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Home , Acetylation, Diallyl disulfide

ANTICANCER RESEARCH 28 : 2791-2800 (2008)

Diallyl (DADS) Induces in Human 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 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 , 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 cells (5) and it abrogation of mitochondrial membrane potential (Δψm) and was suggested as an antiproliferative agent in cancer cleavage of Bid (t-Bid). DADS increased the levels of therapy based on a pivotal role for (5). , and Bax, but caused a decrease in the level of Bcl-2. DADS affects 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 (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 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 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 , 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 ( 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). 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) , 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% (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 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. c of Ca Ski cells. The total were collected from Ca Ski cells Approximately 2×10 5 Ca Ski cells/well were grown in 6-well plates treated with or without 25 μM of DADS for 6, 12, 24, 48 and 72 h. and 0, 10, 25, 50, 75 and 100 μM DADS were individually added to P53, p212, Bax, Bcl-2, caspase-3, AIF, MMP-1, -7 and -9, Bid, t- the cells for 48 h prior to staining. Cells were washed three times Bid and cytochrome c expression were examined using sodium with PBS and fixed with 4% paraformaldehyde. Fixed cells were dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and washed with PBS and stained with 4,6-diamidino-2-phenylindole Western blot as described previously (17, 18). (DAPI, 1 μg/ml; Sigma) for 30 min. Stained cells were then examined under fluorescen ce microscopy, photographed and Statistical analysis. Student’s t-test was used to analyze the apoptotic cells identified (15). differences between the DADS-treated and control groups. DNA laddering fragmentation. Approximately 1×10 6 Ca Ski cells/well were grown in 6-well plates and treated with DADS at 25 Results μM for 12, 24 and 48 h. DNA was isolated from DADS-treated and non-treated cells and examined in 0.8% agarose gel electrophoresis, Effects of DADS on the morpholog y and cell viability of Ca Ski then photographed under fluorescence microscopy as described cells. DADS (25 μM) induced morphological changes on Ca Ski previously (16). cells revealed both necrotic and apoptotic cell death (Figure 1A).

2792 Lin et al : DADS Induces Apoptosis in Human Cervical Cancer Cells

Figure 1. Morphological changes and percentages of viable Ca Ski cells after DADS treatment. Ca Ski cells (2×10 5 cells/well; 12-well plates) were plated in RPMI-1640 + 10% fetal bovine serum (FBS) with different concentrations of DADS for 0, 6, 12, 24, 48 and 72 h. The morphological changes of Ca Ski cells were photographed under phase-contrast light microscopy (A). The cells were collected by centrifugation and the viable cells were determined using flow cytometry (B and C) as described in Materials and Methods. *Significantly different from control at p<0.05.

Fluorescence microscopy (Figure 1A) indicated that a proportion Induction of apoptosis by DADS in Ca Ski cells using DAPI of the cells showed swelling and cell membrane lysis, indicating staining. Apoptosis of Ca Ski cells brought about by DADS necrosis, but other cells showed shrinkage and nuclear treatment was detected using DAPI staining after 48 h of condensation, suggesting apoptosis of the cells. DADS reduced continuous exposure to DADS and determined and the proportion of viable Ca Ski cells in a dose- (Figure 1B) and photographed using fluorescence microscopy (Figure 3). The time-dependent manner (Figure 1C). results indicated that DADS induced apoptosis in a concentration-dependent manner. Flow cytometric analysis of the effects of DADS in Ca Ski cell apoptosis. The Ca Ski cells were incubated with Induction of DNA fragmentation by DADS in Ca Ski cells. different concentrations of DADS for 48 hours and apoptosis DNA fragmentation of Ca Ski cells treated with 25 μM was examined. Representative profiles and the percentage of DADS was examined using DNA gel electrophoresis. The apoptosis are presented in Figure 2A and B. DADS induced results indicated that DADS induced DNA fragmentation apoptosis in a dose-dependent manner. (apoptosis) in a time-dependent manner (Figure 4).

2793 ANTICANCER RESEARCH 28 : 2791-2800 (2008)

Figure 2. Flow cytometric analysis of the effects of DADS on Ca Ski cell apoptosis. Ca Ski cells were incubated with different concentrations of DADS for 48 h and apoptosis was examined using flow cytometry as described in Materials and Methods. Representative profiles (A) and the percentage of apoptosis (B) are presented. Data represent the mean ± S.D. of three experiments. *Significantly different from control at p<0.05.

the DADS-treated group compared to the control group (Figure 6). Levels of ROS remained higher in the DADS- treated group than that in the control group for at least 5 hours (Figure 6).

Detection of cytoplasmic Ca 2+ levels in Ca Ski cells after treatment with DADS. Cytoplasmic Ca 2+ levels increased in the DADS-treated group compared to that in the control group. On exposure to DADS at 25 μM, cytoplasmic Ca 2+ increased by approximately 24% in 2 h, reaching a maximum at 6 h, and started to decrease at 8 h (Figure 7). Effects of DADS on caspase-3 activity in Ca Ski cells. The caspase-3 activity was determined using flow cytometric Detection of mitochondrial membrane potential (Δψm) in analysis after exposure to 25 μM DADS for 0, 12, 24 and 48 h. Ca Ski cells after treatment with DADS. Mitochondrial The increase of caspase-3 activity in Ca Ski cells treated with membrane potential (Δψm) was lower in the DADS-treated 25 μM DADS was enhanced from 24 to 48 hours (Figure 5). group than that in the control group. The decrease of Δψm in the examined Ca Ski cells was generally proportional to the Detection of reactive oxygen species (ROS) in Ca Ski cells duration of DADS treatment from 2 to 24 hours at 25 μM, after treatment with DADS. The levels of ROS increased in revealing a time-dependent effect (Figure 8).

2794 Lin et al : DADS Induces Apoptosis in Human Cervical Cancer Cells

Figure 3. DAPI staining analysis of the effects of DADS on Ca Ski cell apoptosis. Ca Ski cells were incubated with diffe rent concentrations of DADS for 48 h and apoptosis was determined using DAPI staining as described in Materials and Methods. Data represent mean ± S.D. of three experiments. *Significantly different from control of p<0.05.

Effects of BAPTA on MMP and cytoplasmic Ca 2+ in DADS- treated Ca Ski cells. The results from flow cytometric analysis indicated that pre-treatment with BAPTA (a chelator) greatly influenced the levels of Δψm and cytoplasmic Ca 2+ in DADS-treated Ca Ski cells (Figure 9). BAPTA significantly suppressed the increase of cytoplasmic Ca 2+ induced by DADS treatment (Figure 9A). The decrease in Δψm levels was inhibited by BAPTA pre-treatment in DADS-treated cells (Figrue 9B).

Western blotting for the effects of DADS on p53, p27, AIF, pro- caspase-3, Bax, Bid, t-Bid and Bcl-2 in Ca Ski cells. The results from Western blotting analysis are shown in Figure 10A and B. DADS promoted the levels of p53, p27, AIF, BAX and t-Bid but decreased the expression of pro-caspase-3, Bid and Bcl-2.

Discussion

In the present study, we showed that DADS induced morphological changes and reduced the percentage of viable cells in the Ca Ski cell line in a dose- and time-dependent manner. Based on the data from flow cytometric analysis, DADS may induce cell death through either necrosis or Figure 4. DNA fragmentation determination of the apoptotic effects of apoptosis in Ca Ski cells. The IC 50 of DADS for Ca Ski DADS on Ca Ski cells. Ca Ski cells were incubated with 25 μM DAD cells was 25 μM in our study, hence this concentration was for 12, 24 and 48 h, and DNA fragmentation was determined using DNA used for further experiments . ROS, Ca 2+ , Δψm and gel electrophoresis as described in Materials and Methods.

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Figure 6. Flow cytometric analysis of reactive oxygen species (ROS) in human Ca Ski cells. Approximately 5×10 5 Ca Ski cells/ml were treated with 25 μM DADS for 0, 2, 4 and 5 h. The zero concentration was defined as the control. ROS were stained by DCFH-DA dye and determined as described in the Materials and Methods. *Significant difference between the DADS-treated cells and the control, p<0.05.

Figure 5. Flow cytometric assays of the effects of DADS on caspase-3 activity in Ca Ski cells. Ca Ski cells were incubated with 25 μM DADS for 0, 12, 24 and 48 h and harvested for caspase-3 activity determination as described in Materials and Methods. Representative profiles (A) and data (B) are presented. Data represent the mean ± S.D. of three experiments. *Significantly different from control of p<0.05.

Figure 7. Flow cytometric analysis of cytoplasmic Ca 2+ levels in human Ca Ski cells. Approximately 5×10 5 Ca Ski cells/ml were treated with 25 μM DADS for 0, 0.15, 1, 3, 4, 6 and 8 h. The zero concentration was apoptosis-associated protein levels indicated that DADS defined as the control. On exposure to DADS, the cytoplasmic Ca 2+ may induce apoptosis through ROS production, Ca 2+ increased from 0.15 to 6 h. *Significant difference between DADS- treated cells and control, p<0.05. release, followed by a change of the ratio of Bax/Bcl-2 then affecting the levels of Δψm, promoting the activation of caspase-3 and finally leading to apoptosis (Figure 11). This finding is in agreement with Park et al. , who demonstrated SH-SY5Y (21). Apparently, ROS play an important role in that DADS induced apoptosis in HCT-15 human colon DADS-induced apoptosis. cancer cells through the sequential mechanism of Ca 2+ In the present study, DADS-induced apoptosis in Ca Ski homeostasis disruption, accumulation of H 2O2 and resulting cells was based on the following findings: (i) occurrence of caspase-3 activation (19). Other investigators also reported sub-G1 group by flow cytometry (Figure 2); (ii) that DADS induced apoptosis of human leukemia HL-60 demonstration of DAPI staining of cells under fluorescence cells through the generation of H 2O2 and subsequent microscopy (Figure 3), (iii) fragmentation of DNA in gel activation of caspase-3 (20). DADS had also been reported electrophoresis (Figure 4) and (iv) increase/decrease in to increase the levels of ROS in human neuroblastoma cell apoptosis-associated proteins confirmed by Western blotting

2796 Lin et al : DADS Induces Apoptosis in Human Cervical Cancer Cells

Figure 8. Flow cytometric analysis of mitochondrial membrane potential (Δψm) in human Ca Ski cells. Approximately 5×10 5 Ca Ski cells/ml were treated with 25 μM DADS for 0, 0.15, 1, 6 and 24 h. The zero concentration was defined as the control. Levels of Δψm were detected using DiOC 6 dye as described in the Materials and Methods. *Significant difference between DADS-treated cells and control, p<0.05.

(Figure 10). Our results from phase-contrast microscopy examination also showed that DADS induced Ca Ski cell shrinkage, condensation and loss of cell-to-cell contact (Figure 1). The induction of apoptosis by DADS in an animal model in vivo for cervical cancer needs to be Figure 9. Effects of the calcium chelator BAPTA on Δψm of DADS- investigated in the future. treated Ca Ski cells. Ca Ski cells were pre-treated with BAPTA for 3 h We also used flow cytometry to demonstrate that DADS then treated with DADS before cells were harvested for Δψm determination as described in Materials and Methods. A, Levels of promoted the activation of caspase-3 in Ca Ski cells and this cytoplasmic Ca 2+ (%); B, levels of Δψm (%). Data represent mean ± effect was time dependent (Figure 5). Caspases play a S.D. of three experiments. *Significantly different from control of p<0.05. critical role in the process of apoptosis and can be grouped into apoptotic ‘initiators’, such as caspase-8, and apoptotic ‘effectors’, such as caspase-3, based on caspase substrate We also used flow cytometry to demonstrate that DADS specificities and target proteins (22). Adding a caspase-3 induced ROS production in Ca Ski cells (Figure 6). ROS play inhibitor, Ac-DEVD-CHO, led to a decrease in apoptosis in a key role in cell apoptosis and many chemopreventive agents Ca Ski cells treated with DADS (data not shown). Our induce apoptosis in part through generation of ROS and results indicated that DADS-induced apoptosis involved a disruption of redox homeostasis (25, 26). Pretreat ment with caspase-3-mediated mechanism. catalase (a scavenger of ROS) resulted in a decrease of ROS DADS induced dysfunction of mitochondria based on the levels and led to a decrease is the percentage of apoptosis in difference in Δψm levels in Ca Ski cells (Figure 8). DADS-treated Ca Ski cells (data not shown). Therefore, our Mitochondria play an important role in the regulation of findings demonstrated that DADS induced apoptosis in Ca Ski apoptosis (23, 24). Our results from Western blotting also cells through mitochondria- and ROS-dependent pathways. showed that DADS promoted Bax, but inhibited the We also used flow cytometry to demonstrate that DADS expression of Bcl-2 (Figure 10). In other words, DADS induced an increase in cytoplasmic Ca 2+ levels in Ca Ski cells changed the ratio of Bax/Bcl-2 which led to changes of the (Figure 7). Pre-treatment with BAPTA led to a blockage of levels of Δψm. Our results also showed that DADS the increase of cytoplasmic Ca 2+ and a protection against promoted t-Bid which can cause release of cytochrome c Δψm reduction (Figure 9A and B). It was reported that from the mitochondri a leading to caspase-3 activation and endoplasmic reticulum (ER) stress caused increase d release of finally causing apoptosis. Therefore, a mitochondria- cytoplasmic Ca 2+ from the ER to the cytosol (27). Therefore, dependent pathway is suggested to be involved in DADS- we may suggest that Ca 2+ plays a critical role in DADS- induced apoptosis in Ca Ski cells. induced mitochondria-dependent apoptosis in Ca Ski cells.

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Figure 11. Proposed model of DADS mechanism of action for induction of apoptosis in Ca Ski cells. DADS increased the production of ROS and Ca 2+ , promoted Bax, inhibited Bcl-2 and reduced Δψm levels, leading Figure 10. Representative Western blot showing changes in the levels of to increased caspase-3 activity before causing apoptosis in Ca Ski cells. p53, p27, AIF, pro-caspase-3, Bax, Bid, t-Bid and Bcl-2 in Ca Ski cells. Approximately 5×10 6 Ca Ski cells/ml were treated with DADS (25 μM) for 0, 6, 12, 24, 48 and 72 h, and harvested for protein level determin ation 3 Morris CR, Chen SC, Zhou L, Schopfer LM, Ding X and by Western blotting as described in Materials and Methods. Panel A: p53, Mirvish SS: Inhibition by allyl sulfides and phenethyl p27, AIF, pro-caspase-3; Panel B: Bax, Bid, t-Bid and Bcl-2. of methyl- n-pentylnitrosamine depentylation by rat esophageal microsomes, human and rat CYP2E1, and rat CYP2A3. Nutr Cancer 48 : 54-63, 2004. 4 Wen J, Zhang Y, Chen X, Shen L, Li GC and Xu M: Acknowledgements Enhancement of diallyl disulfide-induced apoptosis by inhibitors of MAPKs in human HepG2 hepatoma cells. Biochem Pharmacol 68 : 323-331, 2004. This work was supported by grant NSC93-2320-B-039-033 and 5 Filomeni G, Aquilano K, Rotilio G and Ciriolo MR: Reactive SC94-2320-B-039-018 from the National Science Council of oxygen species-dependent c-Jun NH -terminal /c-Jun Taiwan, R.O.C. 2 signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide. Cancer Res 63 : 5940-5949, 2003. References 6 Druesne N, Pagniez A, Mayeur C, Thomas M, Cherbuy C, Duee PH, Martel P and Chaumontet C: Diallyl disulfide (DADS) 1 Sundaram SG and Milner JA: Impact of organosulfur increases histone acetylation and p21 (waf1/cip1) expression in components in garlic on canine mammary tumor cells in culture. human colon tumor cell lines. Carcinogenesis 25 : 1227-1236, 2004. Cancer Letter 74 : 85-90, 1993. 7 Chung JG: Effects of garlic components diallyl sulfide and 2 Belman S, Solomon T, Segal A, Block E and Barany G: Inhibition diallyl disulfide on arylamine N- activity in of soybean lipoxygenase and mouse skin tumor promotion by human bladder tumour cells. Drug Chem Toxicol 22 : 343-358, and garlic components. J Biochem Toxicol 4: 151-160, 1989. 1999.

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