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Primary tubulovillous adenomas are scarcely encountered in the urinary tract, and most of these tumors arise from the intestinal segments of urinary reconstruction. We report the extremely rare case of tubulovillous adenomas primarily occurring in the urinary bladder in this article. A 50-year-old female patient initially presented with painless gross hematuria for several times within 1 month. Image studies, including abdominal ultrasonography and computed tomography, demonstrated one urinary bladder tumor with suspicious perivesical invasion. The diagnosis was made by a transurethral resection and histopathology, and no involvement of muscularis propria was confirmed. Although the pathogenesis and clinical behavior are not well understood, tubulovillous adenomas may have a tendency to undergo malignant transformation into the form of invasive adenocarcinomas. Complete resection and rigorous follow-up are strongly recommended.

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.

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

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.

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.

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.

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 the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2+]i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. This Ca2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2+]i rise, suggesting that thapsigargin released Ca2+ from the endoplasmic reticulum. Inhibition of p...

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.

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.

The current study explored whether capsazepine changed basal cytosolic free Ca2+ concentrations ([Ca2+]i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura-2 as a Ca2+-selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was partially reduced by 40% by removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura-2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine-induced Ca2+ influx was unchanged by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 100 µM capsazepine-induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin-induced Ca2+ release. Inhibition of phospholipase C with U73122 did not change capsazepine-induced [Ca2+]i rises. Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley-Liss, Inc.

The effect of nortriptyline, a tricyclic antidepressant, on Ca2+ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca2+]i levels in suspended PC3 cells using fura-2 as a Ca2+-sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was partially reduced by removing extracellular Ca2+, indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Nortriptyline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by activation of protein kinase C, but not by inhibition of L-type Ca2+ channels. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin nearly abolished nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that nortriptyline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline-induced [Ca2+]i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca2+-independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca2+]i rises by causing the phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via the protein kinase C-sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration-dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley-Liss, Inc.

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.

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 concentrationsor =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.

The effect of MK-886 (3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid), a compound widely used to inhibit leukotriene synthesis, on cytosolic free Ca2+ concentrations ([Ca2+]i) in osteosarcoma cells has not been explored. This study examined whether MK-886 altered [Ca2+]i levels in suspended MG63 human osteosarcoma cells using fura-2. MK-886 at 0.1 μM and above increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. MK-886 induced Mn2+ quenching of fura-2 fluorescence, implicating Ca2+ entry. MK-886-induced Ca2+ influx was inhibited by store-operated Ca2+ entry inhibitors, nifedipine, econazole, and SKF96365; and by the protein kinase C modulators, phorbol 12-myristate 13-acetate (PMA) and GF109203X. In Ca2+-free medium, after pretreatment with 5 μM MK-886, 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were abolished; conversely, thapsigargin pretreatment nearly abolished MK-886-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change MK-886-induced [Ca2+]i rises. Collectively, in MG63 osteosarcoma cells, MK-886 induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via protein kinase C-regulated store-operated Ca2+ entry. Drug Dev Res 69: 49–57, 2008. © 2008 Wiley-Liss, Inc.

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. Mann–Whitney 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.

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 NH(2)-terminal protein kinase (JNK) and p38. 4. It 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 micromol/L, 20 micromol/L or 50 micromol/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.

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.

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca(2+)](i) and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations > or = 1 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). The econazole-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and protein kinase C modulators. In Ca(2+)-free medium, after pretreatment with 20 microM econazole, [Ca(2+)](i) rises induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with 2 microM U73122 did not change econazole-induced [Ca(2+)](i) rises. At concentrations between 10 and 80 microM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 microM econazole was not reversed by prechelating cytosolic Ca(2+) with BAPTA. This shows that in SIRC cells econazole induces [Ca(2+)](i) rises by causing Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca(2+)](i) rise.

The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation, and Ca2+ regulation in MG63 human osteosarcoma cells. AC (25–50 µg/ml) did not affect cell viability, but at 100–200 µg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25–200 µg/ml did not alter basal [Ca2+]i, but at 25 µg/ml decreased [Ca2+]i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25 µg/ml) pretreatment failed to alter bradykinin- and thapsigargin-induced effects on viability, but potentiated ATP- and histamine-induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho-JNK and phospho-p38 mitogen-activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho-ERK MAPK that was inhibited by 200 µg/ml AC. The ERK-specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71–78, 2007. © 2007 Wiley-Liss, Inc.

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 prostatec- tomy specimens were stained using anti-p21 WAF1 and anti-p27 KIP1 antibodies. Biochemical relapse was defined as 2 con- secutive 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: p21 WAF1 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. (J Chin Med Assoc 2007;70(1):11-15)

The effect of the carcinogen safrole on intracellular Ca2+ movement has not been explored in osteoblast-like cells. This study examined whether safrole could alter Ca2+ handling and viability in MG63 human osteosarcoma 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 130 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 450 microM. The Ca2+ signal was reduced by 30% by removing extracellular Ca2+. Addition of Ca2+ after safrole had depleted intracellular Ca2+ induced Ca2+ influx, suggesting that safrole caused Ca2+ entry. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca2+; and pretreatment with thapsigargin inhibited most of the safrole-induced [Ca2+]i increases. Inhibition of phospholipase C with U73122 did not affect safrole-induced Ca2+ release; whereas activation of protein kinase C with phorbol ester enhanced safrole-induced [Ca2+]i increase. Trypan exclusion assays revealed that incubation with 65 microM safrole for 30 min did not kill cells, but incubation with 650 microM safrole for 10-30 min nearly killed all cells. Flow cytometry demonstrated that safrole evoked apoptosis in a concentration-dependent manner. Safrole-induced cytotoxicity was not reversed by chelation of Ca2+ with BAPTA. Collectively, the data suggest that in MG63 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release mainly from the endoplasmic reticulum in a phospholipase C-independent manner. The safrole response involved Ca2+ influx and is modulated by protein kinase C. Furthermore, safrole can cause apoptosis in a Ca2+-independent manner.

To investigate the effect of vitamin D receptor (VDR) FokI polymorphism on susceptibility to prostate cancer and the outcome of the disease in a Taiwanese population, we genotyped a total of 416 prostate cancer patients, 502 age-matched male controls and 189 non age-matched symptomatic benign prostatic hyperplasia. Although we did not find a significant association between VDR FokI genotypes and overall prostate cancer risk, we found that in men aged less than or equal to the median age of 73 years with VDR FokI F allele specifically had an increased risk of prostate cancer with a marginal significant trend (OR, 2.08; 95% CI, 1.00–4.34, p for trend = 0.056). The FF genotype was also highly associated with more aggressive prostate cancer (Gleason score 8–10) (OR, 2.47; 95% CI, 1.20–5.08) than did the Ff and ff genotypes. After adjusting other covariates, we found that in patients who had localized prostate cancer for which a radical prostatectomy was performed (n = 131), the VDR FokI FF genotype was associated with worse prostate-specific antigen (PSA) recurrence-free survival (hazard ratio = 3.25, 95% CI = 1.32–8.00, p = 0.010). Our findings suggest that the VDR FF genotype may increase the risk of early-onset prostate cancer and is associated with more aggressive disease. Furthermore, the VDR polymorphism could be used as a prognostic marker for localized prostate cancer after radical prostatectomy. © 2006 Wiley-Liss, Inc.

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 Ca 2 + concentrations ([Ca 2 + ] 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. 2. At a concentration of 0.1 μmol/L, econazole started to increase [Ca 2 + ] i in a concentration-dependent manner. The econazole-induced increase in [Ca 2 + ] i was reduced by 48% by removal of extracellular Ca 2 + , suggesting that the econazole-induced increase in [Ca 2 + ] i was composed of extracellular Ca 2 + influx and intracellular Ca 2 + . 3. This econazole-induced Ca 2 + influx was via an L-type Ca 2 + channel-like pathway. In Ca 2 + -free medium, 1 μmol/L thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2 + -ATPase, caused a monophasic increase in [Ca 2 + ] i , after which the effect of econazole to increase [Ca 2 + ] i was substantially inhibited. Conversely, pretreatment with 5 μmol/L econazole to deplete intracellular Ca 2 + stores totally prevented thapsigargin from releasing more Ca 2 + . 4. The phospholipase C (PLC) inhibitor U73122 (2 μmol/L) abolished the increase in [Ca 2 + ] i induced by 10 μmol/L ATP (a Ca 2 + mobilizer that needs inositol 1,4,5-trisphosphate). 5. Overnight incubation with 1-30 μmol/L econazole inhibited proliferation of PC3 cells in a concentration-dependent manner. 6. These findings suggest that, in PC3 cells, econazole increases [Ca 2 + ] i by stimulating Ca 2 + influx into cells and Ca 2 + release from the endoplasmic reticulum via a PLC-independent mechanism. Econazole is cytotoxic at submicromolar concentrations.

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

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 325 microM 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 650 microM safrole, 1 microM 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 2 microM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 1 microM safrole did not alter cell proliferation, but incubation with 10-1000 microM safrole increased cell proliferation by 60+/-10%. This increase was not reversed by pre-chelating Ca2+ with 10 microM 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.

The effect of miconazole, an anti-fungal drug, on cytoplasmic free Ca 2+ concentrations ([Ca 2+ ] i ) in human osteosarcoma cells (MG63) was explored by using the Ca 2+ -sensitive dye fura-2. Miconazole acted in a concentration-dependent manner with an EC 50 of 75 μM. The Ca 2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca 2+ reduced 50% of the signal. In Ca 2+ -free medium, the [Ca 2+ ] i rise induced by 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) was completely inhibited by pretreatment with 20 μM miconazole. Pretreatment with thapsigargin partly inhibited miconazole-induced Ca 2+ release. The miconazole-induced Ca 2+ release was not changed by inhibition of phospholipase C with 2 μM U73122. By using tetrazolium as a fluorescent probe, it was shown that 10–100 μM miconazole decreased cell proliferation rate in a concentration-dependent manner. Collectively, this study shows that miconazole induces [Ca 2+ ] i rises in human osteosarcoma cells via releasing Ca 2+ mainly from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca 2+ influx. Furthermore, miconazole may be cytotoxic to the cells at higher concentrations.

The differences in the intracellular Ca2+ responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca2+ signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per µl in inactive and normal groups did not differ, whereas the number in active SLE patients was smaller than the other two groups by 60%. The intracellular free Ca2+ levels ([Ca2+]i) in response to stimulation of four endogenous Ca2+ mobilizing hormones, 100 µM arachidonic acid (AA), 10 µM ADP, 10 nM platelet activation factor (PAF) and 1 µM thrombin, were investigated using the Ca2+-sensitive fluorescent dye, fura-2. The AA-induced [Ca2+]i rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca2+]i rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca2+]i rises in active SLE groups appears to be caused by defective Ca2+ influx and Ca2+ releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca2+, whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca2+, and the AA-induced Ca2+ release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca2+]i rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca2+ signaling in platelets from active SLE patients are altered in response to particular stimulators. In these regards, platelets from inactive SLE patients appear to be similar to those from normal humans.

The tumor suppressor gene p53 and its downstream effector p21 are thought to play major roles in the development of human malignancy. Polymorphic variants of p53 at codon 72, and p21 at codon 31, have been found to be associated with cancer susceptibility, but few studies have investigated their effect on prostate cancer risk. In this case-control study, we investigated the association of p53 codon 72 and p21 codon 31 polymorphisms with prostate cancer risk in a Taiwanese population. In total, 200 patients with prostate cancer, 247 age-matched male controls, and 181 non–age-matched symptomatic benign prostatic hyperplasia (BPH; American Urological Association symptom score ≥ 8 and prostate volume > 20 gm) recruited from two medical centers in southern Taiwan were genotyped. Overall, we found no significant association between p53 polymorphism and risk of prostate cancer. However, for p21 polymorphism, the frequencies of p21 Ser/Ser, Ser/Arg and Arg/Arg were 52 (26.0%), 85 (42.5%), 63 (31.5%) in case patients, 48 (26.5%), 82 (45.3%), 51 (28.2%) in BPH patients, and 76 (30.8%), 119 (48.2%), 52 (21.1%) in controls, respectively. Among the prostate cancer cases and controls, subjects with Arg/Arg genotype were found to have a 1.78-fold increased risk [95% confidence interval (CI), 1.06-3.01] of developing prostate cancer compared with those having the Ser/Ser genotype, after adjusting for other potential covariates. This significant association was slightly stronger [odds ratio (OR), 2.13; 95% CI, 1.16-3.92] in younger men (≤ 72 years; n = 99 and 126 for cases and controls, respectively) and correlated with localized disease stage (OR, 1.96; 95 % CI, 1.15-3.35) and moderately differentiated prostate cancer (OR, 2.04; 95% CI, 1.17-3.53). In addition, the Arg/Arg genotype was associated with BPH risk in those with large prostate volumes (> 50 mL) compared with those having the Ser/Ser genotype [OR, 2.29; 95% CI, 1.07-4.98]. Our findings suggest that the p21 codon 31 polymorphism may be associated with the development of prostate enlargement and cancer.

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca 2+ concentrations ([Ca 2+ ] i ) was explored by using the Ca 2+ -sensitive probe fura-2. Maprotiline at concentrations greater than 100 μM caused a rapid rise in [Ca 2+ ] i in a concentration-dependent manner (EC 50 = 200 μM). Maprotiline-induced [Ca 2+ ] i rise was reduced by 50% by removal of extracellular Ca 2+ . Maprotiline-induced [Ca 2+ ] i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. 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 increasing effect of maprotiline on [Ca 2+ ] i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca 2+ ] i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca 2+ ] i by stimulating extracellular Ca 2+ influx and also by causing intracellular Ca 2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.

Introduction: To determine the role of prostate-specific antigen (PSA) level and age-specific PSA reference ranges for detecting prostate cancer in Chinese. Material and Methods: We retrospectively reviewed the medical records of all 50- to 79-year-old men who had their first-time prostatic biopsy done at our institute between January 1998 and December 2000. We evaluated the effect on biopsy rate and cancer detection if the PSA cutoff was shifted from 4.0 ng/ml to the age-specific reference ranges. Also, we tried to develop age-adjusted PSA cutoffs for Chinese. Results: Among 1,236 men enrolled, 252 (20.4%) were diagnosed as prostate cancers. The positive predictive value was 22.2% for PSA >4.0 ng/ml. Using the traditional age-specific PSA reference ranges 134 (10.8%) biopsies could be avoided but five (1.98%) cancers would be missed. We proposed age-specific PSA reference ranges for Chinese as: 0–4.0 ng/ml for men in their 50s and 0–6.0 ng/ml for men between 60 and 79 years. Had we used these alternative cutoffs, 150 (12.1%) biopsies could be saved, of them 3 were cancers. Conclusions: The age-specific PSA reference ranges may help to avoid 12% of biopsies but 1.2% of prostate cancers would have gone undetected. Among population with low incidence of prostate cancer as Chinese, minimizing unnecessary biopsies might be more important issue than maximizing cancer detection rate.

The effect of thimerosal, a reactive oxidant, on cytoplasmic free Ca 2π concentrations ([Ca 2π ] i ) in Madin Darbycanine kidney (MDCK) cells was explored by using the Ca 2π -sensitive dye fura-2. Thimerosal acted in a concentration-dependent manner with an EC 50 of 0.5 mM. The Ca 2π signal comprised a gradual rise and a sustained elevation. Removalof extracellular Ca 2π reduced 80% of the signal. In Ca 2π -free medium, the [Ca 2π ] i rise induced by 1 mM thapsigargin (anendoplasmic reticulum Ca 2π pump inhibitor) was completely inhibited by pretreatment with 5 mM thimerosal. The thi-merosal (5 mM)-induced Ca 2π release was not changed by inhibition of phospholipase C with 2 mM U73122. Collectively,this study shows that thimerosal induced [Ca 2π ] i rises in renal tubular cells via releasing store Ca 2π from the endoplasmicreticulum Ca 2π stores in a manner independent of phospholipase C activity. Thimerosal, a sulfhydryl reagent, is used as an antisepticand preservative. The ability of thimerosal to act on sulfhy-dryl group is related to the presence of mercury. Thimersalcauses a release of Ca

The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca 2+ concentration ([Ca 2+ ] i ) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 μM) caused a rapid and transient plateau [Ca 2+ ] i rise in a concentration-dependent manner (EC 50 =0.5 μM). The L-NASPA-induced [Ca 2+ ] i rise was partly reduced by removal of extracellular Ca 2+ but was not altered by L-type voltage-gated Ca 2+ channel blockers. In Ca 2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, induced a [Ca 2+ ] i rise, after which the increasing effect of L-NASPA on [Ca 2+ ] i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca 2+ ] i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca 2+ ] i rise. Overnight incubation with 1 μM L-NASPA did not affect cell proliferation, but 10-20 μM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca 2+ ] i in MG63 cells by evoking both extracellular Ca 2+ influx and intracellular Ca 2+ release, and is cytotoxic at higher concentrations.

We developed a convenient, self-administered 8-item stone recurrence predictive score (SRPS) to predict the recurrence of calcium oxalate stones.An 8-item SRPS to predict stone recurrence was developed based on general patient data, including age, sex, urine volume, smoking, wine drinking, family history, stone number and history of gouty arthritis. Mean age of the 204 studied patients with calcium oxalate stones +/- SD was 59.4 +/- 14.5 years (range 24 to 83). The male-to-female ratio was 3:1. Of the patients 115 were recurrent stone formers and 89 were single stone formers. We compared all available general data in the recurrence and control groups.Family history, stone number, gouty arthritis and SRPS were independent risk factors for stone recurrence. Mean SRPS in recurrent and single stone formers was 7.6 +/- 3.1 and 5.1 +/- 2.0, respectively (p = 0). An increase in SRPS had a significant positive correlation with stone recurrence (r2 = 0.859, p0.0001). At an SRPS cutoff of 7 or greater we achieved 61.7% sensitivity and 75.3% specificity to predict stone recurrence. At an SRPS of 11 or greater we found that 100% of patients had recurrent stones.With the introduction of the 8-item SRPS we provide a simple, convenient and reliable tool to predict calcium oxalate stone recurrence. Due to the characteristics of the high incidence of stone recurrence thorough metabolic evaluation may be justified in patients with an SRPS of 7 or greater and preventive measures are highly recommended in those with an SRPS of 11 or greater.

We evaluated the prognostic significance of p53, bcl-2 and E-cadherin immunoreactivity for organ confined prostate cancer after radical prostatectomy.The medical records on 70 pT2 prostatic adenocarcinomas were analyzed retrospectively. Radical prostatectomy specimens were stained using antip53 (DO7), antibcl-2 (124) (Dako, Glostrup, Denmark) and antiE-cadherin (HECD-1) (RD Systems, Abingdon, United Kingdom) antibodies. Biochemical relapse was defined as 2 consecutive elevations in serum prostatic specific antigen (PSA) higher than 0.2 ng/ml. The prognostic significance of Gleason grade, PSA, and p53, bcl-2 and E-cadherin expression was assessed.While p53 immunoreactivity was identified in 16 patients (22.9%), only 3 tumors (4.3%) expressed bcl-2. Aberrant E-cadherin expression was noted in 39 tumors (55.7%). At a median followup of 36.5 months 21 patients (30%) experienced biochemical relapse. There was a significantly higher biochemical failure rate in patients with abnormal bcl-2 and E-cadherin expression (log rank test p = 0.024 and 0.003, respectively). On multivariate analysis bcl-2 and E-cadherin contributed independently to the prediction of PSA relapse (p = 0.017 and 0.005, respectively).We noted that bcl-2 and aberrant E-cadherin expression are independent factors predicting biochemical relapse in stage pT2 prostatic cancers.

In human osteosarcoma MG63 cells, the effect of desipramine, an antidepressant, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Desipramine (>10 µmol/l) caused a rapid and sustained rise of [Ca2+]i in a concentration-dependent manner (EC50 = 200 µmol/l). Desipramine-induced [Ca2+]i rise was prevented by 80% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of desipramine on [Ca2+]i was abolished; also, pretreatment with desipramine partly reduced thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not affect desipramine-induced [Ca2+]i rise. Overnight incubation with 10 µmol/l desipramine did not alter cell proliferation, but killed 32 and 89% of cells at concentrations of 100 and 200 µmol/l, respectively. These findings suggest that desipramine rapidly increases [Ca2+]i in osteoblasts by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at high concentrations.

The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca(2+)]i in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to interact with oestrogens leading to modulation of bone metabolism, the present study was aimed at exploring whether tamoxifen could alter Ca(2+) signaling in human osteoblast-like MG63 cells. Cytosolic free Ca(2+) levels were recorded by using the Ca(2+)-sensitive dye fura-2. Tamoxifen induced a sustained [Ca(2+)]i increase at concentrations above 1 microM with an EC(50) of 8 microM. Removal of extracellular Ca(2+) reduced the response by 40%, suggesting that tamoxifen induced both Ca(2+) influx and store Ca(2+) release. Tamoxifen-induced Ca(2+) influx was confirmed as tamoxifen caused Mn(2+) influx-induced quench of fura-2 fluorescence. In Ca(2+)-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca(2+)]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), and by 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m-chlorophenylhydrazone only reduced 64% of tamoxifen-induced [Ca(2+)]i increases. Addition of 2 microM U73122 to inhibit phospholipase C activity abolished the [Ca(2+)]i increase induced by 1 microM histamine, a phospholipase C-dependent Ca(2+) mobilizer, without affecting 10 microM tamoxifen-induced Ca(2+) release. The [Ca(2+)]i increase induced by 10 microM tamoxifen was not altered by 10 microM of nifedipine, verapamil and diltiazem. Together, the data show that tamoxifen induced a lasting increase in [Ca(2+)]i in human osteoblast-like cells by causing Ca(2+) influx and releasing Ca(2+) from multiple stores in a phospholipase C-independent manner.

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca(2+) indicator. Clomiphene at concentrations between 10-50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The [Ca(2+)](i) signal was biphasic with an initial rise and a slow decay. Ca(2+) removal inhibited the Ca(2+) signal by 41%. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with clomiphene in Ca(2+)-free medium, confirming that clomiphene induced Ca(2+) entry. In Ca(2+)-free medium, pretreatment with 50 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store Ca(2+) release. Conversely, pretreatment with 50 microM clomiphene in Ca(2+)-free medium abolished the [Ca(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced Ca(2+)release was unaltered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 microM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca(2+)](i) increases in PC3 cells by releasing store Ca(2+) from multiple stores in an phospholipase C-independent manner, and by activating Ca(2+) influx; and clomiphene was of mild cytotoxicity.

The effects of triethyltin on Ca 2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca 2+ ] i at concentrations larger than 3 μM with an EC 50 of 30 μM. Within 5 min, the [Ca 2+ ] i signal was composed of a gradual rise and a sustained phase. The [Ca 2+ ] i signal was reduced by half by removing extracellular Ca 2+ . The triethyltin-induced [Ca 2+ ] i increases were inhibited by 40% by 10 μM nifedipine, nimodipine and nicardipine, but were not affected by 10 μM of verapamil or diltiazem. In Ca 2+ -free medium, pretreatment with thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, reduced 200 μM triethyltin-induced Ca 2+ increases by 50%. Pretreatment with U73122 (2 μM) to inhibit phospholipase C did not alter 200 μM triethyltin-induced [Ca 2+ ] i increases. Incubation with triethyltin at a concentration that did not increase [Ca 2+ ] i (1 μM) in Ca 2+ -containing medium for 3 min potentiated ATP (10 μM)- or bradykinin (1 μM)-induced [Ca 2+ ] i increases by 41±3% and 51±2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca 2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca 2+ ] i ; and at lower concentrations it potentiated agonists-induced [Ca 2+ ] i increases.

The effect of 17β-estradiol on intracellular Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney cells was investigated by using the fluorescent dye fura-2. 17β-Estradiol (5–100 µmol/l) induced instantaneous increases in [Ca2+]i in a concentration-dependent manner. Ca2+ removal inhibited 45 ± 15% of the Ca2+ signal. In Ca2+-free medium, pretreatment with 50 µmol/l 17β-estradiol abolished the [Ca2+]i increases induced by 2 µmol/l carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), 1 µmol/l thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) and 50 µmol/l brefeldin A (an antibiotic which disperses the Golgi complex), but pretreatment with brefeldin A, CCCP and thapsigargin only partly inhibited the 17β-estradiol-induced [Ca2+]i signal. Adding 3 mmol/lCa2+ increased [Ca2+]i in cells pretreated with 5–100 µmol/l 17β-estradiol in Ca2+-free medium. Pretreatment with 1 µmol/l U73122 to abolish the formation of inositol-1,4,5-trisphosphate inhibited 50% of the Ca2+ release induced by 50 µmol/l 17β-estradiol. 17β-Estradiol (20 µmol/l) also increased [Ca2+]i in human bladder cancer cells and prostate cancer cells. Collectively, this study shows that 17β-estradiol evoked a significant internal Ca2+ release and external Ca2+ entry possibly in a nongenomic manner.

In human osteosarcoma MG63 cells, the effect of the neuroprotective drug riluzole on the intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2. Riluzole (50–500 µmol/l) caused a rapid and sustained plateau increase in [Ca2+]i in a concentration-dependent manner (EC50 = 150 µmol/l). The riluzole-induced rise in [Ca2+]i was prevented by 58 and 20% by extracellular Ca2+ removal and nifedipine, respectively, but was not changed by La3+ and verapamil. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase, caused a monophasic increase in [Ca2+]i, after which the increasing effect of riluzole on [Ca2+]i was attenuated by 84%; also, pretreatment with riluzole abolished the thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, abrogated the ATP (but not riluzole)-induced rise in [Ca2+]i. A low concentration (6 µmol/l) of riluzole selectively potentiated the bradykinin (but not ATP and histamine)-induced increase in [Ca2+]i. These results suggest that riluzole rapidly increases [Ca2+]i by stimulating both the extracellular Ca2+ influx via a nifedipine-sensitive pathway and intracellular Ca2+ release from the ER via an as yet unidentified mechanism(s).

Introduction: A nationwide survey was conducted to investigate the prevalence of upper urinary calculi in Taiwan. Materials and Methods: A postal questionnaire was mailed to 27,758 people, 0.2% of the adults in Taiwan. Results: Of the 4,588 valid respondents, 440 had at least one episode of upper urinary calculus disease. The overall prevalence was 9.6% (14.5% in males and 4.3% in females). Men were more prone to nephrolithiasis than women (age-adjusted prevalence of 12.2% in men and 3.1% in women, p = 0). The ‘stone belt’ was localized in the Midwest region of Taiwan. A gender- and age-adjusted multivariate analysis revealed that alcohol consumption and family history of kidney stone were significant risk factors for stone occurrence. Compared with general population, the odds ratios for stone disease in inhabitants whose father, mother and both parents with stone history were 3.44 [95% confidence interval (CI), 2.33–5.07], 4.79 (95% CI, 2.85–8.07) and 10.40 (95% CI, 3.75–28.84), respectively. The subtropical temperature and gradually higher socioeconomic standards of living may contribute to the high prevalence. Inhabitants in the Midwest region have higher risk to develop stones. Conclusions: Further studies are needed to investigate the exact cause of these regional variations of stone prevalence. Nevertheless, the present study provides an additional piece of information on worldwide epidemiology of urolithiasis.

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, SKF96365, 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.

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.