Ureteroscope-assisted double-J stenting following laparoscopic ureterolithotomy.

The aim of this study was to examine the feasibility of ureteroscope-assisted double-J stenting following laparoscopic ureterolithotomy and to evaluate the effects of retrograde ureteroscopic access exerted on the sutured ureterotomy site. From January 2002 to December 2011, 30 patients with proximal ureteral stone underwent ureteroscopic double-J stenting of the ureter following retroperitoneal laparoscopic ureterolithotomy. Patient demographics and perioperative parameters, including the degree of hydronephrosis, urine leakage, and drainage time, were retrospectively reviewed. These data were compared with those of 30 consecutive patients who received open ureterolithotomy and intracorporeal ureteral double-J stenting. In addition, a PubMed search was conducted and the related literature on the placement of a ureteral stent was reviewed. Twenty-eight patients successfully underwent ureteral double-J stenting with ureteroscopic access. No malposition of the ureteral stent was identified in the ureteroscopic group, but two patients in the intracorporeal group required postoperative adjustment of the stent. Residual stone fragments were found during stent placement in three patients in the ureteroscopic group and holmium:yttrium-aluminum-garnet laser lithotripsy was immediately performed. There was no significant difference in postoperative outcomes or complication rates between the two groups. Ureteroscope-assisted ureteral double-J stenting is a simple and safe alternative allowing intraluminal navigation along the entire ureter, correct stent placement, and prompt treatment of residual stone fragments, without radiation exposure. In addition, ureteral disruption and urinary extravasation may not be concerns for ureteroscopic access with continuous normal saline irrigation.

Mechanisms of resveratrol-induced changes in [Ca(2+)]i and cell viability in PC3 human prostate cancer cells.

Resveratrol is a natural compound that affects cellular Ca(2+) homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca(2+) concentrations ([Ca(2+)]i) and viability in PC3 human prostate cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i and WST-1 was used to measure viability. Resveratrol-evoked [Ca(2+)]i rises concentration-dependently. The response was reduced by removing extracellular Ca(2+). Resveratrol-evoked Ca(2+) 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 Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca(2+)]i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca(2+)]i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca(2+)]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 Ca(2+) 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 [Ca(2+)]i by evoking phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry, via protein kinase C-regulated mechanisms. Resveratrol at 1-10 µM also caused Ca(2+)-dependent cell proliferation.

Effect of BayK 8644 on [Ca²âº]i and viability in PC3 human prostate cancer cells.

The effect of BayK 8644 on cytosolic Ca²âº concentrations ([Ca²âº]i) and viability in PC3 human prostate cancer cells was explored. Fura-2 was applied to measure [Ca²âº]i. BayK 8644 at 1-50 µM induced a [Ca2²âº]i rise concentration-dependently. The response was reduced by removing extracellular Ca²âº. BayK 8644-evoked Ca²âº entry was inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished BayK 8644-induced [Ca²âº]i rise. Inhibition of phospholipase C did not alter BayK 8644-induced [Ca²âº]i rise. BayK 8644 killed cells in a concentrationdependent manner, 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). Collectively, in PC3 human prostate cancer cells, BayK 8644 induced a [Ca²âº]i rise by evoking phospholipase C-independent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via protein kinase C-sensitive store-operated Ca²âº channels (and/or T-type Ca²âº channels). At high concentrations, BayK 8644 caused cell death.

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

The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 µM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also 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 50-100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) 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 [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.

3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells.

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). DIM at concentrations of 40-80 µM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca(2+)](i) rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca(2+)](i) rise. At concentrations of 10-50 µM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 µM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca(2+)](i) rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM caused 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.

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

The effect of the natural essential oil thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in human glioblastoma cells was examined. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Thymol at concentrations of 400-1000 µM 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+) signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca(2+)](i) rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca(2+)](i) rise. At concentrations of 200-800 µM, 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/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 µM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis.

Mixed epithelial and stromal tumor of the kidney with elevated serum level of cancer antigen 125.

Mixed epithelial and stromal tumor of the kidney is a newly categorized lesion, with few reported cases. We report a rare case of a 45-year-old woman with a palpable abdominal mass and elevated serum level of serum cancer antigen 125, who was not receiving hormones or contraceptive agents. Abdominal magnetic resonance imaging revealed a large multilocular cystic tumor that arose in the left central kidney. Nephrectomy was performed under the initial impression of cystic renal cell carcinoma; however, a diagnosis of mixed epithelial and stromal tumor was confirmed according to pathological and immunohistochemical findings. Serum level of cancer antigen 125 returned to normal after 1 month postoperatively and no recurrence was found in the following 18 months.

The mechanism of sertraline-induced [Ca(2+) ](i) rise in human PC3 prostate cancer cells.

The effect of sertraline, an antidepressant, on cytosolic-free Ca(2+) levels ([Ca(2+) ](i) ) in human cancer cells is unclear. This study examined whether sertraline altered basal [Ca(2+) ](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca(2+) -sensitive fluorescent probe. At concentrations of 10-150 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 the suppression of store-operated Ca(2+) channels or by the modulation of protein kinase C activity. In Ca(2+) -free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone nearly abolished sertraline-induced Ca(2+) release. Conversely, pre-treatment 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 inhibited sertraline-induced [Ca(2+) ](i) rise. At 20-30 µM, sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was enhanced by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM. Annexin V-FITC data suggest that sertraline (20 and 30 µM) evoked apoptosis in a concentration-dependent manner. Together, in PC3 human prostate cancer cells, sertraline induced [Ca(2+) ](i) rises by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and via multiple Ca(2+) influx pathways that involve store-operated Ca(2+) channels. Sertraline also induced apoptosis that was not triggered by [Ca(2+) ](i) rise.

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 [Ca²âº](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²âº-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca²âº](i) in a concentration-dependent fashion. The Ca²âº signal was reduced partly by removing extracellular Ca²âº indicating that Ca²âº entry and release both contributed to the [Ca²âº](i) rise. This Ca²âº influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²âº channels or by modulation of protein kinase C activity. In Ca²âº-free medium, pretreatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²âº release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²âº](i) rise, suggesting that thapsigargin released Ca²âº from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²âº](i) rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²âº 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 [Ca²âº](i) rises by causing phospholipase C-independent Ca²âº release from the endoplasmic reticulum and Ca²âº influx via phospholipase A2-sensitive Ca²âº channels. Thapsigargin also induced cell death via Ca²âº-dependent pathways and Ca²âº-independent apoptotic pathways.

Effect of diallyl disulfide on Ca2+ movement and viability in PC3 human prostate cancer cells.

The effect of diallyl disulfide (DADS) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca(2+)](i) in PC3 cells by using fura-2. DADS at 50-1000 µM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by removing Ca(2+). DADS-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, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca(2+)](i) rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca(2+)](i) rise. At 500-1000 µM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500 µM) induced apoptosis in a Ca(2+)-independent manner. Annexin V/PI staining further shows that 10 µM and 500 µM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca(2+)](i) rise probably by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. DADS induced Ca(2+)-dependent cell death, ROS production, and Ca(2+)-independent apoptosis.

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.

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells.

The effect of calmidazolium on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca2+]i and caused cell death in HA59T cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations > or =1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1.5 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Calmidazolium induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to L-type Ca2+ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), calmidazolium-induced [Ca2+]i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change calmidazolium-induced [Ca2+]i rises. At concentrations between 1 and 15 microM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via protein kinase C-regulated Ca2+ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.

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.

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.

Mechanisms of AM404-induced [Ca(2+)](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.

Expression of vascular endothelial growth factor in Taiwanese benign and malignant prostate tissues.

BACKGROUND: The expression of vascular endothelium growth factor (VEGF) has been correlated to the grading and stage of prostate cancers. However, data regarding Taiwanese prostate cancer patients are lacking. The aim of the present study was to examine VEGF expression in our radical prostatectomy specimens. METHODS: Fifty-one radical prostatectomy specimens with prostate cancer (15 stage pT2N0, 25 pT3N0, 11 pT2-4 N1) were stained using goat anti-human VEGF polyclonal antibody (AB-293NA; R&D Systems Inc., Minneapolis, MN, USA). The VEGF expression in malignant and nonmalignant prostate tissues was compared. The correlations of VEGF immunoreactivity with Gleason scores and pathologic stages were examined. MannWhitney U test was used for comparison of preoperative prostate-specific antigen levels between patients with and without VEGF expression. RESULTS: Positive VEGF staining was observed in 80.4% of malignant epithelia, 39.2% of peritumoral stroma, 68.6% of benign hyperplastic glands, and 25.5% of adjacent stroma. There was no difference in VEGF expression between malignant and nonmalignant areas. Advanced disease had significantly higher frequency of stroma but not epithelium VEGF staining as compared to organ-confined disease (p = 0.002 and p = 0.412, respectively). The Gleason 7 and higher tumors had significantly higher frequency of VEGF staining in stroma but not glandular epithelium (p = 0.041 and p = 0.353, respectively). Tumors with positive epithelium VEGF staining had significantly higher PSA levels (21.3 18.1 vs. 10.8 6.8 ng/mL; p = 0.013). CONCLUSION: There was no difference in VEGF immunoreactivity between malignant and benign prostatic epithelium in Taiwanese. High Gleason grade tumors and advanced disease had significantly higher frequency of VEGF expression in stroma but not glandular epithelium. Tumors with positive epithelium VEGF staining had significantly higher PSA levels.

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 microM 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 microM ketoconazole, thapsigargin-(1 microM)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change ketoconazole-induced [Ca2+]i rises. At concentrations between 5 and 100 microM, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 microM 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.

The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells.

The effect of the carcinogen safrole on intracellular Ca2+ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca2+ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 33 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 400 microM. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca2+ after safrole had depleted intracellular Ca(2+)-induced dramatic Ca2+ influx, suggesting that safrole caused store-operated Ca2+ entry. In Ca(2+)-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca 2+. Inhibition of phospholipase C with 2 microM U73122 did not affect safrole-induced Ca2+ release. Trypan blue exclusion assays revealed that incubation with 650 microM safrole for 30 min did not kill cells, but killed 70% of cells after incubation for 60 min. Collectively, the data suggest that in MDCK cells, safrole induced a [Ca2+] increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca2+ influx via store-operated Ca2+ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.