Jeng-Yu Tsai

Epidemiological studies on the prevalence of upper urinary calculi in Taiwan.

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.

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.

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.

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

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

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced, partly, by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quenching of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca2+ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C–dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca2+ channels.

Effect of MK-886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK-886 on cytosolic-free Ca(2+) concentrations ([Ca(2+)](i)) and viability in human PC3 prostate cancer cells. [Ca(2+)](i) in suspended cells was measured by using fura-2. MK-886 at concentrations of 1 microM and above increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 20 microM. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). MK-886 evoked Mn(2+) quenching of fura-2 fluorescence, implicating Ca(2+) entry. MK-886-induced Ca(2+) influx was inhibited by store-operated Ca(2+) entry inhibitors nifedipine, econazole and SKF96365. In Ca(2+)-free medium, after pre-treatment with 10 microM MK-886, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor)-induced [Ca(2+)](i) rises were abolished; and conversely, thapsigargin pre-treatment abolished MK-886-induced [Ca(2+)](i) rises. Inhibition of phospholipase C with U73122 did not alter MK-886-induced [Ca(2+)](i) rises. MK-886 at concentrations of 1-100 microM concentration-dependently decreased cell viability with an IC(50) value of 60 microM. The cytotoxic effect of MK-886 was not inhibited by pre-chelating cytosolic Ca(2+) with BAPTA/AM. Together, in PC3 cells, MK-886 induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum; and Ca(2+) influx via store-operated Ca(2+) channels. Independently, MK-886 was cytotoxic to cells in a Ca(2+)-independent manner.

Role of p21WAF1 and p27KIP1 in Predicting Biochemical Recurrence for Organ-confined Prostate Adenocarcinoma

Background: Both p21WAF1 and p27KIP1 have been reported as prognostic markers predicting biochemical failure for prostate cancers. We examined the expression and prognostic significance of p21WAF1 and p27KIP1 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‐p21WAF1 and anti‐p27KIP1 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 p21WAF1 and p27KIP1 expression was assessed. Results: p21WAF1 immunoreactivity was found in 19 patients (35.8%). Twenty‐nine tumors (54.7%) had decreased p27KIP1 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: p21WAF1 and p27KIP1 expression have no role in predicting biochemical relapse for stage pT2 prostate cancers.

Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca(superscript 2+) regulation of OC2 human oral cancer cells. AC at a concentration of 25μM induced an increase in cell viability, but AC at concentrations ≥50μg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200μg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25μg/ml) did not alter basal [Ca(superscript 2+)](subscript i), but decreased the [Ca2(superscript +)](subscript i) increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25μg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinininduced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargininduced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca(superscript 2+)](subscript i), induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca2+ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca2+ concentrations ([Ca2+]i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Celecoxib-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A2 inhibitor, aristolochic acid. In Ca2+-free medium, 30 μM of celecoxib failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca2+ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca2+ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca2+]i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca2+ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca2+]i rises by causing phospholipase C–independent Ca2+ release from the ER and Ca2+ influx via non-L-type, phospholipase A2-regulated Ca2+ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.

Mechanisms of AM404-induced [Ca2+]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.