Effect of Thymol on Ca 2+ Homeostasis and Viability in PC3 Human Prostate Cancer Cells

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1 Chinese Journal of Physiology 6(1): 32-4, 217 DOI: 1.477/CJP.217.BAF447 Effect of Thymol on Ca 2+ Homeostasis and Viability in PC3 Human Prostate Cancer Cells Jeng-Hsien Yeh 1, 2, Chiang-Ting Chou 3, 4, I-Shu Chen 5, Ti Lu 6, Ko-Long Lin 7, Chia-Cheng Yu 5, Wei-Zhe Liang 8, Hong-Tai Chang 5, Chun-Chi Kuo 9, Chin-Man Ho 8, Wen-Teng Chang 2, Pochuen Shieh 1, and Chung-Ren Jan 8 1 Pathology and Laboratory Medicine Department, Kaohsiung Veterans General Hospital, Kaohsiung Department of Biological Science and Technology, Chung Hwa College of Medical Technology, Tainan Department of Nursing, Division of Basic Medical Sciences, Chang Gung Institute of Technology, Chia-Yi Chronic Diseases and Health Promotion Research Center, Chang Gung Institute of Technology, Chia-Yi Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung Department of Psychiatry, Kaohsiung Veterans General Hospital, Kaohsiung Department of Rehabilitation, Kaohsiung Veterans General Hospital, Kaohsiung Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung Department of Nursing, Tzu Hui Institute of Technology, Pingtung and 1 Department of Pharmacy, Tajen University, Pingtung 9741, Taiwan, Republic of China Abstract Thymol is a phenolic compound that affects physiology in different cell models. However, whether affects Ca 2+ homeostasis in prostate cancer cells is unknown. The action of this compound on cytosolic Ca 2+ concentrations ([Ca 2+ ] i ) and viability in PC3 human prostate cancer cells was explored. The results show that at concentrations of - μm caused [Ca 2+ ] i rises in a concentrationdependent manner. Removal of extracellular Ca 2+ reduced s effect by approximately 8%. Thymol-induced Ca 2+ entry was confirmed by Mn 2+ entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 3% by Ca 2+ entry modulators (nifedipine, econazole, SKF96365), and the protein kinase C (PKC) inhibitor GF1923X. In Ca 2+ -free medium, treatment with the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin abolished -induced [Ca 2+ ] i rises. Treatment with also abolished thapsigargin-induced [Ca 2+ ] i rises. Thymol-induced Ca 2+ release from the endoplasmic reticulum was abolished by the phospholipase C (PLC) inhibitor U Thymol at -9 µm decreased cell viability, which was not reversed by pretreatment with the Ca 2+ chelator 1,2-bis(2-aminophenoxy) ethane-n,n,n,n -tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, induced [Ca 2+ ] i rises by inducing PLC-dependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via PKC-sensitive store-operated Ca 2+ channels and other unknown channels. Thymol also induced Ca 2+ -dissociated cell death. Key Words: Ca 2+, endoplasmic reticulum, fura-2, prostate cancer cells, Corresponding authors: [1] Dr. Pochuen Shieh, Department of Pharmacy, Tajen University, Pingtung 9741, Taiwan, R.O.C. and [2] Dr. Chung-Ren Jan, Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, R.O.C. Tel: ext. 9, Fax: , crjan@isca.vghks.gov.tw Received: June 7, 216; Revised: July 22, 216; Accepted: August 11, by The Chinese Physiological Society and Airiti Press Inc. ISSN :

2 Effect of Thymol on PC3 Cells 33 Introduction Thymol is a natural essential oil present in many plants. Thymus ciliatus is the highest containing species of the genus (2, 8, 22). Thymol is also a major component of thyme and oregano and has medical uses in oral care products as an astringent and antibiotic (7, 17, 23). Thymol has many physiological effects in different cells. In terms of Ca 2+ signaling, suppressed spontaneous contractile activity of the smooth muscles (3), inhibited skeletal type sarcoplasmic reticulum Ca 2+ -ATPase and ryanodine receptors (28), relaxed rat isolated aorta (24), activated human transient receptor potential cation channel subfamily A member 1 (htrpa1) (19), and induced Ca 2+ mobilization and ion currents in pituitary GH3 cells (29). Regarding cytotoxicty, protected Chinese hamster lung fibroblasts from radiation-induced cytotoxicity (1), and inhibited suicidal erythrocyte death (2). Furthermore, changed flight motor activity and wing beat frequency in the blowfly Phaenicia sericata (35). However, the effect of on Ca 2+ homeostasis and associated physiological effects in prostate cancer cells has not been explored. Ca 2+ is a key second messenger in many biological responses. Cytosolic free Ca 2+ concentrations ([Ca 2+ ] i ) can be increased upon a stimulation. This results in many pathophysiological cellular processes (4). An uncontrolled Ca 2+ signal may alter secretion, contraction, protein activity, apoptosis, and proliferation, etc. (6). Since Ca 2+ signaling is so important, cells have complex mechanisms to regulate Ca 2+ influx and release. In order to understand the physiological significance of this Ca 2+ signal, it is important to elucidate the underlying mechanisms. The aim of this study was to examine the effect of on [Ca 2+ ] i and viability, and to elucidate the underlying pathways in PC3 human prostate cancer cells. This cell line is a useful model for prostate research. Furthermore, the PC3 cell was used because it produces measurable [Ca 2+ ] i rises upon pharmacological stimulation. It has been shown that in this cell, [Ca 2+ ] i rises and death can be evoked by stimulation with chemicals such as diindolylmethane (34), celecoxib (36), and resveratrol (9). In this study, fura-2 was used as a Ca 2+ -sensitive dye to measure [Ca 2+ ] i. The [Ca 2+ ] i rises were characterized, the concentration-response plots were established, and the pathways underlying -evoked Ca 2+ entry and Ca 2+ release were explored. The cytotoxic effect of and its relationship with Ca 2+ was assessed. Chemicals Materials and Methods The reagents for cell culture were purchased from Gibco (Gaithersburg, MD, USA). Aminopolycarboxylic acid/acetoxy methyl (fura-2/am) and 1,2- bis(2-aminophenoxy)ethane-n,n,n,n -tetraacetic acid/ acetoxy methyl (BAPTA/AM) were from Molecular Probes (Eugene, OR, USA). Thymol and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. Cell Culture PC3 human prostate cancer cells obtained from Bioresource Collection and Research Center (Taiwan) were cultured in RPMI-164 medium supplemented with 1% heat-inactivated fetal bovine serum, U/ml penicillin and μg/ml streptomycin. Solutions Used in [Ca 2+ ] i Measurements Ca 2+ -containing medium (ph 7.4) had 14 mm NaCl, 5 mm KCl, 1 mm MgCl 2, 2 mm CaCl 2, 1 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 5 mm glucose. Ca 2+ -free medium (ph 7.4) contained 14 mm NaCl, 5 mm KCl, 3 mm MgCl 2,.3 mm ethylene glycol tetraacetic acid (EGTA), 1 mm HEPES, and 5 mm glucose. Thymol was dissolved in ethanol as a 1 M stock solution. The other chemicals were dissolved in water, ethanol or dimethyl sulfoxide (DMSO). The concentration of organic solvents in the experimental solutions did not exceed.1%, and did not affect viability or basal [Ca 2+ ] i. [Ca 2+ ] i Measurements The [Ca 2+ ] i was measured as previously described (9, 34, 36). Confluent cells grown on 6 cm dishes were trypsinized and made into a suspension in culture medium at a concentration of 1 6 cells/ml. Cell viability was determined by trypan blue exclusion. The viability was greater than 95% after the treatment. Cells were subsequently loaded with 2 μm fura-2/am for 3 min at 25 C in the same medium. After loading, cells were washed with Ca 2+ -containing medium twice and was made into a suspension in Ca 2+ -containing medium at a concentration of 1 7 cells/ml. Fura-2 fluorescence measurements were performed in a water-jacketed cuvette (25 C) with continuous stirring; the cuvette contained 1 ml of medium and 1 million cells. Fluorescence was monitored with a Shimadzu RF-531PC spectrofluorophotometer immediately after.1 ml cell suspension was added to.9 ml Ca 2+ -containing or Ca 2+ - free medium, by recording excitation signals at 34 nm and 38 nm and emission signal at 51 nm at 1-sec intervals. During the recording, reagents were added to the cuvette by pausing the recording for 2 sec to open and close the cuvette-containing chamber. For calibration

3 34 Yeh, Chou, Chen, Lu, Lin, Yu, Liang, Chang, Kuo, Ho, Chang, Shieh and Jan of [Ca 2+ ] i, after completion of the experiments, the detergent Triton X- (.1%) and CaCl 2 (5 mm) were added to the cuvette to obtain the maximum fura-2 fluorescence. Then the Ca 2+ chelator EGTA (1 mm) was added to chelate Ca 2+ in the cuvette to obtain the minimum fura-2 fluorescence. Control experiments showed that cells bathed in a cuvette had a viability of 95% after 2 min of fluorescence measurements. [Ca 2+ ] i was calculated as previously described (13). Mn 2+ quenching of fura-2 fluorescence was performed in Ca 2+ -containing medium containing μm MnCl 2. MnCl 2 was added to cell suspension in the cuvette 3 sec before the fluorescence recoding was started. Data were recorded at excitation signal at 36 nm (Ca 2+ -insensitive) and emission signal at 51 nm at 1-sec intervals as described previously (21). Cell Viability Assays Viability was assessed as previously described (9, 34, 36). The measurement of cell viability was based on the ability of cells to cleave tetrazolium salts by dehydrogenases. Increases in the amount of developed color directly correlated with the number of live cells. Assays were performed according to manufacturer s instructions (Roche Molecular Biochemical, Indianapolis, IN, USA). Cells were seeded in 96-well plates at a concentration of 1, cells/well in culture medium for 24 h in the presence of. The cell viability detecting tetrazolium reagent 4-[3-[4-lodophenyl]- 2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1; 1 μl pure solution) was added to samples after treatment, and cells were incubated for 3 min in a humidified atmosphere. In experiments using BAPTA/AM to chelate cytosolic Ca 2+, cells were treated with 5 µm BAPTA/AM for 1 h prior to incubation with. The cells were washed once with Ca 2+ -containing medium and incubated with/without for 24 h. The absorbance of samples (A 4 ) was determined using an enzymelinked immunosorbent assay (ELISA) reader. Absolute optical density was normalized to the absorbance of unstimulated cells in each plate and expressed as a percentage of the control value. Statistics Data are reported as mean ± standard error of the mean (SEM) of three separate experiments. Data were analyzed by one-way analysis of variances (ANOVA) using the Statistical Analysis System (SAS, SAS Institute Inc., Cary, NC, USA). Multiple comparisons between group means were performed by post-hoc analysis using the Tukey s honestly significantly difference (HSD) procedure. P <.5 was considered significant. Effect of Thymol on [Ca 2+ ] i Results The effect of on basal [Ca 2+ ] i was examined. Fig. 1A shows that the basal [Ca 2+ ] i level was ± 2 nm. At concentrations between and μm, induced [Ca 2+ ] i rises in a concentration-dependent manner in Ca 2+ -containing medium. At a concentration of μm, evoked [Ca 2+ ] i rises that attained to net increases of 175 ± 3 nm (n = 3) followed by a slow decay phase. The Ca 2+ response saturated at μm tymol because at a concentration of 2 μm, evoked a similar response as that induced by μm (not shown). Fig. 1B shows that in Ca 2+ -free medium, - μm induced concentrationdependent rises in [Ca 2+ ] i. In the absence of extracellular Ca 2+, µm induced [Ca 2+ ] i rises of 49 ± 3 nm. Fig. 1C shows the concentration-response plots of -induced [Ca 2+ ] i rises. The EC value was 721 ± 1 μm in Ca 2+ -containing or 987 ± 2 μm in Ca 2+ -free medium, respectively, by fitting to a Hill equation. Removal of Ca 2+ reduced the -induced [Ca 2+ ] i rises by approximately 8%. Thymol-Induced Mn 2+ Influx Experiments were performed to confirm that -evoked [Ca 2+ ] i rises involved Ca 2+ influx. Mn 2+ enters cells through similar mechanisms as Ca 2+ but quenches fura-2 fluorescence at all excitation wavelengths (21). Therefore, quenching of fura-2 fluorescence excited at the Ca 2+ -insensitive excitation wavelength of 36 nm by Mn 2+ implicates Ca 2+ influx. Because -induced Ca 2+ response saturated at μm, in the following experiments the response induced by μm was used as control. Fig. 2 shows that μm evoked an instant decrease in the 36 nm excitation signal that reached a maximum value of 6 ± 2 arbitrary units at 6 sec. This suggests that Ca 2+ influx participated in -evoked [Ca 2+ ] i rises. The Pathway of Thymol-Induced Ca 2+ Entry Experiments were conducted to explore the Ca 2+ entry pathway of -induced [Ca 2+ ] i rises. Nifedipine and the store-operated Ca 2+ entry inhibitors: econazole (.5 μm) and SKF96365 (5 μm); phorbol 12-myristate 13 acetate (PMA; 1 nm; a protein kinase C [PKC] activator); and GF1923X (2 μm; a PKC inhibitor) were applied 1 min before μm. Except PMA, GF1923X, nifedipine, econazole or SKF96365 significantly inhibited -induced [Ca 2+ ] i rises by approximately 3% (P <.5) (Fig. 3).

4 Effect of Thymol on PC3 Cells 35 A 3 a, µm B 2 2 c, µm b, µm c, µm d, µm b, µm a, µm e, µm 2 2 d, µm 2 2 C Percentage of µm Thymol-induced Response (area under curve) Thymol (µm) Fig. 1. Effect of on [Ca 2+ ] i in fura-2-loaded cells. (A) Thymol was added at 25 sec. The concentration of was indicated. The experiments were performed in Ca 2+ -containing medium. (B) A concentration-response plot of -induced [Ca 2+ ] i rises in Ca 2+ -free medium. (C) Concentration-response plots of -induced [Ca 2+ ] i rises. Y axis is the percentage of the net (baseline subtracted) area under the curve (25-2 sec) of the [Ca 2+ ] i rises induced by µm in Ca 2+ -containing medium. Data are mean ± SEM of three separate experiments. P <.5 compared to control. +Ca 2+ -Ca 2+ Sources of Thymol-Induced Ca 2+ Release In most cell types including PC3 cells, the endoplasmic reticulum has been shown to be the main Ca 2+ store (6). Thus the role of the endoplasmic reticulum in -evoked Ca 2+ release in PC3 cells was explored. The experiments were conducted in Ca 2+ -free medium to exclude the involvement of Ca 2+ influx. Fig. 4A shows that addition of 1 μm thapsigargin (31), a selective endoplasmic reticulum Ca 2+ pump inhibitor, induced [Ca 2+ ] i rises of 52 ± 2 nm. Addition of μm at sec did not induce [Ca 2+ ] i rises. Conversely, Fig. 4B shows that after μm -induced [Ca 2+ ] i rises, addition of 1 μm thapsigargin at sec also failed to induce [Ca 2+ ] i rises. Therefore, it suggests that the mechanism of Ca 2+ release from the endoplasmic reticulum activated by appears to be similar to that of thapsigargin. A Role of Phospholipase C (PLC) in Thymol-Induced [Ca 2+ ] i Rises PLC is one of the crucial enzymes that regulate the release of Ca 2+ from Ca 2+ stores. Because released Ca 2+ from the endoplasmic reticulum, the role of PLC in this process was examined. The PLC inhibitor U73122 (32), was applied to explore if the activation of this enzyme was required for -evoked Ca 2+ release. Cellular activation by many agonists results in the stimulation of PLC and the subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate to IP 3 and diacylglycerol (DAG) (4, 6). Each of these two molecules exerts a specific effect on the cell. The increased DAG concentration leads to the activation

5 36 Yeh, Chou, Chen, Lu, Lin, Yu, Liang, Chang, Kuo, Ho, Chang, Shieh and Jan 36 nm Fluorescence (arbitrary unit) a, control b, + Fig. 2. Effect of on Ca 2+ influx by measuring Mn 2+ quenching of fura-2 fluorescence. Experiments were performed in Ca 2+ -containing medium. MnCl 2 ( μm) was added to cells 1 min before fluorescence measurements. The y axis is fluorescence intensity (in arbitrary units) measured at the Ca 2+ -insensitive excitation wavelength of 36 nm and the emission wavelength of 51 nm. Trace a: control, without. Trace b: ( μm) was added as indicated. Data are mean ± SEM of three separate experiments. Percentage of Control Thymol econazole+thymol SKF96365+Thymol PMA+Thymol Nifedipine+Thymol GF1923X+Thymol Fig. 3. Effect of Ca 2+ channel modulators on -induced [Ca 2+ ] i rises. The experiments were performed in Ca 2+ -containing medium. In modulator-treated groups, the modulator was added 1 min before ( μm). The concentration was 1 nm for phorbol 12-myristate 13-acetate (PMA), 2 μm for GF1923X, 1 μm for nifedipine,.5 μm for econazole, 5 μm for SKF Data are expressed as the percentage of control (1 st column) that is the area under the curve (25-2 sec) of μm -induced [Ca 2+ ] i rises, and are mean ± S.E.M. of three separate experiments. P <.5 compared to the 1 st column. A B TG TG Fig. 4. Intracellular Ca 2+ stores of -induced Ca 2+ release. Experiments were performed in Ca 2+ -free medium. (A) (B) Thapsigargin (TG, 1 μm) and ( μm) were added at time points indicated. Data are mean ± SEM of three separate experiments. of PKC while IP 3 binds to the IP 3 receptor (IP 3 R) located on the endoplasmic reticulum, thereby inducing Ca 2+ release from this store (4-6). Fig. 5A shows that ATP (1 μm) caused [Ca 2+ ] i rises of 91 ± 2 nm. ATP is a PLC-dependent agonist of [Ca 2+ ] i rises in most cell types (12), and therefore was the reason to use it as a tool to examine whether U73122 effectively inhibited the activity of PLC. Fig. 5B shows that incubation with

6 Effect of Thymol on PC3 Cells 37 A B Percentage of Control Thymol (control) ATP U73122 ATP U73122+ATP 2 μm U73122 did not change basal [Ca 2+ ] i but abolished ATP-induced [Ca 2+ ] i rises. This suggests that U73122 effectively suppressed PLC activity. The data also show that incubation with 2 μm U73122 did not alter basal [Ca 2+ ] i but abolished μm -induced [Ca 2+ ] i rises. U73343 (2 μm), a U73122 analogue, did not have an effect (not shown). Effect of Thymol on Cell Viability U73122+ATP+Thymol U73122+Thymol Fig. 5. Effect of U73122 on -induced Ca 2+ release. Experiments were performed in Ca 2+ -free medium. (A) ATP (1 μm) was added as indicated. (B) U73122 (2 μm), ATP, and ( μm) were added as indicated. Data are mean ± SEM of three separate experiments. P <.5 compared to first bar (control). Control is the area under the curve of μm induced [Ca 2+ ] i rises (25-22 sec). Percentage of Control 12 Treatment with Thymol Treatment with Thymol and BAPTA/AM (5B) µm Thymol 5B + 3 µm Thymol Control 5 µm BAPTA/AM (5B) µm Thymol 5B + µm Thymol Cells were treated with -9 μm for 24 h, and tetrazolium assay was performed. In the presence of, cell viability decreased in a concentrationdependent manner between -9 μm (Fig. 6). The next question was whether -induced cytotoxicity was related to preceding [Ca 2+ ] i rises. The intracellular Ca 2+ chelator BAPTA/AM (5 μm) (33) was used to prevent [Ca 2+ ] i rises during pretreatment. At μm, did not evoke [Ca 2+ ] i rises in BAPTA/ AM-treated cells (not shown). Fig. 6 shows that 5 μm BAPTA/AM loading did not alter control cell viability. In the presence of -9 μm, BAPTA/AM loading failed to prevent -induced cell death. This suggests that -induced cell death was not caused by preceding [Ca 2+ ] i rises. Discussion µm Thymol 5B + µm Thymol 7 µm Thymol 5B + 7 µm Thymol 9 µm Thymol # 5B + 9 µm Thymol Fig. 6. Thymol-induced Ca 2+ -independent cell death. Cells were treated with -9 μm for 24 h, and the cell viability assay was performed. Data are mean ± SEM of three separate experiments. Each treatment had six replicates (wells). Data are expressed as percentage of control that is the increase in cell numbers in -free groups. Control had 1,987 ± 799 cells/ well before experiments, and had 13,557 ± 725 cells/ well after incubation for 24 h. P <.5 compared to control. In each group, the Ca 2+ chelator BAPTA/AM (5 μm) was added to cells followed by treatment with in Ca 2+ -containing medium. Cell viability assay was subsequently performed. # P <.5 compared to the pairing group. Ca 2+ signaling regulates physiology in most cell types including human prostate cancer cells (4, 6). Previous studies showed that the phenolic compound affected Ca 2+ signaling in various cell models such as MDCK canine renal tubular cells (11), MG63

7 38 Yeh, Chou, Chen, Lu, Lin, Yu, Liang, Chang, Kuo, Ho, Chang, Shieh and Jan human osteosarcoma cells (1) and DBTRG-5MG human glioblastoma cells (14). However, whether affected Ca 2+ homeostasis in PC3 cells is unclear. The results show that concentrationdependently induced [Ca 2+ ] i rises and cell death in PC3 cells. The data suggest that elevated [Ca 2+ ] i by depleting intracellular Ca 2+ stores and inducing Ca 2+ entry from extracellular medium. Removal of extracellular Ca 2+ reduced the -induced [Ca 2+ ] i rise throughout the measurement interval of 2 sec, implying that Ca 2+ entry happened during the whole stimulation period. Mn 2+ -induced quench of fura-2 fluorescence also confirmed that induced Ca 2+ entry. The mechanism of -induced Ca 2+ entry and release was explored. The data suggest that induced Ca 2+ entry was via store-operated Ca 2+ entry based on the inhibition of nifedipine, econazole or SKF Previous reports have suggested that these chemicals have been used as blockers of store-operated Ca 2+ entry (25) in different cell models (15, 16, 26); although so far no compounds were available as selective inhibitors for this type of Ca 2+ entry. During [Ca 2+ ] i rises, activation of PLC may produce IP 3 and DAG, which activate PKC, thus the effect of modulation of PKC activity on -induced [Ca 2+ ] i rises was explored. Inhibition of PKC suppressed -evoked [Ca 2+ ] i rises while activation of PKC activity had no effect. This suggests that normal PKC activity is required for -induced [Ca 2+ ] i rises. Regulation of PKC activity has been shown to modulate storeoperated Ca 2+ entry in different cells (4, 6, 25). Because these blockers only inhibited -induced [Ca 2+ ] i rises by 3% whereas Ca 2+ entry contributed to induced [Ca 2+ ] i rises by 8%, the other pathways of Ca 2+ entry is unknown. One possible Ca 2+ channels that prostate cancer cells may have include the transient receptor potential cation channel (TRPC) proteins that are Ca 2+ -permeable, nonselective cation channels with various functions (27). So far, there are also no selective blockers for TRPC channels. Another question was regarding the Ca 2+ stores involved in -induced Ca 2+ release. The thapsigargin-sensitive endoplasmic reticulum stores might be the dominant one because thapsigargin pretreatment abolished -induced [Ca 2+ ] i rises; and conversely, pretreatment also abolished thapsigargin-induced Ca 2+ release. The data further show that the Ca 2+ release was via a PLC-dependent mechanism, given the release was abolished when PLC activity was inhibited. Thus the data suggest that induced Ca 2+ release from the endoplasmic reticulum in a PLC-dependent pathway. In terms of cytotoxic effects of on cancer cells, at an IC value of 7 μm, has been shown to induce cytotoxicity in Hep-2 human laryngeal carcinoma cells (3). Our study shows that (- 9 μm) was also cytotoxic to PC3 cells in a concentration-dependent manner. Ca 2+ overloading is known to initiate processes leading to alteration in cell viability (4, 6). Because induced both [Ca 2+ ] i rises and cell death in PC3 cells, it is important to know whether the death occurred in a Ca 2+ - dependent manner. Our data show that -induced cell death was not reversed when cytosolic Ca 2+ was chelated by BAPTA/AM. This implies that in this case, -induced cell death was not triggered by [Ca 2+ ] i rises. Although in the presence of 9 μm 5 μm BAPTA has a protective effect on cell viability, at μm or combination of μm and 5 μm BAPTA both completely caused cell death (not shown). Therefore, at higher concentrations (e.g. more than 9 μm) of, 5 μm BAPTA did not have a protective effect on cell viability. Previous studies showed that induced [Ca 2+ ] i rises through different pathways in MDCK cells (11), MG63 cells (1) and DBTRG-5MG cells (14). In MDCK cells, 2- μm induced [Ca 2+ ] i rises by inducing Ca 2+ release from the endoplasmic reticulum and Ca²+ entry via PKC-sensitive store-operated Ca²+ channels (11). In MG63 cells, 2- μm induced [Ca 2+ ] i rises by inducing PLCdependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via PKC-sensitive store-operated Ca 2+ channels (1). In DBTRG-5MG cells, 4- μm induced [Ca 2+ ] i rises by inducing PLC- and PKC-dependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via non store-operated Ca 2+ channels (14). Our data show that - μm induced [Ca 2+ ] i rises by inducing PLC- and PKCdependent Ca 2+ release from the endoplasmic reticulum and Ca 2+ entry via store-operated Ca 2+ channels in PC3 cells. Therefore, it appears that the mechanism of the effect of on [Ca 2+ ] i was similar among MDCK, MG63, DBTRG-5MG and PC3 cells. Furthermore, at a concentration of 2-6 μm caused cytotoxicity in MDCK cells (11), MG63 cells (1), DBTRG- 5MG cells (14), but at a higher concentration range of -9 μm in PC3 cells. Because various cancer cell types derived from different origins may have different mechanisms of cytotoxicity, depending on the physiological function of this particular cell. Therefore, the effect of on cytotoxicity may depend on cell types and concentrations. Several studies were performed to explore the plasma level of in animal or adults. The plasma level of may reach ~ µm (5, 18). This level may be expected to go much higher in patients with liver or kidney disorders or taking higher doses. Our data show that at a concentration of µm induced slight cell death. Therefore, our data may be clinically relevant in some groups of patients. Together, the data show that induced PLC-

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