Shiuh-Inn Liu

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca2+ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca2+ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at a concentration of 325xa0μM started to increase [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 40% by removing extracellular Ca2+, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca2+-free medium, after pretreatment with 650xa0μM safrole, 1xa0μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) barely induced a [Ca2+]i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca2+]i rise. Neither inhibition of phospholipase C with 2xa0μM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 1xa0μM safrole did not alter cell proliferation, but incubation with 10–1000xa0μM safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca2+ with 10xa0μM of the Ca2+ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca2+]i rise by causing release of stored Ca2+ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca2+ influx via nifedipine-sensitive Ca2+ entry. Furthermore, safrole can enhance cell growth in a Ca2+-independent manner.

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

1.u2002Econazole 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.

M-3M3FBS-Induced Ca 2+ Movement and Apoptosis in HA59T Human Hepatoma Cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca² ⁺ concentrations ([Ca² ⁺ ]i ) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca² ⁺ ]i levels in suspended cells by using fura-2 as a Ca² ⁺ -sensitive fluorescent dye. M-3M3FBS at concentrations of 10- 50 μM increased [Ca² ⁺ ]i in a concentration-dependent fashion. The Ca² ⁺ signal was reduced partly by removing extracellular Ca² ⁺ . M-3M3FBS-induced Ca² ⁺ influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca² ⁺ -free medium, 50 μM m-3M3FBS pretreatment inhibited the [Ca² ⁺ ]i rise induced by the endoplasmic reticulum Ca² ⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca² ⁺ ]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca² ⁺ ]i rise. At concentrations between 10 and 40 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca² ⁺ with 1,2-bis(2- aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. M-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca² ⁺ ]i rise by inducing phospholipase C-independent Ca² ⁺ release from the endoplasmic reticulum and Ca² ⁺ entry via protein kinase C-sensitive store-operated Ca² ⁺ channels. M-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.

Short waves-induced enhancement of proliferation of human chondrocytes: involvement of extracellular signal-regulated map-kinase (erk).

1 Short‐wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short‐wave exposure on the proliferation of cultured human chondrocytes. 2 Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3 Given the importance of mitogen‐activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal‐regulated kinase (ERK), c‐Jun NH2‐terminal protein kinase (JNK) and p38. 4.u2002It was found that the level of phosphorylated ERK (phospho‐ERK 1 and ERK 2) increased significantly within 5–120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0–240 min. 5 Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 µmol/L, 20 µmol/L or 50 µmol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves‐induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

Rise of (Ca 2+ )i and Apoptosis Induced by M-3M3FBS in SCM1 Human Gastric Cancer Cells

M-3M3FBS (2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide is a presumed phospholipase C activator which induced Ca²⁺ movement and apoptosis in different cell models. How- ever, the effect of m-3M3FBS on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in SCM1 human gastric cancer cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca²⁺]i levels in suspended cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. M-3M3FBS at concentrations between 5-50 μM increased [Ca²⁺]i in a concentration-dependent manner. The Ca²⁺ signal was reduced by half by removing extracellular Ca²⁺. M-3M3FBS-induced Ca²⁺ influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca²⁺-free medium, 50 μM m-3M3FBS pretreatment inhibited the [Ca²⁺]i rise induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca²⁺]i rise. Suppression of inositol 1,4,5-trisphosphate production with U73122 did not change m-3M3FBS- induced [Ca²⁺]i rise. At concentrations between 25 and 50 μM m-3M3FBS killed cells in a concentration- dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca²⁺ with acetoxy-methyl ester of bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid (BAPTA/AM). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis at 25 and 50 μM. M-3M3FBS also increased levels of superoxide. Together, in human gastric cancer cells, m-3M3FBS induced a [Ca²⁺]i rise by inducing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. M-3M3FBS induced cell death that might involve apoptosis via reactive oxygen species production.

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

The effect of ketoconazole on cytosolic free Ca2 + concentrations ([Ca2 +]i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca2 + levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca2 +]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 μ M and above increased [Ca2 +]i in a concentration-dependent manner. The Ca2 + signal was reduced partly by removing extracellular Ca2 +. The ketoconazole-induced Ca2 + influx was insensitive to L-type Ca2 + channel blockers and protein kinase C modulators. In Ca2 +-free medium, after pretreatment with 50 μ M ketoconazole, thapsigargin-(1 μ M)-induced [Ca2 +]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2 +]i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change ketoconazole-induced [Ca2 +]i rises. At concentrations between 5 and 100 μ M, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 μ M ketoconazole was not reversed by prechelating cytosolic Ca2 + with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca2 +]i rises by causing Ca2 + release from the endoplasmic reticulum and Ca2 + influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca2 +]i rise.

Effect of BayK 8644 on [Ca^(2+)]i and Viability in PC3 Human Prostate Cancer Cells

The effect of BayK 8644 on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in PC3 human prostate cancer cells was explored. Fura-2 was applied to measure [Ca²⁺]i. BayK 8644 at 1-50 μM induced a [Ca2²⁺]i rise concentration-dependently. The response was reduced by removing extracellular Ca²⁺. BayK 8644-evoked Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished BayK 8644-induced [Ca²⁺]i rise. Inhibition of phospholipase C did not alter BayK 8644-induced [Ca²⁺]i rise. BayK 8644 killed cells in a concentrationdependent manner, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid/acetoxy methyl (BAPTA/AM). Collectively, in PC3 human prostate cancer cells, BayK 8644 induced a [Ca²⁺]i rise by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels (and/or T-type Ca²⁺ channels). At high concentrations, BayK 8644 caused cell death.

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.

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 ≥ 1∝ 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∝ DES, 1∝ 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∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM 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 μ M)-induced [Ca2+]i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M 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.

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.