RESUMO
Matrix metalloproteinase-2 (MMP-2) is constitutively expressed in vascular smooth muscle cells (VSMCs) and up-regulated in atherosclerotic lesion by various stimuli, such as oxidized low-density lipoprotein (oxLDL). Calcium-sensing receptor (CaSR) is also expressed in VSMCs, but it remains unclear whether CaSR is associated with overproduction of MMP-2 in VSMCs. In this study, the expression of MMP-2 was detected by real-time PCR and Western blot analysis, and the gelatinolytic activity of MMP-2 was measured using gelatin zymography. Our results showed that oxLDL enhanced MMP-2 expression and activity in rat aortic VSMCs in a time- and dose-dependent manner. In addition, CaSR expression was up-regulated by oxLDL. Manipulating CaSR function in these cells by NPS2390 (an antagonist of CaSR) or GdCl(3) (an agonist of CaSR) affected the oxLDL-induced MMP-2 production. In VSMCs, oxLDL stimulated the rapid activation of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, as determined by Western blot analysis. Phosphorylation of Akt and MMP-2 production stimulated by oxLDL were attenuated by LY294002 (a specific inhibitor of PI3K). Activation of Akt was suppressed by NPS2390 but enhanced by GdCl(3). In contrast, oxLDL had no stimulatory effect on the phosphorylation of JNK, and pretreatment with SP600125 (an inhibitor of JNK) produced no significant effect on oxLDL-induced MMP-2 production. These results suggest that CaSR mediates oxLDL-induced MMP-2 production in VSMCs via PI3K/Akt signal pathway.
Assuntos
Lipoproteínas LDL/metabolismo , Metaloproteinase 2 da Matriz/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacologia , Animais , Antracenos/farmacologia , Aorta/metabolismo , Aterosclerose/metabolismo , Células Cultivadas , Cromonas/farmacologia , Gadolínio/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Morfolinas/farmacologia , Músculo Liso Vascular/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Detecção de Cálcio/biossínteseRESUMO
1. Calcium-sensing receptors (CaSR) exist in a variety of tissues. In 2010, we first identified its functional expression in Buffalo rat liver (BRL) cells and demonstrated that the activation of CaSR was involved in an increased intracellular calcium through the Gq subunit-phospholipase C-inositol triphosphate pathway. However, its role and related mechanism in hepatic ischaemia/reperfusion (I/R) injury is still unclear. 2. Therefore, in the present study, BRL cells were incubated in ischaemia-mimetic solution for 4 h, then reincubated in the normal culture medium for 10 h to establish a simulated I/R model. We assayed the apoptotic ratio of BRL cells by flow cytometry and Hoechst 33342 staining; analyzed the expression of CaSR, cytochrome c (Cyt-c), caspase-3, Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), and p38 by Western blotting; and measured the concentration of intracellular calcium by laser-scanning confocal microscopy. 3. The results showed that simulated I/R increased the expression of CaSR and induced apoptosis in BRL cells. GdCl(3), a specific activator of CaSR, further increased CaSR expression, intracellular calcium, and apoptosis in BRL cells during I/R. The activation of CaSR downregulated Bcl-2 expression, upregulated Cyt-c, caspase-3, and Bax expressions, and promoted p38 and ERK-1/2 phosphorylation. 4. In conclusion, increased CaSR expression plays a vital role in apoptosis induced by I/R injury, in which its mechanism is related with calcium overload and the activation of the mitochondrial and mitogen-activated protein kinase apoptotic pathways. The regulation of CaSR activity might serve as a novel pharmacological target to prevent and treat liver disease.
Assuntos
Apoptose/fisiologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Apoptose/genética , Cálcio/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Citocromos c/genética , Citocromos c/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Inositol Polifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Endogâmicos BUF , Receptores de Detecção de Cálcio/genética , Traumatismo por Reperfusão/genética , Transdução de Sinais/fisiologia , Fosfolipases Tipo C/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The expression and function of calcium-sensing receptor (CaSR) in differentiated THP-1 (human acute monocytic leukemia cell line) cells are unknown currently. This study investigated above-mentioned issues using TRAP staining, immunofluorescence staining, Western blotting, ELISA, and Laser Confocal Scanning Microscopy techniques. We found that CaSR protein was expressed, and mainly located in the membrane and cytoplasm in differentiated THP-1 cells. Elevated extracellular calcium or GdCl(3) (an agonist of CaSR) raised intracellular calcium concentration. And this increase was inhibited or abolished by NPS2390 (an inhibitor of CaSR), U73122 (a specific inhibitor of phospholipase C, PLC) or thapsigargin (a Ca(2+)-ATPase inhibitor). The extracellular GdCl(3) elevation stimulated both of IL-1beta and TNFalpha release, and this effect of GdCl(3) was inhibited by NPS2390. In conclusion, CaSR is functionally expressed in differentiated THP-1 cells, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC- inositol triphosphate (IP3) pathway and commits to cytokine secretion. These results suggest that CaSR might be involved in a variety of pathological processes mediated by activated monocyte-macrophages.
Assuntos
Cálcio/metabolismo , Diferenciação Celular , Leucemia Monocítica Aguda/metabolismo , Leucemia Monocítica Aguda/patologia , Receptores de Detecção de Cálcio/metabolismo , Anti-Inflamatórios/farmacologia , Western Blotting , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Gadolínio/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Transdução de Sinais , Células Tumorais Cultivadas , Fosfolipases Tipo C/metabolismoRESUMO
BACKGROUND: Polyamines and nitric oxide (NO) have been involved in the pathogenesis of cardiac hypertrophy. NO can regulate cardiac ion channels by direct actions on G-proteins and adenyl cyclase. The present study was undertaken to elucidate the molecular mechanism of interactions with polyamines and NO in cardiac hypertrophy. METHODS: Cardiaomyocyte hypertrophy was induced by angiotensinII (AngII). Hypertrophy was estimated by cell-surface area, atrial natriuretic peptide (ANP) mRNA expression, and the immunofluorescence of phalloidin. Pretreatment with alpha-difluoromethylornithine (DFMO) was done to deplete putrescine; KT5823 pretreatment was carried out to block the nitric oxide/cGMP-dependent protein kinase type-I (NO/PKG-I) pathway. Expressions of endothelial nitric oxide synthase (eNOS), PKG-I, c-fos and c-myc were analyzed by western blotting and immunofluorescence. The intracellular concentration of free calcium ([Ca2+](i)) was determined by confocal laser scanning microscopy. RESULTS: Hypertrophy of cardiomyocytes was induced by AngII, this caused an increase in putrescine, spermidine and total polyamine pool in association with a decreased level of NO. Expressions of eNOS and PKG-I were down-regulated, [Ca2+](i) was increased, and expressions of c-Fos and c-Myc upregulated. DFMO reversed these changes induced by AngII. CONCLUSIONS: Downregulation of polyamines inhibits cardiomyocyte hypertrophy, which is closely related to [Ca2+](i) and the NO/PKG-I pathway.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Angiotensinas/farmacologia , Animais , Fator Natriurético Atrial/genética , Fator Natriurético Atrial/metabolismo , Cálcio/metabolismo , Carbazóis/farmacologia , Crescimento Celular , Proteína Quinase Dependente de GMP Cíclico Tipo I , Regulação para Baixo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
OBJECTIVE: To investigate the anti-tumor effect and the mechanism of down-regulation of HER - 2 by cabamazepine in SKBR - 3 cells , a breast cancer cell line with HER - 2 over - expression. METHODS: Western blotting was performed to evaluate the Her-2 expression level. The mRNA level of HER-2 was detected by RT-PCR. Immunoprecipitation was applied to detect the chaperon function and acetylation level of HSP90. The viability of cells was tested by MTT assay. RESULTS: Cabamazepine treatment down-regulated HER-2 expression. Only HER-2 protein level decrease was observed with 10 micromol/L cabamazepine treatment, but both protein and mRNA expressions were inhibited by 100 micromol/L cabamazepine. Cabamazepine treatment could induce a higher acetylation level of HSP90 and destroy its chaperon function. Cabamazepine exerted synergism with Herceptin in promoting HER-2 protein degradation and synergism or potentiation with Herceptin or 17-AAG in inhibition of proliferation. CONCLUSION: Cabamazepine can reduce the expression of HER-2 and show a synergistic effect with Herceptin or 17-AAG. There may be potential benefits of carbamazepine for cancer therapy in future. HER-2;
Assuntos
Carbamazepina/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Receptor ErbB-2/metabolismo , Acetilação/efeitos dos fármacos , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Benzoquinonas/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Inibidores de Histona Desacetilases , Humanos , Lactamas Macrocíclicas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TrastuzumabRESUMO
BACKGROUND & OBJECTIVE: Carbamazepine, which has been used as an anti-epileptic drug in clinic for many years, is currently recognized as a histone deacetylase inhibitor (HDI), most of which showed anti-tumor characteristics. This study was to investigate the inhibitory effect of carbamazepine on estrogen dependent breast cancer cell lines with estrogen receptor alpha (ERalpha) expression and further explore the underlying mechanisms. METHODS: Sulforhodamine B viability assay was used to evaluate the viability of various cells treated with different drugs. Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were performed to detect the protein and mRNA expression of ERalpha and Cyclin D1. Immunofluorescence assay was employed to observe HER-2 expression in MCF-7RT cells, which were resistant to tamoxifen. Immunoprecipitation was performed to detect the chaperon function and acetylation level of Hsp90. RESULTS: Carbamazepine treatment could inhibit the proliferation of MCF-7 and T47D cells stimulated by estradiol (P<0.01). Carbamazepine and 4-hydroxytamoxifen (4-OHT) demonstrated a synergic effect on the inhibition of proliferation of MCF-7 cells stimulated by estradiol (q=1.00). Cabamazepine reversed the proliferation of MCF-7RT cells stimulated by 4-hydroxytamoxifen (P<0.01). Carbamazepine treatment could decrease the expression of ERalpha and Cyclin D1 at protein and mRNA level in ERalpha-positive cells and could reduce HER-2 expression in MCF-7RT cells. The decrease of ERalpha and Cyclin D1 expression was inhibited by MG132, an inhibitor of 26S proteosome. Carbamazepine treatment elevated the acetylation level of Hsp90 and disrupted its chaperon function. CONCLUSIONS: Carbamazepine shows significant anti-proliferation effect in ERalpha-positive breast cancer cell lines and this might be due to the enhancement of proteosome-mediated degradation of ERalpha and Cyclin D1 by carbamazepine. Furthermore, carbamazepine could reverse HER-2 dependent drug resistance to 4-OHT by reducing HER-2 expression.
Assuntos
Neoplasias da Mama/patologia , Carbamazepina/farmacologia , Proliferação de Células/efeitos dos fármacos , Ciclina D1/biossíntese , Receptor alfa de Estrogênio/biossíntese , Acetilação/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Estradiol/metabolismo , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/metabolismo , Inibidores de Histona Desacetilases , Humanos , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
Successful application of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL) has been attracting worldwide interest, but the exact mechanism for the action of As2O3 remains somewhat obscure. In the present work, we show for the first time that As2O3 facilitates the DIDS-sensitive anion transport activity of band 3 protein in red blood cells (RBCs) isolated from normal adults and APL patients. To elucidate the effect of As2O3 on band 3 protein, constructs encoding the full length of the band 3 transmembrane domain (mdb3) and its C-terminal deletion forms were transfected into yeast cells by a yeast display system. The results demonstrate that deletion of the C-terminal 16 residues of mdb3 (mdb3-d16) does not affect anion transport activity of mdb3 or its sensitivity to DIDS, but decreases its sensitivity to As2O3 in the yeast cell. More intriguingly, the forced expression of intact mdb3 by transfection significantly induces cell apoptosis in HeLa cells, to a higher degree than in cells transfected with mdb3-d16 or empty vector. Expression of activated caspase 3 in HeLa cells also indicates that the C-terminal 16 residues are important for mdb3-mediated apoptosis in cells treated with As2O3. Our results provide the first evidence that As2O3 enhances the anion transport activity of band 3 and the action is related with the C-terminal 16 residues of the protein.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Arsenicais/uso terapêutico , Eritrócitos/efeitos dos fármacos , Leucemia Promielocítica Aguda/tratamento farmacológico , Óxidos/uso terapêutico , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Adulto , Proteína 1 de Troca de Ânion do Eritrócito/genética , Antineoplásicos/farmacologia , Trióxido de Arsênio , Arsenicais/farmacologia , Eritrócitos/metabolismo , Feminino , Deleção de Genes , Células HeLa , Humanos , Técnicas In Vitro , Leucemia Promielocítica Aguda/sangue , Masculino , Óxidos/farmacologia , Transfecção , Leveduras/genéticaRESUMO
BACKGROUND & OBJECTIVE: Membrane domain of band 3 protein (mdb3) mediates transmembrane exchange of chloride and bicarbonate, and regulates intracellular pH. It has been found recently that abnormality of CI(-)/HCO3(-) exchange, which mainly leads to change of intracellular pH, may be involved in cell proliferation and apoptosis. This study was to explore expression of band 3 protein on erythrocytes, and its impact on proliferation of K562 cells. METHODS: Anion transport activity of band 3 protein on erythrocytes of 8 malignant tumor patients was measured using SPQ fluorescent probe. Expression of band 3 protein was detected by Western blot. Plasmid pYD1-mdb3 was constructed, and transfected into K562 cells. Cl- transport activity and proliferation of K562 cells were detected after transfection. RESULTS: Of the 8 patients, 7 showed increase of anion transport activity on erythrocytes, 5 showed increase of band 3 protein expression. To some extent, expression of mdb3 enhanced proliferation of K562 cells. CONCLUSION: Expression of band 3 protein is enhanced on erythrocytes of some malignant tumors, and might be a candidate marker of malignant tumors.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/biossíntese , Membrana Eritrocítica/metabolismo , Neoplasias/metabolismo , Adulto , Proteína 1 de Troca de Ânion do Eritrócito/genética , Transporte Biológico , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562/citologia , Células K562/metabolismo , Distribuição Aleatória , TransfecçãoRESUMO
By using the C-terminal 112-residue of band 3 to screen the K562 cDNA library, we find that the p16 interacts with band 3, which was confirmed both in yeast and in mammalian cells. Functional experiments show that p16 facilitates the movement of band 3 to plasma membrane with increased anion transport activity in 293t cells. Moreover, expression of endogenous p16 in 293t cells was increased at 24 and 36 h after transfection with band 3. Our findings provide a novel regulation pathway for both band 3 and p16.