentrations of shikonin for 6 h. By contrast, treatment with shikonin for 24 h significantly induced apoptosis, as evidenced by the results of TUNEL assay and flow cytometry analysis in proportion to the concentration. Disruption of the mito- chondrial membrane potential was observed in gastric cancer cells that were treated with shikonin for 6 and 24 h. Pretreatment with necrostatin-1 recovered cell death and mitochondrial membrane potential in the 6 h shikonin treatment, but not in the 24 h shikonin treatment. Western blot results reveal enhanced p38 phosphorylation, downregulated AKT phosphorylation, and increased caspase3 and PARP cleavage in cells that were treated with shikonin for 24 h, but not in cells treated for 6 h. Shikonin also triggered reactive oxygen species (ROS) generation both in the 6 and 24 h treatments. Pretreatment with N-acetylcysteine blocked shikonin-induced cell death. In summary, our findings suggest that shikonin, which may function as a promising agent in the treatment of gastric cancers, sequentially triggered necrosis or apoptosis through ROS generation in gastric cancer cells. Ó 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Shikonin, one of the major bioactive components of Lithosper- mum erythrorhizon, is widely used in traditional Chinese medicine in treating measles, sore throat, and burns [1]. Shikonin has been shown to exert multiple biological effects, such as the inhibition of PMA-induced cyclooxygenase 2 expression [2], induction of glu- cose uptake in 3T3L1 cells and skeletal muscle cells [2,3], and inhi- bition of lipopolysaccharide-mediated tumor necrosis factor a release via the proteasome pathway [4]. Recently, shikonin has been extensively studied for its anti-tu- mor activities. Shikonin administration inhibits tumor growth and prolongs the life span of sarcoma S180-bearing mice [5]. Treat- ment with shikonin also activates the caspase pathway, which in- duces the apoptosis of several human cancer cells, such as leukemia HL-60 cells [6], colorectal cancer cells [7], and osteosar- coma [8]. Shikonin blocks the signal mediated by the epithelial growth factor receptor and decreases extracellular signal-regu- lated kinase (ERK) activities, which can diminish cell proliferation in human epidermoid carcinoma cells [9]. In hepatoma cells, shiko- nin triggers apoptosis through increased reactive oxygen species (ROS) production, and subsequently downregulates AKT and RIP1/NFjB activities [10]. Chen et al. showed that shikonin also elevates ROS generation, decreases glutathione concentration, and disrupts the mitochondrial membrane potential, which results in apoptosis in human glioma cells [11]. Han et al. demonstrated that shikonin treatment causes drug-resistant cancer cells to un- dergo necroptotic death [12]. In addition, these authors also showed that pretreatment with antioxidant agents, such as N-acetylcysteine (NAC), pifithrin-a, or cyclosporin A, abolishes shikonin-induced cell death [11]. Chen et al. demonstrated that http://dx.doi.org/10.1016/j.cbi.2014.01.008 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved. ⇑ Corresponding authors at: Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Sec 1, Jianguo, N. Rd., Taichung 40201, Taiwan. Tel.: +886 2 24730022x11670; fax: +886 4 23248195 (C.-J. Wang). Tel.: +886 2 24730022x11682; fax: +886 4 23248195 (L.-S. Hsu). E-mail addresses: wcj@csmu.edu (C.-J. Wang), lshsu405@yahoo.com.tw (L.-S. Hsu). 1 These authors contributed eqully as first author. Chemico-Biological Interactions 211 (2014) 44–53 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint