RESUMEN
The effect of the natural product diindolylmethane on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 20-50 µM induced [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). Diindolylmethane-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 50-100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca(2+)](i) rise in PC3 cells by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive store-operated Ca(2+) channels. Diindolylmethane caused cell death in which apoptosis may participate.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Neoplasias de la Próstata/metabolismo , Calcio/metabolismo , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Fura-2/farmacología , Humanos , Indoles/farmacología , Masculino , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca(2+) is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca(2+) concentrations ([Ca(2+)](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 [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Celecoxib-induced Ca(2+) influx was not blocked by L-type Ca(2+) entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A(2) inhibitor, aristolochic acid. In Ca(2+)-free medium, 30 µM of celecoxib failed to induce a [Ca(2+)](i) rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca(2+) pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca(2+)](i) rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca(2+) with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the ER and Ca(2+) influx via non-L-type, phospholipase A(2)-regulated Ca(2+) channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.
Asunto(s)
Calcio/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Pirazoles/farmacología , Sulfonamidas/farmacología , Canales de Calcio Tipo L/metabolismo , Celecoxib , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/química , Fura-2/química , Humanos , Masculino , Fosfolipasas A2/metabolismo , Neoplasias de la Próstata/patología , Pirazoles/administración & dosificación , Sulfonamidas/administración & dosificaciónRESUMEN
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.
Asunto(s)
Anticarcinógenos/farmacología , Calcio/metabolismo , Indoles/farmacología , Osteosarcoma/tratamiento farmacológico , Anticarcinógenos/administración & dosificación , Apoptosis/efectos de los fármacos , Canales de Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Homeostasis , Humanos , Indoles/administración & dosificación , Osteosarcoma/patología , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Glioblastoma/patología , Homeostasis/efectos de los fármacos , Timol/farmacología , Apoptosis/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fura-2/metabolismo , Humanos , Manganeso/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
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.
Asunto(s)
Antidepresivos/farmacología , Calcio/metabolismo , Neoplasias de la Próstata/metabolismo , Sertralina/farmacología , Apoptosis , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Masculino , Manganeso/metabolismo , Neoplasias de la Próstata/patología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²âº](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²âº-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca²âº](i) in a concentration-dependent fashion. The Ca²âº signal was reduced partly by removing extracellular Ca²âº indicating that Ca²âº entry and release both contributed to the [Ca²âº](i) rise. This Ca²âº influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²âº channels or by modulation of protein kinase C activity. In Ca²âº-free medium, pretreatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²âº release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²âº](i) rise, suggesting that thapsigargin released Ca²âº from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²âº](i) rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²âº with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²âº](i) rises by causing phospholipase C-independent Ca²âº release from the endoplasmic reticulum and Ca²âº influx via phospholipase A2-sensitive Ca²âº channels. Thapsigargin also induced cell death via Ca²âº-dependent pathways and Ca²âº-independent apoptotic pathways.