Chun-Chi Kuo

Effect of Thymol on Ca2+ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

Aims: The effect of the natural product thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was examined. Methods: The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Results: Thymol at concentrations of 200–1,000 µmol/l induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited the thymol-induced [Ca²⁺]_i rise. Incubation with thymol also inhibited the thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished the thymol-induced [Ca²⁺]_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 Ca²⁺ 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 [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis via mitochondrial pathways.

Mechanism of maprotiline-induced apoptosis: role of [Ca2+](i), ERK, JNK and caspase-3 signaling pathways.

Antidepressants are generally used for treatment of various mood and anxiety disorders. Several studies have shown the anti-tumor and cytotoxic activities of some antidepressants, but the underlying mechanisms were unclear. Maprotiline is a tetracyclic antidepressant and possesses a highly selective norepinephrine reuptake ability. We found that maprotiline decreased cell viability in a concentration- and time-dependent manner in Neuro-2a cells. Maprotiline induced apoptosis and increased caspase-3 activation. The activation of caspase-3 by maprotiline appears to depend on the activation of JNK and the inactivation of ERK. Maprotiline also induced [Ca(2+)](i) increases which involved the mobilization of intracellular Ca(2+) stored in the endoplasmic reticulum. Pretreatment with BAPTA/AM, a Ca(2+) chelator, suppressed maprotiline-induced ERK phosphorylation, enhanced caspase-3 activation and increased maprotiline-induced apoptosis. In conclusion, maprotiline induced apoptosis in Neuro-2a cells through activation of JNK-associated caspase-3 pathways. Maprotiline also evoked an anti-apoptotic response that was both Ca(2+)- and ERK-dependent.

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.

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.

Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca(superscript 2+) regulation of OC2 human oral cancer cells. AC at a concentration of 25μM induced an increase in cell viability, but AC at concentrations ≥50μg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200μg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25μg/ml) did not alter basal [Ca(superscript 2+)](subscript i), but decreased the [Ca2(superscript +)](subscript i) increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25μg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinininduced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargininduced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca(superscript 2+)](subscript i), induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

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

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

Effect of Methoxychlor on Ca2+ Handling and Viability in OC2 Human Oral Cancer Cells

Abstract:  The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca2+ concentrations ([Ca2+]i) in human oral cancer cells (OC2) by 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 70% by removing extracellular Ca2+. Methoxychlor‐induced Ca2+ entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca2+‐free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished methoxychlor‐induced [Ca2+]i rise. Incubation with methoxychlor also inhibited thapsigargin‐ or BHQ‐induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor‐induced [Ca2+]i rise. At 5–20 μM, methoxychlor killed cells in a concentration‐dependent manner. 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 methoxychlor (10 and 20 μM) evoked apoptosis in a concentration‐dependent manner. Together, in human OC2, methoxychlor induced a [Ca2+]i rise probably by inducing phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via phospholipase A2‐sensitive channels. Methoxychlor induced cell death that may involve apoptosis.

Effect of Nortriptyline on Ca(superscript 2+) Handling in SIRC Rabbit Corneal Epithelial Cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca(superscript 2+) concentrations ([Ca(superscript 2+)](subscript i)) in corneal epithelial cells, [Ca(superscript 2+)](subscript i) levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca(superscript 2+)-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 μM increased [Ca(superscript 2+)](subscript i) in a concentration-dependent manner. The Ca(superscript 2+) signal was reduced partly by removing extracellular Ca(superscript 2+). Nortriptyline-induced Ca(superscript 2+) influx was inhibited by the store-operated Ca(superscript 2+) channel blockers econazole and SKF another endoplasmic reticulum Ca(superscript 2+) pump inhibitor) nearly abolished nortriptyline-induced [Ca(superscript 2+)](subscript i) rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca(superscript 2+)](subscript i) rise by 75%. Taken together, nortriptyline induced [Ca(superscript 2+)](subscript i) rises in SIRC cells by causing phospholipase C-dependent Ca(superscript 2+) release from the endoplasmic reticulum and Ca(superscript 2+) influx via store-operated Ca(superscript 2+) channels.

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 m-3M3FBS on Ca(superscript 2+) Movement in PC3 Human Prostate Cancer Cells

The effect of 2, 4, 6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca(superscript 2+) concentrations ([Ca(superscript 2+)](subscript i)) in PC3 human prostate cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca(superscript 2+)](subscript i) levels in suspended PC3 cells by using fura-2 as a Ca(superscript 2+)-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-50μM increased [Ca(superscript 2+)](subscript i) in a concentration-dependent manner. The Ca(superscript 2+) signal was reduced by 60% by removing extracellular Ca(superscript 2+). M-3M3FBS-induced Ca(superscript 2+) influx was inhibited by the store-operated Ca(superscript 2+) channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca(superscript 2+)-free medium, 30μM m-3M3FBS pretreatment greatly inhibited the [Ca(superscript 2+)](subscript i) rise induced by the endoplasmic reticulum Ca(superscript 2+) pump inhibitor thapsigargin or BHQ. Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid reduced the major part of m-3M3FBS-induced [Ca(superscript 2+)](subscript i) rise. Inhibition of phospholipase C with U73122 did not much alter m-3M3FBS-induced [Ca(superscript 2+)](subscript i) rise. Collectively, in PC3 cells, m-3M3FBS induced [Ca(superscript 2+)+](subscript i) rises by causing phospholipase C-independent Ca(superscript 2+) release from the endoplasmic reticulum and Ca(superscript 2+) influx via store-operated Ca(superscript 2+) channels.