Wei-Chuan Liao

Effect of thimerosal on Ca2+ movement and viability in human oral cancer cells

The effect of thimerosal on cytosolic free Ca2+ concentrations ([Ca2+]i ) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca2+]i levels in suspended OC2 cells using fura-2. Thimerosal at concentrations between 1and 50 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca 2+. Thimerosal-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors and protein kinase C modulators (phorbol 12-myristate 13-acetate [PMA] and GF109203X). In Ca2+-free medium, 50 μM thimerosal failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca2+]i rises. At concentrations between 5 and 10 μM, thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8 μM thimerosal was potentiated by prechelating cytosolic Ca2+ with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetomethyl (BAPTA/ AM). Flow cytometry data suggested that 1—7 μM thimerosal-induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, thimerosal-induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx through non—L-type Ca2+ channels. Thimerosal killed cells in a concentration-dependent manner through apoptosis.

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent 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 MK-886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

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 Ca(2+) concentrations ([Ca(2+)](i)) and viability in human PC3 prostate cancer cells. [Ca(2+)](i) in suspended cells was measured by using fura-2. MK-886 at concentrations of 1 microM and above increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 20 microM. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). MK-886 evoked Mn(2+) quenching of fura-2 fluorescence, implicating Ca(2+) entry. MK-886-induced Ca(2+) influx was inhibited by store-operated Ca(2+) entry inhibitors nifedipine, econazole and SKF96365. In Ca(2+)-free medium, after pre-treatment with 10 microM MK-886, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were abolished; and conversely, thapsigargin pre-treatment abolished MK-886-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with U73122 did not alter MK-886-induced [Ca(2+)](i) rises. MK-886 at concentrations of 1-100 microM concentration-dependently decreased cell viability with an IC(50) value of 60 microM. The cytotoxic effect of MK-886 was not inhibited by pre-chelating cytosolic Ca(2+) with BAPTA/AM. Together, in PC3 cells, MK-886 induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum; and Ca(2+) influx via store-operated Ca(2+) channels. Independently, MK-886 was cytotoxic to cells in a Ca(2+)-independent manner.

The mechanism of sertraline-induced [Ca2+]i rise in human OC2 oral cancer cells

Effect of sertraline, an antidepressant, on cytosolic free Ca2+ levels ([Ca2+]i) in human cancer cells is unclear. This study examined if sertraline altered basal [Ca2+]i levels in suspended OC2 human oral cancer by using fura-2 as a Ca2+-sensitive fluorescent probe. At concentrations of 10−100 μM, sertraline induced a [Ca2+]i rise in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholipase A2, inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change sertraline-induced [Ca2+]i rise. Together, in human oral cancer cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.

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.

Mechanisms of AM404-induced [Ca2+]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 > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. 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 microM 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 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM 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.

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells was explored. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Diindolylmethane at concentrations of 20–50 µM induced [Ca2+]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca2+. Diindolylmethane-evoked Ca2+ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca2+, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca2+]i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca2+]i rise. At concentrations of 50–100 µM, diindolylmethane 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/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca2+]i rise in PC3 cells by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via phospholipase A2-sensitive store-operated Ca2+ channels. Diindolylmethane caused cell death in which apoptosis may participate.

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2+]i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. This Ca2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2+]i rise, suggesting that thapsigargin released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2+]i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca2+ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels. Thapsigargin also induced cell death via Ca2+-dependent pathways and Ca2+-independent apoptotic pathways.

Effect of phenethyl isothiocyanate on Ca2+ movement and viability in MDCK canine renal tubular cells

The effect of the natural compound phenethyl isothiocyanate (PEITC) on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in MDCK renal cells is unknown. This study explored whether PEITC changed [Ca2+]i in MDCK cells using the Ca2+-sensitive fluorescent dye fura-2. PEITC at 200–700 μM increased [Ca2+]i in a concentration-dependent manner. The signal was reduced by removing extracellular Ca2+. PEITC-induced Ca2+ influx was inhibited by nifedipine, econazole, SK&F 96365 and protein kinase C modulators. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited PEITC-induced rise in [Ca2+]i. Incubation with PEITC also inhibited TG or BHQ-induced rise in [Ca2+]i. Inhibition of phospholipase C with U73122 abolished PEITC-induced rise in [Ca2+]i. At 15–75 μM, PEITC decreased viability. The cytotoxic effect of PEITC was enhanced by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester. Annexin V-FITC data suggest that 20 and 50 μM PEITC induced apoptosis. At 10 and 15 μM, PEITC did not increase reactive oxygen species (ROS) production. Together, in renal tubular cells, PEITC-induced rise in [Ca2+]i by inducing phospholipase C-dependent Ca2+ release from endoplasmic reticulum and Ca2+ entry via store-operated Ca2+ channels. PEITC induced apoptosis in a concentration-dependent, ROS/Ca2+-independent manner.