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.     

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

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

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 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 melamine on [Ca2+]i and viability in PC3 human prostate cancer cells

Melamine is thought to be an endocrine disrupter that affects physiology in cells. This study examined the effect of melamine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Melamine evoked [Ca²⁺]_i rises concentration-dependently. Melamine-evoked Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365, GF109203X and phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited melamine-evoked [Ca²⁺]_i rise. Conversely, treatment with melamine abolished thapsigargin-evoked [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter melamine-evoked [Ca²⁺]_i rise. Melamine at 500–800 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). Collectively, our data suggest that in PC3 cells, melamine induced [Ca²⁺]_i rises by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ entry. Melamine also caused Ca²⁺-independent cell death.     

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 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.     

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.     

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA_A and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca²⁺ concentration ([Ca²⁺]_i). As Ca²⁺ triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca²⁺ homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca²⁺]_i imaging experiments with fura-2 demonstrated that lindane (≥ 10 μM) rapidly increases basal exocytosis and basal [Ca²⁺]_i. Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca²⁺ channels (VGCC). On the other hand, lindane (≥ 3 μM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca²⁺]_i and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca²⁺ influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic.These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca²⁺]_i and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca²⁺]_i and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.     

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 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.     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

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.     

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 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.     

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca²⁺ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of cells were measured using fura-2 as a fluorescent Ca²⁺ probe. Safrole at a concentration of 325 M started to increase [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 40% by removing extracellular Ca²⁺, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca²⁺-free medium, after pretreatment with 650 M safrole, 1 M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) barely induced a [Ca²⁺]_i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca²⁺]_i rise. Neither inhibition of phospholipase C with 2 M U73122 nor modulation of protein kinase C activity affected safrole-induced Ca²⁺ release. Overnight incubation with 1 M safrole did not alter cell proliferation, but incubation with 10–1000 M safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca²⁺ with 10 M of the Ca²⁺ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca²⁺]_i rise by causing release of stored Ca²⁺ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca²⁺ influx via nifedipine-sensitive Ca²⁺ entry. Furthermore, safrole can enhance cell growth in a Ca²⁺-independent manner.     

Effect of nortriptyline on cytosolic Ca2+ regulation and viability in PC3 human prostate cancer cells

The effect of nortriptyline, a tricyclic antidepressant, on Ca²⁺ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca²⁺]_i levels in suspended PC3 cells using fura‐2 as a Ca²⁺‐sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca²⁺]_i in a concentration‐dependent fashion. The Ca²⁺ signal was partially reduced by removing extracellular Ca²⁺, indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺]_i rise. Nortriptyline induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by activation of protein kinase C, but not by inhibition of L‐type Ca²⁺ channels. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor, thapsigargin nearly abolished nortriptyline‐induced Ca²⁺ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor‐induced [Ca²⁺]_i rise, suggesting that nortriptyline released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline‐induced [Ca²⁺]_i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca²⁺‐independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca²⁺]_i rises by causing the phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via the protein kinase C‐sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration‐dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley‐Liss, Inc.     

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 2,5-dimethylphenol on Ca2+ movement and viability in PC3 human prostate cancer cells

The phenolic compound 2,5-dimethylphenol is a natural product. 2,5-Dimethylphenol has been shown to affect rat hepatic and pulmonary microsomal metabolism. However, the effect of 2,5-dimethylphenol on Ca^2+?signaling and cyotoxicity has never been explored in any culture cells. This study explored the effect of 2,5-dimethylphenol on cytosolic free Ca^2+?levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500?μM and 1000?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca^2+?signal was inhibited by approximately half by the removal of extracellular Ca²⁺. 2,5-Dimethylphenol-induced Ca^2+?influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pretreatment with the protein kinase C (PKC) inhibitor GF109203X, nifedipine or the store-operated Ca^2+?entry inhibitors (econazole or SKF96365) inhibited 2,5-dimethylphenol-induced Ca^2+?signal in Ca²⁺-containing medium by ～30%. Treatment with the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin in Ca²⁺-free medium abolished 2,5-dimethylphenol-induced [Ca²⁺]_i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca²⁺]_i rises by ～80%. 2,5-Dimethylphenol killed cells at concentrations of 350–1000?μM in a concentration-dependent fashion. Chelation of cytosolic Ca^2+?with 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid/AM (BAPTA/AM) did not prevent 2,5-dimethylphenol’s cytotoxicity. Together, in PC3 cells, 2,5-dimethylphenol induced [Ca²⁺]_i rises that involved Ca^2+?entry through PKC-regulated store-operated Ca^2+?channels and PLC-dependent Ca^2+?release from the endoplasmic reticulum. 2,5-Dimethylphenol induced cytotoxicity in a Ca²⁺-independent manner.     

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.