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Phenethyl isothiocyanate induces Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells
Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 895–901
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Phenethyl isothiocyanate induces Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells Chung-Ren Jan a, Chung-Yi Chen b, Shu-Chi Wang c, Soong-Yu Kuo b,* a
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung 83102, Taiwan c School of Post-Baccalaureate Chinese Medicine, I-Shou University, Kaohsiung 82445, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2011 Received in revised form 29 March 2011 Accepted 5 April 2011 Available online 14 May 2011
The effect of phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in prostate cells has not been explored. This study examined whether PEITC altered [Ca2+]i levels in suspended human prostate PC3 cancer cells loaded with the Ca2+-sensitive dye fura-2. PEITC caused [Ca2+]i rises in a concentration-dependent manner with an EC50 of 192 mM. Removing extracellular Ca2+ reduced the Ca2+ signal by 42%. PEITC-induced [Ca2+]i rise in Ca2+-containing medium was not affected by modulation of protein kinase C activity, but was inhibited by 90% by the phospholipase A2 inhibitor aristolochic acid (20 mM). In Ca2+-free medium, the PEITC-induced [Ca2+]i rise was changed by depleting store Ca2+ with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, PEITC pretreatment abolished thapsigargin-induced [Ca2+]i rise. Chelation of cytosolic Ca2+ with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, PEITC induced a [Ca2+]i increase by causing Ca2+ release from endoplasmic reticulum in a phospholipase A2-dependent fashion and by inducing Ca2+ influx. PEITC decreased cell viability in a concentration-dependent, Ca2+-independent manner. Moreover, the effect of allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC) and PEITC on Ca2+ signaling has also been compared. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: [Ca2+]i Cruciferous vegetable Fura-2 PC3 Phenethyl isothiocyanate (PEITC) Allyl isothiocyanate (AITC) Benzyl isothiocyanate (BITC)
1. Introduction Cancer chemopreventive properties of cruciferous vegetables are attributed to organic isothiocyanates (ITCs) that are present naturally as thioglucoside conjugates (glucosinolates) in a variety of edible crucifers such as broccoli, cauliflower, cabbage and so forth [1]. Dietary ITCs are formed by hydrolysis of corresponding glucosinolates through enzymatical mediation of endogenous myrosinase, which is released by disruption of the plant cell during processing (cutting or chewing) [1,2]. ITCs have a common basic skeleton but differ in their terminal R-group, which can be an alkyl, alkenyl, alkylthioalkyl, aryl, beta-hydroxyalkyl or indolylmethyl group. At least 120 different glucosinolates have been identified [2]. The widely studied ITCs include allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC). PEITC is one of the most extensively studied ITCs, and recent epidemiological and experimental studies show that PEITC and other ITCs may possess promising cancer chemopreventive activities [3,4]. In regard to prostate cells, PEITC has been found
* Corresponding author. Tel.: +886 7 7811151x6200; fax: +886 7 7863667. E-mail address: [email protected] (S.-Y. Kuo).
to reduce tumor cell growth by affecting signaling pathways, arresting cell cycle and causing apoptotic cell death in vivo [5–7] or in vitro [8,9]. However, the molecular mechanism underlying the Ca2+ signal is still unclarified in prostate. More recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death. A well-tuned increase in cytosolic free Ca2+ levels ([Ca2+]i) plays a key role in most cell types, and can trigger many pathophysiological responses [10,11], but a unregulated increase in [Ca2+]i is cytotoxic [12]. Thus it is central to examine the effect of a reagent on Ca2+ signaling in order to understand its in vitro action. Elevated [Ca2+]i can trigger downstream deleterious events including upregulation of the Ca2+-dependent protease calpain, mitochondrial malfunction, and cytochrome c release for activation of caspase cascade leading to cytoskeletal disorder and apoptosis [13]. Moreover, perturbation of intracellular Ca2+ homeostasis during ER stress leading to improper protein folding and oxidative stress that are known to cause cell death [14]. In this study, by using fura-2 as a fluorescent Ca2+ probe it has been demonstrated that PEITC altered Ca2+ signaling in human prostate PC3 cancer cells and decreased cell viability. PC3 cells were chosen because previous studies suggest that this cell line produced a robust [Ca2+]i rise in response to different reagents.
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.04.009
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Also, PC3 cells have properties similar to human prostate cancer cells and have been widely used as a system for investigation of human prostate cells [15]. Many endogenous and exogenous agents can stimulate PC3 cells by causing a [Ca2+]i rise, such as estrogens [16], histamine [17], and clomiphene [18]. The present study demonstrates that PEITC evoked a [Ca2+]i rise in a concentration-dependent manner in PC3 cells. The time course and the concentration–response relationship, the Ca2+ sources of the Ca2+ signal, and the role of phospholipase A2 in the signal have been investigated. The effect of PEITC on cell viability and the dependence/independence of this cytotoxicity on Ca2+ have also been examined by using the tetrazolium assay and the Ca2+ chelator BAPTA, respectively. In addition, the effect of ITCs (AITC, BITC and PEITC) on Ca2+ signaling has also been compared. 2. Materials and methods 2.1. Cell culture Human PC3 prostate cancer cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were kept at 37 8C in 5% CO2-containing humidified air. 2.2. Solutions Ca2+-containing medium contained (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; glucose 5, pH 7.4. Ca2+-free medium
contained similar components as Ca2+-containing medium except that Ca2+ was substituted with 0.3 mM EGTA. PEITC was dissolved in dimethyl sulfoxide and kept at -20 8C as a 0.03 M stock, and was diluted to the final concentration before experiments. The concentration of the organic solvent(s) in the [Ca2+]i measurements and cell viability assay did not exceed 0.1% and did not alter baseline [Ca2+]i and cell viability (n = 4). 2.3. [Ca2+]i measurements The fluorescent Ca2+ indicator fura-2/AM was used as ascribed previously [19]. Trypsinized cells (106 ml1) were allowed to recover in culture medium for 1 h before being loaded with 2 mM fura-2/acetyl methyl for 30 min at 25 8C in the same medium. The cells were washed and resuspended in Ca2+-containing medium. Fura-2 fluorescence measurements were performed in a waterjacketed cuvette (25 8C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) by alternatively 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 [20]. Mn2+ quench of fura-2 fluorescence was performed in Ca2+-containing medium containing 50 mM MnCl2, by recording the excitation signal at 360 nm (Ca2+insensitive) and emission signal at 510 nm at 1-sec intervals as described previously [21].
Fig. 1. Effects of PEITC on [Ca2+]i in PC3 cells. (A) Concentration-dependent effects of PEITC. The experiments were performed in Ca2+-containing medium. PEITC was added at 30 s and was present throughout the measurement of 250 s. (B) PEITC-induced changes in the 340 nm and 380 nm excitation wavelength signals (emission wavelength 510 nm). PEITC (300 mM) was added at 30 s. (C) Effect of extracellular Ca2+ removal on PEITC-induced [Ca2+]i rise. The concentration of PEITC was indicated. (D) Concentration–response plots of PEITC-induced [Ca2+]i rises in Ca2+-containing medium (filled bars) and Ca2+-free medium(free bars). Data were mean SEM of 3–5 replicates.
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2.4. Cell viability assay The experiments were performed using 4-[3-[4-lodophenyl]-24(4-nitrophenyl)-] 2H-5-tetrazolio-1,3-benzene disulfonate (WST-1), a fluorescent cell viability reagent. The assay was based on cleavage of the tetrazolium salt WST-1 by active mitochondria to produce a soluble colored formazan salt [22]. Since the conversion is operated only by live cells, it directly correlated with cell numbers. Cells were plated at 1 104 in 96-well microtiter plates (Corning Costar Italia, Milan, Italy). Twenty-four hours after plating, at 70% confluence, the growth medium was removed and replaced with the test solutions (100 ml) containing different concentrations of PEITC. After 4-h exposure, the reaction medium was removed and the cells were washed twice with culture medium, then 100 ml culture medium and 10 ml WST-1 were added to each well. The cells were incubated for 2 h at 37 8C in a humidified atmosphere with 5% CO2, then the microplate was thoroughly shaken for 1 min and the absorbance was measured at 450 nm using a microtiter reader (model MRX II DYNEX Technologies, Chantily, VA, USA). In experiments using BAPTA to chelate intracellular Ca2+, fura-2-loaded cells were treated with 20 mM BAPTA/AM for 1 h. The cells were washed once with Ca2+containing medium and incubated with or without 300 mM PEITC for 4 h. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control. 2.5. Chemical reagents The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). WST-1 was from Roche Molecular Biochemical (Indianapolis, IN, USA). Phenylethyl isothiocyanate (PEITC) and other reagents were from Sigma (St. Louis, MO, USA). 2.6. Statistics Data are reported as means SEM of five experiments. Data were analyzed by one-way or two-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS1, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s honestly significant difference (HSD) procedure. P < 0.05 was considered significant. 3. Results
Fig. 2. Effect of PEITC on Ca2+ influx by measuring Mn2+ quench of fura-2 fluorescence. Experiments were performed in Ca2+-containing medium. MnCl2 (50 mM) was added to cells before fluorescence measurements. The y axis is the fluorescence intensity (in arbitrary units) measured at the Ca2+-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Control: no PEITC was present. Trace: PEITC (300 mM) was added at the time point indicated by the arrow. Data are mean SEM of five experiments.
PEITC response. Fig. 1(C) shows that removal of extracellular Ca2+ partly suppressed the PEITC-induced [Ca2+]i increase. The concentration–response relationship of PEITC-induced [Ca2+]i increase in the presence and absence of extracellular Ca2+ was shown in Fig. 1(D). Ca2+ removal inhibited the [Ca2+]i increase caused by 300 mM PEITC by 42% in terms of the maximum value (n = 5; P < 0.05). Mn2+ enters cells through similar pathways as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths [21]. Thus, quench of fura-2 fluorescence excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+ indicates Ca2+ influx. Fig. 2 shows that 300 mM PEITC induced an immediate decrease in the 360 nm excitation signal (compared to control; n = 5; P < 0.05). The decrease became greater with time and reached a maximal value of 66 2 arbitrary units (n = 5) at 90 s. This suggests that PEITC-induced [Ca2+]i rise involved Ca2+ influx from extracellular space. 3.3. Effects of Ca2+ blockers on PEITC-induced increases in [Ca2+]i In Ca2+-containing medium, PEITC (300 mM)-induced [Ca2+]i rise was not significantly changed by pretreatment with 1 mM nifedipine, nimodipine, verapamil or diltiazem (Fig. 3; n = 5; P > 0.05). Also, econazole and SKF96365 did not inhibit 300 mM PEITC-induced [Ca2+]i rise (Fig. 3).
3.1. Effect of PEITC on [Ca2+]i in PC3 cells In Ca2+-containing medium, at concentrations 100 mM, PEITC caused an immediate increase in [Ca2+]i in a concentrationdependent manner. Fig. 1(A) shows the responses induced by 100–300 mM PEITC. The basal [Ca2+]i was 31 3 nM (n = 5). The [Ca2+]i rise induced by 300 mM PEITC expressed an initial rise that reached a net (baseline subtracted) maximum of 138 4 nM at 100 s (n = 5), followed by a gradual decay that reached a sustained phase with a net value of 116 3 nM at the time point of 250 s. Fig. 1(B) shows that 300 mM PEITC induced an increase in the 340 nm excitation signal accompanied by a corresponding decrease in the 380 nm excitation signal. This suggests that the rises in fura-2 340/ 380 ratio signals induced by PEITC reported changes in [Ca2+]i, instead of an artifact. 3.2. Sources of Ca2+ for the PEITC-induced increase in [Ca2+]i. Experiments were performed to evaluate the relative contribution of extracellular Ca2+ entry and stored Ca2+ release in the
Fig. 3. Lack of effect of Ca2+ entry blockers on PEITC-induced [Ca2+]i rise. These chemicals were added 1 min prior to PEITC (300 mM) treatment in Ca2+-containing medium. The data are presented as percentage of control which was the [Ca2+]i rise induced by 300 mM PEITC. Data were means SEM of 3–5 replicates.
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Fig. 4. Effect of phospholipase A2 inhibitor, protein kinase A inhibitor, and protein kinase C modulators on PEITC-induced [Ca2+]i rises. (A) Trace a: PEITC (300 mM) was added at 30 s. Trace b: aristolochic acid (1 mM) was added to cells 1 min before PEITC. (B) These chemicals were added 1 min prior to PEITC (300 mM) treatment in Ca2+-containing medium. The data are presented as percentage of control which was the [Ca2+]i rise induced by 300 mM PEITC. Data were means SEM of 3–5 replicates.
3.4. Modulation of protein kinases and phospholipases on PEITCinduced [Ca2+]i elevation To see whether PEITC-induced Ca2+ signal was modulated by protein kinases and phospholipases, H-89 (10 mM), an inhibitor of protein kinase A; aristolochic acid (20 mM), an inhibitor of phospholipase A2; phorbol 12-myristate 13-acetate (PMA; 1 nM), a protein kinase C activator; and GF109203X (2 mM), a protein kinase C inhibitor were applied to cells for 1 min before addition of 300 mM PEITC. Fig. 4(A) shows that pretreatment of cells with 20 mM aristolochic acid decreased PEITC (300 mM)induced [Ca2+]i rise to 9% (n = 5; P < 0.05). H-89, PMA and GF109203X failed to alter the PEITC-induced [Ca2+]i rise in Ca2+containing medium (Fig. 4(B)). 3.5. Intracellular Ca2+ stores for PEITC-induced Ca2+ release In Ca2+-free medium, when PC3 cells were pretreated with 1 mM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+/ATP pump) [23], [Ca2+]i gradually increased to a net maximum value of 105 3 nM (Fig. 5(A)). The [Ca2+]i rise declined to 30 1 nM at 500 s. In this condition, 300 mM PEITC was added at 500 s and failed to induce a [Ca2+]i rise. Fig. 5(B) shows that addition of 300 mM PEITC induced a [Ca2+]i rise of 103 3 nM. The area under the curve of this response was smaller than that observed in Ca2+containing medium (Fig. 1(A)) by 42 3% (n = 5; P < 0.05). The PEITC response in Ca2+-free medium was not only smaller than that observed in Ca2+-containing medium in the peak value but also in the
later sustained phase. This suggests that PEITC induced both extracellular Ca2+ influx and Ca2+ release from stores throughout the measurement period. Fig. 5(B) shows that after PEITC treatment for 460 s, addition of thapsigargin failed to induce a [Ca2+]i rise (n = 5). 3.6. Lack of Effect of Chelating Ca2+ with BAPTA on PEITC-induced cell death The effect of 4 h incubation with PEITC on the viability of PC3 cells was explored. Fig. 6(A) shows that, in the presence of 25, 50, 100, 200, and 300 mM PEITC, the cell number decreased in a concentration-dependent manner (n = 5; P < 0.05). The next issue was whether the PEITC-induced cytotoxicity was caused by a preceding [Ca2+]i rise. The intracellular Ca2+ chelator BAPTA was applied to prevent a [Ca2+]i rise during PEITC pretreatment. It was found that 300 mM PEITC failed to cause a [Ca2+]i rise in 20 mM BAPTA-loaded cells (Fig. 6(B); n = 5). Fig. 6C shows that BAPTA loading did not significantly alter control cell viability. In the presence of 300 mM PEITC, cell viability was reduced to 18 3%. In the presence of BAPTA, the PEITC-induced decrease in cell viability was further decreased to 17 3% (n = 5; P < 0.05). 3.7. Structure activity relationship To explore the role of the structure moiety in ITCs-induced [Ca2+]i rises, the effects of equimolar concentrations of ITC analogs on [Ca2+]i were examined. Fig. 7(A) shows that in Ca2+-containing medium, [Ca2+]i was not significantly increased when cells were
Fig. 5. Intracellular sources of PEITC-induced [Ca2+]i rises. All experiments were performed in Ca2+-free medium. Reagents were applied at the time points indicated by arrows. The concentrations of drugs were 1 mM for thapsigargin and 300 mM for PEITC. Traces were means SEM of 3–5 replicates.
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Fig. 6. A tetrazolium assay of effect of PEITC on viability of PC3 cells. (A) Cells were treated with 0–300 mM of PEITC for 4 h, and the tetrazolium assay was performed. Data are expressed as percentage of control that is the increase in cell number in PEITC-free groups. *P < 0.05 compared to control. (B) BAPTA loading completely inhibited PEITC-induced [Ca2+]i rises. PEITC (300 mM) was added at 30 s to 20 mM BAPTA/AM-treated cells bathed in Ca2+-containing medium. (C) Independence of PEITC-induced cell death on preceding [Ca2+]i rises. BAPTA loading did not significantly alter cell viability in the absence or presence of 300 mM PEITC. *P < 0.05 compared to control. *P < 0.05 compared to the third column. Data are mean SEM of five experiments. Each treatment had six replicates (wells).
treated with 300 mM AITC. BITC-induced [Ca2+]i rise was 55.6 1.2% of PEITC-induced responses (n = 5; P < 0.05). Fig. 7(B) compares the net (baseline subtracted) maximum value of the three ITCs-induced [Ca2+]i rises. The order of the magnitude of ITCs-caused [Ca2+]i rises was: AITC < BITC < PEITC. 4. Discussion This study has explored the effect of the cruciferous ITCs on [Ca2+]i and viability in human prostate cancer PC3 cells. The data suggest that PEITC evoked a concentration-dependent [Ca2+]i rise in Ca2+-containing medium. The Ca2+ signal was contributed both by Ca2+ influx and release from intracellular Ca2+ store, because
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removing extracellular Ca2+ reduced the [Ca2+]i signal by 42%. Probably, the generation of Ca2+ signal is determined by interaction of external Ca2+ entry such as L-type Ca2+-channels [11]. However, the PEITC-induced Ca2+ influx was not sensitive to L-type Ca2+ channel blockers. Although Ca2+ entry has been observed in PC3 cells in response to different stimuli [15,17], the pathways underlying the Ca2+ entry is unclear. No store-operated Ca2+ entry or L-type Ca2+ channels have been observed. Our data also show that aristolochic acid, a phospholipase A2 inhibitor [24], significantly inhibited 300 mM PEITC-induced [Ca2+]i rise. Evidence shows that phospholipase A2 activity is associated with Ca2+ fluxes. It is reported that activation of PLA2 is required prior to the influx of extracellular Ca2+ into the CYP2E1expressing HepG2 cells [25]. Ca2+ overload was also observed by snake PLA2 neurotoxins in nerve terminals of cultured neurons [26]. Most importantly, recent evidence shows that PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta [27], and enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers [28]. Additionally, phospholipase A2 mediates store-operated Ca2+ entry in rat cerebellar granule cells [29]. Thus, these reports are consistent with our data that phospholipase A2 activity was required for PEITC-induced Ca2+ signal in PC3 cells. Because activation of PLC produces IP3 and diacylglycerol, which activates protein kinase C, the effect of modulation of protein kinase C activity on PEITC-induced [Ca2+]i rise was explored. Neither activation nor inhibition of protein kinase C altered PEITC-induced [Ca2+]i rise. In PC3 cells, thapsigargin/IP3-sensitive endoplasmic reticulum Ca2+ stores are dominant intracellular Ca2+ stores [17,18]. Ryanodine-sensitive Ca2+ stores are not reported. PEITC and thapsigargin appeared to share a common endoplasmic reticulum Ca2+ store, because the PEITC response was mostly inhibited by depletion of endoplasmic reticulum Ca2+ store with thapsigargin, and conversely, thapsigargin failed to release more Ca2+ after PEITC treatment. Similar observation was also examined in the manipulation of [6]-gingerol and diallyl sulfide (DAS) [30,31]. The endoplasmic reticulum is one of the major intracellular Ca2+ stores and the organelle where proteins and lipids are synthesized and modified [32,33]. Ca2+ dyshomeostasis of endoplasmic reticulum, protein misfolding, or oxidative stress can lead to endoplasmic reticulum stress-induced cell death [33,34]. Reactive disulfide compounds are a group of oxidizing agents that can produce reactive oxygen species which are thought to oxidize cell membrane and lead to cell injury and death during inflammation, aging, radiation, ischemia-reperfusion of heart, kidney, liver, intestine and brain [35–37]. Cells exposed to some sulfhydryl agents are recognized to change the cellular redox state and key enzymes involved in cell function and growth. Disruption of this homeostasis is mostly followed by dysregulation in [Ca2+]i and cell growth [38,39]. Therefore, the antiproliferative effects of PEITC to cells may relate to modulation of thiols in cytoplasm and membrane. Incubation with PEITC inhibited cell proliferation in a concentration-dependent manner starting at 50 mM PEITC. Although Ca2+ overloading can trigger cell death, not all forms of cell death are linked to Ca2+. The BAPTA experiments revealed that chelation of cytosolic Ca2+ during PEITC stimulation did not prevent PEITCinduced cell death, but actually worsened it. Thus it is clear that PEITC-induced cytotoxicity was not caused by Ca2+ overloading. The extent of [Ca2+]i rises were correlated to the steric structure of cruciferous isothiocyanates and were in the order of PEITC > BITC > AITC. Recent studies have revealed that the structural analog of PEITC may play a critical role in the biological activities of dietary ITCs [7]. These results suggest that even a subtle change in the ITC structure could have a significant impact on its effect on Ca2+ signaling. Allyl-ITC was relatively less effective
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Fig. 7. Effect of AITC, BITC and PEITC on [Ca2+]i. (A) ITCs (300 mM) was added at 30 s and were present throughout the measurement of 250 s. Traces a, b and c represent AITC, BITC and PEITC, respectively. The experiments were performed in Ca2+-containing medium. (B) The y axis is the percentage of control response that was the net area under the curve (30–250 s) of the [Ca2+]i rise induced by PEITC (trace c in A). Results are expressed as the percentage of control which is the response induced by PEITC. Data are mean SEM of five experiments. *P < 0.05 compared with control.
against Ca2+ handling of PC3 cells compared with aromatic ITCs. In addition, the alkyl chain length has a marked effect on the activity of aromatic ITCs against proliferation of PC3 cells. Thus, the extent of [Ca2+]i rise caused by benzyl-ITC is about 47% compared with PEITC examined in the present study. Together, this study suggests that in PC3 cells, PEITC induced a [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase A2-dependent, protein kinase C-independent fashion, and by inducing Ca2+ influx. PEITC decreased cell viability in a Ca2+-independent, concentration-dependent manner. The antiproliferative activity is related to the ITC structure. These effects may play a crucial role in the physiological action of PEITC. Acknowledgements This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS98-100) to Jan CR, and National Science Council (NSC93-2311-B-242-002) to Kuo SY. References [1] Conaway CC, Yang YM, Chung FL. Isothiocyanates as cancer chemopreventive agents: their biological activities and metabolism in rodents and humans. Curr Drug Metab 2002;3:233. [2] Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001;56:5. [3] Higdon JV, Delage B, Williams DE, Dashwood RH. Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol Res 2007;55:224. [4] Khor TO, Keum YS, Lin W, Kim JH, Hu R, Shen G. Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice. Cancer Res 2006;66:613. [5] Xiao D, Choi S, Lee YJ, Singh SV. Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells. Mol Carcinogen 2005;43:130. [6] Xiao D, Zeng Y, Choi S, Lew KL, Nelson JB, Singh SV. Caspase-dependent apoptosis induction by phenethyl isothiocyanate, a cruciferous vegetablederived cancer chemopreventive agent, is mediated by Bak and Bax. Clin Cancer Res 2005;11:2670. [7] Xiao D, Lew KL, Zeng Y, Xiao H, Marynowski SW, Dhir R. Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential. Carcinogen 2006;27:2223. [8] Xiao D, Johnson CS, Trump DL, Singh SV. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells. Mol Cancer Ther 2004;3:567. [9] Butler R, Mitchell SH, Tindall DJ, Young CY. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferatoractivated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth Differ 2000;11:49. [10] Clapham DE. Calcium signaling. Cell 1995;80:259.
[11] Berridge MJ. Elementary and global aspects of calcium signaling. J Exp Biol 1997;200:315. [12] Berridge MJ. The endoplasmic reticulum: a multifunctional signaling organelle. Cell Calcium 2002;32:235. [13] Karmakar S, Weinberg MS, Banik NL, Patel SJ, Ray SK. Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane. Neuroscience 2006;141:1265. [14] Welihinda AA, Tirasophon W, Kaufman RJ. The cellular response to protein misfolding in the endoplasmic reticulum. Gene Expr 1999;7:293. [15] Hsu SS, Chen WC, Lo YK, Cheng JS, Yeh JH, Cheng HH, et al. Effect of maprotiline on Ca2+ movement in human neuroblastoma cells. Life Sci 2004;75:1105. [16] Huang JK, Jan CR. Mechanism of estrogens-induced increases in intracellular Ca2+ in PC3 human prostate cancer cells. Prostate 2001;47:141. [17] Lee KC, Chang HT, Chou KJ, Tang KY, Wang JL, Lo YK, et al. Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells. Pharmacol Res 2001;44:547. [18] Jiann BP, Lu YC, Chang HT, Huang JK, Jan CR. Effect of clomiphene on Ca2+ movement in human prostate cancer cells. Life Sci 2002;70:3167. [19] Jan CR, Chen CH, Wang SC, Kuo SY. Effect of methylglyoxal on intracellular calcium levels and viability in renal tubular cells. Cell Signal 2005;17:847. [20] Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440. [21] Merritt JE, Jacob R, Hallam TJ. Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils. J Biol Chem 1989;264:1522. [22] Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly watersoluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull 1996;19:1518. [23] Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 1990; 87:2466. [24] Rosenthal MD, Vishwanath BS, Franson RC. Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils. J Biol Chem 1989;264:17069. [25] Caro AA, Cederbaum AI. Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells. J Biol Chem 2003;278:33866. [26] Tedesco E, Rigoni M, Caccin P, Grishin E, Rossetto O, Montecucco C. Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins. Toxicon 2009;54:138. [27] Boittin FX, Gribi F, Serir K, Be´ny JL. Ca2+. Am J Physiol Heart Circ Physiol 2008;295:H2466. [28] Boittin FX, Petermann O, Hirn C, Mittaud P, Roulet OM, Dorchies E, et al. Ca2+independent phospholipase A2 enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers. J Cell Sci 2006;119:3733. [29] Singaravelu K, Lohr C, Deitmer JW. Calcium-independent phospholipase A2 mediates store-operated calcium entry in rat cerebellar granule cells. Cerebellum 2008;7:467. [30] Chen CY, Chen CH, Kung CH, Kuo SH, Kuo SY. [6]-Gingerol induces Ca2+ mobilization in Madin-Darby canine kidney cells. J Nat Prod 2008;71:137. [31] Chen CH, Su SJ, Chang KL, Huang MW, Kuo SY. The garlic ingredient diallyl sulfide induces Ca2+ mobilization in Madin-Darby canine kidney cells. Food Chem Toxicol 2009;47:2344. [32] Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer 2004;4:966. [33] Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calciumapoptosis link. Nat Rev Mol Cell Biol 2003;4:552.
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Short communication
Phenethyl isothiocyanate induces Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells Chung-Ren Jan a, Chung-Yi Chen b, Shu-Chi Wang c, Soong-Yu Kuo b,* a
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan Department of Medical Laboratory Science and Biotechnology, School of Medical and Health Sciences, Fooyin University, Kaohsiung 83102, Taiwan c School of Post-Baccalaureate Chinese Medicine, I-Shou University, Kaohsiung 82445, Taiwan b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2011 Received in revised form 29 March 2011 Accepted 5 April 2011 Available online 14 May 2011
The effect of phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in prostate cells has not been explored. This study examined whether PEITC altered [Ca2+]i levels in suspended human prostate PC3 cancer cells loaded with the Ca2+-sensitive dye fura-2. PEITC caused [Ca2+]i rises in a concentration-dependent manner with an EC50 of 192 mM. Removing extracellular Ca2+ reduced the Ca2+ signal by 42%. PEITC-induced [Ca2+]i rise in Ca2+-containing medium was not affected by modulation of protein kinase C activity, but was inhibited by 90% by the phospholipase A2 inhibitor aristolochic acid (20 mM). In Ca2+-free medium, the PEITC-induced [Ca2+]i rise was changed by depleting store Ca2+ with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, PEITC pretreatment abolished thapsigargin-induced [Ca2+]i rise. Chelation of cytosolic Ca2+ with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, PEITC induced a [Ca2+]i increase by causing Ca2+ release from endoplasmic reticulum in a phospholipase A2-dependent fashion and by inducing Ca2+ influx. PEITC decreased cell viability in a concentration-dependent, Ca2+-independent manner. Moreover, the effect of allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC) and PEITC on Ca2+ signaling has also been compared. ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: [Ca2+]i Cruciferous vegetable Fura-2 PC3 Phenethyl isothiocyanate (PEITC) Allyl isothiocyanate (AITC) Benzyl isothiocyanate (BITC)
1. Introduction Cancer chemopreventive properties of cruciferous vegetables are attributed to organic isothiocyanates (ITCs) that are present naturally as thioglucoside conjugates (glucosinolates) in a variety of edible crucifers such as broccoli, cauliflower, cabbage and so forth [1]. Dietary ITCs are formed by hydrolysis of corresponding glucosinolates through enzymatical mediation of endogenous myrosinase, which is released by disruption of the plant cell during processing (cutting or chewing) [1,2]. ITCs have a common basic skeleton but differ in their terminal R-group, which can be an alkyl, alkenyl, alkylthioalkyl, aryl, beta-hydroxyalkyl or indolylmethyl group. At least 120 different glucosinolates have been identified [2]. The widely studied ITCs include allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC). PEITC is one of the most extensively studied ITCs, and recent epidemiological and experimental studies show that PEITC and other ITCs may possess promising cancer chemopreventive activities [3,4]. In regard to prostate cells, PEITC has been found
* Corresponding author. Tel.: +886 7 7811151x6200; fax: +886 7 7863667. E-mail address: [email protected] (S.-Y. Kuo).
to reduce tumor cell growth by affecting signaling pathways, arresting cell cycle and causing apoptotic cell death in vivo [5–7] or in vitro [8,9]. However, the molecular mechanism underlying the Ca2+ signal is still unclarified in prostate. More recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death. A well-tuned increase in cytosolic free Ca2+ levels ([Ca2+]i) plays a key role in most cell types, and can trigger many pathophysiological responses [10,11], but a unregulated increase in [Ca2+]i is cytotoxic [12]. Thus it is central to examine the effect of a reagent on Ca2+ signaling in order to understand its in vitro action. Elevated [Ca2+]i can trigger downstream deleterious events including upregulation of the Ca2+-dependent protease calpain, mitochondrial malfunction, and cytochrome c release for activation of caspase cascade leading to cytoskeletal disorder and apoptosis [13]. Moreover, perturbation of intracellular Ca2+ homeostasis during ER stress leading to improper protein folding and oxidative stress that are known to cause cell death [14]. In this study, by using fura-2 as a fluorescent Ca2+ probe it has been demonstrated that PEITC altered Ca2+ signaling in human prostate PC3 cancer cells and decreased cell viability. PC3 cells were chosen because previous studies suggest that this cell line produced a robust [Ca2+]i rise in response to different reagents.
1876-1070/$ – see front matter ß 2011 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jtice.2011.04.009
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Also, PC3 cells have properties similar to human prostate cancer cells and have been widely used as a system for investigation of human prostate cells [15]. Many endogenous and exogenous agents can stimulate PC3 cells by causing a [Ca2+]i rise, such as estrogens [16], histamine [17], and clomiphene [18]. The present study demonstrates that PEITC evoked a [Ca2+]i rise in a concentration-dependent manner in PC3 cells. The time course and the concentration–response relationship, the Ca2+ sources of the Ca2+ signal, and the role of phospholipase A2 in the signal have been investigated. The effect of PEITC on cell viability and the dependence/independence of this cytotoxicity on Ca2+ have also been examined by using the tetrazolium assay and the Ca2+ chelator BAPTA, respectively. In addition, the effect of ITCs (AITC, BITC and PEITC) on Ca2+ signaling has also been compared. 2. Materials and methods 2.1. Cell culture Human PC3 prostate cancer cells were obtained from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were kept at 37 8C in 5% CO2-containing humidified air. 2.2. Solutions Ca2+-containing medium contained (in mM): NaCl 140; KCl 5; MgCl2 1; CaCl2 2; Hepes 10; glucose 5, pH 7.4. Ca2+-free medium
contained similar components as Ca2+-containing medium except that Ca2+ was substituted with 0.3 mM EGTA. PEITC was dissolved in dimethyl sulfoxide and kept at -20 8C as a 0.03 M stock, and was diluted to the final concentration before experiments. The concentration of the organic solvent(s) in the [Ca2+]i measurements and cell viability assay did not exceed 0.1% and did not alter baseline [Ca2+]i and cell viability (n = 4). 2.3. [Ca2+]i measurements The fluorescent Ca2+ indicator fura-2/AM was used as ascribed previously [19]. Trypsinized cells (106 ml1) were allowed to recover in culture medium for 1 h before being loaded with 2 mM fura-2/acetyl methyl for 30 min at 25 8C in the same medium. The cells were washed and resuspended in Ca2+-containing medium. Fura-2 fluorescence measurements were performed in a waterjacketed cuvette (25 8C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million cells. Fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) by alternatively 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 [20]. Mn2+ quench of fura-2 fluorescence was performed in Ca2+-containing medium containing 50 mM MnCl2, by recording the excitation signal at 360 nm (Ca2+insensitive) and emission signal at 510 nm at 1-sec intervals as described previously [21].
Fig. 1. Effects of PEITC on [Ca2+]i in PC3 cells. (A) Concentration-dependent effects of PEITC. The experiments were performed in Ca2+-containing medium. PEITC was added at 30 s and was present throughout the measurement of 250 s. (B) PEITC-induced changes in the 340 nm and 380 nm excitation wavelength signals (emission wavelength 510 nm). PEITC (300 mM) was added at 30 s. (C) Effect of extracellular Ca2+ removal on PEITC-induced [Ca2+]i rise. The concentration of PEITC was indicated. (D) Concentration–response plots of PEITC-induced [Ca2+]i rises in Ca2+-containing medium (filled bars) and Ca2+-free medium(free bars). Data were mean SEM of 3–5 replicates.
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2.4. Cell viability assay The experiments were performed using 4-[3-[4-lodophenyl]-24(4-nitrophenyl)-] 2H-5-tetrazolio-1,3-benzene disulfonate (WST-1), a fluorescent cell viability reagent. The assay was based on cleavage of the tetrazolium salt WST-1 by active mitochondria to produce a soluble colored formazan salt [22]. Since the conversion is operated only by live cells, it directly correlated with cell numbers. Cells were plated at 1 104 in 96-well microtiter plates (Corning Costar Italia, Milan, Italy). Twenty-four hours after plating, at 70% confluence, the growth medium was removed and replaced with the test solutions (100 ml) containing different concentrations of PEITC. After 4-h exposure, the reaction medium was removed and the cells were washed twice with culture medium, then 100 ml culture medium and 10 ml WST-1 were added to each well. The cells were incubated for 2 h at 37 8C in a humidified atmosphere with 5% CO2, then the microplate was thoroughly shaken for 1 min and the absorbance was measured at 450 nm using a microtiter reader (model MRX II DYNEX Technologies, Chantily, VA, USA). In experiments using BAPTA to chelate intracellular Ca2+, fura-2-loaded cells were treated with 20 mM BAPTA/AM for 1 h. The cells were washed once with Ca2+containing medium and incubated with or without 300 mM PEITC for 4 h. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and was expressed as a percentage of the control. 2.5. Chemical reagents The reagents for cell culture were from Gibco (Gaithersburg, MD, USA). Fura-2/AM was from Molecular Probes (Eugene, OR, USA). WST-1 was from Roche Molecular Biochemical (Indianapolis, IN, USA). Phenylethyl isothiocyanate (PEITC) and other reagents were from Sigma (St. Louis, MO, USA). 2.6. Statistics Data are reported as means SEM of five experiments. Data were analyzed by one-way or two-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS1, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post hoc analysis using the Tukey’s honestly significant difference (HSD) procedure. P < 0.05 was considered significant. 3. Results
Fig. 2. Effect of PEITC on Ca2+ influx by measuring Mn2+ quench of fura-2 fluorescence. Experiments were performed in Ca2+-containing medium. MnCl2 (50 mM) was added to cells before fluorescence measurements. The y axis is the fluorescence intensity (in arbitrary units) measured at the Ca2+-insensitive excitation wavelength of 360 nm and the emission wavelength of 510 nm. Control: no PEITC was present. Trace: PEITC (300 mM) was added at the time point indicated by the arrow. Data are mean SEM of five experiments.
PEITC response. Fig. 1(C) shows that removal of extracellular Ca2+ partly suppressed the PEITC-induced [Ca2+]i increase. The concentration–response relationship of PEITC-induced [Ca2+]i increase in the presence and absence of extracellular Ca2+ was shown in Fig. 1(D). Ca2+ removal inhibited the [Ca2+]i increase caused by 300 mM PEITC by 42% in terms of the maximum value (n = 5; P < 0.05). Mn2+ enters cells through similar pathways as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths [21]. Thus, quench of fura-2 fluorescence excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+ indicates Ca2+ influx. Fig. 2 shows that 300 mM PEITC induced an immediate decrease in the 360 nm excitation signal (compared to control; n = 5; P < 0.05). The decrease became greater with time and reached a maximal value of 66 2 arbitrary units (n = 5) at 90 s. This suggests that PEITC-induced [Ca2+]i rise involved Ca2+ influx from extracellular space. 3.3. Effects of Ca2+ blockers on PEITC-induced increases in [Ca2+]i In Ca2+-containing medium, PEITC (300 mM)-induced [Ca2+]i rise was not significantly changed by pretreatment with 1 mM nifedipine, nimodipine, verapamil or diltiazem (Fig. 3; n = 5; P > 0.05). Also, econazole and SKF96365 did not inhibit 300 mM PEITC-induced [Ca2+]i rise (Fig. 3).
3.1. Effect of PEITC on [Ca2+]i in PC3 cells In Ca2+-containing medium, at concentrations 100 mM, PEITC caused an immediate increase in [Ca2+]i in a concentrationdependent manner. Fig. 1(A) shows the responses induced by 100–300 mM PEITC. The basal [Ca2+]i was 31 3 nM (n = 5). The [Ca2+]i rise induced by 300 mM PEITC expressed an initial rise that reached a net (baseline subtracted) maximum of 138 4 nM at 100 s (n = 5), followed by a gradual decay that reached a sustained phase with a net value of 116 3 nM at the time point of 250 s. Fig. 1(B) shows that 300 mM PEITC induced an increase in the 340 nm excitation signal accompanied by a corresponding decrease in the 380 nm excitation signal. This suggests that the rises in fura-2 340/ 380 ratio signals induced by PEITC reported changes in [Ca2+]i, instead of an artifact. 3.2. Sources of Ca2+ for the PEITC-induced increase in [Ca2+]i. Experiments were performed to evaluate the relative contribution of extracellular Ca2+ entry and stored Ca2+ release in the
Fig. 3. Lack of effect of Ca2+ entry blockers on PEITC-induced [Ca2+]i rise. These chemicals were added 1 min prior to PEITC (300 mM) treatment in Ca2+-containing medium. The data are presented as percentage of control which was the [Ca2+]i rise induced by 300 mM PEITC. Data were means SEM of 3–5 replicates.
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Fig. 4. Effect of phospholipase A2 inhibitor, protein kinase A inhibitor, and protein kinase C modulators on PEITC-induced [Ca2+]i rises. (A) Trace a: PEITC (300 mM) was added at 30 s. Trace b: aristolochic acid (1 mM) was added to cells 1 min before PEITC. (B) These chemicals were added 1 min prior to PEITC (300 mM) treatment in Ca2+-containing medium. The data are presented as percentage of control which was the [Ca2+]i rise induced by 300 mM PEITC. Data were means SEM of 3–5 replicates.
3.4. Modulation of protein kinases and phospholipases on PEITCinduced [Ca2+]i elevation To see whether PEITC-induced Ca2+ signal was modulated by protein kinases and phospholipases, H-89 (10 mM), an inhibitor of protein kinase A; aristolochic acid (20 mM), an inhibitor of phospholipase A2; phorbol 12-myristate 13-acetate (PMA; 1 nM), a protein kinase C activator; and GF109203X (2 mM), a protein kinase C inhibitor were applied to cells for 1 min before addition of 300 mM PEITC. Fig. 4(A) shows that pretreatment of cells with 20 mM aristolochic acid decreased PEITC (300 mM)induced [Ca2+]i rise to 9% (n = 5; P < 0.05). H-89, PMA and GF109203X failed to alter the PEITC-induced [Ca2+]i rise in Ca2+containing medium (Fig. 4(B)). 3.5. Intracellular Ca2+ stores for PEITC-induced Ca2+ release In Ca2+-free medium, when PC3 cells were pretreated with 1 mM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+/ATP pump) [23], [Ca2+]i gradually increased to a net maximum value of 105 3 nM (Fig. 5(A)). The [Ca2+]i rise declined to 30 1 nM at 500 s. In this condition, 300 mM PEITC was added at 500 s and failed to induce a [Ca2+]i rise. Fig. 5(B) shows that addition of 300 mM PEITC induced a [Ca2+]i rise of 103 3 nM. The area under the curve of this response was smaller than that observed in Ca2+containing medium (Fig. 1(A)) by 42 3% (n = 5; P < 0.05). The PEITC response in Ca2+-free medium was not only smaller than that observed in Ca2+-containing medium in the peak value but also in the
later sustained phase. This suggests that PEITC induced both extracellular Ca2+ influx and Ca2+ release from stores throughout the measurement period. Fig. 5(B) shows that after PEITC treatment for 460 s, addition of thapsigargin failed to induce a [Ca2+]i rise (n = 5). 3.6. Lack of Effect of Chelating Ca2+ with BAPTA on PEITC-induced cell death The effect of 4 h incubation with PEITC on the viability of PC3 cells was explored. Fig. 6(A) shows that, in the presence of 25, 50, 100, 200, and 300 mM PEITC, the cell number decreased in a concentration-dependent manner (n = 5; P < 0.05). The next issue was whether the PEITC-induced cytotoxicity was caused by a preceding [Ca2+]i rise. The intracellular Ca2+ chelator BAPTA was applied to prevent a [Ca2+]i rise during PEITC pretreatment. It was found that 300 mM PEITC failed to cause a [Ca2+]i rise in 20 mM BAPTA-loaded cells (Fig. 6(B); n = 5). Fig. 6C shows that BAPTA loading did not significantly alter control cell viability. In the presence of 300 mM PEITC, cell viability was reduced to 18 3%. In the presence of BAPTA, the PEITC-induced decrease in cell viability was further decreased to 17 3% (n = 5; P < 0.05). 3.7. Structure activity relationship To explore the role of the structure moiety in ITCs-induced [Ca2+]i rises, the effects of equimolar concentrations of ITC analogs on [Ca2+]i were examined. Fig. 7(A) shows that in Ca2+-containing medium, [Ca2+]i was not significantly increased when cells were
Fig. 5. Intracellular sources of PEITC-induced [Ca2+]i rises. All experiments were performed in Ca2+-free medium. Reagents were applied at the time points indicated by arrows. The concentrations of drugs were 1 mM for thapsigargin and 300 mM for PEITC. Traces were means SEM of 3–5 replicates.
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Fig. 6. A tetrazolium assay of effect of PEITC on viability of PC3 cells. (A) Cells were treated with 0–300 mM of PEITC for 4 h, and the tetrazolium assay was performed. Data are expressed as percentage of control that is the increase in cell number in PEITC-free groups. *P < 0.05 compared to control. (B) BAPTA loading completely inhibited PEITC-induced [Ca2+]i rises. PEITC (300 mM) was added at 30 s to 20 mM BAPTA/AM-treated cells bathed in Ca2+-containing medium. (C) Independence of PEITC-induced cell death on preceding [Ca2+]i rises. BAPTA loading did not significantly alter cell viability in the absence or presence of 300 mM PEITC. *P < 0.05 compared to control. *P < 0.05 compared to the third column. Data are mean SEM of five experiments. Each treatment had six replicates (wells).
treated with 300 mM AITC. BITC-induced [Ca2+]i rise was 55.6 1.2% of PEITC-induced responses (n = 5; P < 0.05). Fig. 7(B) compares the net (baseline subtracted) maximum value of the three ITCs-induced [Ca2+]i rises. The order of the magnitude of ITCs-caused [Ca2+]i rises was: AITC < BITC < PEITC. 4. Discussion This study has explored the effect of the cruciferous ITCs on [Ca2+]i and viability in human prostate cancer PC3 cells. The data suggest that PEITC evoked a concentration-dependent [Ca2+]i rise in Ca2+-containing medium. The Ca2+ signal was contributed both by Ca2+ influx and release from intracellular Ca2+ store, because
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removing extracellular Ca2+ reduced the [Ca2+]i signal by 42%. Probably, the generation of Ca2+ signal is determined by interaction of external Ca2+ entry such as L-type Ca2+-channels [11]. However, the PEITC-induced Ca2+ influx was not sensitive to L-type Ca2+ channel blockers. Although Ca2+ entry has been observed in PC3 cells in response to different stimuli [15,17], the pathways underlying the Ca2+ entry is unclear. No store-operated Ca2+ entry or L-type Ca2+ channels have been observed. Our data also show that aristolochic acid, a phospholipase A2 inhibitor [24], significantly inhibited 300 mM PEITC-induced [Ca2+]i rise. Evidence shows that phospholipase A2 activity is associated with Ca2+ fluxes. It is reported that activation of PLA2 is required prior to the influx of extracellular Ca2+ into the CYP2E1expressing HepG2 cells [25]. Ca2+ overload was also observed by snake PLA2 neurotoxins in nerve terminals of cultured neurons [26]. Most importantly, recent evidence shows that PLA2 controls endothelial store-operated Ca2+ entry and vascular tone in intact aorta [27], and enhances store-operated Ca2+ entry in dystrophic skeletal muscle fibers [28]. Additionally, phospholipase A2 mediates store-operated Ca2+ entry in rat cerebellar granule cells [29]. Thus, these reports are consistent with our data that phospholipase A2 activity was required for PEITC-induced Ca2+ signal in PC3 cells. Because activation of PLC produces IP3 and diacylglycerol, which activates protein kinase C, the effect of modulation of protein kinase C activity on PEITC-induced [Ca2+]i rise was explored. Neither activation nor inhibition of protein kinase C altered PEITC-induced [Ca2+]i rise. In PC3 cells, thapsigargin/IP3-sensitive endoplasmic reticulum Ca2+ stores are dominant intracellular Ca2+ stores [17,18]. Ryanodine-sensitive Ca2+ stores are not reported. PEITC and thapsigargin appeared to share a common endoplasmic reticulum Ca2+ store, because the PEITC response was mostly inhibited by depletion of endoplasmic reticulum Ca2+ store with thapsigargin, and conversely, thapsigargin failed to release more Ca2+ after PEITC treatment. Similar observation was also examined in the manipulation of [6]-gingerol and diallyl sulfide (DAS) [30,31]. The endoplasmic reticulum is one of the major intracellular Ca2+ stores and the organelle where proteins and lipids are synthesized and modified [32,33]. Ca2+ dyshomeostasis of endoplasmic reticulum, protein misfolding, or oxidative stress can lead to endoplasmic reticulum stress-induced cell death [33,34]. Reactive disulfide compounds are a group of oxidizing agents that can produce reactive oxygen species which are thought to oxidize cell membrane and lead to cell injury and death during inflammation, aging, radiation, ischemia-reperfusion of heart, kidney, liver, intestine and brain [35–37]. Cells exposed to some sulfhydryl agents are recognized to change the cellular redox state and key enzymes involved in cell function and growth. Disruption of this homeostasis is mostly followed by dysregulation in [Ca2+]i and cell growth [38,39]. Therefore, the antiproliferative effects of PEITC to cells may relate to modulation of thiols in cytoplasm and membrane. Incubation with PEITC inhibited cell proliferation in a concentration-dependent manner starting at 50 mM PEITC. Although Ca2+ overloading can trigger cell death, not all forms of cell death are linked to Ca2+. The BAPTA experiments revealed that chelation of cytosolic Ca2+ during PEITC stimulation did not prevent PEITCinduced cell death, but actually worsened it. Thus it is clear that PEITC-induced cytotoxicity was not caused by Ca2+ overloading. The extent of [Ca2+]i rises were correlated to the steric structure of cruciferous isothiocyanates and were in the order of PEITC > BITC > AITC. Recent studies have revealed that the structural analog of PEITC may play a critical role in the biological activities of dietary ITCs [7]. These results suggest that even a subtle change in the ITC structure could have a significant impact on its effect on Ca2+ signaling. Allyl-ITC was relatively less effective
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Fig. 7. Effect of AITC, BITC and PEITC on [Ca2+]i. (A) ITCs (300 mM) was added at 30 s and were present throughout the measurement of 250 s. Traces a, b and c represent AITC, BITC and PEITC, respectively. The experiments were performed in Ca2+-containing medium. (B) The y axis is the percentage of control response that was the net area under the curve (30–250 s) of the [Ca2+]i rise induced by PEITC (trace c in A). Results are expressed as the percentage of control which is the response induced by PEITC. Data are mean SEM of five experiments. *P < 0.05 compared with control.
against Ca2+ handling of PC3 cells compared with aromatic ITCs. In addition, the alkyl chain length has a marked effect on the activity of aromatic ITCs against proliferation of PC3 cells. Thus, the extent of [Ca2+]i rise caused by benzyl-ITC is about 47% compared with PEITC examined in the present study. Together, this study suggests that in PC3 cells, PEITC induced a [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase A2-dependent, protein kinase C-independent fashion, and by inducing Ca2+ influx. PEITC decreased cell viability in a Ca2+-independent, concentration-dependent manner. The antiproliferative activity is related to the ITC structure. These effects may play a crucial role in the physiological action of PEITC. Acknowledgements This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS98-100) to Jan CR, and National Science Council (NSC93-2311-B-242-002) to Kuo SY. References [1] Conaway CC, Yang YM, Chung FL. Isothiocyanates as cancer chemopreventive agents: their biological activities and metabolism in rodents and humans. Curr Drug Metab 2002;3:233. [2] Fahey JW, Zalcmann AT, Talalay P. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 2001;56:5. [3] Higdon JV, Delage B, Williams DE, Dashwood RH. Cruciferous vegetables and human cancer risk: epidemiologic evidence and mechanistic basis. Pharmacol Res 2007;55:224. [4] Khor TO, Keum YS, Lin W, Kim JH, Hu R, Shen G. Combined inhibitory effects of curcumin and phenethyl isothiocyanate on the growth of human PC-3 prostate xenografts in immunodeficient mice. Cancer Res 2006;66:613. [5] Xiao D, Choi S, Lee YJ, Singh SV. Role of mitogen-activated protein kinases in phenethyl isothiocyanate-induced apoptosis in human prostate cancer cells. Mol Carcinogen 2005;43:130. [6] Xiao D, Zeng Y, Choi S, Lew KL, Nelson JB, Singh SV. Caspase-dependent apoptosis induction by phenethyl isothiocyanate, a cruciferous vegetablederived cancer chemopreventive agent, is mediated by Bak and Bax. Clin Cancer Res 2005;11:2670. [7] Xiao D, Lew KL, Zeng Y, Xiao H, Marynowski SW, Dhir R. Phenethyl isothiocyanate-induced apoptosis in PC-3 human prostate cancer cells is mediated by reactive oxygen species-dependent disruption of the mitochondrial membrane potential. Carcinogen 2006;27:2223. [8] Xiao D, Johnson CS, Trump DL, Singh SV. Proteasome-mediated degradation of cell division cycle 25C and cyclin-dependent kinase 1 in phenethyl isothiocyanate-induced G2-M-phase cell cycle arrest in PC-3 human prostate cancer cells. Mol Cancer Ther 2004;3:567. [9] Butler R, Mitchell SH, Tindall DJ, Young CY. Nonapoptotic cell death associated with S-phase arrest of prostate cancer cells via the peroxisome proliferatoractivated receptor gamma ligand, 15-deoxy-delta12,14-prostaglandin J2. Cell Growth Differ 2000;11:49. [10] Clapham DE. Calcium signaling. Cell 1995;80:259.
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