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1.
Chin J Physiol ; 64(4): 202-209, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34472451

RESUMO

Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 µM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 µM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 µM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.


Assuntos
Neoplasias da Mama , Ácido gama-Linolênico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Estresse Oxidativo , Ácido gama-Linolênico/metabolismo
2.
Eur J Pharmacol ; 904: 174115, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33901459

RESUMO

In this report we examined the effects of lidocaine on Ca2+ homeostasis of neuronal cells using microfluorimetric measurement of cytosolic Ca2+ with fura 2 as probe. In mouse neuroblastoma N2A cells, 10 mM lidocaine caused Ca2+ release from the cyclopiazonic acid (CPA)-dischargeable pool and abolished ATP-triggered Ca2+ release. Lidocaine-triggered Ca2+ release was not affected by xestospongin C (XeC), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor. N2A cells did not have functional ryanodine receptors (RYR) (absence of caffeine response) and we used differentiated NG108-15 cells (presence of caffeine response) for further experiments. Caffeine-triggered Ca2+ release was unaffected by a brief lidocaine exposure, but was eliminated after a prolonged treatment of lidocaine, suggesting lidocaine abolished caffeine action possibly not by interfering caffeine binding but via Ca2+ store depletion. Lidocaine-elicited Ca2+ release was unaffected by XeC or a high concentration of ryanodine, suggesting Ca2+ release was not via IP3R or RYR. Lidocaine did not affect nigericin-dischargeable lysosomal Ca2+ stores. Lastly, we observed that lidocaine suppressed CPA-induced store-operated Ca2+ influx in both N2A cells and differentiated NG108-15 cells. Our results suggest two novel actions of lidocaine in neuronal cells, namely, depletion of Ca2+ store (via an IP3R- and RYR-independent manner) and suppression of store-operated Ca2+ influx.

3.
Fundam Clin Pharmacol ; 34(2): 213-221, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31618480

RESUMO

Quercetin is a flavonol polyphenol widely found in many vegetables, grains, and fruits. Quercetin has been shown to inhibit proliferation and invasion of various glioma cells and is regarded as a potential anticancer agent against glioma. However, whether and how this drug could affect brain blood vessels and endothelial cells (EC) are less understood. Further, there is hitherto no report on how quercetin affects brain EC Ca2+ homeostasis. In this report, we investigated the effects of quercetin on Ca2+ homeostasis in mouse brain bEnd.3 EC. We demonstrated that quercetin raised cytosolic Ca2+ level in a concentration-dependent manner. Quercetin-triggered Ca2+ signal composed of both internal Ca2+ release and extracellular Ca2+ influx. Quercetin caused Ca2+ release from the endoplasmic reticulum, and consistently, inhibition of inositol 1,4,5-trisphosphate receptor (IP3R) by xestospongin C (XeC) suppressed quercetin-triggered Ca2+ release. Quercetin also caused Ca2+ release from lysosomes, an observation in concordance with the inhibition of quercetin-triggered Ca2+ release by trans-Ned-19, a blocker of two-pore channels. As quercetin depleted intracellular Ca2+ storage, it suppressed ATP-induced Ca2+ release and thereby blunted ATP-triggered Ca2+ signaling. In addition, quercetin co-treatment significantly suppressed ATP-stimulated nitric oxide release. Our work therefore showed, for the first time, quercetin perturbed intracellular Ca2+ stores and strongly suppressed ATP-triggered response in bEnd.3 cells.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Quercetina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Endoteliais/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Quercetina/administração & dosagem
4.
Naunyn Schmiedebergs Arch Pharmacol ; 392(7): 823-832, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30826858

RESUMO

Ca2+-sensing receptors (CaSR) are G protein-coupled receptors which are activated by a rise in extracellular Ca2+. CaSR activation has been known to inhibit parathyroid hormone release and stimulate calcitonin release from parathyroid glands and thyroid parafollicular C cells, respectively. The roles of CaSR in other cell types including endothelial cells (EC) are much less understood. In this work, we demonstrated protein and functional expression of CaSR in mouse cerebral EC (bEND.3). Unexpectedly, CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by edelfosine or U73122 but strongly suppressed by SK&F 96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB), suggesting involvement of TRPV and TRPC channels but not Gq-phospholipase C. Acute application of NPS2143, a negative allosteric modulator of CaSR, suppressed CaSR response. However, a 40-min NPS2143 pre-treatment surprisingly enhanced CaSR response. After 4-24 h of application, this enhancement faded away and suppression of CaSR response was observed again. Similar results were obtained when La3+ and Sr2+ were used as CaSR agonists. The transient NPS 2143 enhancement effect was abolished by SB203580, a p38 inhibitor. Consistently, NPS 2143 triggered a transient p38 activation. Taken together, results suggest that in bEND.3 cells, NPS 2143 caused acute suppression of CaSR response, but then elicited a transient enhancement of CaSR response in a p38-dependent manner. NPS 2143 effects on CaSR in bEND.3 cells therefore depended on drug exposure time. These findings warrant cautious use of this agent as a CaSR modulator and potential cardiovascular drug.


Assuntos
Encéfalo/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Naftalenos/farmacologia , Receptores de Detecção de Cálcio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Camundongos , Fatores de Tempo
5.
Clin Exp Pharmacol Physiol ; 45(8): 788-796, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29498086

RESUMO

Gossypol, a polyphenolic dialdehyde toxin isolated from cotton seed, has anti-cancer properties and has recently shown some success in the treatment of glioma. Its effects on brain neurons and blood vessels are poorly understood. In this work we examined the effects of gossypol on cytosolic Ca2+ concentration ([Ca2+ ]i ) of mouse brain bEND.3 endothelial cells. Cell viability tests revealed that after 3 hour and 18 hour exposures, 10 µmol/L gossypol caused 23% and 65% cell death, respectively; 3 µmol/L gossypol caused no and 21% cell death, respectively. [Ca2+ ]i was raised concentration-dependently by 1-10 µmol/L gossypol. We then explored the Ca2+ signalling triggered by 3 µmol/L gossypol, which inflicted minimal toxicity: the Ca2+ signal was composed largely of Ca2+ influx and to a small extent, intracellular Ca2+ release. Such Ca2+ influx was much larger than store-operated Ca2+ influx triggered by maximal Ca2+ pool depletion. The Ca2+ influx triggered by 3 and 10 µmol/L gossypol caused NO release and cell death, respectively. Gossypol also triggered influx of Mn2+ and Na+ , but not Ni2+ and Co2+ . Gossypol-triggered Ca2+ signal was inhibited only by 14% and 37% by 100 µmol/L La3+ and 10 µmol/L nimodipine, respectively; and not suppressed at all by 5 mmol/L Ni2+ . Gossypol-triggered Ca2+ signal was suppressed by 78% by 30 µmol/L ruthenium red, suggesting gossypol may act on TRPV channels. Our results suggest gossypol triggered opening of a non-selective cation pore, possibly a member of the TRPV family.


Assuntos
Cálcio/metabolismo , Cobalto/metabolismo , Células Endoteliais/efeitos dos fármacos , Gossipol/farmacologia , Níquel/metabolismo , Sódio/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Ativação do Canal Iônico , Camundongos , Óxido Nítrico/metabolismo , Permeabilidade
6.
Respir Physiol Neurobiol ; 225: 19-30, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26702732

RESUMO

Acute lung injury (ALI) is a severe, life-threatening medical condition whose pathogenesis is linked to neutrophil infiltration of the lung. Activation and recruitment of neutrophils to the lung is mostly attributed to the production of chemokines NO, IL-6, for instance. This study aims to investigate lobeline ability in reducing NO production, and nitric oxide synthase (iNOs) expression. Lobeline was tested by inhibiting phosphorylation of mitogen-activated protein kinases (MAPKs), NF-κB and IκBα in LPS-stimulated RAW 264.7 cells. When RAW 264.7 macrophages were given lobeline with LPS, a significant concentration-dependent inhibition of NO production was detected. In vivo tests, mice were either treated with normal saline, 10mg/kg dexmethasone or 5, 10, 20mg/kg lobeline intraperitoneally, and after an hour, the administration of 5mg/kg of LPS was given intratracheally. External performance, cytokines, MAPK pathways and antioxidative enzymes (AOEs) were also carried out to evaluate the effects of these drugs. This is the first investigation in which lobeline was found to effectively inhibit acute lung edema, which may provide a potential target for treating ALI. Lobeline may utilize MAPKs pathways as well as AOEs activity to attenuate LPS-induced nonspecific pulmonary inflammation.


Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Lobelina/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Lesão Pulmonar Aguda/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos
7.
Arch Toxicol ; 89(1): 57-72, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25069801

RESUMO

Antagonizing the action of the pregnane X receptor (PXR) may have important clinical implications for preventing inducer-drug interactions and improving therapeutic efficacy. We identified a widely distributed isothiocyanate, allyl isothiocyanate (AITC), which acts as an effective antagonist of the nuclear receptor pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3). HepG2 cells were used to assay reporter function, mRNA levels, and protein expression. Catalytic activities of the PXR and CAR target genes, CYP3A4 and CYP2B6, respectively, were also assessed in differentiated HepaRG cells. Protective effects of AITC on rifampin-induced cytotoxicity were observed, and transient transfection assays showed that AITC was able to effectively attenuate the agonist effects of rifampin and CITCO on human PXR and CAR activity, respectively. AITC-mediated reduction in the transcriptional activity of PXR and CAR correlated well with the suppression of CYP3A4 and CYP2B6 expression in HepG2 cells, which reflected the reduced catalytic activities of both of these genes following AITC treatment in differentiated HepaRG cells. Furthermore, AITC disrupts the co-regulations of PXR with several important co-regulators. Furthermore, the antagonist effect of AITC against PXR was found in HepaRG cells upon addition of acetaminophen (APAP) and amiodarone, indicating that AITC protects cells from drug-induced cytotoxicity. Taken together, our results show that AITC inhibits the transactivation effects of PXR and CAR and reduces the expression and function of CYP3A4 and CYP2B6. Additionally, AITC reversed the cytotoxic effects of APAP and amiodarone induced by PXR ligand. Results from this study suggest that AITC could be a powerful agent for reducing potentially dangerous interactions between transcriptional inducers of CYP enzymes and therapeutic drugs.


Assuntos
Acetaminofen/toxicidade , Amiodarona/toxicidade , Isotiocianatos/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores de Esteroides/antagonistas & inibidores , Acetaminofen/metabolismo , Amiodarona/metabolismo , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP2B6/genética , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Receptor de Pregnano X , Receptores Citoplasmáticos e Nucleares/genética , Receptores de Esteroides/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional , Transfecção
8.
Planta Med ; 80(13): 1097-106, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25197954

RESUMO

The growing interest in the use of natural herbal products and dietary supplements to treat and prevent diseases raises the question of medicinal drug safety. Allyl isothiocyanate, a hydrolysis product of a glucosinolate, sinigrin, has multiple beneficial properties, and based on this fact, allyl isothiocyanate-containing dietary supplements have been developed. To date, no studies of the effects of this compound on the cytochrome P450 2C9 have been reported. In this study, we found that allyl isothiocyanate reduced catalytic activity, messenger ribonucleic acid, and protein expression of cytochrome P450 2C9 in HepaRG cells. An investigation of the transcriptional activity of the pregnane X receptor and the constitutive androstane receptor revealed that allyl isothiocyanate disrupted the transcriptional coregulation effects of the pregnane X receptor/constitutive androstane receptor with several important coregulators and interfered with the assembly of transcriptional complexes of the cytochrome P450 2C9 pregnane X receptor/constitutive androstane receptor-response element. The decrease of cytochrome P450 2C9 expression and activity mediated by allyl isothiocyanate suggested that this agent could alter the metabolism of drugs metabolized by cytochrome P450 2C9. This may cause food/dietary supplement-drug interactions or alter the therapeutic effects, and even the toxicity of drugs coadministered with allyl isothiocyanate. Since the consumption of allyl isothiocyanate-containing food/dietary supplements continues to increase, it is important to predict and ultimately avoid interactions with concomitant drugs. It is required that these possible pharmacokinetic interactions be characterized and the recommendations available to patients and healthcare professionals be improved.


Assuntos
Inibidores do Citocromo P-450 CYP2C9/farmacologia , Citocromo P-450 CYP2C9/metabolismo , Isotiocianatos/farmacologia , Linhagem Celular Tumoral , Inibidores do Citocromo P-450 CYP2C9/química , Inibidores do Citocromo P-450 CYP2C9/isolamento & purificação , Interações Ervas-Drogas , Humanos , Isotiocianatos/química , Isotiocianatos/isolamento & purificação , Receptor de Pregnano X , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo
9.
J Med Virol ; 85(7): 1206-14, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23918539

RESUMO

This study aimed to evaluate whether genetic polymorphisms of the inducible nitric oxide synthase (iNOS) gene NOS2A could be associated with a sustained virological response (SVR) among patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2) who were treated with peginterferon plus ribavirin (PEG-IFNα-RBV). We analyzed the associations between SVR to PEG-IFNα-RBV therapy and two single nucleotide polymorphisms (SNPs) in NOS2A. This study included Taiwanese Chinese patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) with or without a SVR. Among the NOS2A SNPs examined, the combination of genotypes A/A and A/G of rs2248814 was inversely correlated with SVR in patients infected with HCV-1 (P = 0.0048), particularly in males (P = 0.0281). This effect was not observed in patients infected with HCV-2. The AC NOS2A haplotype comprising two SNPs (rs2248814 and rs2072324) was found to be associated with SVR, and its presence may decrease the chances for a successful outcome of treatment of patients infected with HCV-1 (P = 0.0053). HCV-1 infected patients who carried the A-C diplotype will have a lower success rate of achieving a SVR (P = 0.0117). In addition, a multivariate logistic regression model for predicting a SVR revealed that the presence of the A-C diplotype interactively affected the outcome of PEG-IFNα-RBV treatment. The presence of NOS2A SNPs and the association with SVR showed that NOS2A polymorphisms may influence the therapeutic outcomes of patients infected with HCV-1 under standard of care treatment.


Assuntos
Antivirais/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Interferon-alfa/uso terapêutico , Óxido Nítrico Sintase Tipo II/genética , Polietilenoglicóis/uso terapêutico , Polimorfismo de Nucleotídeo Único , Ribavirina/uso terapêutico , Adulto , Idoso , Quimioterapia Combinada/métodos , Feminino , Genótipo , Hepatite C Crônica/virologia , Humanos , Interferon alfa-2 , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Taiwan , Resultado do Tratamento
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