RESUMO
Unfractionated heparin (UFH) is the main anticoagulant used in intensive care unit. The anticoagulant effect is monitored by activated partial thrombin time (aPTT) and anti-Xa activity (anti-Xa) measurement. However, delayed centrifugation induces platelet factor 4 (PF4) release and anti-Xa decrease. Several studies have concluded that aPTT and anti-Xa measurement should be performed within 2 h in citrated anticoagulant but may be delayed longer in Citrate Theophylline Adenosine and Dypiridamol (CTAD) anticoagulant. The objective of this study was to compare the stability of both aPTT and anti-Xa in citrate and CTAD samples, and to determine the effect of delayed centrifugation on both aPTT, anti-Xa results, and PF4 release in citrate samples only. aPTT and anti-Xa were measured in citrate and CTAD anticoagulant samples from 93 patients. Delayed centrifugation was performed in citrate samples from 31 additional patients, with hourly aPTT and anti-Xa measurement from 1 to 6 h. In 14 of these last patients, PF4 release was also evaluated with Human CXCL4/PF4 Quantikine ELISA Kit. We observed a significant correlation between citrate and CTAD anticoagulant for aPTT (r2 = 0.94) and anti-Xa (r2 = 0.95). With Bland-Altman correlation, a minor bias was observed for anti-Xa (- 0.025 ± 0.041). Delayed centrifugation in citrated anticoagulant showed an excellent concordance from 1 to 4 h for aPTT (- 4.0 ± 5.3 s) and anti-Xa (1.10-9 ± 0.058 UI/ml) measurements. Moreover, PF4 release was not different between 1 h (31.5 ± 14.7 ng/ml) and 4 h (33.8 ± 11.8 ng/ml). We have demonstrated that anti-Xa measurement for unfractionated heparin should be done 4 h in citrated plasma and that CTAD was not better than citrate. However, these initial findings require confirmation using other aPTT and calibrated anti-Xa assays.
Assuntos
Preservação de Sangue/métodos , Citratos/farmacologia , Dipiridamol/farmacologia , Monitoramento de Medicamentos/métodos , Heparina/farmacocinética , Anticoagulantes/farmacologia , Testes de Coagulação Sanguínea , Centrifugação , Heparina/uso terapêutico , Humanos , Inibidores de Fosfodiesterase/farmacologia , Teofilina , Fatores de TempoRESUMO
Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. The major ZEN metabolites are α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). In the present study, we investigated the underlying mechanism of the toxicity induced by ZEN, α-ZOL and ß-ZOL in cardiac cells (H9c2). We show that treatment with ZEN or its metabolites induces the activation of the mitochondrial pathway of apoptosis as characterized by an increase in ROS generation, a loss of mitochondrial transmembrane potential (ΔΨm) and an activation of caspases. Besides, we demonstrate that these mycotoxins promote the activation of autophagy before the onset of apoptosis. Indeed, we observed that a short-time (6h) treatment with ZEN, α-ZOL or ß-ZOL, increased the level of Beclin-1 and LC3-II and induced the accumulation of the CytoID® autophagy detection probe. Moreover, the inhibition of autophagy by Chloroquine significantly increased cell death induced by ZEN, α-ZOL or ß-ZOL, suggesting that the activation of autophagy serves as a cardioprotective mechanism against these mycotoxins. In addition, we found that the inhibition (EX527) or the knockdown of SIRT1 (siRNA) significantly increased apoptosis induced by ZEN or its derivatives, whereas SIRT1 activation with RSV greatly prevents the cytotoxic effects of these mycotoxins. By contrast, when autophagy was inhibited by CQ, the activation of SIRT1 by RSV had no protection against the cardiotoxicity of ZEN or its metabolites, suggesting that SIRT1 protects cardiac cells by an autophagy-dependent pathway.
Assuntos
Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Sirtuína 1/fisiologia , Zearalenona/toxicidade , Zeranol/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Morte Celular , Linhagem Celular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Zeranol/toxicidadeRESUMO
Patulin (PAT) is a secondary metabolite produced by several species of the genera of Penicillium, Aspergillus, and Byssochlamys that can be found in rotting fruits, especially in apples and apple-based products. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. The mechanism underlying such toxicity has been linked to the induction of apoptosis which occurred with reactive oxygen species production and endoplasmic reticulum (ER) stress induction. This study aimed to evaluate the effect of the two common dietary compounds Quercetin (QUER), a natural flavonoid, and Crocin (CRO), a natural carotenoid, on PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We showed that antioxidant properties of QUER and CRO help to prevent ER stress activation and lipid peroxidation as evidenced by the reduction in GRP78 and GADD34 expressions and the decrease in malondialdehyde production. Furthermore, we demonstrated their ability to re-establish the loss of the mitochondrial membrane potential to inhibit caspase 3 activation and DNA fragmentation. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1851-1858, 2016.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Carotenoides/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Patulina/toxicidade , Quercetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Caspase 3/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Ativação Enzimática , Células HCT116 , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteína Fosfatase 1/metabolismoRESUMO
BACKGROUND: CaMKII activation is proarrhythmic in heart failure where myocardium is stretched. However, the arrhythmogenic role of CaMKII in stretched ventricle has not been well understood. OBJECTIVE: We tested abnormal impulse inducibility by stretch current in myocytes isolated from CaMKIIδ knockout (KO) mouse left ventricle (LV) where CaMKII activity is reduced by ≈ 62%. METHODS AND RESULTS: Action potentials were recorded by whole-cell patch clamp, and abnormal impulses were induced in LV myocytes by a simulation of stretch-activated channel (SAC) current. SAC activation failed to induce abnormal impulses in wild type (WT) myocytes but steadily produced early after-depolarizations and automaticity in KO myocytes in which an increase in L-type calcium channel (LTCC) current (I(Ca)) and a reduction of sarcoplasmic reticulum Ca(2+) leak and action potential duration (APD) were observed. The abnormal impulses were not suppressed by CaMKII inhibitor AIP whereas a low concentration of nifedipine eliminated abnormal impulses without shortening APD, implicating I(Ca) in promoting stretch-induced abnormal impulses. In addition, APD prolongation by LTCC opener S(-)Bay K 8644 or isoproterenol facilitated abnormal impulse induction in WT ventricular myocytes even in the presence of CaMKII inhibitor AIP, whereas APD prolongation by K(+) channel blocker 4-aminopyridine promoted abnormal impulses in KO myocytes but not in WT myocytes. CONCLUSION: I(Ca) activation plays a central role in stretch-induced abnormal impulses and APD prolongation is arrhythmogenic only when I(Ca) is highly activated. At increased I(Ca) activation, CaMKII inhibition cannot suppress abnormal impulse induction.
Assuntos
Arritmias Cardíacas/enzimologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Ventrículos do Coração/enzimologia , Mecanorreceptores/metabolismo , Miócitos Cardíacos/enzimologia , Potenciais de Ação , Agonistas Adrenérgicos beta/farmacologia , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Cálcio/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Ativação Enzimática , Potenciais Evocados , Ventrículos do Coração/efeitos dos fármacos , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Retículo Sarcoplasmático/metabolismo , Fatores de TempoRESUMO
Calmodulin-dependent protein kinase II (CaMKII) has been proposed to be a therapeutic target for heart failure (HF). However, the cardiac effect of chronic CaMKII inhibition in HF has not been well understood. We have tested alterations of Ca(2+) handling, excitation-contraction coupling, and in vivo ß-adrenergic regulation in pressure-overload HF mice with CaMKIIδ knockout (KO). HF was produced in wild-type (WT) and KO mice 1 wk after severe thoracic aortic banding (sTAB) with a continuous left ventricle (LV) dilation and reduction of ejection fraction for up to 3 wk postbanding. Cardiac hypertrophy was similar between WT HF and KO HF mice. However, KO HF mice manifested exacerbation of diastolic function and reduction in cardiac reserve to ß-adrenergic stimulation. Compared with WT HF, L-type calcium channel current (I(Ca)) density in KO HF LV was decreased without changes in I(Ca) activation and inactivation kinetics, whereas I(Ca) recovery from inactivation was accelerated and Ca(2+)-dependent I(Ca) facilitation, a positive staircase blunted in WT HF, was recovered. However, I(Ca) response to isoproterenol was reduced. KO HF myocytes manifested dramatic decrease in sarcoplasmic reticulum (SR) Ca(2+) leak and slowed cytostolic Ca(2+) concentration decline. Sarcomere shortening was increased, but relaxation was slowed. In addition, an increase in myofilament sensitivity to Ca(2+) and the slow skeletal muscle troponin I-to-cardiac troponin I ratio and interstitial fibrosis and a decrease in Na/Ca exchange function and myocyte apoptosis were observed in KO HF LV. CaMKIIδ KO cannot suppress severe pressure-overload-induced HF. Although cellular contractility is improved, it reduces in vivo cardiac reserve to ß-adrenergic regulation and deteriorates diastolic function. Our findings challenge the strategy of CaMKII inhibition in HF.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Insuficiência Cardíaca/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Agonistas Adrenérgicos beta/farmacologia , Animais , Aorta Torácica/fisiologia , Apoptose/efeitos dos fármacos , Western Blotting , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Citosol/efeitos dos fármacos , Citosol/metabolismo , Fibrose/patologia , Insuficiência Cardíaca/diagnóstico por imagem , Ventrículos do Coração/efeitos dos fármacos , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Contração Miocárdica/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Miofibrilas/fisiologia , Inibidores de Proteínas Quinases/toxicidade , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/fisiologia , Sarcômeros/efeitos dos fármacos , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/fisiologia , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , UltrassonografiaRESUMO
BACKGROUND: Transient Receptor Potential (TRP) channels are expressed in many solid tumors. However, their expression in breast cancer remains largely unknown. Here, we investigated the profile expression of 13 TRP channels in human breast ductal adenocarcinoma (hBDA) and performed a correlation between their overexpression and pathological parameters. METHODS: The TRP channels expression was determined by RT-PCR in hBDA tissue, in human breast cancer epithelial (hBCE) primary culture and in MCF-7 cell line. The TRP protein level was evaluated by immunohistochemistry in hBDA tissue samples of 59 patients. RESULTS: TRPC1, TRPC6, TRPM7, TRPM8, and TRPV6 channels were overexpressed in hBDA compared to the adjacent non-tumoral tissue. Most interestingly, TRPC1, TRPM7 and TRPM8 expression strongly correlated with proliferative parameters (SBR grade, Ki67 proliferation index, and tumor size), and TRPV6 was mainly overexpressed in the invasive breast cancer cells. Using laser capture microdissection, we found that TRPV6 expression was higher in invasive areas, compared to the corresponding non-invasive ones. Moreover, TRPV6 silencing inhibited MDA-MB-231 migration and invasion, and MCF-7 migration. CONCLUSION: TRP channels are aberrantly expressed in hBDA, hBCE primary cultures, and cell lines, and associated with pathological parameters. The high expression of TRP channels in tumors suggests the potential of these channels for diagnostic, prognosis and/or therapeutic approaches in human breast ductal adenocarcinoma.
Assuntos
Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Canais de Potencial de Receptor Transitório/metabolismo , Neoplasias da Mama/metabolismo , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Proteínas Serina-Treonina Quinases , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPC/metabolismo , Canal de Cátion TRPC6 , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
BACKGROUND: The calcium-permeable cation channel TRPM8 (melastatin-related transient receptor potential member 8) is over-expressed in several cancers. The present study aimed at investigating the expression, function and potential regulation of TRPM8 channels by ER alpha (estrogen receptor alpha) in breast cancer. METHODS: RT-PCR, Western blot, immuno-histochemical, and siRNA techniques were used to investigate TRPM8 expression, its regulation by estrogen receptors, and its expression in breast tissue. To investigate the channel activity in MCF-7 cells, we used the whole cell patch clamp and the calcium imaging techniques. RESULTS: TRPM8 channels are expressed at both mRNA and protein levels in the breast cancer cell line MCF-7. Bath application of the potent TRPM8 agonist Icilin (20 microM) induced a strong outwardly rectifying current at depolarizing potentials, which is associated with an elevation of cytosolic calcium concentration, consistent with established TRPM8 channel properties. RT-PCR experiments revealed a decrease in TRPM8 mRNA expression following steroid deprivation for 48 and 72 hours. In steroid deprived medium, addition of 17-beta-estradiol (E2, 10 nM) increased both TRPM8 mRNA expression and the number of cells which respond to Icilin, but failed to affect the Ca2+ entry amplitude. Moreover, silencing ERalpha mRNA expression with small interfering RNA reduced the expression of TRPM8. Immuno-histochemical examination of the expression of TRPM8 channels in human breast tissues revealed an over-expression of TRPM8 in breast adenocarcinomas, which is correlated with estrogen receptor positive (ER+) status of the tumours. CONCLUSION: Taken together, these results show that TRPM8 channels are expressed and functional in breast cancer and that their expression is regulated by ER alpha.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Canais de Cátion TRPM/metabolismo , Adenocarcinoma/genética , Western Blotting , Neoplasias da Mama/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Potenciais da Membrana , Técnicas de Patch-Clamp , Pirimidinonas/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/genética , Fatores de TempoRESUMO
Unfractionated heparin (UFH) is the main anticoagulante used in intensive care unit. The anticoagulant effect is monitored by activated partial thrombin time (aPTT) and anti-Xa activity (anti-Xa) measurement. However, delayed centrifugation induces platelet factor 4 (PF4) release and anti-Xa decrease. Several studies have concluded that aPTT and anti-Xa measurement should be performed within 2 hours in citrated anticoagulant but may be delayed longer in citrate theophylline adenosine and dypiridamol (CTAD) anticoagulant. The objective of this study was to compare the stability of both aPTT and anti-Xa in citrate and CTAD samples, and to determine the effect of delayed centrifugation on both aPTT, anti-Xa results, and PF4 release in citrate samples only. METHODS: aPTT and anti-Xa were measured in citrate and CTAD anticoagulant samples from 93 patients. Delayed centrifugation was performed in citrate samples from 31 additional patients, with hourly aPTT and anti-Xa measurement from 1 to 6 hours. In 14 of these last patients, PF4 release was also evaluated with Human CXCL4/PF4 Quantikine ELISA Kit. RESULTS: We observed a significant correlation between citrate and CTAD anticoagulant for aPTT (r2=0.94) and anti-Xa (r2=0.95). With Bland-Altman correlation, a minor bias was observed for anti-Xa (-0.025±0.041). Delayed centrifugation in citrated anticoagulant showed an excellent concordance from 1 to 4 hours for aPTT (-4.0±5.3 s) and anti-Xa (1.10-9±0.058 UI/mL) measurements. Moreover, PF4 release was not different between 1 hour (31.5±14.7 ng/mL) and 4 hours (33.8±11.8 ng/mL). CONCLUSION: We have demonstrated that anti-Xa measurement for unfractionated heparin should be done 4 hours in citrated plasma and that CTAD was not better than citrate. However, these initial findings require confirmation using other aPTT and calibrated anti-Xa assays.
Assuntos
Anticoagulantes/farmacologia , Coleta de Amostras Sanguíneas/métodos , Monitoramento de Medicamentos/métodos , Heparina/uso terapêutico , Adenosina/química , Adenosina/farmacologia , Anticoagulantes/química , Anticoagulantes/uso terapêutico , Testes de Coagulação Sanguínea/métodos , Preservação de Sangue/métodos , Centrifugação/métodos , Fracionamento Químico/métodos , Ácido Cítrico/química , Ácido Cítrico/farmacologia , Dipiridamol/química , Dipiridamol/farmacologia , Fator Xa/metabolismo , Inibidores do Fator Xa/análise , Inibidores do Fator Xa/sangue , Heparina/análise , Humanos , Tempo de Tromboplastina Parcial , Teofilina/química , Teofilina/farmacologia , Tempo de Trombina , Fatores de TempoRESUMO
Many recent studies have demonstrated the involvement of endoplasmic reticulum (ER) stress in the development of cardiac diseases and have suggested that modulation of ER stress response could be cardioprotective. Previously, we demonstrated that the deacetylase Sirtuin 1 (SIRT1) attenuates ER stress response and promotes cardiomyocyte survival. Here, we investigated whether and how autophagy plays a role in SIRT1-afforded cardioprotection against ER stress. The results revealed that protective autophagy was initiated before cell death in response to tunicamycin (TN)-induced ER stress in cardiac cells. SIRT1 inhibition decreased ER stress-induced autophagy, whereas its activation enhanced autophagy. In response to TN- or isoproterenol-induced ER stress, mice deficient for SIRT1 exhibited suppressed autophagy along with exacerbated cardiac dysfunction. At the molecular level, we found that in response to ER stress (i) the extinction of eEF2 or its kinase eEF2K not only reduced autophagy but further activated cell death, (ii) inhibition of SIRT1 inhibited the phosphorylation of eEF2, (iii) eIF2α co-immunoprecipitated with eEF2K, and (iv) knockdown of eIF2α reduced the phosphorylation of eEF2. Our results indicate that in response to ER stress, SIRT1 activation promotes cardiomyocyte survival by enhancing autophagy at least through activation of the eEF2K/eEF2 pathway.
Assuntos
Autofagia , Quinase do Fator 2 de Elongação/metabolismo , Estresse do Retículo Endoplasmático , Sirtuína 1/metabolismo , Animais , Autofagia/efeitos dos fármacos , Quinase do Fator 2 de Elongação/antagonistas & inibidores , Quinase do Fator 2 de Elongação/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Tunicamicina/farmacologiaRESUMO
Because transient receptor potential (TRP) channels have been implicated in tumor progression, we have investigated the potential role of TRPM7 channel in breast cancer cell proliferation. Under whole cell patch clamp, a Mg(2+)-inhibited cationic (MIC) current was observed in MCF-7 cells. This current was characterized by an inward current and a strong outward rectifying current that were both inhibited in a concentration-dependent manner by the presence of intracellular Mg(2+) or Mg(2+)-ATP. The inward current was reduced by La(3+), and the outward current was sensitive to 2-aminoethoxydiphenyl borate (2-APB), spermine, La(3+), and flufenamic acid. Importantly, a similar MIC current was also recorded in the primary culture of human breast cancerous epithelial cells (hBCE). Moreover, TRPM7 transcripts were found in both hBCE and MCF-7 cells. In MCF-7 cells, the MIC current was inhibited by TRPM7 small interfering RNA. Interestingly, we found that cell proliferation and intracellular Ca(2+) concentration were also reduced by TRPM7 silencing in MCF-7 cells. TRPM7 channels were also found in both human breast cancer and healthy tissues. Importantly, TRPM7 channel was overexpressed in grade III breast cancer samples associated with important Ki67 or tumor size. Our findings strongly suggest that TRPM7 is involved in the proliferative potentiality of breast cancer cells, probably by regulating Ca(2+) influx.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Canais de Cátion TRPM/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Magnésio/metabolismo , Manganês , Potenciais da Membrana/fisiologia , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/genética , Técnicas de Cultura de TecidosRESUMO
Aims: Endoplasmic reticulum (ER) stress has recently emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms by which ER stress leads to cardiac dysfunction remain poorly understood. Methods and results: In this study, we evaluated the early cardiac effects of ER stress induced by tunicamycin (TN) in mice. Echocardiographic analysis indicated that TN-induced ER stress led to a significant impairment of the cardiac function. Electron microscopic observations revealed that ultrastructural changes of cardiomyocytes in response to ER stress manifested extensively at the level of the reticular membrane system. Smooth tubules of sarcoplasmic reticulum in connection with short sections of rough ER were observed. The presence of rough instead of smooth reticulum was increased at the interfibrillar space, at the level of dyads, and in the vicinity of mitochondria. At the transcriptional level, ER stress resulted in a substantial decrease in the expression of the major regulator of mitochondrial biogenesis PGC-1α and of its targets NRF1, Tfam, CS, and COXIV. At the functional level, ER stress also induced an impairment of mitochondrial Ca2+ uptake, an alteration of mitochondrial oxidative phosphorylation, and a metabolic remodelling characterized by a shift from fatty acid to glycolytic substrate consumption. Conclusions: Our findings show that ER stress induces cytoarchitectural and metabolic alterations in cardiomyocytes and provide evidences that ER stress could represent a primary mechanism that contributes to the impairment of energy metabolism reported in most cardiac diseases.
Assuntos
Estresse do Retículo Endoplasmático , Cardiopatias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ácidos Graxos/metabolismo , Glicólise , Cardiopatias/induzido quimicamente , Cardiopatias/patologia , Cardiopatias/fisiopatologia , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Camundongos , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Fator 1 Relacionado a NF-E2/genética , Fator 1 Relacionado a NF-E2/metabolismo , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , TunicamicinaRESUMO
BACKGROUND: TRP channels have been shown to be involved in tumour generation and malignant growth. However, the expression of these channels in breast cancer remains unclear. Here we studied the expression and function of endogenous TRPC6 channels in a breast cancer cell line (MCF-7), a human breast cancer epithelial primary culture (hBCE) and in normal and tumour breast tissues. METHODS: Molecular (Western blot and RT-PCR), and immunohistochemical techniques were used to investigate TRPC6 expression. To investigate the channel activity in both MCF-7 cells and hBCE we used electrophysiological technique (whole cell patch clamp configuration). RESULTS: A non selective cationic current was activated by the oleoyl-2-acetyl-sn-glycerol (OAG) in both hBCE and MCF-7 cells. OAG-inward current was inhibited by 2-APB, SK&F 96365 and La3+. TRPC6, but not TRPM7, was expressed both in hBCE and in MCF-7 cells. TRPC3 was only expressed in hBCE. Clinically, TRPC6 mRNA and protein were elevated in breast carcinoma specimens in comparison to normal breast tissue. Furthermore, we found that the overexpression of TRPC6 protein levels were not correlated with tumour grades, estrogen receptor expression or lymph node positive tumours. CONCLUSION: Our results indicate that TRPC6 channels are strongly expressed and functional in breast cancer epithelial cells. Moreover, the overexpression of these channels appears without any correlation with tumour grade, ER expression and lymph node metastasis. Our findings support the idea that TRPC6 may have a role in breast carcinogenesis.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Humanas/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Canais de Cátion TRPC/biossíntese , Western Blotting , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/citologia , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPC/fisiologia , Canal de Cátion TRPC6RESUMO
Over the past decade, endoplasmic reticulum (ER) stress has emerged as an important mechanism involved in the pathogenesis of cardiovascular diseases including heart failure. Cardiac therapy based on ER stress modulation is viewed as a promising avenue toward effective therapies for the diseased heart. Here, we tested whether sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, participates in modulating ER stress response in the heart. Using cardiomyocytes and adult-inducible SIRT1 knockout mice, we demonstrate that SIRT1 inhibition or deficiency increases ER stress-induced cardiac injury, whereas activation of SIRT1 by the SIRT1-activating compound STAC-3 is protective. Analysis of the expression of markers of the three main branches of the unfolded protein response (i.e., PERK/eIF2α, ATF6 and IRE1) showed that SIRT1 protects cardiomyocytes from ER stress-induced apoptosis by attenuating PERK/eIF2α pathway activation. We also present evidence that SIRT1 physically interacts with and deacetylates eIF2α. Mass spectrometry analysis identified lysines K141 and K143 as the acetylation sites on eIF2α targeted by SIRT1. Furthermore, mutation of K143 to arginine to mimic eIF2α deacetylation confers protection against ER stress-induced apoptosis. Collectively, our findings indicate that eIF2α deacetylation on lysine K143 by SIRT1 is a novel regulatory mechanism for protecting cardiac cells from ER stress and suggest that activation of SIRT1 has potential as a therapeutic approach to protect the heart against ER stress-induced injury.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Sirtuína 1/metabolismo , Acetilação , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Carbazóis/farmacologia , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/genética , Proteínas de Choque Térmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Tunicamicina/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL) are the major metabolites of Zearalenone (ZEN) and are known to induce many toxic effects. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in α- and ß-ZOL-mediated toxicity in human kidney cells (HEK293) and evaluated the effect of a common dietary compound Crocin (CRO), from saffron. We show that α- and ß-ZOL treatment induces ER stress as evidenced by the upregulation of the 78 kDa glucose-regulated protein (GRP78) and the Growth arrest and DNA damage-inducible protein (GADD34). Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm) and activation of caspases. We also demonstrate that the antioxidant properties of CRO help to prevent ER stress and reduce α- and ß-ZOL-induced apoptosis in HEK293 cells. Our results suggest that saffron consumption might be helpful to prevent α- and ß-ZOL-induced ER stress and toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Carotenoides/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Zeranol/análogos & derivados , Antioxidantes/metabolismo , Caspases/metabolismo , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Zeranol/química , Zeranol/toxicidadeRESUMO
Zearalenone (ZEN) and its metabolites are found in many food products and are known to induce many toxic effects. The major ZEN metabolites are α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). The mechanisms by which they mediate their cytotoxic effects are not well known and seem to differ depending on the type of cells. We investigated the possible underlying mechanism in α-ZOL and ß-ZOL-induced toxicity in HCT116 cells. We showed that cell treatment with α-ZOL/ß-ZOL generated endoplasmic reticulum (ER) stress and activated the Unfolded Protein Response (UPR) as evidenced by XBP1 mRNA splicing and up-regulation of GADD34, GRP78, ATF4 and CHOP. Apoptosis was triggered by ZEN metabolites-induced ER stress, and executed through a mitochondria-dependent pathway, characterized by a loss of mitochondrial transmembrane potential (ΔΨm), a downstream generation of O2â¢(-) and caspase 3 activation. Cellular deficiency of the pro-apoptotic proteins Bax and Bak protected cells against α/ß-ZOL-induced toxicity. However, treatment with α-ZOL or ß-ZOL combined with Quercetin (QUER), a common dietary flavonoid with well-known antioxidant activity, significantly reduced damage induced by α and ß-ZOL in all tested markers. We concluded that QUER protects against the cellular toxicity of α and ß-ZOL.×.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Quercetina/farmacologia , Zearalenona/farmacologia , Zeranol/análogos & derivados , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Análise de Variância , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteína Fosfatase 1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , Zeranol/farmacologiaRESUMO
BACKGROUND: Previous studies suggest that CaMKII activity is required for frequency-dependent acceleration of relaxation (FDAR) in ventricular myocytes. We propose that the underlying mechanism involves CaMKII-dependent regulation of myofilament Ca(2+) sensitivity. METHODS AND RESULTS: Cardiac function was measured in mice using murine echo machine. [Ca(2+)]i and sarcomere length were measured by IonOptix Ca(2+) image system. Increasing pacing rate from 0.5 to 4 Hz in left ventricular myocytes induced frequency-dependent myofilament Ca(2+) desensitization (FDMCD) and FDAR. Acute inhibition of PKA or PKC had no effect, whereas CaMKII inhibition abolished both FDMCD and FDAR. Co-immunoprecipitation of CaMKII and troponin I (TnI) has been detected and CaMKII inhibition significantly reduced serine residue phosphorylation of TnI. Finally, chronic inhibition of CaMKII in vivo reduced TnI phosphorylation and abolished both FDAR and FDMCD, leading to impaired diastolic function. CONCLUSIONS: Our results suggest that CaMKII-dependent TnI phosphorylation is involved in FDMCD and the consequent FDAR and that CaMKII inhibition removes this mechanism and thus induces diastolic dysfunction.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Miofibrilas/metabolismo , Aceleração , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.
Assuntos
Apoptose/efeitos dos fármacos , Carotenoides/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Quercetina/farmacologia , Zearalenona/farmacologia , Chaperona BiP do Retículo Endoplasmático , Células HCT116 , Células HEK293 , HumanosRESUMO
Patulin (PAT) is a toxic metabolite produced by several filamentous fungi of the genera of Penicillium, Aspergillus, and Byssochlamys. PAT is the most common mycotoxin found in apples and apple-based products including juice, compotes, cider, and baby food. Exposure to this mycotoxin has been reported to induce intestinal and kidney injuries. This study investigated the mechanism of PAT-induced toxicity in human colon carcinoma (HCT116) and embryonic kidney cells (HEK293). We demonstrated that PAT activated endoplasmic reticulum (ER) and unfolded protein response as evidenced by up-regulation of GRP78 and GADD34, splicing of XBP1 mRNA, and expression of the proapoptotic factor CHOP. This ER stress response was accompanied by the induction of the mitochondrial apoptotic pathway. Apoptosis occurred with ROS production, drop in mitochondrial membrane potential and caspase activation. Further, we showed that deficiency of the proapoptotic protein Bax or Bak protected cells against PAT-induced apoptosis. The treatment of cells with the ROS scavenger N-acetyl cysteine inhibits the ER stress response and prevents mitochondrial apoptosis. Collectively, our data provide new mechanistic insights in the signaling pathways of the cell death induced by PAT and demonstrate that PAT induces cytotoxicity through a ROS-dependent mechanism involving ER stress and activation of mitochondrial apoptotic pathway in human intestinal and kidney cells.