Chiang-Ting Chou

Desipramine-Induced Apoptosis in Human Pc3 Prostate Cancer Cells: Activation of Jnk Kinase and Caspase-3 Pathways and a Protective Role of Ca2+ (I) Elevation

The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+]i) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+]i rises may slow down or alleviate desipramine-induced cytotoxicity.

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.

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract Resveratrol is a natural compound that affects cellular Ca2+ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i and WST-1 was used to measure viability. Resveratrol-evoked [Ca2+]i rises concentration-dependently. The response was reduced by removing extracellular Ca2+. Resveratrol-evoked Ca2+ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca2+]i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca2+]i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca2+]i. Previous studies showed that resveratrol between 10 and 100 µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10 μM) increased cell viability, which was abolished by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10 µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca2+]i by evoking phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10 µM also caused Ca2+-dependent cell proliferation.

EFFECTS OF ECONAZOLE ON Ca2+ LEVELS IN AND THE GROWTH OF HUMAN PROSTATE CANCER PC3 CELLS

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura‐2 and tetrazolium as fluorescent dyes.

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.

DESIPRAMINE-INDUCED Ca2+-INDEPENDENT APOPTOSIS IN MG63 HUMAN OSTEOSARCOMA CELLS: DEPENDENCE ON P38 MITOGEN-ACTIVATED PROTEIN KINASE-REGULATED ACTIVATION OF CASPASE 3

1 It has been shown that the antidepressant desipramine is able to induce increases in [Ca2+]i and cell death in MG63 human osteosacroma cells, but whether apoptosis is involved is unclear. In the present study, the effect of desipramine on apoptosis and the underlying mechanisms were explored. It was demonstrated that desipramine induced cell death in a concentration‐ and time‐dependent manner. 2 Cells treated with 100–800 mmol/L desipramine showed typical apoptotic features, including an increase in sub‐diploid nuclei and activation of caspase 3, indicating that these cells underwent apoptosis. Immunoblotting revealed that 100 mmol/L desipramine activated extracellular signal‐regulated kinase (ERK), c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK). Although pretreatment of cells with 20 mmol/L PD98059 (an ERK inhibitor) or 20 mmol/L SP600125 (an inhibitor of JNK) did not inhibit cell death, the addition of 20 mmol/L SB203580 (a p38 MAPK inhibitor) partially rescued cells from apoptosis. Desipramine‐induced caspase 3 activation required p38 MAPK activation. 3 Pretreatment of cells with BAPTA/AM (20 mmol/L) to prevent desipramine‐induced increases in [Ca2+]i did not protect cells from death. 4 The results of the present study suggest that, in MG63 human osteosarcoma cells, desipramine causes Ca2+‐independent apoptosis by inducing p38 MAPK‐associated activation of caspase 3.

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.

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability in OC2 human oral cancer cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The tamoxifen-induced Ca2+ influx was sensitive to blockade of L-type Ca2+ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca2+]i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca2+]i rises, in a nongenomic manner, by causing Ca2+ release from the endoplasmic reticulum, and Ca2+ influx from L-type Ca2+ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca2+]i rise.

Econazole induces increases in free intracellular Ca2+ concentrations in human osteosarcoma cells.

Econazole is an antifungal drug with different in vitro effects. However, econazoles effect on osteoblast-like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 microM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca+ -ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not econazole-induced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry.

Effect of the pesticide, deltamethrin, on Ca2+ signaling and apoptosis in OC2 human oral cancer cells

Abstract Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Although deltamethrin has been shown to induce cytosolic free Ca2+ concentration ([Ca2+]i) rises and apoptosis in different cancer cells, there is no information concerning the effects of deltamethrin on oral cancer. This study explored the effects of deltamethrin on [Ca2+]i and viability in OC2 human oral cancer cells. Deltamethrin, at concentrations of 5–10 μM, increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Deltamethrin-induced [Ca2+]i rise was not inhibited by econazole, SK&F96365, phorbol 12-myristate 13 acetate (PMA) or GF109203X, but was inhibited by nifedipine. In Ca2+-free medium, 10-μM deltamethrin pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitor, 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with BHQ inhibited deltamethrin-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with phospholipase C (PLC) inhibitor U73122 did not suppress deltamethrin-induced Ca2+ release. At concentrations between 20 and 100 μM, deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxymethyl. Deltamethrin-induced cell death was not caused by a preceding [Ca2+]i rise. Annexin V/propidium iodide staining data suggest that deltamethrin (40–60 μM) induced apoptosis in a concentration-dependent manner. To conclude, in OC2 cells, deltamethrin evoked a [Ca2+]i rise by inducing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry by nifedipine-sensitive Ca2+ channels. Further, deltamethrin induced Ca2+-independent cell death might involve apoptosis.