Diallyl disulfide induces Ca(2+) mobilization in human colon cancer cell line SW480

Diallyl disulfide induces Ca

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 organosulfur compounds of garlic, is recognized as a group of potential chemopreventive compounds. In this study, we examines the early signaling effects of DADS on human colorectal cancer cells SW480 loaded with Ca2?-sensitive dye fura-2. It was found that DADS caused an immediate and sustained rise of [Ca2?]iin a concentration-dependent manner (EC50= 232 lM). DADS also induced a [Ca2?]i elevation when extracellular Ca2? was removed, but the magnitude was reduced by 45%. Depletion of intracellular Ca2? stores with 2 lM carbonylcyanide m-chloro-phenylhydrazone, a mitochondrial uncoupler, didn’t affect DADS’s effect. In Ca2?-free medium, the DADS-induced [Ca2?]irise was abolished by depleting stored Ca2? with 1 lM thapsigargin (an endoplasmic reticulum Ca2? pump inhibitor). DADS-caused [Ca2?]i rise in Ca2?-containing medium was not affected by modulation of protein kinase C activity. The DADS-induced Ca2?influx was blocked by nicardipine (10 lM). U73122, an inhibitor of phospholi-pase C, abolished ATP (but not DADS)-induced [Ca2?]i rise. These findings suggest that DADS induced a signifi-cant rise in [Ca2?]i in SW480 colon cancer cells by stimulating both extracellular Ca2? influx and thapsigar-gin-sensitive intracellular Ca2? release via as yet uniden-tified mechanisms.

Keywords Ca2?signaling Diallyl disulfide (DADS)  Fura-2 Garlic  SW480

Introduction

Garlic has been commonly used in foodstuff and medicines for improving health. Laboratory studies in animals and cell lines indicate that sulfur-containing compounds released upon processing (cutting or chewing) of Allium vegetable have diverse anti-carcinogenic properties involving multi-ple cellular events: proliferation, drug metabolism, apop-tosis, gene expression, redox status or inter-cellular communication (Knowles and Milner2001; Wu et al.2001,

2002). Enhanced dietary intake of garlic is closely related with reduced cancer incidence (Hussain et al.1990; Milner

1996). Diallyl disulfide (DADS), the most prevalent oil soluble organosulfur compound (OSC) in processed garlic, inhibits the cancer cell proliferation in various types of human cancers, such as breast cancer (Nakagawa et al.

2001), colon cancer (Sundarm and Milner 1996), lung cancer (Sakamoto et al.1997), leukemia (Kwon et al.2002), and neuroblastoma (Filomeni et al.2003).

Recent studies showed that DADS may inhibit the cell 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-induced apoptosis accompanied with an increase in intra-cellular-free calcium concentration ([Ca2?]i) has been reported in various cell culture models including colon cancer cells (Park et al. 2002), mouse–rat hybrid retina ganglion cells (Lin et al.2006), neuroblastoma (Karmakar et al.2007), and glioblastoma (Das et al.2007). It is sug-gested that an intracellular Ca2? chelator (BAPTA) can suppress DADS-evoked [Ca2?]i elevation and ROS C.-Y. Chen Y.-T. Tseng  S.-Y. Kuo (&)

Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, 151 Chinhsueh Rd, Ta-Liao District,

Kaohsiung City 83102, Taiwan e-mail: [email protected] C.-F. Huang

Department of Biological Science and Technology, I-Shou University, Kaohsiung City 82445, Taiwan

production can prevent caspase 3 activation and apoptosis. However, despite the accumulation of data, the molecular mechanism underlying the Ca2?signal is still unexplored. It is known that Ca2?ions serve as a ubiquitous second messenger in all eukaryotic cells (Clapham 1995). The resting [Ca2?]i is maintained at levels less than 0.1 lM, about four orders of magnitude lower than in the extra-cellular solution (1–2 mM), but extra-cellular excitation induces a transient [Ca2?]irise up to several mM, or to even higher levels in tiny cellular compartments. These transient fluc-tuations of [Ca2?]i(termed ‘‘Ca2?signal’’) trigger or reg-ulate various intracellular events. It is well established that cellular Ca2? overload, or perturbation of intracellular [Ca2?]i level, may cause cytotoxicity and result in either apoptosis, necrosis, or autophagy. Usually, the generation of Ca2?signal is determined by interaction of (1) external Ca2? entry (2) Ca2? release from intracellular compart-ments (Ca2? stores) (3) cytoplasmic Ca2? buffering by Ca2? binding proteins, and (4) subsequent Ca2? removal from the cytoplasm due to transmembrane Ca2? efflux or sequestration by intracellular Ca2? stores located in organelles (Blaustein1988).

The effect of DADS on the profile of Ca2?signaling in human colon cancer SW480 cells has been unexplored. Colorectal cancer is the third most frequent and second most lethal in the United States (Jemal et al. 2007). Therefore, there is a need to search more effective che-motherapeutic agents that can be used to remedy the patients who have failed to respond under traditional che-motherapy. This study was performed to elucidate the molecular mechanism of Ca2? in DADS-affected human colorectal tumorigenesis. Using fura-2 as a fluorescent Ca2? indicator, we report for the first time that DADS induced a significant and prolonged [Ca2?]i increase and cytotoxicity in human colorectal cancer cells. The con-centration–response relationship, the Ca2? sources of the Ca2?signal, and the role of protein kinase A/C in the signal have been investigated.

Materials and methods Chemical reagents

The reagents for cell culture were from Gibco (Gaithers-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) and U73343 (1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-2,5-pyrrolidine-dione) were from Bio-mol (Plymouth Meeting, PA, USA). Diallyl disulfide (DADS) was purchased from Fluka Chemical Co., dis-solved in dimethyl sulfoxide (DMSO) and stored at -20°C.

The other reagents were obtained from Sigma (St. Louis, MO, USA).

Cell culture

The SW480 cells were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium. The media were supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 lg/ml streptomycin. Cells were kept at 37°C in 5% CO2-containing humidified air.

Optical measurements of [Ca2?]i

The fluorescence Ca2? indicator fura-2/AM was used as ascribed previously (Jan et al. 2005). Trypsinized cells (106/ml) were allowed to recover in the culture medium for 1 h before been loading with 2 lM fura-2/AM for 30 min at 25°C in the same medium. The cells were washed and resuspended in Ca2?-containing medium. Cells were trea-ted with vehicle (0.1% DMSO), 50, 100, 200, 300, 400, and 500 lM DADS for the indicated times. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was moni-tored with a Shimadzu RF-5301PC spectrofluorophotom-eter (Kyoto, Japan) by recording excitation signals at 340 and 380 nm and emission signal at 510 nm at 1-s intervals. Maximum and minimum fluorescence values were obtained by adding 0.1% Triton X-100 and 10 mM EGTA sequentially at the end of each experiment. [Ca2?]i was calculated as described previously assuming a Kd of 155 nM (Grynkiewicz et al.1985).

Ca2?-containing medium contains (mM): NaCl, 140; KCl, 5; MgCl2, 1; CaCl2, 2; HEPES, 5; D-glucose, 5; pH 7.4. In Ca2?-free medium, 2 mM Ca2? was substituted with 0.1 mM EGTA.

Statistics

All data are reported as means ± SEM of several separate experiments. Data were analyzed by analysis of variances (ANOVA). Multiple comparisons between group means were determined by using Student’s t test, and a P value of \0.05 was considered statistically significant.

Results

Effect of DADS on [Ca2?]iin colorectal cancer cells DADS at concentrations between 0 and 500 lM increased [Ca2?]i in a concentration-dependent manner in the

presence of extracellular Ca2?. Figure1a shows typical recordings of the [Ca2?]i increase induced by 0–500 lM DADS. At a concentration of 1 lM, DADS had no effect (i.e., equivalent to baseline, 0 lM). The [Ca2?]iinduced by 0–500 lM comprised an immediate rise and a sustained phase within 250 s. At a concentration of 500 lM, the [Ca2?]i increase had a net value of 124 ± 3 nM at 90 s. Figure1c (filled circles) shows the concentration–response curve of DADS-induced responses. The rising speed of the Ca2?signal was slower in response to lower concentrations of DADS.

Experiments were performed to evaluate the relative contribution of extracellular Ca2? entry and stored Ca2? release in the DADS response. Figure1b shows that removal of extracellular Ca2? partly suppressed the DADS-induced [Ca2?]i increase. The concentration– response relationship of DADS-induced [Ca2?]iincrease in the presence and absence of extracellular Ca2?was shown in Fig.1c. Ca2? removal inhibited the [Ca2?]i increase caused by 500 lM DADS by 26% in terms of the maxi-mum value (n = 5; P \ 0.05).

Sources of Ca2?for the DADS-induced increase in [Ca2?]i

To investigate the possible mechanisms of this calcium elevation in the calcium-free condition, several inhibitors of intracellular Ca2? stores were used. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is a mitochondrial uncoupler and has been shown to release Ca2? from mitochondria in different cells (Jan et al.2005; Vaur et al.

2000). Thapsigargin is an endoplasmic reticulum Ca2? pump inhibitor (Thastrup et al.1990). Figure2a shows that in Ca2?-free medium, application of CCCP (2 lM) induced a [Ca2?]i increase with a net maximum value of 25 ± 2 nM (n = 5), suggesting that mitochondrial Ca2? was mobilized. Thapsigargin (1 lM) was added afterward and induced a [Ca2?]iincrease with a net maximum value of 92 ± 2 nM (n = 5). DADS (500 lM), subsequently added at the time point of 500 s, did not induce a [Ca2?]i increase as shown in Fig.1b. To evaluate the contribution

of the stored Ca2? release in the DADS-mediated Ca2? signal, CCCP or thapsigargin was conducted in parallel experiments. Figure2b shows that in Ca2?-free medium, after preincubation with DADS (500 lM) for 250 s, sub-sequent addition of 2 lM CCCP induced a [Ca2?]i rise with a net value of 26 ± 2 nM (P \ 0.05) which was the same as the control CCCP response shown in Fig.2a.

Time (sec) 0 50 100 150 200 250 300 [C a 2+ ]i (nM) 0 50 100 150 [DADS] (µM) 100 50 300 400 500 200 Time (sec) [Ca 2+ ]i (nM) 0 50 100 150 [DADS] (µM) 500 400 300 200 100 50 A B [Diallyl disulfide] (µM) 0 100 200 300 400 500 600 % co n tro l 0 20 40 60 80 100 120 _C

*

*

*

*

*

Fig. 1 Effects of DADS on [Ca2?]iin SW480 colon cancer cells. aThe concentration-dependent effects of DADS on intracellular Ca2? content in Ca2?containing medium. The concentration of DADS was 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 DADS on intracellular Ca2? _{content in Ca}2? _{free medium. The} concentration of DADS was indicated. c Dose–response plots of DADS-induced [Ca2?_]

iincreases in Ca2?-containing medium (filled circles) and Ca2?-free medium (open circles). The data are presented as the percentage of control which is the net [Ca2?]iincrease induced by 500 lM DADS in Ca2?-containing medium. Data are mean ± SEM of five experiments. *P \ 0.05 compared to open circles

Time (sec) [Ca 2+ ](nM)i 0 50 100 150 200 CCCP Time (sec) [Ca 2+ ] i (n M ) 0 50 100 150 200 CCCP diallyl disulfide Time (sec) 0 100 200 300 400 500 600 [Ca 2+ ] (nM)i 0 50 100 150 200

diallyl disulfide _thapsigargin diallyl disulfide Time (sec) [Ca 2+ ] (nM)i 0 50 100 150 200 diallyl disulfide Time (sec) 0 200 400 600 800 [C a 2+ ]i (nM) 0 50 100 150 200 diallyl disulfide Thapsigargin CCCP A B C thapsigargin D E 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 100 200 300 400 500 600

Fig. 2 Intracellular sources of DADS-induced [Ca2?]i increases. All experiments were performed in Ca2?-free medium. Reagents were applied at the time indicated by arrows. a–e The concentration of reagents was 2 lM CCCP, 500 lM DADS, and 1 lM thapsigargin. Data are means ± SEM of five experiments Time (sec) 0 100 200 300 400 500 [Ca 2+ ]i (n m ) 0 50 100 150 200 Time (sec) 0 100 200 300 400 500 [Ca 2+ ]i (n m ) 0 50 100 150 200 ATP U73122 ATP diallyl disulfide A B

Fig. 3 Lack of involvement of phospholipase C on DADS-induced [Ca2?]iincreases. aATP (10 lM) was added at 30 s. b U73122 (2 lM), ATP (10 lM), DADS (500 lM) were added at 30, 250, and 280 s, respectively. All experiments were performed in Ca2?-free medium. Data are

means ± SEM of five experiments

Conversely, Fig.2c shows that after depleting the mito-chondrial Ca2? store with CCCP (2 lM), addition of 500 lM DADS induced a [Ca2?]i elevation that was indistinguishable from the control response shown in Fig.2b in kinetics and magnitude. Thus, it appears that mitochondrial Ca2?stores may be not involved in DADS-induced Ca2?release.

Figure2d shows that after preincubation with DADS (500 lM) for 250 s, subsequent addition of 1 lM thapsi-gargin also didn’t induced a [Ca2?]i rise like the control thapsigargin response shown in Fig.2a. In contrast, Fig.2e shows that application of 1 lM thapsigargin, caused a [Ca2?]i increase that comprised an initial increase and a gradual decay toward baseline. The net maximum [Ca2?]i value was 86 ± 4 nM (n = 5). After depleting the endo-plasmic reticulum Ca2?store with thapsigargin, addition of 500 lM DADS did not induce a [Ca2?]iincrease as shown in Fig.2d. This Ca2? signal most likely reflected endo-plasmic reticulumic Ca2?release.

Lack effect of phospholipase C on DADS-induced increases in [Ca2?]i

Previous studies have shown that stored Ca2? can be released by pathways dependent or independent on phos-pholipase C-associated IP3 formation (Chen et al. 2010,

2009). We therefore chose to explore whether IP3 is required for DADS-induced Ca2?release. The control ATP (10 lM)-induced [Ca2?]i had a net peak value of 88 ± 3 nM (n = 5) (Fig.3a). Figure3b shows that in Ca2?-free medium, addition of 2 lM U73122 to suppress phospholipase C activity (Thompson et al.1991) did not alter basal [Ca2?]i but abolished the [Ca2?]i increase induced by ATP (10 lM) (n = 5), an IP3-dependent Ca

2? mobilizer (Jan et al.1998). Conversely, U73343 (10 lM),

an inactive U73122 analog (Thompson et al.1991), did not affect ATP-induced [Ca2?]i increases. This suggests that U73122 effectively suppressed IP3 formation. Figure3b further shows that 500 lM DADS added after ATP induced a [Ca2?]i increase that was similar to the control DADS response shown in Fig.1b (n = 5).

Effects of Ca2?blockers on DADS-induced increases in [Ca2?]i

To test whether DADS-induced Ca2? release can be reversed by Ca2? entry blockers, the effects of different L-type Ca2?entry blockers on DADS-induced [Ca2?]irises

C a 2+ el ev at ion (% contr o l) 0 20 40 60 80 100 120 140 control

nicardipine nifedipine verapamil diltiazem

*

Fig. 4 Effect of Ca2? channel blockers on DADS-induced [Ca2?]i increases. All experiments were performed in Ca2?-containing medium. a Trace a: DADS (500 lM) was added at 30 s. Trace b: nicardipine (10 lM) was added to cells 1 min before DADS. b The

data are presented as the percentage of control which is the net area under the curve (30–250 s) of the [Ca2?]i increase induced by 500 lM DADS (trace a in a). Data are means ± SEM of five experiments. *P \ 0.05 compared to control

Control GF109203X PMA H89 0 20 40 60 80 100 120 % control

Fig. 5 Effect of protein kinase A inhibitor and C modulator on DADS-induced [Ca2?]i elevation. Experiments were performed in Ca2?-containing medium. PMA (10 nM), GF 109203X (2 lM), or H-89 (10 lM) were added 1 min prior to 500 lM DADS. Data are expressed as the percentage of control that is the net area under the curve of 500 lM DADS-induced [Ca2?]irise (30–250 s interval), and are means ± SEM of five experiments

were examined. Figure4a shows that in Ca2?-containing medium, pretreatment with 10 lM nicardipine inhibited 500 lM DADS-induced [Ca2?]i elevation by 78.9% (n = 5; P \ 0.05). However, the Ca2?influx component of the DADS response was not affected by diltiazem, nifedi-pine, and verapamil (n = 5, Fig.4b).

Modulation of protein kinases on DADS-induced [Ca2?]ielevation

The roles of protein kinase A and C on DADS-induced [Ca2?]i elevation were also investigated. Figure5 shows that 500 lM DADS-induced [Ca2?]irises were not altered by pretreatment with 10 lM H-89 (an inhibitor of protein kinase A), 10 nM phorbol myristate acetate (PMA, a pro-tein kinase C activator) or 2 lM GF109203X (a propro-tein kinase C inhibitor) (n = 5).

Discussion

DADS induces apoptosis and cytotoxicity in different human colorectal cancer cell lines (Sundarm and Milner

1996; Robert et al. 2001; Yang et al. 2009; Liao et al.

2009). Reports show that DADS inhibits the cell cycle of cancer cells at G2/M phase and induces apoptosis via the mitochondrial pathway with ROS generation (Park et al.

2002; Yang et al.2009). DADS also causes the increase of cytosolic Ca2? levels ([Ca2?]i) (Park et al. 2002; Yang et al.2009). However, the detailed mechanism of DADS-mediated Ca2? elevation is poorly understood. In this study, the effects of DADS on [Ca2?]i profile in colon cancer cells have been investigated for the first time. The results suggest that DADS caused a significant concentra-tion-dependent, sustained increase in [Ca2?]i. In Ca2? -medium, the [Ca2?]i increases induced by DADS were prolonged and did not decay during the 5 min of mea-surement. Intracellular Ca2?is involved in the modulation of virtually many cell functions. Omnipresent and sus-tained [Ca2?]iincreases are thought to alter numerous cell functions (Annunziato et al. 2003; Orrenius et al. 2003; Montell 2005). Garlic-derived OSCs may significantly affect cell physiology by changing Ca2? signaling and stimulating Ca2?-coupled bioactive molecules. The results show that the [Ca2?]i increase was contributed by both intracellular Ca2? release and extracellular Ca2? influx, because the signal was partly suppressed by Ca2?removal. Regarding the Ca2? stores of the DADS response, the store Ca2? in the mitochondria did not appear to play a significant role since depletion of mitochondrial Ca2?with CCCP did not affect DADS-induced Ca2? release. The thapsigargin-sensitive endoplasmic reticulumic store appears to play an important role because the

DADS-induced Ca2? release was significantly inhibited by depletion of the endoplasmic reticulumic Ca2? store with thapsigargin. Similar observation was also examined in the manipulation of [10]-gingerol (Chen et al. 2009). The endoplasmic reticulum (ER) is one of major intracellular calcium stores and the organelle where proteins and lipids are synthesized and modified (Ma and Hendershot 2004; Orrenius et al.2003). Ca2?dyshomeostasis of ER, protein misfolding, or oxidative stress can lead to ER stress-induced cell death (Orrenius et al.2003; Zhang et al.2006). An elevation in [Ca2?]i by oxidants may activate Ca2? -dependent enzymes such as proteases, nucleases, and phospholipases (Goldhaber and Qayyum2000; Chakraborti et al. 1999) to facilitate mitochondrial oxidative stress leading to cell death. How DADS releases Ca2? stores is unclear, but the process seems to be independent of IP3 because suppression of phospholipase C activity did not affect DADS-induced Ca2? release.

The DADS-induced Ca2? rise appears to be via a pathway sensitive to nicardipine, since nicardipine-sensi-tive DADS-induced Ca2? influx was not controlled via conventional L-type Ca2? channels because it was not inhibited by diltiazem and verapamil. This is consistent with a previous study showing that SW480 cells are non-excitable (Chen et al.2009). In Ca2?-free medium, DADS-induced [Ca2?]i elevation displayed a smaller [Ca2?]i increase throughout the measurement of 250 s. This sug-gests that extracellular Ca2? influx contributes not only to the initial increase, but also to the prolonged phase of DADS-induced [Ca2?]i increase in Ca2?-containing med-ium. In non-excitable cells, a possible Ca2?influx pathway is called store-operated Ca2? entry, a process triggered by depletion of Ca2?stores (Putney1986). But this possibility was not explored due to the lack of selective pharmaco-logical inhibitors for this Ca2? influx (McFadzean and Gibson 2002). Thus, it remains possible that Ca2? entry mechanisms other than depletion-activated channels may be important in Ca2? influx in non-excitable cells.

Recently, a group of reactive disulfide agents have been found to produce ROS which are thought to oxidize cell membrane and lead to cell injury and death during aging, inflammation, c-radiation, ischemia–reperfusion of heart, kidney, liver, intestine, and brain (Jacobsen et al. 1994; Weinberg and Venkatachalam 1991; Wang and Joseph

2000). Cells exposed to those sulfhydryl agents are rec-ognized to change the cellular redox state and key enzymes involved in cell function and growth. Disruption of this homeostasis is mostly followed by unregulated [Ca2?]i elevation and cell death (Kuo et al.2003a,b). Probably, the anti-proliferative effects of DADS to cells may relate to modulation of thiols in cytoplasm and cell membranes.

In conclusion, this study shows that DADS induces a significant increase in [Ca2?]i in SW480 cells. In SW480

cells, DADS caused Ca2? signal in a concentration-dependent manner by evoking phospholipase C-inconcentration-dependent Ca2?release from ER and also by causing extracellular Ca2? influx. These signaling effects may play a crucial role in the physiological action of DADS.

Acknowledgments This work was supported by grants from National Science Council (NSC93-2311-B-242-002) to Kuo SY.

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