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Diallyl disulfide induces Ca2+ mobilization in human colon cancer cell line SW480

Chung-Yi Chen • Chien-Fu Huang • Ya-Ting Tseng • Soong-Yu Kuo

Abstract Diallyl disulfide (DADS), one of the major Keywords Ca2? signaling Diallyl disulfide (DADS) of , is recognized as a group Fura-2 Garlic SW480 of potential chemopreventive compounds. In this study, we examines the early signaling effects of DADS on human cells SW480 loaded with Ca2?-sensitive Introduction dye fura-2. It was found that DADS caused an immediate 2? and sustained rise of [Ca ]i in a concentration-dependent Garlic has been commonly used in foodstuff and medicines 2? manner (EC50 = 232 lM). DADS also induced a [Ca ]i for improving health. Laboratory studies in animals and cell elevation when extracellular Ca2? was removed, but the lines indicate that -containing compounds released magnitude was reduced by 45%. Depletion of intracellular upon processing (cutting or chewing) of vegetable Ca2? stores with 2 lM carbonylcyanide m-chloro- have diverse anti-carcinogenic properties involving multi- phenylhydrazone, a mitochondrial uncoupler, didn’t affect ple cellular events: proliferation, drug metabolism, apop- DADS’s effect. In Ca2?-free medium, the DADS-induced tosis, gene expression, redox status or inter-cellular 2? 2? [Ca ]i rise was abolished by depleting stored Ca with communication (Knowles and Milner 2001; Wu et al. 2001, 1 lM thapsigargin (an endoplasmic reticulum Ca2? pump 2002). Enhanced dietary intake of garlic is closely related 2? 2? inhibitor). DADS-caused [Ca ]i rise in Ca -containing with reduced cancer incidence (Hussain et al. 1990; Milner medium was not affected by modulation of protein kinase 1996). Diallyl disulfide (DADS), the most prevalent oil C activity. The DADS-induced Ca2? influx was blocked by soluble organosulfur compound (OSC) in processed garlic, nicardipine (10 lM). U73122, an inhibitor of phospholi- inhibits the cancer cell proliferation in various types of 2? pase C, abolished ATP (but not DADS)-induced [Ca ]i human cancers, such as breast cancer (Nakagawa et al. rise. These findings suggest that DADS induced a signifi- 2001), colon cancer (Sundarm and Milner 1996), lung 2? cant rise in [Ca ]i in SW480 colon cancer cells by cancer (Sakamoto et al. 1997), leukemia (Kwon et al. 2002), stimulating both extracellular Ca2? influx and thapsigar- and neuroblastoma (Filomeni et al. 2003). gin-sensitive intracellular Ca2? release via as yet uniden- Recent studies showed that DADS may inhibit the cell tified mechanisms. cycle of cancer cells at G2/M phase and induce apoptosis via the mitochondrial pathway through modulation of the bcl-2 family (Hong et al. 2000; Lin et al. 2006). DADS- C.-Y. Chen Y.-T. Tseng S.-Y. Kuo (&) induced apoptosis accompanied with an increase in intra- Department of Medical Laboratory Science and Biotechnology, 2? cellular-free concentration ([Ca ]i) has been School of Medical and Health Sciences, Fooyin University, 151 Chinhsueh Rd, Ta-Liao District, reported in various cell culture models including colon Kaohsiung City 83102, Taiwan cancer cells (Park et al. 2002), mouse–rat hybrid retina e-mail: [email protected] ganglion cells (Lin et al. 2006), neuroblastoma (Karmakar et al. 2007), and glioblastoma (Das et al. 2007). It is sug- C.-F. Huang 2? Department of Biological Science and Technology, gested that an intracellular Ca chelator (BAPTA) can 2? I-Shou University, Kaohsiung City 82445, Taiwan suppress DADS-evoked [Ca ]i elevation and ROS production can prevent caspase 3 activation and apoptosis. The other reagents were obtained from Sigma (St. Louis, However, despite the accumulation of data, the molecular MO, USA). mechanism underlying the Ca2? signal is still unexplored. It is known that Ca2? ions serve as a ubiquitous second Cell culture messenger in all eukaryotic cells (Clapham 1995). The 2? resting [Ca ]i is maintained at levels less than 0.1 lM, The SW480 cells were obtained from the American Type about four orders of magnitude lower than in the extra- Culture Collection. Cells were cultured in Dulbecco’s cellular solution (1–2 mM), but cellular excitation induces modified Eagle’s medium. The media were supplemented 2? a transient [Ca ]i rise up to several mM, or to even higher with 10% heat-inactivated fetal calf serum, 100 units/ml levels in tiny cellular compartments. These transient fluc- penicillin, and 100 lg/ml streptomycin. Cells were kept at 2? 2? tuations of [Ca ]i (termed ‘‘Ca signal’’) trigger or reg- 37°Cin5%CO2-containing humidified air. ulate various intracellular events. It is well established that 2? 2? cellular Ca overload, or perturbation of intracellular Optical measurements of [Ca ]i 2? [Ca ]i level, may cause cytotoxicity and result in either apoptosis, necrosis, or autophagy. Usually, the generation The fluorescence Ca2? indicator fura-2/AM was used as of Ca2? signal is determined by interaction of (1) external ascribed previously (Jan et al. 2005). Trypsinized cells Ca2? entry (2) Ca2? release from intracellular compart- (106/ml) were allowed to recover in the culture medium for ments (Ca2? stores) (3) cytoplasmic Ca2? buffering by 1 h before been loading with 2 lM fura-2/AM for 30 min Ca2? binding proteins, and (4) subsequent Ca2? removal at 25°C in the same medium. The cells were washed and from the cytoplasm due to transmembrane Ca2? efflux or resuspended in Ca2?-containing medium. Cells were trea- sequestration by intracellular Ca2? stores located in ted with vehicle (0.1% DMSO), 50, 100, 200, 300, 400, and organelles (Blaustein 1988). 500 lM DADS for the indicated times. Fura-2 fluorescence The effect of DADS on the profile of Ca2? signaling in measurements were performed in a water-jacketed cuvette human colon cancer SW480 cells has been unexplored. (25°C) with continuous stirring; the cuvette contained 1 ml Colorectal cancer is the third most frequent and second of medium and 0.5 million cells. Fluorescence was moni- most lethal in the United States (Jemal et al. 2007). tored with a Shimadzu RF-5301PC spectrofluorophotom- Therefore, there is a need to search more effective che- eter (Kyoto, Japan) by recording excitation signals at 340 motherapeutic agents that can be used to remedy the and 380 nm and emission signal at 510 nm at 1-s intervals. patients who have failed to respond under traditional che- Maximum and minimum fluorescence values were motherapy. This study was performed to elucidate the obtained by adding 0.1% Triton X-100 and 10 mM EGTA 2? 2? molecular mechanism of Ca in DADS-affected human sequentially at the end of each experiment. [Ca ]i was colorectal tumorigenesis. Using fura-2 as a fluorescent calculated as described previously assuming a Kd of 155 Ca2? indicator, we report for the first time that DADS nM (Grynkiewicz et al. 1985). 2? 2? induced a significant and prolonged [Ca ]i increase and Ca -containing medium contains (mM): NaCl, 140; cytotoxicity in human colorectal cancer cells. The con- KCl, 5; MgCl2, 1; CaCl2, 2; HEPES, 5; D-, 5; pH centration–response relationship, the Ca2? sources of the 7.4. In Ca2?-free medium, 2 mM Ca2? was substituted Ca2? signal, and the role of protein kinase A/C in the signal with 0.1 mM EGTA. have been investigated. Statistics

Materials and methods All data are reported as means ± SEM of several separate experiments. Data were analyzed by analysis of variances Chemical reagents (ANOVA). Multiple comparisons between group means were determined by using Student’s t test, and a P value of The reagents for cell culture were from Gibco (Gaithers- \0.05 was considered statistically significant. burg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). U73122 (1-(6-((17b-3- methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) Results and U73343 (1-(6-((17b-3-methoxyestra-1,3,5(10)-trien- 2? 17-yl)amino)hexyl)-2,5-pyrrolidine-dione) were from Bio- Effect of DADS on [Ca ]i in colorectal cancer cells mol (Plymouth Meeting, PA, USA). Diallyl disulfide (DADS) was purchased from Fluka Chemical Co., dis- DADS at concentrations between 0 and 500 lM increased 2? solved in (DMSO) and stored at -20°C. [Ca ]i in a concentration-dependent manner in the 2? 150 Fig. 1 Effects of DADS on [Ca ]i in SW480 colon cancer cells. c A a The concentration-dependent effects of DADS on intracellular Ca2? 2? content in Ca containing medium. The concentration of DADS was [DADS] (µM) indicated. The experiments were performed in Ca2?-containing medium. DADS was added at 30 s and was present throughout the measurement of 250 s. b The concentration-dependent effects of 100 500 DADS on intracellular Ca2? content in Ca2? free medium. The 400

concentration of DADS was indicated. c Dose–response plots of (nM) i 300 2? 2? filled ] 200 DADS-induced [Ca ]i increases in Ca -containing medium ( 2+ circles) and Ca2?-free medium (open circles). The data are presented 2? [Ca 50 as the percentage of control which is the net [Ca ]i increase induced 2? 100 by 500 lM DADS in Ca -containing medium. Data are mean ± 50 SEM of five experiments. *P \ 0.05 compared to open circles DADS presence of extracellular Ca2?. Figure 1a shows typical 0 0 50 100 150 200 250 300 recordings of the [Ca2?] increase induced by 0–500 lM i Time (sec) DADS. At a concentration of 1 lM, DADS had no effect 2? (i.e., equivalent to baseline, 0 lM). The [Ca ]i induced by 150 B 0–500 lM comprised an immediate rise and a sustained phase within 250 s. At a concentration of 500 lM, the 2? [Ca ]i increase had a net value of 124 ± 3nMat90s. µ Figure 1c (filled circles) shows the concentration–response 100 [DADS] ( M) curve of DADS-induced responses. The rising speed of the 500 400 (nM)

2? i Ca signal was slower in response to lower concentrations ] 300 2+ of DADS. 200 Experiments were performed to evaluate the relative [Ca 50 100 contribution of extracellular Ca2? entry and stored Ca2? 50 release in the DADS response. Figure 1b shows that DADS removal of extracellular Ca2? partly suppressed the 2? 0 DADS-induced [Ca ]i increase. The concentration– 0 50 100 150 200 250 300 response relationship of DADS-induced [Ca2?] increase in i Time (sec) the presence and absence of extracellular Ca2? was shown 2? 2? in Fig. 1c. Ca removal inhibited the [Ca ]i increase 120 C caused by 500 lM DADS by 26% in terms of the maxi- mum value (n = 5; P \ 0.05). 100 *

2? * Sources of Ca for the DADS-induced increase 80 2? in [Ca ]i * 60 To investigate the possible mechanisms of this calcium % control * elevation in the calcium-free condition, several inhibitors 40 of intracellular Ca2? stores were used. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is a mitochondrial 20 uncoupler and has been shown to release Ca2? from * mitochondria in different cells (Jan et al. 2005; Vaur et al. 0 2000). Thapsigargin is an endoplasmic reticulum Ca2? 0 100 200 300 400 500 600 [Diallyl disulfide] (µM) pump inhibitor (Thastrup et al. 1990). Figure 2a shows that in Ca2?-free medium, application of CCCP (2 lM) 2? 2? 2? induced a [Ca ]i increase with a net maximum value of of the stored Ca release in the DADS-mediated Ca 25 ± 2nM(n = 5), suggesting that mitochondrial Ca2? signal, CCCP or thapsigargin was conducted in parallel was mobilized. Thapsigargin (1 lM) was added afterward experiments. Figure 2b shows that in Ca2?-free medium, 2? and induced a [Ca ]i increase with a net maximum value after preincubation with DADS (500 lM) for 250 s, sub- 2? of 92 ± 2nM(n = 5). DADS (500 lM), subsequently sequent addition of 2 lM CCCP induced a [Ca ]i rise 2? added at the time point of 500 s, did not induce a [Ca ]i with a net value of 26 ± 2nM(P \ 0.05) which was the increase as shown in Fig. 1b. To evaluate the contribution same as the control CCCP response shown in Fig. 2a. Fig. 2 Intracellular sources of 200 2? A DADS-induced [Ca ]i increases. All experiments were 2? performed in Ca -free 150 medium. Reagents were applied at the time indicated by arrows. (nM) a–e The concentration of i ] 100 diallyl disulfide reagents was 2 lM CCCP, 2+

500 lM DADS, and 1 lM [Ca thapsigargin. Data are 50 means ± SEM of five experiments Thapsigargin CCCP 0 0 200 400 600 800 Time (sec)

200 B 200 C

150 150

diallyl disulfide (nM) (nM) i i ]

] 100 100 2+ 2+ [Ca [Ca

50 CCCP 50

diallyl disulfide CCCP 0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Time (sec) Time (sec)

200 D 200 E

150 150

thapsigargin (nM) (nM) i i ] 100 ] 100

2+ 2+ diallyl disulfide [Ca [Ca

50 50

diallyl disulfide thapsigargin 0 0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 Time (sec) Time (sec)

Fig. 3 Lack of involvement of 200 AB200 phospholipase C on DADS- 2? induced [Ca ]i increases. a ATP (10 lM) was added at 150 150 30 s. b U73122 (2 lM), ATP (10 lM), DADS (500 lM) were ATP (nm) (nm)

added at 30, 250, and 280 s, i i ] 100 ] 100 respectively. All experiments 2+ 2+ 2? were performed in Ca -free Ca [Ca [ medium. Data are U73122 ATP means ± SEM of five 50 50 experiments diallyl disulfide 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (sec) Time (sec) Conversely, Fig. 2c shows that after depleting the mito- 120 chondrial Ca2? store with CCCP (2 lM), addition of 2? 500 lM DADS induced a [Ca ]i elevation that was 100 indistinguishable from the control response shown in Fig. 2b in kinetics and magnitude. Thus, it appears that 80 mitochondrial Ca2? stores may be not involved in DADS- induced Ca2? release. 60

Figure 2d shows that after preincubation with DADS % control (500 lM) for 250 s, subsequent addition of 1 lM thapsi- 40 2? gargin also didn’t induced a [Ca ]i rise like the control thapsigargin response shown in Fig. 2a. In contrast, Fig. 2e 20 shows that application of 1 lM thapsigargin, caused a 2? 0 [Ca ]i increase that comprised an initial increase and a 2? PMA H89 gradual decay toward baseline. The net maximum [Ca ]i Control value was 86 ± 4nM(n = 5). After depleting the endo- GF109203X 2? plasmic reticulum Ca store with thapsigargin, addition of Fig. 5 Effect of protein kinase A inhibitor and C modulator on 2? 2? 500 lM DADS did not induce a [Ca ]i increase as shown DADS-induced [Ca ]i elevation. Experiments were performed in 2? in Fig. 2d. This Ca2? signal most likely reflected endo- Ca -containing medium. PMA (10 nM), GF 109203X (2 lM), or 2? H-89 (10 lM) were added 1 min prior to 500 lM DADS. Data are plasmic reticulumic Ca release. expressed as the percentage of control that is the net area under the 2? curve of 500 lM DADS-induced [Ca ]i rise (30–250 s interval), and Lack effect of phospholipase C on DADS-induced are means ± SEM of five experiments 2? increases in [Ca ]i an inactive U73122 analog (Thompson et al. 1991), did not 2? 2? Previous studies have shown that stored Ca can be affect ATP-induced [Ca ]i increases. This suggests that released by pathways dependent or independent on phos- U73122 effectively suppressed IP3 formation. Figure 3b pholipase C-associated IP3 formation (Chen et al. 2010, further shows that 500 lM DADS added after ATP 2? 2009). We therefore chose to explore whether IP3 is induced a [Ca ]i increase that was similar to the control required for DADS-induced Ca2? release. The control ATP DADS response shown in Fig. 1b(n = 5). 2? (10 lM)-induced [Ca ]i had a net peak value of 88 ± 3nM(n = 5) (Fig. 3a). Figure 3b shows that in Effects of Ca2? blockers on DADS-induced increases 2? 2? Ca -free medium, addition of 2 lM U73122 to suppress in [Ca ]i phospholipase C activity (Thompson et al. 1991) did not 2? 2? 2? alter basal [Ca ]i but abolished the [Ca ]i increase To test whether DADS-induced Ca release can be 2? 2? induced by ATP (10 lM) (n = 5), an IP3-dependent Ca reversed by Ca entry blockers, the effects of different 2? 2? mobilizer (Jan et al. 1998). Conversely, U73343 (10 lM), L-type Ca entry blockers on DADS-induced [Ca ]i rises

150 AB140

120 l) o 100 a ontr

100 c %

( 80 (nM) i ] on 2+

ati 60 v e [Ca 50 b el 40 2+ a C 20 *

0 0 0 50 100 150 200 250 control diltiazem Time nicardipine nifedipine verapamil

2? 2? Fig. 4 Effect of Ca channel blockers on DADS-induced [Ca ]i data are presented as the percentage of control which is the net area 2? 2? increases. All experiments were performed in Ca -containing under the curve (30–250 s) of the [Ca ]i increase induced by medium. a Trace a: DADS (500 lM) was added at 30 s. Trace b: 500 lM DADS (trace a in a). Data are means ± SEM of five nicardipine (10 lM) was added to cells 1 min before DADS. b The experiments. *P \ 0.05 compared to control were examined. Figure 4a shows that in Ca2?-containing induced Ca2? release was significantly inhibited by medium, pretreatment with 10 lM nicardipine inhibited depletion of the endoplasmic reticulumic Ca2? store with 2? 500 lM DADS-induced [Ca ]i elevation by 78.9% thapsigargin. Similar observation was also examined in the (n = 5; P \ 0.05). However, the Ca2? influx component of manipulation of [10]- (Chen et al. 2009). The the DADS response was not affected by diltiazem, nifedi- endoplasmic reticulum (ER) is one of major intracellular pine, and verapamil (n = 5, Fig. 4b). calcium stores and the organelle where proteins and are synthesized and modified (Ma and Hendershot 2004; Modulation of protein kinases on DADS-induced Orrenius et al. 2003). Ca2? dyshomeostasis of ER, protein 2? [Ca ]i elevation misfolding, or can lead to ER stress- induced cell death (Orrenius et al. 2003; Zhang et al. 2006). 2? 2? The roles of protein kinase A and C on DADS-induced An elevation in [Ca ]i by oxidants may activate Ca - 2? [Ca ]i elevation were also investigated. Figure 5 shows dependent enzymes such as proteases, nucleases, and 2? that 500 lM DADS-induced [Ca ]i rises were not altered phospholipases (Goldhaber and Qayyum 2000; Chakraborti by pretreatment with 10 lM H-89 (an inhibitor of protein et al. 1999) to facilitate mitochondrial oxidative stress kinase A), 10 nM phorbol myristate acetate (PMA, a pro- leading to cell death. How DADS releases Ca2? stores is tein kinase C activator) or 2 lM GF109203X (a protein unclear, but the process seems to be independent of IP3 kinase C inhibitor) (n = 5). because suppression of phospholipase C activity did not affect DADS-induced Ca2? release. The DADS-induced Ca2? rise appears to be via a Discussion pathway sensitive to nicardipine, since nicardipine-sensi- tive DADS-induced Ca2? influx was not controlled via DADS induces apoptosis and cytotoxicity in different conventional L-type Ca2? channels because it was not human colorectal cancer cell lines (Sundarm and Milner inhibited by diltiazem and verapamil. This is consistent 1996; Robert et al. 2001; Yang et al. 2009; Liao et al. with a previous study showing that SW480 cells are non- 2009). Reports show that DADS inhibits the cell cycle of excitable (Chen et al. 2009). In Ca2?-free medium, DADS- 2? 2? cancer cells at G2/M phase and induces apoptosis via the induced [Ca ]i elevation displayed a smaller [Ca ]i mitochondrial pathway with ROS generation (Park et al. increase throughout the measurement of 250 s. This sug- 2002; Yang et al. 2009). DADS also causes the increase of gests that extracellular Ca2? influx contributes not only to 2? 2? cytosolic Ca levels ([Ca ]i) (Park et al. 2002; Yang the initial increase, but also to the prolonged phase of 2? 2? et al. 2009). However, the detailed mechanism of DADS- DADS-induced [Ca ]i increase in Ca -containing med- mediated Ca2? elevation is poorly understood. In this ium. In non-excitable cells, a possible Ca2? influx pathway 2? 2? study, the effects of DADS on [Ca ]i profile in colon is called store-operated Ca entry, a process triggered by cancer cells have been investigated for the first time. The depletion of Ca2? stores (Putney 1986). But this possibility results suggest that DADS caused a significant concentra- was not explored due to the lack of selective pharmaco- 2? 2? 2? tion-dependent, sustained increase in [Ca ]i.InCa - logical inhibitors for this Ca influx (McFadzean and 2? 2? medium, the [Ca ]i increases induced by DADS were Gibson 2002). Thus, it remains possible that Ca entry prolonged and did not decay during the 5 min of mea- mechanisms other than depletion-activated channels may surement. Intracellular Ca2? is involved in the modulation be important in Ca2? influx in non-excitable cells. of virtually many cell functions. Omnipresent and sus- Recently, a group of reactive disulfide agents have been 2? tained [Ca ]i increases are thought to alter numerous cell found to produce ROS which are thought to oxidize cell functions (Annunziato et al. 2003; Orrenius et al. 2003; membrane and lead to cell injury and death during aging, Montell 2005). Garlic-derived OSCs may significantly inflammation, c-radiation, ischemia–reperfusion of heart, affect cell physiology by changing Ca2? signaling and kidney, liver, intestine, and brain (Jacobsen et al. 1994; stimulating Ca2?-coupled bioactive molecules. The results Weinberg and Venkatachalam 1991; Wang and Joseph 2? show that the [Ca ]i increase was contributed by both 2000). Cells exposed to those sulfhydryl agents are rec- intracellular Ca2? release and extracellular Ca2? influx, ognized to change the cellular redox state and key enzymes because the signal was partly suppressed by Ca2? removal. involved in cell function and growth. Disruption of this 2? 2? Regarding the Ca stores of the DADS response, the homeostasis is mostly followed by unregulated [Ca ]i store Ca2? in the mitochondria did not appear to play a elevation and cell death (Kuo et al. 2003a, b). Probably, the significant role since depletion of mitochondrial Ca2? with anti-proliferative effects of DADS to cells may relate to CCCP did not affect DADS-induced Ca2? release. The modulation of thiols in cytoplasm and cell membranes. thapsigargin-sensitive endoplasmic reticulumic store In conclusion, this study shows that DADS induces a 2? appears to play an important role because the DADS- significant increase in [Ca ]i in SW480 cells. In SW480 cells, DADS caused Ca2? signal in a concentration- Knowles LM, Milner JA (2001) Possible mechanism by which allyl dependent manner by evoking phospholipase C-independent sulfides suppress neoplastic cell proliferation. J Nutr 131:1061S– 2? 2? 1066S Ca release from ER and also by causing extracellular Ca Kuo SY, Ho CM, Chen WC, Jan CR (2003a) Sulfhydryl Modification influx. These signaling effects may play a crucial role in the by 4, 40-dithiodipyridine induces calcium mobilization in human physiological action of DADS. osteoblast-like cells. Arch Toxicol 77:630–637 Kuo SY, Jiann BP, Lu YC, Chang HT, Chen WC, Huang JK, Jan CR 0 Acknowledgments This work was supported by grants from (2003b) Thiol oxidation by 2,2 -dithiodipyridine induced cal- National Science Council (NSC93-2311-B-242-002) to Kuo SY. cium mobilization in MG63 human osteosarcoma cells. Life Sci 72:1733–1743 Kwon KB, Yoo SJ, Ryu DG, Yang JY, Rho HW, Kim JS, Park JW, Kim HR, Park BH (2002) Induction of apoptosis by diallyl References disulfide through activation of caspase-3 in human leukemia HL-60 cells. Biochem Pharmacol 63:41–47 Annunziato L, Amoroso S, Pannaccione A, Cataldi M, Pignataro G, Liao QJ, Su J, He J, Song Y, Tang HL, Su Q (2009) Effect of diallyl D’Alessio A, Sirabella R, Secondo A, Sibaud L, Di Renzo GF disulfide on cell cycle arrest of human colon cancer SW480 cells. (2003) Apoptosis induced in neuronal cells by oxidative stress: Ai Zheng 28:138–141 role played by caspases and intracellular calcium ions. Toxicol Lin HL, Yang JS, Yang JH, Fan SS, Chang WC, Li YC, Chung JG Lett 139:125–133 (2006) The role of Ca2? on the DADS-induced apoptosis Blaustein MP (1988) Calcium transport and buffering in neurons. inmouse-rat hybrid retina ganglion cells (N18). Neurochem Res Trends Neurosci 11:438–443 31:383–393 Chakraborti T, Das S, Mondal M, Roychoudhury S, Chakraborti S Ma Y, Hendershot LM (2004) The role of the unfolded protein (1999) Oxidant, mitochondria and calcium: an overview. Cell response in tumour development: friend or foe? Nat Rev Cancer Signal 11:77–85 4:966–977 2? Chen CY, Li YW, Kuo SY (2009) Effect of [10]-gingerol on [Ca ]i McFadzean I, Gibson A (2002) The developing relationship between and cell death in human colorectal cancer cells. Molecules receptor-operated and store-operated calcium channels in smooth 14:959–969 muscle. Br J Pharmacol 135:1–13 Chen CY, Yang YH, Kuo SY (2010) Effect of [6]- on Milner JA (1996) Garlic: its anticarcinogenic and antitumorigenic cytosolic Ca2? levels and proliferation in human oral cancer properties. Nutr Rev 54:S82–S86 cells (OC2). J Nat Prod 73:1370–1374 Montell C (2005) The latest waves in calcium signaling. Cell Clapham DE (1995) Calcium signaling. Cell 80:259–268 122:157–163 Das A, Banik NL, Ray SK (2007) Garlic compounds generate reactive Nakagawa H, Tsuta K, Kiuchi K, Senzaki H, Tanaka K, Hioki A, oxygen species leading to activation of stress kinases and Tsubura A (2001) Growth inhibitory effects of diallyl disulfide proteases for apoptosis in human glioblastoma T98G on human breast cancer cell lines. Carcinogenesis 22:891–897 and U87MG cells. Cancer 110:1083–1095 Orrenius S, Zhivotovsky B, Nicotera P (2003) Regulation of cell Filomeni G, Aquilano K, Rotilio G, Ciriolo MR (2003) Reactive death: the calcium-apoptosis link. Nat Rev Mol Cell Biol oxygen species-dependent c-JunNH2-terminal kinase/c-Jun sig- 4:552–565 naling cascade mediates neuroblastoma cell death induced by Park EK, Kwon KB, Park KI, Park BH, Jhee EC (2002) Role of Ca2? diallyl disulfide. Cancer Res 63:5940–5949 in diallyl disulfide induced apoptotic cell death of HCT-15 cells. Goldhaber JI, Qayyum MS (2000) Oxygen free radicals and Exp Mol Med 34:250–257 excitation-contraction coupling. Antioxid Redox Signal 2:55–64 Putney JW (1986) A model for receptor-regulated calcium entry. Cell Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Calcium 7:1–12 Ca2? indicators with greatly improved fluorescence properties. Robert V, Mouille´ B, Mayeur C, Michaud M, Blachier F (2001) J Biol Chem 260:3440–3450 Effects of the garlic compound diallyl disulfide on the metab- Hong YS, Ham YA, Choi JH, Kim J (2000) Effects of allyl sulfur olism, adherence and cell cycle of HT-29 colon carcinoma cells: compounds and garlic extract on the expression of Bcl-2, Bax, evidence of sensitive and resistant sub-populations. Carcinogen- and p53 in non small cell lung cancer cell lines. Exp Mol Med esis 22:1155–1161 32:127–134 Sakamoto K, Lawson LD, Milner JA (1997) Allyl sulfides from garlic Hussain SP, Jannu LN, Rao AR (1990) Chemopreventive action of suppress the in vitro proliferation of human A549 lung tumor garlic on methylcholanthrene-induced carcinogenesis in the cells. Nutr Cancer 29:152–156 uterine cervix of mice. Cancer Lett 49:175–180 Sundarm SG, Milner JA (1996) Diallyl disulfide induces apoptosis of Jacobsen WK, Schell RM, Matsumura JS, Cole DJ, Stier GR, Martin human colon tumor cells. Carcinogenesis 17:669–673 RD, Fandrich BL (1994) Nitrendipine and superoxide dismutase Thastrup O, Cullen PT, Drobak BK, Hanley MR, Dawson AP in ischemic renal injury. Ren Fail 16:697–705 (1990) Thapsigargin, a tumor promotor, discharges intracellu- Jan CR, Ho CM, Wu SN, Huang JK, Tseng CJ (1998) Mechanism of lar calcium stores by specific inhibition of the endoplasmic inhibition of extracellular ATP-evoked calcium reticulum calcium ATPase. Proc Natl Acad Sci USA mobilization in MDCK cells. Life Sci 62:533–543 87:2466–2470 Jan CR, Chen CH, Wang SC, Kuo SY (2005) Effect of Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher on intracellular calcium levels and viability in renal tubular cells. SK (1991) The aminosteroid U73122 inhibits muscarinic Cell Signal 17:847–855 receptor sequestration and phosphoinositide hydrolysis in Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ (2007) Cancer SK-N-SH neuroblastoma cells. J Biol Chem 266:23856–23862 statistics. CA Cancer J Clin 57:43–66 Vaur S, Sartor P, Dufy-Barbe L (2000) Calcium store depletion Karmakar S, Banik NL, Patel SJ, Ray SK (2007) Garlic compounds induced by mitochondrial uncoupling in prostatic cells. Gen induced calpain and intrinsic caspase cascade for apoptosis in Physiol Biophys 19:265–278 human malignant neuroblastoma SH-SY5Y cells. Apoptosis Wang H, Joseph JA (2000) Mechanisms of hydrogen-induced calcium 12:671–684 dysregulation in PC12 cells. Free Radic Biol Med 28:1222–1231 Weinberg JM, Venkatachalam MA (1991) Role of calcium channel Yang JS, Chen GW, Hsia TC, Ho HC, Ho CC, Lin MW, Lin SS, Yeh blockers in protection against experimental renal injury. Am J RD, Ip SW, Lu HF, Chung JG (2009) Diallyl disulfide induces Med 90:21S–26S apoptosis in human colon cancer cell line (COLO 205) through Wu CC, Sheen LY, Chen HW, Tsai SJ, Lii CK (2001) Effects of the induction of reactive oxygen species, endoplasmic reticulum organosulfur compounds from on the antioxidation stress, caspases casade and mitochondrial-dependent pathways. system in rat liver and red blood cells. Chem Toxicol Food Chem Toxicol 47:171–179 39:563–569 Zhang Y, Soboloff J, Zhu Z, Berger SA (2006) Inhibition of Ca2? Wu CC, Sheen LY, Chen HW, Kuo WW, Tsai SJ, Lii CK (2002) influx is required for mitochondrial reactive oxygen species- Differential effects of garlic oil and its three major organosulfur induced endoplasmic reticulum Ca2? depletion and cell death in components on the hepatic detoxification system in rats. J Agric leukemia cells. Mol Pharmacol 70:1424–1434 Food Chem 50:378–383