RESUMEN
Gomisin A is a dietary lignan compound isolated from the fruit of Schisandra chinensis and has many pharmacological properties, including hepato-protective, anti-diabetic, and anti-oxidative activities. However, the benefit of gomisin A is still not well understood. The action of gomisin A is diverse. However, the effect of gomisin A on Ca2+ signaling in prostate cancer cells is unknown. Ca2+ is a pivotal second envoy that triggers and regulates cellular processes such as apoptosis, fertilization, energy transduction, secretion, and protein activation. The goal of this study was to explore the action of gomisin A on [Ca2+]i and cytotoxicity in PC3 prostate cancer cells. Gomisin A at 100-200 µM provoked [Ca2+]i raises. 20% of the response was reduced by removing external Ca2+. The Ca2+ influx provoked by gomisin A was suppressed by 20% by store-caused Ca2+ entry suppressors: econazole, SKF96365, nifedipine; also by phorbol 12-myristate 13 acetate and GF109203X. Without external Ca2+, gomisin A-caused [Ca2+]i raises were abolished by thapsigargin. In contrast, gomisin A suppressed the [Ca2+]i raises caused by thapsigargin. U73122 fell short to change gomisin A-caused [Ca2+]i responses. Gomisin A (20-100 µM) elicited cytotoxicity in a dose-associated fashion. Blockade of [Ca2+] elevations with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl failed to inhibit cytotoxicity of gomisin A. Collectively, gomisin A evoked [Ca2+]i raises and provoked cytotoxicity in a Ca2+-dissociated fashion in prostate cancer cells.
Asunto(s)
Lignanos , Neoplasias de la Próstata , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Ciclooctanos , Dioxoles , Humanos , Lignanos/farmacología , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
Hepatotoma is the leading type of primary liver cancer in adults and third cause of death in the world. Hydroxytyrosol is a natural phenol existing in olive (Olea europaea L.). Hydroxytyrosol is the chief ingredient of olive oil, which was early deemed to be the most robust antioxidant in olive oil. Hydroxytyrosol is known to inhibit various types of cancer by different methods. This study was aimed to delineate the action of hydroxytyrosol on viability and [Ca2+]i in HepG2 hepatoma cells. Fura-2 was used to detect [Ca2+]i, and WST-1 assays were applied to explore cell cytotoxicity. Hydroxytyrosol elicited [Ca2+]i raises. Eliminating external Ca2+ diminished the Ca2+ signal by 30%. Hydroxytyrosol-evoked Ca2+ influx was diminished by 20% by three inhibitors of store-operated Ca2+ channels and by a protein kinase C activator and an inhibitor. In the absence of Ca2+, thapsigargin eradicated hydroxytyrosol-provoked [Ca2+]i raises. Suppression of phospholipase C (PLC) with U73122, a PLC inhibitor, did not inhibit hydroxytyrosol-elicited [Ca2+]i raises. Hydroxytyrosol reduced cell viability. This cytotoxic action was not reversed by preincubation with BAPTA/AM, a cytosolic Ca2+ binder. In sum, in HepG2 hepatoma cells, hydroxytyrosol elicited [Ca2+]i raises by provoking PLC-unrelated discharge of Ca2+ from ER and Ca2+ influx through PKC-sensitive store-operated Ca2+ entry. In addition, hydroxytyrosol elicited Ca2+-dissociated cytotoxicity.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Olea , Apoptosis , Calcio/metabolismo , Señalización del Calcio , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular , Etanol , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Olea/metabolismo , Fenoles , Alcohol Feniletílico/análogos & derivados , Fosfolipasas de Tipo C/metabolismoRESUMEN
Thioridazine, belonging to first-generation antipsychotic drugs, is a prescription used to treat schizophrenia. However, the effect of thioridazine on intracellular Ca2+ concentration ([Ca2+]i) and viability in human liver cancer cells is unclear. This study examined whether thioridazine altered Ca2+ signaling and viability in HepG2 human hepatocellular carcinoma cells. Ca2+ concentrations in suspended cells were measured using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Thioridazine at concentrations of 25-100 µM induced [Ca2+]i rises. Ca2+ removal reduced the signal by 20%. Thioridazine (100 µM) induced Mn2+ influx suggesting of Ca2+ entry. Thioridazine-induced Ca2+ entry was inhibited by 20% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and by three inhibitors of store-operated Ca2+ channels: nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) abolished thioridazine-evoked [Ca2+]i rises. On the other hand, thioridazine preincubation completely inhibited the [Ca2+]i rises induced by TG. Furthermore, U73122 totally suppressed the [Ca2+]i rises induced by thioridazine via inhibition of phospholipase C (PLC). Regarding cytotoxicity, at 30-80 µM, thioridazine reduced cell viability in a concentration-dependent fashion. This cytotoxicity was not prevented by preincubation with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM) (a Ca2+ chelator). To conclude, thioridazine caused concentration-dependent [Ca2+]i rises in HepG2 human hepatoma cells by inducing Ca2+ release from the endoplasmic reticulum via PLC-associated pathways and Ca2+ influx from extracellular medium through PKC-sensitive store-operated Ca2+ entry. In addition, thioridazine induced cytotoxicity in a Ca2+-independent manner.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Apoptosis , Calcio , Señalización del Calcio , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular , Humanos , Tioridazina , Fosfolipasas de Tipo CRESUMEN
Peroxisome proliferator-activated receptor coactivator-1 alpha (PGC-1α) is a transcriptional coactivator that regulates energy metabolism and mitochondrial biogenesis. Recently, mitochondrial dysfunction has been indicated as an established risk factor for the development of renal fibrosis. However, whether PGC-1α is involved in the pathogenesis of renal fibrosis is unknown. In this study, we treated NRK-49F (normal rat kidney fibroblast) cells with transforming growth factor-beta 1 (TGF-ß1) for 24â¯h to establish an in vitro fibrosis model. TGF-ß1 induced the upregulation of type I collagen, fibronectin, TGF-ß receptor I (TGFß-RI), TGFß-RII, Smad4, and pSmad2/3, as well as PGC-1α. NRK-49F cells transfected with pcDNA-PGC-1α showed significantly increased expression of fibronectin and type I collagen, as revealed by western blot assay. Interestingly, transfection with PGC-1α-siRNA caused a stark reversal of TGF-ß1-induced cellular fibrosis, with concomitant suppression of fibronectin and type I collagen, as revealed by western blot and immunofluorescence assays. Moreover, SB431542 (TGFß-RI), LY294002 (PI3K/Akt), and SB203580 (p38 MAPK), inhibitors of TGF-ß-associated pathways, markedly suppressed TGF-ß1-induced PGC-1α upregulation. These results implicate a role of PGC-1α in renal interstitial fibrosis mediated via the TGFß-RI, PI3K/Akt, and p38 MAPK pathways. Our findings that PGC-1α-siRNA downregulates fibronectin and type I collagen suggest that it can be used as a novel molecular treatment for renal fibrosis.
Asunto(s)
Antifibrinolíticos/farmacología , Fibrosis/tratamiento farmacológico , Enfermedades Renales/tratamiento farmacológico , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , ARN Interferente Pequeño/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibrosis/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Enfermedades Renales/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Transfección/métodos , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND/PURPOSE: Far-infrared (FIR) therapy is a safe and noninvasive source for medical applications. Animal study has shown the effects of FIR in promoting nerve repair. However, the cellular mechanism is not well known. Nerve growth factor (NGF) treated neuron-like PC12 cells for neurite outgrowth have been widely employed as the in vitro model for neural regeneration. METHODS: In this study, we tried to evaluate the potential of FIR in promoting neurite outgrowth and related mechanism by using NGF-treated neuron-like PC12 cells as a cellular model. We found that FIR could promote neurites outgrowth of neuron-like PC12 cells at earlier culture period. RESULTS: The neurite outgrowth-enhancing effect of FIR irradiation was more obvious when lower NGF concentration (1 ng/ml and 10 ng/ml) was added into the medium. We also found that FIR had no thermal effects on culture medium. The effects of FIR in promoting neurite outgrowth were dose dependent, and higher power density of FIR provided more effects for improving neurite outgrowth. The mechanism of FIR in promoting neurite outgrowth was through AKT1 pathway. CONCLUSION: The effects of FIR irradiation on promoting neurite outgrowth and neural regeneration of NGF-treated neuron-like PC12 cells are dose dependent and through activation of AKT1 phosphorylation. This study provided important information for understanding the cellular mechanism of FIR in promoting neurite outgrowth and possible neural regeneration for further clinical applications.
Asunto(s)
Rayos Infrarrojos , Proyección Neuronal/efectos de la radiación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Factor de Crecimiento Nervioso/administración & dosificación , Células PC12 , Fosforilación , RatasRESUMEN
Timolol is a medication used widely to treat glaucoma. Regarding Ca2+ signaling, timolol was shown to modulate Ca2+-related physiology in various cell types, however, the effect of timolol on Ca2+ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100-1000 µM induced [Ca2+]i rises. The Ca2+ signal in Ca2+-containing medium was reduced by removal of extracellular Ca2+ by approximately 75%. Timolol (1000 µM) induced Mn2+ influx suggesting of Ca2+ entry. Timolol-induced Ca2+ entry was partially inhibited by three inhibitors of store-operated Ca2+ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished timolol-evoked [Ca2+]i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca2+]i rises. Timolol at concentrations between 200 and 600 µM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca2+]i rises by evoking Ca2+release from the endoplasmic reticulum in a PLC-dependent manner, and Ca2+ influx via PKC-regulated store-operated Ca2+ entry. Timolol also caused cell death that was not linked to preceding [Ca2+]i rises.
Asunto(s)
Agonistas de los Canales de Calcio/toxicidad , Canales de Calcio/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Próstata/efectos de los fármacos , Timolol/toxicidad , Canales de Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Cinética , Masculino , Células PC-3 , Próstata/metabolismo , Próstata/patología , Proteína Quinasa C/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Captopril, an angiotensin-converting enzyme (ACE) inhibitor, induced different Ca²âº signaling responses in various cell models. However, the effect of captopril on Ca²âº homeostasis and cell viability in hepatoma cells is unknown. This study examined whether captopril altered Ca²âº homeostasis and viability in HepG2 human hepatoma cells. Intracellular Ca²âº concentrations in suspended cells were monitored by using the fluorescent Ca²âº-sensitive dye fura-2. Cell viability was examined by using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1). Captopril at concentrations of 500-3000 µM induced [Ca²âº]i rises in a concentration-dependent manner. Ca²âº removal reduced the signal by approximately 15%. Mn²âº has been shown to enter cells through similar mechanisms as Ca²âº but quenches fura-2 fluorescence at all excitation wavelengths. Captopril (3000 µM)-induced Mn²âº influx indirectly suggested that captopril evoked Ca²âº entry. Captopril-induced Ca²âº entry was inhibited by 15% by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and an inhibitor (GF109203X) and three inhibitors of store-operated Ca²âº channels: nifedipine, econazole and SKF96365. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished captopril-evoked [Ca²âº]i rises. Conversely, treatment with captopril abolished BHQ-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 70% of captopril-induced [Ca²âº]i rises. Captopril at concentrations between 150-550 µM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse captopril's cytotoxicity. Together, in HepG2 human hepatoma cells, captopril induced [Ca²âº]i rises and caused cell death that was not triggered by preceding [Ca²âº]i rises.
Asunto(s)
Carcinoma Hepatocelular , Homeostasis , Neoplasias Hepáticas , Apoptosis , Calcio , Señalización del Calcio , Captopril , Línea Celular Tumoral , Supervivencia Celular , Humanos , Fosfolipasas de Tipo CRESUMEN
Niflumic acid, a drug used for joint and muscular pain, affected Ca²âº signaling in different models. However, the effect of niflumic acid on Ca²âº homeostasis and Ca²âº-related physiology in human osteosarcoma cells is unknown. This study examined the effect of niflumic acid on cytosolic free Ca²âº concentrations ([Ca²âº]i) in MG63 human osteosarcoma cells. Intracellular Ca²âº concentrations in suspended cells were monitored by using the fluorescent Ca²âº-sensitive dye fura- 2. Cell viability was examined by using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio- 1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1). In MG63 cells, niflumic acid at concentrations of 250-750 µM evoked [Ca²âº]i rises concentration-dependently. Niflumic acid-evoked Ca²âº entry was confirmed by Mn²âº-induced quenching of fura-2 fluorescence. This entry was inhibited by nifedipine, econazole, SKF96365, the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was not affected by the PKC inhibitor GF109203X. In Ca²âº- free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin (TG) inhibited niflumic acid-evoked [Ca²âº]i rises. Conversely, treatment with niflumic acid abolished TG-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 also partly reduced niflumic acid-evoked [Ca²âº]i rises. Niflumic acid killed cells at 200-500 µM in a concentration-dependent fashion. Chelating cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/ AM (BAPTA/AM) did not reverse niflumic acid-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, niflumic acid induced [Ca²âº]i rises by evoking PLC-dependent Ca²âº release from the endoplasmic reticulum, and Ca²âº entry via PKC-sensitive store-operated Ca²âº entry. Niflumic acid also induced Ca²âº-independent cell death.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Apoptosis , Calcio , Señalización del Calcio , Línea Celular Tumoral , Supervivencia Celular , Humanos , Ácido Niflúmico , Fosfolipasas de Tipo CRESUMEN
Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca2+ concentrations ([Ca2+ ]i ) in renal cells is unclear. This study examined whether TMP altered Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells. TMP at 100-800 µM induced [Ca2+ ]i rises, which were reduced by Ca2+ removal. TMP induced Mn2+ influx implicating Ca2+ entry. TMP-induced Ca2+ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store-operated Ca2+ channels. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited 93% of TMP-evoked [Ca2+ ]i rises. Treatment with TMP abolished BHQ-evoked [Ca2+ ]i rises. Inhibition of phospholipase C (PLC) abolished TMP-induced responses. TMP at 200-1000 µM decreased viability, which was not reversed by pretreatment with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. Together, in MDCK cells, TMP induced [Ca2+ ]i rises by evoking PLC-dependent Ca2+ release from endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. TMP also caused Ca2+ -independent cell death.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Túbulos Renales/metabolismo , Pirazinas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Perros , Túbulos Renales/citología , Células de Riñón Canino Madin DarbyRESUMEN
Thymic stromal lymphopoietin (TSLP) has previously been linked to allergic inflammatory diseases, and tissue fibrosis and organ dysfunction may also arise from such inflammation. It remains unclear, however, whether TSLP plays any role in the occurrence of renal fibrosis, so this study investigated that possibility. An in vitro fibrosis model was established by treating normal rat kidney fibroblast (NRK-49F) cells with transforming growth factor-ß1 (TGF-ß1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. Also, TSLP shRNA was used to silence the effects of TSLP, while an ELISA was conducted to evaluate the fibronectin secretions. The level of fibronectin in the NRK-49F cells was dose- and time-dependently increased by the administration of exogenous TSLP (P<0.05). TSLP also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of Smad7. Interestingly, the application of TSLP shRNA caused a stark reversal of the TGF-ß1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. Taken together, these observations provide insights into how extracellular matrices develop and could thus lead to potential therapeutic interventions for the suppression of renal fibrosis.
Asunto(s)
Citocinas/metabolismo , ARN Interferente Pequeño/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Línea Celular , Fibronectinas/metabolismo , Fibrosis , Modelos Biológicos , Ratas , Transducción de Señal , Proteínas Smad/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
BACKGROUND: Mitochondrial dysfunction is a newly established risk factor for the development of renal fibrosis. Cell survival and injury repair is facilitated by mitochondrial biogenesis. Nuclear respiratory factor 1 (NRF-1) is a transcriptional regulation factor that plays a central role in the regulation of mitochondrial biogenesis. However, the transcription factor of this process in renal fibrosis is unknown. Thus, we hereby discussed the correlations of NRF-1 and renal interstitial fibrosis. MATERIALS AND METHODS: In vitro fibrosis model was established by treatment with transforming growth factor-ß1 (TGF-ß1) in NRK-49F (Normal Rat kidney fibroblast). We investigated the ROS production, mitochondrial biogenesis and fibrogenic marker (e.q. fibronectin) during the progression of renal fibrosis by kit and Western blotting assay. Here, we used that two distinct mechanisms regulate NRF-1 activation and degradation of NRF-1. NRF-1 was transfect by pcDNA-NRF-1 overexpression gene to evaluate the NRF-1 activity of the therapeutic effect in renal fibrosis. In addition, NRF-1 was silenced by shRNA-NRF-1 to evaluate the significance of NRF-1. ELISA was used to evaluate the secreted fibronectin. Immunofluorescence staining was used to assay the in situ expression of proteins (e.g. fibronectin, NRF-1). RESULTS: Under renal fibrosis conditions, TGF-ß1 (5ng/ml) increased ROS. Simultaneously, TGF-ß1-induced extracellular fibronectin by ELISA assay. In addition, TGF-ß1 decreased expression of mitochondrial biogenesis. This is the first time to demonstrate that expression of NRF-1 is significantly decreased in renal fibrosis. However, NRK49F was a transfection with pcDNA-NRF-1 (2µg/ml) expression vector dramatically reverse TGF-ß1-induced cellular fibrosis concomitantly with the suppression of fibronectin (both intracellular and extracellular fibronectin). More importantly, transfection with shRNA-NRF-1 (2µg/ml) significantly increased the expression of fibronectin of both intercellular and extracellular origins in NRK-49F cells. DISCUSSION: These finding suggest that NRF-1 plays a pivotal role on renal cellular fibrosis. Moreover, NRF-1 might act as a novel renal fibrosis antagonist by down-regulating fibrosis signaling in renal fibroblast cells.
Asunto(s)
Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Factor Nuclear 1 de Respiración/metabolismo , Biogénesis de Organelos , Animales , Biomarcadores/metabolismo , Línea Celular , Fibronectinas/metabolismo , Fibrosis , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Methoxsalen is a natural compound found in many seed plants. The effect of methoxsalen on Ca²âº- related physiology in human osteosarcoma is unclear. This study investigated the effect of methoxsalen on cytosolic free Ca²âº concentrations ([Ca²âº]i) in MG63 human osteosarcoma cells. Methoxsalen induced [Ca²âº]i rises concentration-dependently. Methoxsalen-induced Ca²âº entry was confirmed by Mn²âº-induced quench of fura-2 fluorescence. This Ca²âº entry was suppressed by nifedipine, econazole, and SKF96365. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited methoxsalen-evoked [Ca²âº]i rises by 96%. In contrast, incubation with methoxsalen abolished BHQ-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished methoxsalen-induced [Ca²âº]i rises. Methoxsalen was cytotoxic at 300-700 µM in a concentration-dependent fashion. Chelating cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) did not prevent methoxsalen-induced cytotoxicity. Collectively, our data suggest that in MG63 cells, methoxsalen induced [Ca²âº]i rises by evoking PLC-dependent Ca²âº release from the endoplasmic reticulum, and Ca²âº entry via store-operated Ca²âº entry. Methoxsalen also induced Ca²âº- disassociated cell death.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Metoxaleno/farmacología , Osteosarcoma/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Fura-2/metabolismo , Humanos , Fosfolipasas de Tipo C/metabolismoRESUMEN
Protriptyline has been used as an antidepressant. Clinically it has been prescribed in the auxiliary treatment of cancer patients. However, its effect on Ca²âº signaling and related physiology is unknown in renal cells. This study examined the effect of protriptyline on cytosolic free Ca²âº concentrations ([Ca²âº]i) and viability in Madin-Darby canine kidney (MDCK) tubular cells. Protriptyline induced [Ca²âº]i rises concentration-dependently. The response was reduced by 20% by removing extracellular Ca²âº. Protriptyline-induced Ca²âº entry was not altered by protein kinase C (PKC) activity but was inhibited by 20% by three modulators of store-operated Ca²âº channels: nifedipine, econazole and SKF96365. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5- di-tert-butylhydroquinone (BHQ) or thapsigargin partially inhibited protriptyline-evoked [Ca²âº]i rises. Conversely, treatment with protriptyline inhibited partially BHQ or thapsigargin-evoked [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 did not change protriptyline-induced [Ca²âº]i rises. Protriptyline at 5-200 µM decreased cell viability, which was not reversed by pretreatment with the Ca²âº chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/ AM). Together, in MDCK cells, protriptyline induced [Ca²âº]i rises by evoking PLC-independent Ca²âº release from the endoplasmic reticulum and other unknown stores, and Ca²âº entry via PKCinsensitive store-operated Ca²âº entry. Protriptyline also caused Ca²âº-independent cell death.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Supervivencia Celular/fisiología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/fisiología , Protriptilina/administración & dosificación , Animales , Antidepresivos Tricíclicos/administración & dosificación , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Túbulos Renales/citología , Células de Riñón Canino Madin DarbyRESUMEN
Carvacrol, a monoterpenic phenol compound, has been shown to possess various biological effects in different models. However, the effect of carvacrol on intracellular Ca²âº and its related physiology in human prostate cancer is unknown. This study explored the effect of carvacrol on cytosolic free Ca²âº levels ([Ca²âº]i) and viability in PC3 human prostate cancer cells. Fura-2, a Ca²âº- sensitive fluorescent dye, was used to assess [Ca²âº]i. Cell viability was measured by the detecting reagent WST-1. Carvacrol at concentrations of 200-800 µM caused [Ca²âº]i rises in a concentration-dependent manner. Removal of extracellular Ca²âº reduced carvacrol's effect by approximately 60%. Carvacrol-induced Ca²âº entry was confirmed by Mn²âº entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 30% by nifedipine, econazole, SKF96365, and the protein kinase C (PKC) inhibitor GF109203X. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin (TG) abolished carvacrol-induced [Ca²âº]i rises. Treatment with carvacrol also abolished TG-induced [Ca²âº]i rises. Carvacrol-induced Ca²âº release from the endoplasmic reticulum was abolished by inhibition of phospholipase C (PLC). Carvacrol killed cells at concentrations of 200-600 µM in a concentration-dependent fashion. Chelating cytosolic Ca²âº with BAPTA/AM did not prevent carvacrol's cytotoxicity. Together, in PC3 cells, carvacrol induced [Ca²âº]i rises by inducing PLC-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via PKC-sensitive store-operated Ca²âº channels and other unknown channels. Carvacrol also induced Ca²âº-dissociated cell death.
Asunto(s)
Calcio/metabolismo , Monoterpenos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cimenos , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Fosfolipasas de Tipo C/fisiologíaRESUMEN
The effect of protriptyline on Ca2+ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca2+ ]i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50-150 µM) evoked [Ca2+ ]i rises. The Ca2+ entry was inhibited by removal of Ca2+ . Protriptyline-induced Ca2+ entry was confirmed by Mn2+ -induced quench of fura-2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12-myristate 13 acetate did not inhibit Ca2+ entry. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited 40% of protriptyline-induced response. Treatment with protriptyline abolished BHQ-induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline-evoked response by 70%. At 20-40 µM, protriptyline killed cells which was not reversed by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca2+ ]i rises that involved Ca2+ entry through nifedipine-sensitive Ca2+ channels and PLC-dependent Ca2+ release from endoplasmic reticulum. Protriptyline induced Ca2+ -independent cell death.
Asunto(s)
Antidepresivos Tricíclicos/farmacología , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Protriptilina/farmacología , Calcio/agonistas , Cationes Bivalentes , Econazol/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Colorantes Fluorescentes , Fura-2 , Células Hep G2 , Humanos , Hidroquinonas/farmacología , Imidazoles/farmacología , Indoles/farmacología , Transporte Iónico/efectos de los fármacos , Cinética , Maleimidas/farmacología , Manganeso/farmacología , Nifedipino/farmacología , Protriptilina/antagonistas & inhibidores , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
This study explored the effect of deltamethrin, a pesticide, on intracellular free Ca²âº concentration ([Ca²âº]i) in PC3 human prostate cancer cells. Deltamethrin at concentrations between 5 µM and 20 µM evoked [Ca²âº]i rises in a concentration-dependent manner. This Ca²âº signal was inhibited by 22% by removal of extracellular Ca²âº. Nifedipine, econazole, and SKF96365 also inhibited the Ca²âº signal. Treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in Ca²âº-free medium nearly abolished deltamethrin-induced [Ca²âº]i rises. Treatment with deltamethrin also inhibited most of BHQ-induced [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter deltamethrin-evoked [Ca²âº]i rises. Deltamethrin killed cells at concentrations of 20-100 µM in a concentration-dependent fashion. Chelation of cytosolic Ca²âº with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) did not prevent deltamethrin's cytotoxicity. Together, in PC3 human prostate cancer cells, deltamethrin induced [Ca²âº]i rises that involved Ca²âº entry through store-operated Ca²âº channels and PLC-independent Ca²âº release from the endoplasmic reticulum. Deltamethrin induced cytotoxicity in a Ca²âº-independent manner.
Asunto(s)
Calcio/metabolismo , Insecticidas/farmacología , Nitrilos/farmacología , Neoplasias de la Próstata/metabolismo , Piretrinas/farmacología , Apoptosis/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quelantes/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Nitrilos/antagonistas & inhibidores , Piretrinas/antagonistas & inhibidores , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The phenolic compound 2,5-dimethylphenol is a natural product. 2,5-Dimethylphenol has been shown to affect rat hepatic and pulmonary microsomal metabolism. However, the effect of 2,5-dimethylphenol on Ca(2+ )signaling and cyotoxicity has never been explored in any culture cells. This study explored the effect of 2,5-dimethylphenol on cytosolic free Ca(2+ )levels ([Ca(2+)]i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500 µM and 1000 µM evoked [Ca(2+)]i rises in a concentration-dependent manner. This Ca(2+ )signal was inhibited by approximately half by the removal of extracellular Ca(2+). 2,5-Dimethylphenol-induced Ca(2+ )influx was confirmed by Mn(2+)-induced quench of fura-2 fluorescence. Pretreatment with the protein kinase C (PKC) inhibitor GF109203X, nifedipine or the store-operated Ca(2+ )entry inhibitors (econazole or SKF96365) inhibited 2,5-dimethylphenol-induced Ca(2+ )signal in Ca(2+)-containing medium by â¼30%. Treatment with the endoplasmic reticulum Ca(2+ )pump inhibitor thapsigargin in Ca(2+)-free medium abolished 2,5-dimethylphenol-induced [Ca(2+)]i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca(2+)]i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca(2+)]i rises by â¼80%. 2,5-Dimethylphenol killed cells at concentrations of 350-1000 µM in a concentration-dependent fashion. Chelation of cytosolic Ca(2+ )with 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/AM (BAPTA/AM) did not prevent 2,5-dimethylphenol's cytotoxicity. Together, in PC3 cells, 2,5-dimethylphenol induced [Ca(2+)]i rises that involved Ca(2+ )entry through PKC-regulated store-operated Ca(2+ )channels and PLC-dependent Ca(2+ )release from the endoplasmic reticulum. 2,5-Dimethylphenol induced cytotoxicity in a Ca(2+)-independent manner.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Xilenos/farmacología , Canales de Calcio/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Humanos , Masculino , Manganeso/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Tiempo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Dysphagia is common after stroke. Neuromuscular electrical stimulation (NMES) and fiberoptic endoscopic evaluation of swallowing (FEES) for the treatment of dysphagia have gained in popularity, but the combined application of these promising modalities has rarely been studied. We aimed to evaluate whether combined NMES, FEES, and traditional swallowing rehabilitation can improve swallowing functions in stroke patients with moderate to severe dysphagia. Thirty-two patients with moderate to severe dysphagia poststroke (≥3 weeks) were recruited. Patients received 12 sessions of NMES for 1 h/day, 5 days/week within a period of 2-3 weeks. FEES was done before and after NMES for evaluation and to guide dysphagic therapy. All patients subsequently received 12 sessions of traditional swallowing rehabilitation (50 min/day, 3 days/week) for 4 weeks. Primary outcome measure was the Functional Oral Intake Scale (FOIS). Secondary outcome measures included clinical degree of dysphagia, the patient's self-perception of swallowing ability, and the patient's global satisfaction with therapy. Patients were assessed at baseline, after NMES, at 6-month follow-up, and at 2-year follow-up. Twenty-nine patients completed the study. FOIS, degree of dysphagia, and patient's self-perception of swallowing improved significantly after NMES, at the 6-month follow-up, and at the 2-year follow-up (p < 0.001, each compared with baseline). Most patients reported considerable satisfaction with no serious adverse events. Twenty-three of the 29 (79.3 %) patients maintained oral diet with no pulmonary complications at 2-year follow-up. This preliminary case series demonstrated that combined NMES, FEES, and traditional swallowing rehabilitation showed promise for improving swallowing functions in stroke patients with moderate-to-severe dysphagia. The benefits were maintained for up to 2 years. The results are promising enough to justify further studies.
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Trastornos de Deglución/fisiopatología , Trastornos de Deglución/rehabilitación , Terapia por Estimulación Eléctrica , Endoscopía Gastrointestinal , Anciano , Anciano de 80 o más Años , Trastornos de Deglución/etiología , Terapia por Ejercicio , Femenino , Humanos , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Accidente Cerebrovascular/complicaciones , Resultado del TratamientoRESUMEN
The antidepressant, sertraline, has been shown to have diverse in vitro effects. This study examined whether sertraline altered [Ca(2+)](i) in MG63 human osteosarcoma cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. At 50-200 µM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent manner. Ca(2+) response was decreased by removing extracellular Ca(2+), suggesting that Ca(2+) entry and release contributed to the [Ca(2+)](i) signal. Sertraline-induced Ca(2+) entry was inhibited by nifedipine, La(3+), Gd(3+), and SK&F96365. When extracellular Ca(2+) was removed, pretreatment with the endoplasmic reticulum (ER) Ca(2+) pump inhibitor, thapsigargin, or 2,5-di-tert-butylhydroquinone (BHQ) abolished the sertraline-evoked [Ca(2+)](i) rise. Incubation with sertraline also abolished the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished the sertraline-induced [Ca(2+)](i) rise. At 20-30 µM, overnight treatment with sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was not reversed 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 demonstrate that sertraline (30 µM) evoked apoptosis. Sertraline (20 and 30 µM) also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, sertraline evoked a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the ER and Ca(2+) entry by L-type Ca(2+) channels and store-operated Ca(2+) channels. Sertraline induced cell death that may involve apoptosis by mitochondrial pathways.
Asunto(s)
Calcio/metabolismo , Osteosarcoma/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Sertralina/farmacología , Apoptosis/efectos de los fármacos , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Estrenos/farmacología , Humanos , Hidroquinonas/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Pirrolidinonas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación , Sertralina/administración & dosificación , Tapsigargina/farmacología , Fosfolipasas de Tipo C/metabolismoRESUMEN
The exercise capacity and limitation in children with asymptomatic atrial septal defect (ASD) have not been explored thoroughly. The aim of our study was to examine the influence of asymptomatic ASD on exercise capacity in children. Fifty children with asymptomatic ASD who had undergone medical interventions at least 4 years ago and fifty normal children were recruited in this study. The exercise capacity was assessed by the symptom-limited exercise test through the Bruce treadmill protocol. The pulmonary function was also evaluated by the spirometry. Circulatory and ventilatory impairments were respectively reflected by chronotropic incompetence (CI) and ventilatory limitation as measured by the exercise test and spirometry. Eleven (22%) of children with ASD failed to reach the age-predicted peak heart rate during the exercise test. Also, children with ASD had significantly lower oxygen consumption at anaerobic threshold and peak exercise (P < 0.01). The rate of circulatory impairment was significantly higher in children with ASD (P < 0.01). However, the pulmonary function and ventilatory limitation were comparable between these two groups. Within the ASD group, children with CI had significantly worse peak oxygen consumption than their peers without CI (P < 0.01). Our study examined a larger population sample and confirmed that children with asymptomatic ASD, who had previously undergone medical interventions, had significantly worse exercise capacity than normal children. This difference in exercise capacity was mainly related to circulatory impairment. Our findings support the concerns of exercise limitation in ASD children.