ANTICANCER RESEARCH 28 : 2791-2800 (2008) Diallyl Disulfide (DADS) Induces Apoptosis in Human Cervical Cancer Ca Ski Cells via Reactive Oxygen Species and Ca 2+ -dependent Mitochondria-dependent Pathway YUH-TZY LIN 1, JAI-SING YANG 2, SHUW-YUAN LIN 3, TZU-WEI TAN 2, CHIN-CHIN HO 4, TE-CHUN HSIA 5, TSAN-HUNG CHIU 6, CHUN-SHU YU 7, HSU-FENG LU 8, YUEH-SHAN WENG 9 and JING-GUNG CHUNG 9,10 1Department of Nursing and Management, Jen-Teh Junior College of Medicine, Miaoli, Taiwan; Departments of 2Pharmacology and 9Biological Science and Technology, 7School of Pharmacology, China Medical University, Taichung, Taiwan; Department of 5Internal Medicine and 6OBS/GYN, China Medical University Hospital, Taichung, Taiwan; 3Department of Food and Nutrition, Hung-Kuang University, Sha Lu, Taichung Hsien 433, Taiwan 4Department of Nurs ing , Central Taiwan University of Science and Technology, Buzih District, Taiwan; 8Department of Clinical Pathology, Cheng Hsin Rehabilitation Medical Center, Taipei, Taiwan; 10 Department of Biotechnology, Asia University, Wufeng, Taichung County 41354, Taiwan, R.O.C. Abstract. The mechanisms of apoptosis induced by diallyl naturally in garlic, is known to inhibit the growth of disulfide (DADS) were explored in human cervical cancer Ca canine mammary neoplastic CMT-13 cells (1), the Ski cells. Flow cytometric analysis, DNA gel electrophoresis promotion phase of DMBA-induced skin tumors in mice and DAPI staining demonstrated that DADS induced apoptosis (2) and also to inhibit rat and human CYP2E1 and rat in Ca Ski cells. DADS induced apoptosis through the CYP2A3 expressions (3). DADS induced apoptosis in production of reactive oxygen species and Ca 2+ , and induced HepG2 hepatoma (4) and neuroblastoma cells (5) and it abrogation of mitochondrial membrane potential (Δψm) and was suggested as an antiproliferative agent in cancer cleavage of Bid protein (t-Bid). DADS increased the levels of therapy based on a pivotal role for oxidative stress (5). p53, p21 and Bax, but caused a decrease in the level of Bcl-2. DADS affects histone acetylation and this may be DADS also promoted the activities of caspase-3 leading to associated with its protective properties in colon DNA fragmentation, thus indicating that DADS-induced carcinogenesis (6). In our previous studies, we apoptosis is caspase-3 dependent. In addition, DADS induced demonstrated that DADS affects NAT activity and gene an increase in the level of cytochrome c in the cytoplasm, which expression in human cancer cell lines (7-9). Recently, we was released from mitochondria. BAPTA attenuated the Δψm also found that DADS induced signal transducer and abrogation and significantly diminished the occurrence of activator of transcription 1 (STAT 1) expression in human DADS-induced apoptosis in Ca Ski cells. In conclusion, DADS- colon cancer COLO 205 cells (10). induced apoptosis occurs via production of ROS and caspase- Apoptosis, a programmed cell death, occurs under a 3 and a mitochondria-dependent pathway in Ca Ski cells. variety of physiological and pathological conditions that control the development and homeostasis of multicellular Garlic ( Allium sativum L.) is well known for its organisms (11). The induction of apoptosis was recognized chemopreventive potential against cancer, especially colon to be the best strategy for agents to kill cancer cells. The cancer. Diallyl disulfide (DADS), a compound found major apoptotic pathways can be divided into caspase- and mitochondria-dependent pathways. Caspase activation is generally considered to be a key hallmark of apoptosis. Although DADS has been demonstrated to induce apoptosis Correspondence to: Jing-Gung Chung, Ph.D., Department of in colon cancer cells through the ROS-dependent Biological Science and Technology, China Medical University, No 91, mitochondrial death pathway, there is no available Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. Tel: +886 422053366, Fax: +886 422053764, e-mail: jgchung@ mail.cmu.edu.tw information on the effects of DADS in human cervical cancer Ca Ski cells. Therefore, in this study we focused on 2+ Key Words: DADS, apoptosis, caspase-3, cytochrome c, the molecular mechanism and the role of ROS and Ca on mitochondrial membrane potential (Δψm). the induction of apoptosis by DADS in Ca Ski cells. 0250-7005/2008 $2.00+.40 2791 ANTICANCER RESEARCH 28 : 2791-2800 (2008) Materials and Methods Caspase-3 activity determination in Ca Ski cells treated with or without DADS. Approximately 2×10 5 Ca Ski cells/well in 12-well plates with 25 μM DADS were incubat ed for 0, 6, 12, 24 and 48 h. Chemicals and reagents. Diallyl disulfide (DADS) was purchased Cells were harvested before adding 50 μl of 10 μM substrate solution from Fluka Chemika Co. (Bucha, Switzerland). Dimethyl sulfoxide to the cell pellet (1×10 5 cells per sample) and cells were incubated (DMSO), propidium iodide (PI), RNase, trypan blue, Tris-HCl and at 37˚C for 60 min then washed once with 1 ml of ice-cold PBS and Triton X-100 were obtained from Sigma Chemical Co. (St. Louis, re-suspended in 1 ml fresh PBS. Cells were analyzed with a flow MO, USA). BAPTA and potassium phosphates were obtained from cytometer (Becton-Dickinson) equipped with an argon ion laser at Merck Co. (Darmstadt, Germany). Fetal bovine serum (FBS), 488 nm. Caspase-3 activity was determined and analyzed (17, 18) glutamine, penicillin-streptomycin, trypsin-EDTA and RPMI-1640 according to the manufacturer’s instructions. were obtained from Gibco BRL (Grand Island, NY, USA). Caspase-3 activity assay kit was bought from OncoImmunin, Inc (Gaithersburg, Detection of reactive oxygen species (ROS) in Ca Ski cells after MD, USA). treatment with DADS. Approximately 2×10 5 Ca Ski cells/well in 12-well plates were treated with 25 μM DADS for 0, 2, 4 and 5 h. Human cervical epidermoid carcinoma Ca Ski cells. Ca Ski cells were The cells were harvested and washed twice, re-suspended in 500 μl obtained from the Food Industry Research and Development Institute of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (10 μM) (Hsinchu, Taiwan, ROC). The cells were placed into 75 cm 3 tissue and incubated at 37˚C for 30 min, then analyzed to detect the culture flasks and grown at 37˚C under a humidified 5% CO and 95% 2 changes of ROS by flow cytometry (17, 18). air at 1 Atm in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin (10,000 U/ml penicillin and 10 mg/ml Detection of the levels of cytoplasmic Ca 2+ in DADS-treated Ca Ski streptomycin) and 1% L-glutamine. Cells were cultured for several cells. Approximately 2×10 5 Ca Ski cells/well in 12-well plates were generations and the viability of each generation was checked (12). treated with 25 μM DADS for 0, 0.25, 1, 3, 4, 6 and 8 h. The cells were harvested and washed twice, re-suspended in Indo 1/AM Morphological changes and cell viability of Ca Ski cells treated with (3 μg/ml), incubated at 37˚C for 30 min, then analyzed to detect the or without DADS. The Ca Ski cells were plated in 12-well plates at changes of cytoplasmic Ca 2+ levels using flow cytometry (17, 18). a density of 2×10 5 cells/well and grown for 24 h. DADS was individually added to cells at a final concentration of 0, 10, 25, 50, Detection of mitochondrial membrane potential (Δψm) in Ca Ski 75 and 100 μM, while DMSO (solvent) alone was added to the cells after treatment with DADS. Approximately 2×10 5 Ca Ski control group. The cells were grown at 37˚C, 5% CO and 95% air 2 cells/well in 12-well plates were treated with 25 μM DADS for 0, for different periods of time. For morphological changes, cells were 0.25, 1, 6 and 24 h. The cells were harvested and washed twice, re- examined and photographed under phase-contrast light microscopy. suspended in 500 μl of DiOC (4 μmol/l) and incubated at 37˚C for For cell viability, propidium iodi de (PI) stain was used and a flow 6 30 min before being analyzed to detect the changes of mitochondrial cytometric assay was carried out as described elsewhere (12, 13). membrane potential (Δψm) using flow cytometry (17, 18). Flow cytometric analysis for apoptosis. Approximately 5×10 5 Detection of the levels of cytoplasmic Ca 2+ and Δψm in Ca Ski cells cells/well Ca Ski in 6-well plates were incubated with 25 μM DADS pre-treated with BAPTA then treated with DADS. Approximately for 0, 6, 12, 24, 48 and 72 hours. The isolated cells were fixed gently 2×10 5 Ca Ski cells/well in 12-well plates were pre-treated with (drop by drop) by putting 70% ethanol (in PBS) in ice overnight and BAPTA before the addition of 25 μM DADS for 24 hours. The cells were then resuspended in phosphate-buffered saline (PBS) containing were harvested and washed twice, once for apoptosis analysis and 40 μg/m l PI, 0.1 mg/ml RNase (Sigma) and 0.1% Triton X-100 in a the other for re-suspension in Indo 1/AM (3 μg/ml) or DiOC dark room. After 30 minutes at 37˚C, the cells were analyzed with a 6 (4 μmol/L) before being incubated at 37˚C for 30 min and then flow cytometer (Becton Dickinson FACS Calibur, San Jose, CA, analyzed by flow cytometry. USA) equipped with an argon laser at 488 nm. The cell cycle distribution was determined and analyzed (13, 14). Western blotting to examine the effect of DADS on p53, p21, Bax, Bcl-2, caspase-3 AIF, MMP-1, -7 and -9, Bid, tBid and cytochrome DAPI staining for the effects of DADS on apoptosis in Ca Ski cells.