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.

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 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 bisphenol A on Ca(2+) fluxes and viability in Madin-Darby canine renal tubular cells.

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca(2+)](i) levels in suspended MDCK cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced, partly, by removing extracellular Ca(2+). Bisphenol A induced Mn(2+) influx, leading to quenching of fura-2 fluorescence, suggesting Ca(2+) influx. This Ca(2+) influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca(2+) channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca(2+) pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A-induced Ca(2+) release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca(2+)](i) rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca(2+)-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca(2+)](i) rises by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and mitochondria and Ca(2+) influx via phospholipase A2-, protein kinase C-sensitive, store-operated Ca(2+) channels.

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.

Risk factors for individual domains of female sexual function.

INTRODUCTION: Female sexual function contains four major subtypes of desire, arousal, orgasm, and pain. Few studies used validated instruments to determine the dysfunction in these areas and assess their risk factors. AIM: To assess the prevalence of and risk factors for individual components of sexual difficulty in women. METHODS: A self-administered questionnaire containing the Female Sexual Function Index (FSFI) was given to 2,159 woman employees of two hospitals to assess their sexual function and its correlates. MAIN OUTCOME MEASURES: The associations between female sexual difficulty in individual domains defined by the FSFI domain scores and potential risk factors assessed by simple questions. RESULTS: Among the 1,580 respondents, 930 women's data were eligible for analysis with a mean age of 36.1 years (range 20-67). Of them, 43.8% had sexual difficulty in one or more domains, including low desire in 31.3%; low arousal, 18.2%; low lubrication, 4.8%; low orgasmic function, 10.4%; low satisfaction, 7.3%; and sexual pain, 10.5%. Compared with the younger women (20-49 years), the oldest age group (50-67 years) had a significantly higher prevalence in low desire, low arousal, and low lubrication, but not in the other domains. Based on multivariate logistic regression analyses, poor relationship with the partner and perception of partner's sexual dysfunction were major risk factors for low desire, low arousal, low orgasmic function, and low satisfaction. Age and urge urinary incontinence were associated with low lubrication and sexual pain. Most comorbidities were not related to these difficulties, except diabetes being related to low desire. CONCLUSIONS: Relationship factors had substantial impact on female sexual function in desire, arousal, orgasm, and satisfaction. On the other hand, women's lubrication problem and sexual pain were related predominantly with biological factors. These are initial results and future research is needed to confirm them.

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.

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.

Anandamide-induced Ca2+ elevation leading to p38 MAPK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells.

The effect of anandamide on human osteoblasts is unclear. This study examined the effect of anandamide on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca2+ levels in MG63 osteosarcoma cells. Anandamide at 50-200 microM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that anandamide induced expression of ERK, JNK and p38 MAPK. Anandamide-induced cell death and apoptosis were reversed by SB203580, but not by PD98059 and SP600125, suggesting that anandamide's action was via p38 MAPK, but not via ERK and JNK. Anandamide at 1-100 microM induced [Ca2+]i increases. Removal of extracellular Ca2+ decreased the anandamide response, indicating that anandamide induced Ca2+ influx and Ca2+ release. Chelation of intracellular Ca2+ with BAPTA reversed anandamide-induced cell death and p38 MAPK phosphorylation. Collectively, in MG63 cells, anandamide induced [Ca2+]i increases which evoked p38 MAPK phosphorylation. This p38 MAPK phosphorylation subsequently activated caspase-3 leading to apoptosis.

Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells.

Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis.

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.

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.

Role of p21(WAF1) and p27(KIP1) in predicting biochemical recurrence for organ-confined prostate adenocarcinoma.

BACKGROUND: Both p21(WAF1) and p27(KIP1) have been reported as prognostic markers predicting biochemical failure for prostate cancers. We examined the expression and prognostic significance of p21(WAF1) and p27(KIP1) in organ-confined (pT2) prostate cancer patients. METHODS: The medical records of 53 pT2 prostate adenocarcinomas were analyzed retrospectively. Radical prostatectomy specimens were stained using anti-p21(WAF1) and anti-p27(KIP1) antibodies. Biochemical relapse was defined as 2 consecutive elevations in serum prostate specific antigen (PSA) level > 0.2 ng/mL with an interval of more than 3 months. The prognostic significance of p21(WAF1) and p27(KIP1) expression was assessed. RESULTS: p21WAF1 immunoreactivity was found in 19 patients (35.8%). Twenty-nine tumors (54.7%) had decreased p27(KIP1) expression. Both markers were not associated with Gleason scores (p = 1.00 for both). At a median follow-up of 49 months, 15 patients (28.3%) experienced biochemical recurrence. Both p21 and p27 had no prognostic significance in log-rank test (p = 0.98 and p = 0.64, respectively). CONCLUSION: p21(WAF1) and p27(KIP1) expression have no role in predicting biochemical relapse for stage pT2 prostate cancers.

Effect of desipramine on Ca2+ levels and growth in renal tubular cells.

The in vitro effect of desipramine on renal tubular cell is unknown. In Madin-Darby canine kidney (MDCK) cells, the effect of desipramine on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Desipramine (>25 microM) caused a rapid and sustained rise of [Ca2+]i in a concentration-dependent manner (EC50=50 microM). Desipramine-induced [Ca2+]i rise was prevented by 40% by removal of extracellular Ca2+ but was not altered by L-type Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which desipramine failed to release more Ca2+; in addition, pretreatment with desipramine partly decreased thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not change desipramine-induced [Ca2+]i rise. Incubation with 10-100 microM desipramine enhances or inhibits cell proliferation in a concentration- and time-dependent manner. The inhibitory effect of desipramine on proliferation was not extracellular Ca2+-dependent. Apoptosis appears to contribute to desipramine-induced cell death. Together, these findings suggest that desipramine increases baseline [Ca2+]i in renal tubular cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and can cause apoptosis.

NPC-14686 (Fmoc-l-homophenylalanine)-induced CaCa2+ increases and death in human prostate cancer cells.

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca2+ levels ([Ca2+]i) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 100 microM. NPC-14686-induced Ca2+ influx was confirmed by Mn2+ quench of fura-2 fluorescence. The Ca2+ signal was also reduced by removing extracellular Ca2+. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ nearly abolished 200 microM NPC-14686-induced Ca2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca2+ release. The Ca2+ release induced by 200 microM NPC-14686 was not affected by inhibiting phospholipase C with 2 microM U73122. Overnight treatment with 1-500 microM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca2+]i by evoking extracellular Ca2+ influx and releasing intracellular Ca2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.

Effect of capsazepine on cytosolic Ca(2+) levels and proliferation of human prostate cancer cells.

Capsazepine has been widely used as a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on most cell types is unknown. In human PC3 prostate cancer cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was investigated by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 75 microM. Capsazepine-induced [Ca(2+)](i) rise was reduced by 60% by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was contributed by extracellular Ca(2+) influx and intracellular Ca(2+). Consistently, the capsazepine (200 microM)-induced [Ca(2+)](i) rise was decreased by La(3+) by half. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the effect of capsazepine on [Ca(2+)](i) was inhibited by 80%. Conversely, pretreatment with capsazepine partly reduced thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. These findings suggest that in human PC3 prostate cancer cells, capsazepine increases [Ca(2+)](i) by evoking Ca(2+) influx and releasing Ca(2+) from the endoplasmic reticulum via a phospholiase C-independent manner. Overnight incubation with capsazepine (200 microM) killed 37% of cells, which could not be prevented by chelating intracellular Ca(2+) with BAPTA.

Dual effect of flurbiprofen on cell proliferation and agonist-induced Ca(2+) movement in human osteosarcoma cells.

In human MG63 osteosarcoma cells, the effect of flurbiprofen on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and proliferation was explored. The proliferation was enhanced by 20-120 microM flurbiprofen, and was decreased by 140-200 microM flurbiprofen. The effect of flurbiprofen on the increases in cytosolic free Ca(2+) levels ([Ca(2+)](i)) induced by ATP, bradykinin, histamine and thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) ATPase), was examined. In cell preincubated with 20 or 80 microM flurbiprofen, the [Ca(2+)](i) increases induced by all agonists were attenuated. In the presence of 20 microM flurbiprofen, the decreased [Ca(2+)](i) responses with the agonists were attributed to a defective Ca(2+) influx because this decrease was unobserved in agonists-induced [Ca(2+)](i) increases in the absence of extracellular Ca(2+). In the presence of 80 microM flurbiprofen, both the Ca(2+) influx component and the Ca(2+) releasing (from organelles) component were defective. These results suggest that flurbiprofen could alter proliferation and inhibit [Ca(2+)](i) increases.