He-Hsiung Cheng

Effect of Thymol on Ca2+ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

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.

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.

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.

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.

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 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.

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.

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.