Effect of thymol on Ca homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human glioblastoma cells was examined. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Thymol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca²⁺]_i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C- and protein kinase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis.     

Effect of diallyl disulfide on Ca movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca²⁺]_i in PC3 cells by using fura-2. DADS at 50-1000 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. DADS-induced Ca²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca²⁺]_i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca²⁺]_i rise. At 500-1000 μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500 μM) induced apoptosis in a Ca²⁺-independent manner. Annexin V/PI staining further shows that 10 μM and 500 μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca²⁺]_i rise probably by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. DADS induced Ca²⁺-dependent cell death, ROS production, and Ca²⁺-independent apoptosis.     

Effect of capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

The effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 80% by removing extracellular Ca²⁺. Capsacin induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca²⁺ entry blocker La³⁺, but not by store‐operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished capsaicin‐induced Ca²⁺ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca²⁺]_i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca²⁺ entry and Ca²⁺ release. Collectively, in MDCK cells, capsaicin induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2‐regulated, La³⁺‐sensitive Ca²⁺ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of the antidepressant maprotiline on calcium movement and the viability of renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2. Maprotiline (>2.5 µM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ 200 µM). Maprotiline-induced [Ca²⁺]_i rise was reduced by removal of extracellular Ca²⁺ or by addition of La³⁺, but was not altered by voltage-gated Ca²⁺-channel blockers. Maprotiline-induced Mn²⁺ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca²⁺ influx. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of maprotiline on [Ca²⁺]_i was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished [Ca²⁺]_i rise induced by ATP (but not by maprotiline). Overnight incubation with 1–10 µM maprotiline enhanced cell viability, but 20–50 µM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and may modulate cell proliferation in a concentration-dependent manner.     

Effect of diindolylmethane on Ca(2+) movement and viability in HA59T human hepatoma cells

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca²⁺]_i in HA59T cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 1?C50???M evoked a [Ca²⁺]_i rise in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. Diindolylmethane-induced Ca²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 10?C75???M, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N??,N??-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25?C50???M) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca²⁺]_i rise by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Effect of the environmental pollutant bisphenol A dimethacylate (BAD) on Ca2+ movement and viability in OC2 human oral cancer cells

The environmental pollutant bisphenol A dimethacylate (BAD) has been used as a dental composite. The effect of BAD on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in OC2 human oral cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. BAD induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. BAD-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, and SK&F96365. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished BAD-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter BAD-induced [Ca²⁺]_i rise. At 10–30 μM, BAD inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. BAD (20–30 μM) also induced apoptosis. Collectively, in OC2 cells, BAD induced a [Ca²⁺]_i rise by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. BAD also caused apoptosis.     

The garlic ingredient diallyl sulfide induces Ca mobilization in Madin-Darby canine kidney cells

Diallyl sulfide (DAS), one of the major organosulfur compounds (OSCs) of garlic, is recognized as a group of potential chemoproventive compounds. In this study, we examines the early signaling effects of DAS on renal cells loaded with Ca²⁺-sensitive dye fura-2. It was found that DAS caused an immediate and sustained rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ = 2.32 mM). DAS also induced a [Ca²⁺]_i elevation when extracellular Ca²⁺ was removed, but the magnitude was reduced by 45%. Depletion of intracellular Ca²⁺ stores with CCCP, a mitochondrial uncoupler, did not affect DAS’s effect. In Ca²⁺-free medium, the DAS-induced [Ca²⁺]_i rise was abolished by depleting stored Ca²⁺ with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). DAS-caused [Ca²⁺]_i rise in Ca²⁺-containing medium was not affected by modulation of protein kinase C activity. The DAS-induced Ca²⁺ influx was blocked by nicardipine. U73122, an inhibitor of phospholipase C, abolished ATP (but not DAS)-induced [Ca²⁺]_i rise. Additionally, pretreatment with DAS for 24 h decreased cell viability in a concentration-dependent manner. Furthermore, DAS-induced cell death involved apoptotic events. These findings suggest that diallyl sulfide induced a significant rise in [Ca²⁺]_i in MDCK renal tubular cells by stimulating both extracellular Ca²⁺ influx and thapsigargin-sensitive intracellular Ca²⁺ release via as yet unidentified mechanisms.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Carvedilol at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 20 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.     

Effect of the antidepressant paroxetine on Ca2+ movement in PC3 human prostate cancer cells

The effect of the antidepressant paroxetine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in PC3 human prostate cancer cells is unclear. This study explored whether paroxetine changed basal [Ca²⁺]_i levels in suspended PC3 cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Paroxetine at concentrations between 10–150 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 55% by removing extracellular Ca²⁺. Paroxetine‐induced Ca²⁺ influx was inhibited by the store‐operated Ca²⁺ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin, 2,5‐di‐tert‐butylhydroquinone (BHQ), or cyclopiazonic acid (CPA) all abolished paroxetine‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 inhibited paroxetine‐induced [Ca²⁺]_i rise by 80%. Collectively, in PC3 cells, paroxetine induced [Ca²⁺]_i rise by causing phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store‐operated Ca²⁺ channels in a manner regulated by protein kinase C and phospholipase A2. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of timolol on Ca2+ handling and viability in human prostate cancer cells

Timolol is a medication used widely to treat glaucoma. Regarding Ca²⁺ signaling, timolol was shown to modulate Ca²⁺-related physiology in various cell types, however, the effect of timolol on Ca²⁺ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100–1000?μM induced [Ca²⁺]_i rises. The Ca²⁺ signal in Ca²⁺-containing medium was reduced by removal of extracellular Ca²⁺ by approximately 75%. Timolol (1000?μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Timolol-induced Ca²⁺ entry was partially inhibited by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished timolol-evoked [Ca²⁺]_i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca²⁺]_i rises. Timolol at concentrations between 200 and 600?μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca²⁺]_i rises by evoking Ca²⁺release from the endoplasmic reticulum in a PLC-dependent manner, and Ca²⁺ influx via PKC-regulated store-operated Ca²⁺ entry. Timolol also caused cell death that was not linked to preceding [Ca²⁺]_i rises.     

Mechanisms of AM404-induced [Ca]i rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 60 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. AM404 induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, Ni²⁺, nifedipine and verapamil. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), AM404-induced [Ca²⁺]_i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca²⁺]_i rise. At concentrations between 10 and 200 μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 μM AM404 was partly reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca²⁺]_i rise by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via L-type Ca²⁺ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

The involvement of mitochondrial apoptotic pathway in eugenol-induced cell death in human glioblastoma cells

Eugenol, a natural phenolic constituent of clove oil, has a wide range of applications in medicine as a local antiseptic and anesthetic. However, the effect of eugenol on human glioblastoma is unclear. This study examined whether eugenol elevated intracellular free Ca²⁺ levels ([Ca²⁺]_i) and induced apoptosis in DBTRG-05MG human glioblastoma cells. Eugenol evoked [Ca²⁺]_i rises which were reduced by removing extracellular Ca²⁺. Eugenol-induced [Ca²⁺]_i rises were not altered by store-operated Ca²⁺ channel blockers but were inhibited by the PKC inhibitor GF109203X and the transient receptor potential channel melastatin 8 (TRPM8) antagonist capsazepine. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished eugenol-induced [Ca²⁺]_i rises. The phospholipase C (PLC) inhibitor U73122 significantly inhibited eugenol-induced [Ca²⁺]_i rises. Eugenol killed cells which were not reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Eugenol induced apoptosis through increasing reactive oxygen species (ROS) production, decreasing mitochondrial membrane potential, releasing cytochrome c and activating caspase-9/caspase-3. Together, in DBTRG-05MG cells, eugenol evoked [Ca²⁺]_i rises by inducing PLC-dependent release of Ca²⁺ from the endoplasmic reticulum and caused Ca²⁺ influx possibly through TRPM8 or PKC-sensitive channels. Furthermore, eugenol induced the mitochondrial apoptotic pathway.     

Effect of Methoxychlor on Ca(2+) Movement and Viability in MDCK Renal Tubular Cells

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells using the Ca²⁺‐sensitive fluorescent dye fura‐2. Methoxychlor at 5–20 μM increased [Ca²⁺]_i in a concentration‐dependent manner. The signal was reduced by 80% by removing extracellular Ca²⁺. Methoxychlor‐induced Ca²⁺ entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca²⁺‐free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) partly inhibited methoxychlor‐induced [Ca²⁺]_i rise. Incubation with methoxychlor also inhibited thapsigargin‐ or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor‐induced [Ca²⁺]_i rise. At 5–15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid/AM (BAPTA/AM). Annexin V‐FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca²⁺]_i rise by inducing phospholipase C‐dependent Ca²⁺ release from multiple stores and Ca²⁺ entry via protein kinase C‐ and econazole‐sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration‐dependent, Ca²⁺‐independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.     

Effect of capsazepine on [Ca2+]i in MDCK renal tubular cells

The current study explored whether capsazepine changed basal cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura‐2 as a Ca²⁺‐selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was partially reduced by 40% by removing extracellular Ca²⁺. Capsazepine induced Mn²⁺ quench of fura‐2 fluorescence, indirectly implicating Ca²⁺ entry. Capsazepine‐induced Ca²⁺ influx was unchanged by L‐type Ca²⁺ entry inhibitors and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, 100 µM capsazepine‐induced Ca²⁺ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin‐induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change capsazepine‐induced [Ca²⁺]_i rises. Collectively, in MDCK cells, capsazepine induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non‐L‐type Ca²⁺ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley‐Liss, Inc.     

3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells

Abstract: The effect of the natural product 3,3′‐diindolylmethane (DIM) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺‐sensitive fluorescent dye fura‐2 was applied to measure [Ca²⁺]_i. DIM at concentrations of 40–80 μM induced a [Ca²⁺]_i rise in a concentration‐dependent manner. The response was reduced partly by removing Ca²⁺. DIM‐evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished DIM‐induced [Ca²⁺]_i rise. Incubation with DIM also inhibited thapsigargin or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished DIM‐induced [Ca²⁺]_i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration‐dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration‐dependent manner. In sum, in MG63 cells, DIM induced a [Ca²⁺]_i rise by evoking phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C‐sensitive store‐operated Ca²⁺ channels. DIM caused cell death that may involve apoptosis.     

Mechanisms underlying the effect of an oral antihyperglycaemic agent glyburide on calcium ion (Ca2+) movement and its related cytotoxicity in prostate cancer cells

Glyburide is an agent commonly used to treat type 2 diabetes and also affects various physiological responses in different models. However, the effect of glyburide on Ca²⁺ movement and its related cytotoxicity in prostate cancer cells is unclear. This study examined whether glyburide altered Ca²⁺ signalling and viability in PC3 human prostate cancer cells and investigated those underlying mechanisms. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in suspended cells were measured by using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Glyburide at concentrations of 100–1000 μM induced [Ca²⁺]_i rises. Ca²⁺ removal reduced the signal by approximately 60%. In Ca²⁺-containing medium, glyburide-induced Ca²⁺ entry was inhibited by 60% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X), and modulators of store-operated Ca²⁺ channels (nifedipine, econazole and SKF96365). Furthermore, glyburide induced Mn²⁺ influx suggesting of Ca²⁺ entry. In Ca²⁺-free medium, inhibition of phospholipase C (PLC) with U73122 significantly inhibited glyburide-induced [Ca²⁺]_i rises. Treatment with the endoplasmic reticulum (ER) Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished glyburide-evoked [Ca²⁺]_i rises. Conversely, treatment with glyburide abolished BHQ-evoked [Ca²⁺]_i rises. Glyburide at 100–500 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, glyburide induced [Ca²⁺]_i rises by Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and Ca²⁺ release from the ER in a PLC-dependent manner. Glyburide also caused Ca²⁺-independent cell death. This study suggests that glyburide could serve as a potential agent for treatment of prostate cancer.     

Nonylphenol‐induced cytosolic Ca2+ elevation and death in renal tubular cells

Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability in Madin‐Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca²⁺]_i in a concentration‐dependent manner (EC₅₀～0.8 μM). Nonylphenol‐induced Mn²⁺ entry demonstrated Ca²⁺ influx and removal of extracellular Ca²⁺ partly decreased the [Ca²⁺]_i rise. The [Ca²⁺]_i rise was inhibited by the protein kinase C activator, phorbol 13‐myristate acetate (PMA) but not by L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, nonylphenol‐induced [Ca²⁺]_i rise was partly inhibited by pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin‐induced Ca²⁺ release. Nonylphenol‐induced Ca²⁺ release was unaltered by inhibition of phospholipase C. At concentrations of 5–100 μM, nonylphenol killed cells in a concentration‐dependent manner. The cytotoxic effect of 100 μM nonylphenol was not affected by preventing [Ca²⁺]_i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca²⁺]_i increase in MDCK cells via evoking Ca²⁺ entry through protein kinase C‐regulated Ca²⁺ channels, and releasing Ca²⁺ from endoplasmic reticulum and other stores in a phospholipase C‐independent manner. Nonylphenol also killed cells in a Ca²⁺‐independent fashion. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

The mechanism of carvacrol-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in OC2 human oral cancer cells

Carvacrol is one of the main substances of essential oil which triggers intracellular Ca²⁺ mobilization and causes cytotoxicity in diverse cell models. However, the mechanism of carvacrol-induced Ca²⁺ movement and cytotoxicity is not fully understood. This study examined the effect of carvacrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), cell viability and apoptosis in OC2 human oral cancer cells. Carvacrol induced a [Ca²⁺]_i rise and the signal was reduced by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ entry was not altered by store-operated Ca²⁺ channel inhibitors and protein kinase C (PKC) activator, but was inhibited by a PKC inhibitor. In Ca²⁺ -free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited carvacrol-induced [Ca²⁺]_i rise. Conversely, incubation with carvacrol inhibited TG or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. Carvacrol decreased cell viability, which was not reversed when cytosolic Ca²⁺ was chelated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester). Carvacrol-induced apoptosis and activation of reactive oxygen species (ROS) and caspase-3. Together, carvacrol induced a [Ca²⁺]_i rise by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive, non store-operated Ca²⁺ channels. Carvacrol-induced ROS- and caspase-3-associated apoptosis.