Chun-Jen Huang

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.

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca2+ concentration ([Ca2+]i) and viability is largely unknown. This study examined whether melittin alters Ca2+ levels and causes Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 75% by removing extracellular Ca2+. The melittin-induced Ca2+ influx was also implicated by melittin-caused Mn2+ influx. After pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), melittin-induced Ca2+ release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca2+ release. At concentrations of 0.5–20 μM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 μM melittin was nearly completely reversed by prechelating cytosolic Ca2+ with BAPTA. Melittin at 0.5–2 μM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca2+]i rise by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, melittin can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner.

Safrole-Induced Ca2+ Mobilization and Cytotoxicity in Human Pc3 Prostate Cancer Cells

The effect of the carcinogen safrole on intracellular Ca2+ mobilization and on viability of human PC3 prostate cancer cells was examined. Cytosolic free Ca2+ levels ([Ca2+]i) were measured by using fura-2 as a probe. Safrole at concentrations above 10 μM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 350 μ M. The Ca2+ signal was reduced by more than half after removing extracellular Ca2+ but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem, or verapamil. In Ca2+-free medium, after treatment with 650 μM safrole, 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release Ca2+. Neither inhibition of phospholipase C with U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 0.65–65 μM safrole did not affect cell viability, but incubation with 325–625 μM safrole decreased viability. Collectively, the data suggest that in PC3 cells, safrole induced a [Ca2+]i increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion, and by inducing Ca2+ influx. Safrole can decrease cell viability in a concentration-dependent manner.

Fendiline-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in human oral cancer cells.

The effect of fendiline on cytosolic free Ca2+ concentrations ([Ca2+]i) and proliferation has not been explored in human oral cancer cells. This study examined whether fendiline altered Ca2+ levels and caused cell death in OC2 human oral cancer cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Fendiline at concentrations above 10 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The fendiline-induced Ca2+ influx was sensitive to blockade of L-type Ca2+ channel blockers. In Ca2+-free medium, after pretreatment with 50 μM fendiline, 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor)-induced [Ca2+]i rises were inhibited; and conversely, thapsigargin pretreatment nearly abolished fendiline-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 μM U73122 did not change fendiline-induced [Ca2+]i rises. At concentrations between 5 and 25 μM, fendiline killed cells in a concentration-dependent manner. The cytotoxic effect of 15 μM fendiline was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in OC2 cells, fendiline induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from L-type Ca2+ channels. Furthermore, fendiline-caused cytotoxicity was not via a preceding [Ca2+]i rise.

Effect of thimerosal on Ca2+ movement and viability in human oral cancer cells

The effect of thimerosal on cytosolic free Ca2+ concentrations ([Ca2+]i ) in human oral cancer cells (OC2) is unclear. This study explored whether thimerosal changed basal [Ca2+]i levels in suspended OC2 cells using fura-2. Thimerosal at concentrations between 1and 50 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca 2+. Thimerosal-induced Ca2+ influx was not blocked by L-type Ca2+ entry inhibitors and protein kinase C modulators (phorbol 12-myristate 13-acetate [PMA] and GF109203X). In Ca2+-free medium, 50 μM thimerosal failed to induce a [Ca2+]i rise after pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with U73122 did not change thimerosal-induced [Ca2+]i rises. At concentrations between 5 and 10 μM, thimerosal killed cells in a concentration-dependent manner. The cytotoxic effect of 8 μM thimerosal was potentiated by prechelating cytosolic Ca2+ with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate/acetomethyl (BAPTA/ AM). Flow cytometry data suggested that 1—7 μM thimerosal-induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, thimerosal-induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx through non—L-type Ca2+ channels. Thimerosal killed cells in a concentration-dependent manner through apoptosis.

Effect of celecoxib on Ca2+ fluxes and proliferation in MDCK renal tubular cells.

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca2 + concentration ([Ca2 +]i) and proliferation was examined by using the Ca2 +-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca2 +]i in a concentration-dependent manner. Celecoxib-induced [Ca2 +]i increase was partly reduced by removal of extracellular Ca2 +. Celecoxib-induced Ca2 + influx was independently suggested by Mn2 + influx-induced fura-2 fluorescence quench. In Ca2 +-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2 +-ATPase, caused a monophasic [Ca2 +]i increase, after which celecoxib only induced a tiny [Ca2 +]iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca2 +]i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca2 +]i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca2 +]i increase in renal tubular cells by stimulating both extracellular Ca2 + influx and intracellular Ca2 + release and is highly toxic to renal tubular cells in vitro.

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability in OC2 human oral cancer cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The tamoxifen-induced Ca2+ influx was sensitive to blockade of L-type Ca2+ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca2+]i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca2+]i rises, in a nongenomic manner, by causing Ca2+ release from the endoplasmic reticulum, and Ca2+ influx from L-type Ca2+ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca2+]i rise.

Effect of calmidazolium on Ca2+ movement and proliferation in human osteosarcoma cells

1. In human MG63 osteosarcoma cells, the effect of calmidazolium on [Ca2+]i and proliferation was explored using fura‐2 and ELISA, respectively.

Effect of riluzole on Ca2+ movement and cytotoxicity in Madin-Darby canine kidney cells.

Riluzole is a drug used in the treatment of amyotrophic lateral sclerosis; however, its in vitro action is unclear. In this study, the effect of riluzole on intracellular Ca2 - concentration ([Ca2 -]i) in Madin-Darby canine kidney (MDCK) cells was investigated using the Ca2 --sensitive fluorescent dye, fura-2. Riluzole (100 -500 mM) caused a rapid and sustained increase of [Ca2 -]i in a concentration-dependent manner (EC50 = 150 mM). Some 40 and 50% of this [Ca2 -]i increase was prevented by the removal of extracellular Ca2 - and the addition of La3 -, respectively, but was unchanged by dihydropyridines, verapamil and diltiazem. In Ca2 --free medium, thapsigargin -an inhibitor of the endoplasmic reticulum (ER) Ca2 --ATPase -caused a monophasic [Ca2-]i increase, after which the increasing effect of riluzole on [Ca2 -]iwas attenuated by 70%; in addition, pre-treatment with riluzole abolished thapsigargin-induced [Ca2 -]i increases. U73122, an inhibitor of phospholipase C (PLC), abolished ATP (but not riluzole)-induced [Ca2 -]i increases. At concentrations of 250 and 500 mM, riluzole killed 40 and 95% cells, respectively. The cytotoxic effect of riluzole (250 mM) was unaltered by pre-chelating cytosolic Ca2 - with BAPTA. Collectively, in MDCK cells, riluzole rapidly increased [Ca2 -]i by stimulating extra-cellular Ca2 - influx via an La3 --sensitive pathway and intracellular Ca2 - release from the ER via, as yet, unidentified mechanisms. Furthermore, riluzole caused Ca2 --unrelated cytotoxicity in a concentration-depen-dent manner.

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

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