Action of the natural compound gomisin a on Ca2+ movement in human prostate cancer cells.

Gomisin A is a dietary lignan compound isolated from the fruit of Schisandra chinensis and has many pharmacological properties, including hepato-protective, anti-diabetic, and anti-oxidative activities. However, the benefit of gomisin A is still not well understood. The action of gomisin A is diverse. However, the effect of gomisin A on Ca2+ signaling in prostate cancer cells is unknown. Ca2+ is a pivotal second envoy that triggers and regulates cellular processes such as apoptosis, fertilization, energy transduction, secretion, and protein activation. The goal of this study was to explore the action of gomisin A on [Ca2+]i and cytotoxicity in PC3 prostate cancer cells. Gomisin A at 100-200 µM provoked [Ca2+]i raises. 20% of the response was reduced by removing external Ca2+. The Ca2+ influx provoked by gomisin A was suppressed by 20% by store-caused Ca2+ entry suppressors: econazole, SKF96365, nifedipine; also by phorbol 12-myristate 13 acetate and GF109203X. Without external Ca2+, gomisin A-caused [Ca2+]i raises were abolished by thapsigargin. In contrast, gomisin A suppressed the [Ca2+]i raises caused by thapsigargin. U73122 fell short to change gomisin A-caused [Ca2+]i responses. Gomisin A (20-100 µM) elicited cytotoxicity in a dose-associated fashion. Blockade of [Ca2+] elevations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl failed to inhibit cytotoxicity of gomisin A. Collectively, gomisin A evoked [Ca2+]i raises and provoked cytotoxicity in a Ca2+-dissociated fashion in prostate cancer cells.

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol], a natural phenolic compound found in the olive, alters Ca2+ signaling and viability in human HepG2 hepatoma cells.

Hepatotoma is the leading type of primary liver cancer in adults and third cause of death in the world. Hydroxytyrosol is a natural phenol existing in olive (Olea europaea L.). Hydroxytyrosol is the chief ingredient of olive oil, which was early deemed to be the most robust antioxidant in olive oil. Hydroxytyrosol is known to inhibit various types of cancer by different methods. This study was aimed to delineate the action of hydroxytyrosol on viability and [Ca2+]i in HepG2 hepatoma cells. Fura-2 was used to detect [Ca2+]i, and WST-1 assays were applied to explore cell cytotoxicity. Hydroxytyrosol elicited [Ca2+]i raises. Eliminating external Ca2+ diminished the Ca2+ signal by 30%. Hydroxytyrosol-evoked Ca2+ influx was diminished by 20% by three inhibitors of store-operated Ca2+ channels and by a protein kinase C activator and an inhibitor. In the absence of Ca2+, thapsigargin eradicated hydroxytyrosol-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122, a PLC inhibitor, did not inhibit hydroxytyrosol-elicited [Ca2+]i raises. Hydroxytyrosol reduced cell viability. This cytotoxic action was not reversed by preincubation with BAPTA/AM, a cytosolic Ca2+ binder. In sum, in HepG2 hepatoma cells, hydroxytyrosol elicited [Ca2+]i raises by provoking PLC-unrelated discharge of Ca2+ from ER and Ca2+ influx through PKC-sensitive store-operated Ca2+ entry. In addition, hydroxytyrosol elicited Ca2+-dissociated cytotoxicity.

Effect of deoxycholic acid on Ca2+ movement, cell viability and apoptosis in human gastric cancer cells.

Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca(2+) movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i. DOA-evoked [Ca(2+)]i rises concentration dependently. The response was reduced by removing extracellular Ca(2+). DOA-evoked Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca(2+)]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca(2+)]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca(2+)]i rises. At 100-500 µM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 µM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca(2+)]i rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. DOA also caused Ca(2+)-independent apoptosis.

The mechanism of NPC-14686-induced [Ca²âº]i rises and non-Ca²âº-triggered cell death in MG63 human osteosarcoma cells.

NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca²âº concentrations ([Ca²âº]i) and viability in MG63 human osteosarcoma cells was explored. The Ca²âº-sensitive fluorescent dye fura-2 was applied to measure [Ca²âº]i. NPC-14686 at concentrations of 100-500 µM induced a [Ca²âº]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca²âº. NPC-14686 induced Mn²âº influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca²âº entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²âº]i rise. At 20-50 µM, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca²âº 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 NPC-14686 (30-50 µM) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca²âº]i rise by inducing phospholipase C-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via protein kinase C-sensitive store-operated Ca²âº channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways.

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 Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 1-50 µM evoked a [Ca(2+)](i) rise in a concentration-dependent manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 10-75 µM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 µM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.

Effect of thymol on Ca2+ homeostasis and viability in MG63 human osteosarcoma cells.

AIMS: The effect of the natural product thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was examined. METHODS: The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). RESULTS: Thymol at concentrations of 200-1,000 µmol/l induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Thymol-induced Ca(2+) entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited the thymol-induced [Ca(2+)](i) rise. Incubation with thymol also inhibited the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished the thymol-induced [Ca(2+)](i) rise. At concentrations of 100-600 µmol/l, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM. Annexin V/propidium iodide staining data suggest that thymol (200 and 400 µmol/l) induced apoptosis in a concentration-dependent manner. Thymol (200 and 400 µmol/l) also increased levels of reactive oxygen species. CONCLUSIONS: In MG63 cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis via mitochondrial pathways.

Econazole-evoked [Ca2+]i rise and non-Ca2+-triggered cell death in rabbit corneal epithelial cells (SIRC).

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations > or = 1 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The econazole-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 20 microM econazole, [Ca(2+)](i) rises induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change econazole-induced [Ca(2+)](i) rises. At concentrations between 10 and 80 microM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM econazole was not reversed by prechelating cytosolic Ca(2+) with BAPTA. This shows that in SIRC cells econazole induces [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca(2+)](i) rise.

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

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

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

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.

Mechanism of paroxetine-induced cell death in renal tubular cells.

Paroxetine belongs to the family of selective serotonin reuptake inhibitors. Much research has been performed on the in vitro effect of paroxetine; however, the effect of paroxetine on Madin-Darby canine kidney renal tubular cells is unknown. The present study was aimed at exploring how paroxetine affects viability and to examine the underlying mechanisms. Paroxetine (15-200 microM) was shown to reduce cell viability via inducing apoptosis in a concentration-dependent manner. Paroxetine-induced cytotoxicity and apoptosis were not changed by the p38 mitogen-activated protein kinase inhibitor SB203580 and the c-Jun NH2-terminal kinase inhibitor SP600125, but was potentiated by the extracellular signal-regulated kinase inhibitor PD98059; inhibited by GF 109203X, a protein kinase C inhibitor; and potentiated by phorbol 12-myristate 13-acetate, a protein kinase C activator. Paroxetine induced [Ca2+](i) rises; however, pre-treatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester, a Ca2+ chelator, to prevent 20 microM paroxetine-induced [Ca2+](i) rises did not protect cells from death. H-89 (a protein kinase A inhibitor) and U73122 (a phospholipase C inhibitor) failed to alter paroxetine-induced cell death. The results suggest that in Madin-Darby canine kidney cells, paroxetine caused protein kinase C-dependent, Ca2+-independent apoptosis which was potentiated by inhibition of the extracellular signal-regulated kinase pathway.

Diethylstilbestrol-induced estrogen receptor-dependent [Ca2+]i rises and apoptosis in Chinese hamster ovary (CHO) cells.

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations>or=1 proportional, variant increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. In Ca2+-free medium, after pretreatment with 50 proportional, variant DES, 1 proportional, variant thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca2+]i rises. At a concentration of 5 proportional, variant, DES increased cell viability. At concentrations of 100-200 microM, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 microM DES on viability was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 microM)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 microM). ICI-182,780 did not affect 5 microM DES-induced increase in viability but partly reversed 100 microM DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca2+]i rises by stimulating estrogen receptors leading to Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca2+-dependent pathway.