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.     

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.     

Effect of protriptyline on [Ca2+]i and viability in MG63 human osteosarcoma cells

Tricyclic antidepressants (TCA) have been clinically prescribed in the auxiliary treatment of cancer patients. Although protriptyline, a type of TCA, was used primarily in the clinical treatment of mood disorders in cancer patients, the effect of protriptyline on physiology in human osteosarcoma is unknown. This study examined the effect of protriptyline on cytosolic free Ca^2+?concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Protriptyline between 50 and 250?μM evoked [Ca²⁺]_i rises concentration-dependently. Protriptyline induced influx of Mn²⁺, indirectly implicating Ca^2+?influx. Protriptyline-evoked Ca^2+?entry was inhibited by nifedipine by 20% but was not altered by econazole, SKF96365, GF109203X, and phorbol-12-myristate-13-acetate (PMA). In Ca²⁺-free medium, treatment with protriptyline inhibited the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin-evoked [Ca²⁺]_i rises. Conversely, treatment with thapsigargin inhibited 45% of protriptyline-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter protriptyline-evoked [Ca²⁺]_i rises. Protriptyline at 50–250?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). Collectively, our data suggest that in MG63 cells, protriptyline induced [Ca²⁺]_i rises by evoking Ca^2+?release from the endoplasmic reticulum and other stores in a PLC-independent manner, and Ca^2+?entry via a nifedipine-sensitive Ca^2+?pathway. Protriptyline also caused Ca²⁺-independent cell death.     

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 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.     

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.     

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 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 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 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 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.     

Effects of MK‐886, a leukotriene synthesis inhibitor,on [Ca2+]i and apoptosis in MG63 human osteosarcoma cells

The effect of MK‐886 (3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid), a compound widely used to inhibit leukotriene synthesis, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in osteosarcoma cells has not been explored. This study examined whether MK‐886 altered [Ca²⁺]_i levels in suspended MG63 human osteosarcoma cells using fura‐2. MK‐886 at 0.1 μM and above increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK‐886 induced Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors, nifedipine, econazole, and SKF96365; and by the protein kinase C modulators, phorbol 12‐myristate 13‐acetate (PMA) and GF109203X. In Ca²⁺‐free medium, after pretreatment with 5 μM MK‐886, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change MK‐886‐induced [Ca²⁺]_i rises. Collectively, in MG63 osteosarcoma cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via protein kinase C‐regulated store‐operated Ca²⁺ entry. Drug Dev Res 69: 49–57, 2008. © 2008 Wiley‐Liss, Inc.     

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 MK‐886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

Abstract: 3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid (MK‐886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK‐886 on cytosolic‐free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human PC3 prostate cancer cells. [Ca²⁺]_i in suspended cells was measured by using fura‐2. MK‐886 at concentrations of 1 µM and above 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⁺. MK‐886 evoked Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors nifedipine, econazole and SKF96365. In Ca²⁺‐free medium, after pre‐treatment with 10 µM MK‐886, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pre‐treatment abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter MK‐886‐induced [Ca²⁺]_i rises. MK‐886 at concentrations of 1–100 µM concentration‐dependently decreased cell viability with an IC₅₀ value of 60 µM. The cytotoxic effect of MK‐886 was not inhibited by pre‐chelating cytosolic Ca²⁺ with BAPTA/AM. Together, in PC3 cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via store‐operated Ca²⁺ channels. Independently, MK‐886 was cytotoxic to cells in a Ca²⁺‐independent manner.     

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.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel

Summary Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca²⁺ regulated events, we have studied its effects on the compartmentation of free Ca²⁺ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca²⁺ concentration ([Ca²⁺]_i) to micromolar levels. The increase in [Ca²⁺]_i was not transient and a steady rise was observed with time. Omission of Ca²⁺ from the external medium prevented the AM- and DEA-induced rise in [Ca²⁺]_i suggesting that AM and DEA increased the intracellular [Ca²⁺]_i due to increased influx of Ca²⁺ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca²⁺]_i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na⁺ channel blocker, tetrodotoxin. However, the blockade of [Ca²⁺]_i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca²⁺]_i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca²⁺]_i through receptor mediated channel. ⁴⁵Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca²⁺ release from these intracellular organelles. Also, both these drugs did not interfere with inositol 1,4,5-trisphosphate induced Ca²⁺ release from microsomes even at 10 M concentration. These results clearly indicate that both AM and DEA increase intrasynaptosomal [Ca²⁺]_i by an action on receptor mediated channel in plasma membrane, but not due to the release of Ca²⁺ from intracellular storage sites. This initial rise in [Ca²⁺]_i, together with other changes in Ca²⁺ homeostasis, might be responsible for AM and DEA-induced neurotoxicity. Send offprint requests to D. Desaiah at the above address