RESUMO
The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on pyroptosis of macrophages and the underlying molecular mechanisms. RAW264.7 macrophages were treated with AGE-alb (1, 2, 4 and 6 g/L) and control albumin (C-alb, 4 g/L) for 24 h, or preincubated with MCC950 (1 µmol/L) for 1 h and then treated with AGE-alb (4 g/L) for 24 h. Cell viability and caspase-1 activity were measured by MTT and assay kits, respectively. Lactate dehydrogenase (LDH) activity and the levels of interleukin-1ß (IL-1ß) and IL-18 in media were detected. Cell death degree was evaluated by TUNEL and Hoechst 33342/PI staining. The protein levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), procaspase-1 and cleaved caspase-1 were assessed by Western blot. The results showed that AGE-alb treatment caused obvious decrease in cell viability and increases in LDH leakage and the percentages of TUNEL- or PI-positive cells in a concentration-dependent manner. Additionally, AGE-alb promoted IL-1ß and IL-18 secretion, upregulated NLRP3 expression, and increased caspase-1 activity especially at the dose of 4 and 6 g/L. However, MCC950 (an NLRP3 inhibitor) pretreatment inhibited significantly the decrease in cell viability and the increases in LDH leakage and percentages of TUNEL- or PI-positive cells induced by AGE-alb. Furthermore, MCC950 attenuated obviously AGE-alb-induced IL-1ß and IL-18 secretion and caspase-1 activation. These results indicate that AGE-alb may induce macrophage pyroptosis, and the mechanism is at least partially by activating NLRP3-caspase-1 pathway.
Assuntos
Regulação da Expressão Gênica , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Piroptose , Albumina Sérica , Caspase 1 , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos Finais de Glicação Avançada , Interleucina-1beta/genética , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Piroptose/efeitos dos fármacos , Albumina Sérica/farmacologia , Albumina Sérica GlicadaRESUMO
The purpose of this study was to investigate whether activating transcription factor 6 (ATF6), a sensor to endoplasmic reticulum stress (ERS), would mediate advanced glycated albumin (AGE-alb)-induced macrophage apoptosis and to elucidate the possible molecular mechanisms. RAW264.7 macrophages were cultured in vitro and treated with AGE-alb (2, 4 and 6 g/L), normal control albumin or tunicamycin (TM, 4 mg/L) for 24 h. ATF6 small interfering RNA (siRNA) was transfected to RAW264.7 cells by Lipofectamine 2000. Cell viability and apoptosis were determined by MTT method and Annexin V-FITC/propidium iodide apoptosis detection kit, respectively. The activities of lactate dehydrogenase (LDH) in medium and caspase-3 in cells were measured by corresponding detection kits. ATF6 nuclear translocation was detected by Western blot and immunofluorescence cytochemistry. Protein and mRNA levels of C/EBP homologous protein (CHOP, a key-signaling component of ERS-induced apoptosis) were detected by Western blot and real-time fluorescence quantitative PCR, respectively. The results showed that similar to TM, AGE-alb increased the expression of CHOP at both the protein and mRNA levels in a concentration dependent manner. ATF6, as a factor that positively regulates CHOP expression, was activated by AGE-alb in a concentration dependent manner. siRNA-mediated knockdown of ATF6 significantly inhibited AGE-alb-induced macrophage injury, as indicated by the increased cell viability and the decreased LDH release, apoptosis and caspase-3 activation. Additionally, ATF6 siRNA attenuated AGE-alb-induced CHOP upregulation at both the protein and mRNA levels. These results suggest that ATF6 and its downstream molecule CHOP are involved in AGE-alb-induced macrophage apoptosis.
Assuntos
Fator 6 Ativador da Transcrição/fisiologia , Apoptose/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Albumina Sérica/farmacologia , Fator de Transcrição CHOP/fisiologia , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Produtos Finais de Glicação Avançada , Macrófagos/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Albumina Sérica GlicadaRESUMO
This study was designed to explore the protective effect of D4F, an apolipoprotein A-I mimetic peptide, on nuclear factor-κB (NF-κB)-dependent Fas/Fas ligand (FasL) pathway-mediated apoptosis in macrophages induced by oxidized low-density lipoprotein (ox-LDL). Our results showed that ox-LDL induced apoptosis, NF-κB P65 nuclear translocation and the upregulation of Fas/FasL pathway-related proteins, including Fas, FasL, Fas-associated death domain proteins (FADD), caspase-8 and caspase-3 in RAW264.7 macrophages, whereas silencing of Fas blocked ox-LDL-induced macrophage apoptosis. Furthermore, silencing of P65 attenuated macrophage apoptosis and the upregulation of Fas caused by ox-LDL, whereas P65 expression was not significantly affected by treatment with Fas siRNA. D4F attenuated the reduction of cell viability and the increase in lactate dehydrogenase leakage and apoptosis. Additionally, D4F inhibited ox-LDL-induced P65 nuclear translocation and upregulation of Fas/FasL pathway-related proteins in RAW264.7 cells and in atherosclerotic lesions of apoE-/- mice. However, Jo2, a Fas-activating monoclonal antibody, reversed the inhibitory effect of D4F on ox-LDL-induced cell apoptosis and upregulation of Fas, FasL and FADD. These data indicate that NF-κB mediates Fas/FasL pathway activation and apoptosis in macrophages induced by ox-LDL and that D4F protects macrophages from ox-LDL-induced apoptosis by suppressing the activation of NF-κB and the Fas/FasL pathway.
Assuntos
Apolipoproteína A-I/farmacologia , Apoptose/efeitos dos fármacos , Proteína Ligante Fas/metabolismo , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor fas/metabolismo , Animais , Apolipoproteínas E/deficiência , Dieta Hiperlipídica , Células Espumosas/patologia , Lipoproteínas LDL/farmacologia , Camundongos , Transporte Proteico , Células RAW 264.7RESUMO
Autophagy is a cellular catabolic process responsible for removing the injured proteins and organelles via lysosome-dependent pathway, and it plays an important role in maintaining cellular homeostasis. Recent studies have shown that autophagy is activated and implicated in the pathogenesis of atherosclerosis. Autophagy can be triggered by oxidative lipids, cytokines and advanced glycation end products, and exerts protective or detrimental functions in the progression of atherosclerosis. However, the precise role and mechanisms of autophagy in different stages of atherosclerosis are still not fully clarified. This review highlights recent findings regarding autophagy response in vascular cells and its potential contribution to atherogenesis. Additionally, the relationship of autophagy with endoplasmic reticulum stress and whether autophagy could be a new therapeutic target for atherosclerosis are also discussed.
Assuntos
Aterosclerose/fisiopatologia , Autofagia , Estresse do Retículo Endoplasmático , Animais , Humanos , LisossomosRESUMO
The purpose of the present study was to investigate the effect of advanced glycated albumin (AGE-alb) on the activation of caspase-12, a key molecule in endoplasmic reticulum stress (ERS)-associated apoptotic pathway, and to elucidate the underlying molecular mechanisms of macrophage apoptosis. RAW264.7 macrophages were treated with AGE-alb (2, 4 and 6 g/L), control albumin (C-alb, 4 g/L), tunicamycin (TM, 4 mg/L), or pretreated with 4-phenylbutyric acid (PBA, 5 mmol/L) for 1 h and then treated with AGE-alb (4 g/L). After incubation for 24 h, the cell viability and apoptosis were determined by using MTT assay and TUNEL detection kit, respectively. Lactate dehydrogenase (LDH) activity in media was determined by using an assay kit. The protein levels of caspase-12 were examined by Western blot analysis. The results showed that like TM (an ERS inducer), incubation with AGE-alb led to significant decrease in viability and increase in LDH activity in media and apoptotic rate in a dose-dependent manner. In addition, AGE-alb induced activation of caspase-12 especially at the concentration of 4 and 6 g/L (P < 0.01), which was similar to TM. However, PBA (an ERS inhibitor) protected RAW264.7 macrophages from AGE-alb-induced decrease in viability and increases in LDH activity and apoptosis. Moreover, PBA also inhibited the caspase-12 activation induced by AGE-alb (P < 0.05). These results suggest that AGE-alb may induce apoptosis in RAW 264.7 macrophages, and the mechanism may be related to the activation of ERS-associated apoptotic pathway mediated by caspase-12.
Assuntos
Apoptose , Macrófagos , Animais , Caspase 12 , Linhagem Celular Tumoral , Sobrevivência Celular , Estresse do Retículo Endoplasmático , Produtos Finais de Glicação Avançada , Camundongos , Fenilbutiratos , Albumina Sérica , Tunicamicina , Albumina Sérica GlicadaRESUMO
BACKGROUND: Ethanol extract of propolis (EEP), rich in flavones, has been known for various biological activities including antioxidant, antiinflammatory and antibiotic activities. Our previous studies have shown that EEP protects endothelial cells from oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and inhibits atherosclerotic lesion development. In this present study, we explored the protective effect of EEP on ox-LDL-induced cytotoxicity in macrophages and specifically the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis. METHODS: EEP was prepared and the total flavonoids content of EEP was determined by the colorimetric method of Chinese Standard (GB/T 20574-2006). The effects of EEP on lipid accumulation, cytotoxicity and apoptosis in RAW264.7 cells induced by ox-LDL or tunicamycin (TM, an ER stress inducer) were assayed using oil red O staining, MTT assay, flow cytometric analysis and so on. Immunofluorescence, Western blot and real time-PCR analysis were then used to further investigate the molecular mechanisms by which EEP protects macrophages from ox-LDL-induced apoptosis. 4-phenylbutyric acid (PBA), an ER stress inhibitor, was used as a positive control. RESULTS: EEP (7.5, 15 and 30 mg/L) not only attenuated ox-LDL-induced lipid accumulation in RAW264.7 macrophages in a dose-dependent manner but also inhibited the decreased cell viability and the increased lactate dehydrogenase (LDH) leakage, caspase-3 activation and apoptosis induced by ox-LDL or tunicamycin (TM, a classical ER stress inducer), which were similar to 4-phenylbutyric acid (PBA, an inhibitor of ER stress) treatment. In addition, like PBA, EEP significantly suppressed the ox-LDL- or TM-induced activation of ER stress signaling pathway including the phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) as well as upregulation of glucose regulated protein 78 (GRP78) and the pro-apoptotic protein CHOP. Furthermore, EEP significantly suppressed ox-LDL intake by macrophages and the upregulation of CD36 induced by ox-LDL. CONCLUSION: These data indicate that EEP may protect macrophages from ox-LDL-induced apoptosis and the mechanism at least partially involves its ability to suppress the CD36-mediated ox-LDL intake and subsequent activation of ER stress-CHOP signalling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Antígenos CD36/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Lipoproteínas LDL/metabolismo , Macrófagos , Própole/farmacologia , Fator de Transcrição CHOP/metabolismo , Animais , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacosRESUMO
The present study was to investigate whether endoplasmic reticulum stress (ERS) was involved in oxidized low density lipoprotein (ox-LDL)-induced scavenger receptor A1 (SR-A1) upregulation in macrophages. RAW264.7 cells were pretreated with 20 mmol/L of 4-phenylbutyric acid (PBA) for 30 min and then treated with ox-LDL (50 mg/L) for 12 h or stimulated with 2 mg/L tunicamycin (TM) or 2 µmol/L thapsigagin (TG) for 4 h. In addition, RAW264.7 cells were incubated with 0.5, 1 and 2 mg/L TM for 4 h or treated with 2 mg/L TM for 1, 2 and 4 h, respectively. The intracellular total cholesterol (TC) content was measured using a tissue/cell total cholesterol assay kit. The protein and mRNA expressions of SR-A1 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot and real-time PCR, respectively. Dil-ox-LDL uptake was detected using a microplate reader. The results showed that ox-LDL-induced cholesterol accumulation in macrophages was attenuated by PBA, an ERS inhibitor. Ox-LDL caused significant SR-A1 upregulation with concomitant activation of ERS as assessed by upregulation of GRP78, whereas PBA significantly inhibited the ox-LDL-induced SR-A1 upregulation (P < 0.05) and slightly decreased GRP78 expression by 39.3% (P = 0.057). TM, an ERS inducer, upregulated SR-A1 protein expression and ox-LDL uptake in dose- and time-dependent manner, but had no significant effect on SR-A1 mRNA level. However, the TM- or TG-induced SR-A1 upregulation and ox-LDL uptake were significantly mitigated by PBA. These data indicate that ERS plays a critical role in ox-LDL-induced SR-A1 upregulation, which in turn enhances the foam cell formation by uptaking more ox-LDL.
Assuntos
Estresse do Retículo Endoplasmático , Lipoproteínas LDL/farmacologia , Macrófagos/metabolismo , Receptores Depuradores Classe A/metabolismo , Animais , Linhagem Celular , Colesterol/metabolismo , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Regulação para CimaRESUMO
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-inflammatory, antioxidant and antithrombotic properties and plays a protective role against atherosclerosis (AS). The purpose of the present study is to explore the effects of oxidized low density lipoprotein (ox-LDL) on the expression of PEDF in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and incubated with ox-LDL at different concentrations (6.25, 12.5, 25, 50, 100 and 150 mg/L) for 24 h. Apoptosis of endothelial cells were assayed by morphological staining and flow cytometry. The intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Cell viability was assayed by MTT assay. PEDF protein and mRNA expressions in HUVECs were analyzed by Western blot and quantitative real-time PCR, respectively. The results showed that ox-LDL significantly induced apoptosis, reduced cell viability, increased intracellular ROS levels and decreased the PEDF expression in HUVECs in a concentration-dependent manner. Ox-LDL at 50 mg/L obviously decreased the PEDF protein expression compared with control group (P < 0.05), whereas 25 mg/L ox-LDL already markedly reduced the PEDF mRNA expression (P < 0.05). In conclusion, the results suggest that ox-LDL down-regulates the PEDF expression through an increased ox-LDL-induced intracellular production of ROS.
Assuntos
Proteínas do Olho/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Apoptose , Células Cultivadas , Regulação para Baixo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
The purposes of the present study were to investigate the inhibitory effect of quercetin (QUE) preconditioning on endoplasmic reticulum stress (ERS) inducer tunicamycin (TM)-induced apoptosis in RAW264.7 macrophages and the underlying molecular mechanisms. RAW264.7 cells were pretreated with different concentrations (20, 40, and 80 µmol/L) of QUE for 30 min and then treated with TM (5 mg/L) for 12 h. Cell viability and apoptosis were determined using MTT assay and Annexin V-FITC apoptosis detection kit, respectively. The nuclear translocation of activating transcription factor 6 (ATF6) in cells was detected by immunofluorescence analysis and Western blot. Protein and mRNA expressions of C/EBP homologous protein (CHOP) and Bcl-2 were examined by Western blot and real-time PCR, respectively. The results showed that TM reduced cell viability and induced apoptosis in RAW264.7 macrophages. The cytotoxic effects of TM were significantly inhibited by QUE pretreatment at the concentrations of 40 and 80 µmol/L. Interestingly, we found that QUE also significantly suppressed the TM-induced translocation of ATF6, an ERS sensor, from the cytoplasm to the nucleus. In addition, exposure of RAW264.7 macrophages to TM resulted in a significant increase of the expression of CHOP, a transcription factor regulated by ATF6 under conditions of ERS, as well as a decrease of Bcl-2 at transcript and protein levels. QUE blocked these effects in a dose-dependent manner. These data indicate that QUE can protect RAW264.7 cells from TM-induced apoptosis and that the mechanism at least partially involves its ability to inhibit the ATF6-CHOP signaling pathway.
Assuntos
Apoptose , Estresse do Retículo Endoplasmático , Macrófagos/efeitos dos fármacos , Quercetina/farmacologia , Tunicamicina/farmacologia , Fator 6 Ativador da Transcrição/metabolismo , Animais , Sobrevivência Celular , Macrófagos/citologia , Camundongos , Fator de Transcrição CHOP/metabolismoRESUMO
BACKGROUND: A number of effective prevention measures have been introduced in attempts to substantially reduce both the incidence and mortality due to many kinds of cancer. The search for new anti-cancer compounds in foods or in plant medicines is one realistic and promising approach to prevention. Chinese medicines provide a rich pool of novel and efficacious agents for cancer prevention and treatment. Previously it was demonstrated that hyperin extracted from the Manchurian rhododendron leaf reduces the proliferation of many cancer cells. The present study was carried out to evaluate its effects on human endometrial cancer cell viability and apoptosis and to investigate its mechanisms of action in RL952 cells. METHODS: Cell viability was measured using the MTT assay. Intracellular calcium ions were detected using laser-scanning confocal microscopy. The effects of hyperin on apoptosis related proteins in RL952 cells were examined using Western blot analysis. RESULTS: The growth of RL952 cells was inhibited by treatment with hyperin. OD values of caspase-3 and caspase-9 were increased and expression of bcl-2 was increased and bax was decreased in protein levels in RL952 cells after 24 h of hyperin treatment, Moreover, intracellular calcium accumulation occurred in hyperin-treated cells. CONCLUSIONS: These results suggest that hyperin may play an important role in tumor growth suppression by inducing apoptosis in human endometrial cells via a Ca2+-related mitochondrion apoptotic pathway in RL952 cells.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Endométrio/patologia , Mitocôndrias/efeitos dos fármacos , Fitoterapia , Folhas de Planta/química , Quercetina/análogos & derivados , Rhododendron/química , Western Blotting , Cálcio/metabolismo , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , China , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/metabolismo , Feminino , Humanos , Mitocôndrias/metabolismo , Quercetina/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The inhibition of tumor cell growth without toxicity to normal cells is an important target in cancer therapy. One possible way to increase the efficacy of anticancer drugs and to decrease toxicity or side effects is to develop traditional natural products, especially from medicinal plants. Paris polyphylla Smith has shown anti-tumour effects by inhibition of tumor promotion and inducement of tumor cell apoptosis, but mechanisms are still not well understood. The present study was to explore the effect of Paris polyphylla Smith extract (PPSE) on connexin26 and growth control in human esophageal cancer ECA109 cells. METHODS: The effects of PPSE on Connexin26 were examined by RT-PCR, western blot and immunofluorescence; cell growth and proliferation were examined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. RESULTS: PPSE inhibited the growth and proliferation on esophageal cancer ECA109 cells, while increasing the expression of connexin26 mRNA and protein; conversely, PPSE decreased Bcl-2 and increased Bad. CONCLUSION: This study firstly shows that PPSE can increase connexin26 expression at mRNA and protein level, exerting anti-tumour effects on esophageal cacner ECA109 cells via inhibiting cell proliferation and inducing cell apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Conexinas/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Liliaceae/química , Fitoterapia , Extratos Vegetais/farmacologia , Western Blotting , Proliferação de Células/efeitos dos fármacos , Conexina 26 , Conexinas/genética , Neoplasias Esofágicas/tratamento farmacológico , Imunofluorescência , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/metabolismoRESUMO
Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atherosclerotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. The purpose of the present study was to investigate the effects of caveolin-1 (Cav-1) on ER stress-induced apoptosis in cultured macrophages and the underlying mechanisms. RAW264.7 cells were incubated with thapsigargin (TG) to establish ER stress model. And Cav-1 expression was detected by Western blot. After being pretreated with filipin(III), a caveolae inhibitor, RAW264.7 cells were assayed with flow cytometry and confocal laser scanning microscopy to detect cell apoptosis. Moreover, p38 mitogen-activated protein kinase (MAPK) phosphorylation and C/EBP homologous protein (CHOP) expression were detected with Western blot. The results showed that Cav-1 expression was markedly increased at early stage of TG treatment (P < 0.05) and then decreased with prolonged or high dose TG treatments. The increasing of Cav-1 expression induced by TG in RAW264.7 cells was abolished under inhibition of caveolae by filipin(III) (P < 0.05). The effect of TG on apoptosis of RAW264.7 cells was further augmented after pretreatment with filipin(III) (P < 0.05). Western blotting showed that MAPK phosphorylation induced by TG was inhibited by filipin(III) in RAW264.7 cells (P < 0.05), whereas CHOP remained unchanged (P > 0.05). These results suggest that Cav-1 may play a critical role in suppressing ER stress-induced macrophages apoptosis in vitro, and one of the mechanisms may be correlated with the activation of p38 MAPK prosurvival pathway.
Assuntos
Apoptose/efeitos dos fármacos , Caveolina 1/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Macrófagos/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Caveolina 1/genética , Linhagem Celular , Filipina/farmacologia , Sistema de Sinalização das MAP Quinases , Macrófagos/efeitos dos fármacos , Camundongos , Tapsigargina/farmacologia , Fator de Transcrição CHOP/metabolismoRESUMO
The different biological functions were studied in mouse bone marrow-derived endothelial progenitor cells isolated by differential time attachment to obtain the optimal adherent time in this study. Density gradient centrifugation-isolated bone marrow mononuclear cells were seeded on the fibronectin-coated dish. The 1-day cultured unattached cells were seeded on the second dish for 2 more days. Then unattached cells in the second dish were seeded on the third dish. The cells on 3 dishes were defined as 1-day adherent cells, 3-day adherent cells and 3-day unattached cells, respectively. After 20-day culture, the biological functions, such as the percentage of biomarkers, the ability of adhesion, and the ability of forming tubes in vitro were analyzed. The results showed that the percentages of positive CD34, FLK-1, and CD34/FLK-1 expressions in 1-day attached cells were significantly increased compared to those in the 3-day adherent or unattached cells (P < 0.01), which showed the strongest adhesion ability. The expression of eNOS in 1- or 3-day adherent cells was significantly higher than that in 3-day unattached cells (P < 0.01). The expression of VEGF in 3-day adherent cells was significantly higher than that in 1-day adherent cells or 3-day unattached cells (P < 0.01). These results suggest the biological functions of 1-day adherent cells are significantly stronger than that of 3-day adherent or unattached cells. VEGF expression in 3-day adherent cells is higher than that in 1-day adherent cells or 3-day unattached cells. The expression of eNOS in 1-day adherent cells or 3-day adherent cells is higher than that in 3-day unattached cells. The optimal adherent time to obtain mouse bone marrow-derived endothelial progenitor cells is 1-3 d.
Assuntos
Células da Medula Óssea/citologia , Separação Celular/métodos , Células Endoteliais/citologia , Células-Tronco/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Células Endoteliais/metabolismo , Leucócitos Mononucleares/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo III/metabolismo , Células-Tronco/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Ischemic postconditioning (I-postC) is a newly discovered and more amenable cardioprotective strategy capable of protecting the myocardium from ischemia/reperfusion (I/R) injury. Endoplasmic reticulum (ER) is a principal site for secretary protein synthesis and calcium storage. Myocardial I/R causes ER stress and emerging studies suggest that the cardioprotection has been linked to the modulation of ER stress. The aim of the present study was to determine whether cardioprotection of I-postC involves reduction in ER stress through calcineurin pathway. METHODS: In the in vivo model of rat myocardial I/R, myocardial infarct size was measured by triphenyltetrazolium chloride (TTC) staining and apoptosis was detected using terminal eoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Expression of calreticulin, C/EBP homologous protein (CHOP), caspase-12, and activation of caspase-12 in myocardium were detected by Western blotting. The activity and expression of calcineurin in myocardium were also detected. RESULTS: I-postC protected the I/R heart against apoptosis, myocardial infarction, and leakage of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB). I-postC suppressed I/R-induced ER stress, as shown by a decrease in the expression of calreticulin and CHOP, and caspase-12 activation. I-postC downregulated calcineurin activation in myocardium subjected to I/R. CONCLUSION: I-postC protects myocardium from I/R injury by suppressing ER stress and calcineurin pathways are not associated with the I-postC-induced suppression of ER stress-related apoptosis.
Assuntos
Calcineurina/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Pós-Condicionamento Isquêmico , Animais , Apoptose/fisiologia , Western Blotting , Creatina Quinase Forma MB/sangue , Hemodinâmica , L-Lactato Desidrogenase/sangue , Masculino , Infarto do Miocárdio/sangue , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Study of the mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. The mitochondrial-dependent apoptosis pathway is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane, leading to cytochrome C release. This study was to investigate the anti-tumor effects of Dioscin from traditional Chinese anti-snake venom medicine Paris chinensis (PCD) and correlated mechanisms regarding apoptosis in human ovarian cancer SKOV3 cells. METHODS: Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated by flow cytometry and Laser Scanning Confocal Microscope (LSCM) using Annexin-V/PI staining. Intracellular calcium ions were detected using fluorescence microscopy. The expression of apoptosis-related proteins cytochrome C and caspase-3 was measured by immunohistochemical staining. RESULTS: PCD had an anti-proliferation effect on human ovarian cancer SKOV3 cells in a dose- and time-dependent manner. After treatment with PCD, the apoptotic rate significantly increased, and accompanied with the increased levels of caspase-3 and cytochrome C protein in SKOV3 cells. Morphological changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining. Moreover, intracellular calcium accumulation occurred in PCD-treated cells. CONCLUSIONS: The molecular determinants of inhibition of cell proliferation as well as apoptosis of PCD may be associated with the activation of Ca2+-related m itochondrion pathway in SKOV3 cells.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Diosgenina/análogos & derivados , Medicamentos de Ervas Chinesas/farmacologia , Mitocôndrias/metabolismo , Neoplasias Ovarianas/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Citocromos c/metabolismo , Diosgenina/farmacologia , Feminino , Humanos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Neoplasias Ovarianas/patologia , Permeabilidade , Trillium/metabolismoRESUMO
BACKGROUND: Mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Loss of cell cycle control, leading to uncontrolled proliferation, is common in cancer. Therefore, the identification of potent and selective cyclin dependent kinase inhibitors is a priority for anti-cancer drug discovery. There are at least two major apoptotic pathways, initiated by caspase-8 and caspase-9, respectively, which can activate caspase cascades. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. This is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane. Anti-tumor effects of celery seed extract (CSE) and related mechanisms regarding apoptosis were here investigated in human gastric cancer BGC-823 cells. METHODS: CSE was produced by supercritical fluid extraction. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay and apoptosis by flow cytometry using Annexin/PI staining and DAPI staining and a laser scanning confocal microscope (LSCM). Cell cycling was evaluated using PI staining with flow cytometry and expression of cell cycle and apoptosis-related proteins cyclin A, CDK2, bcl-2 and bax was assessed by immunohistochemical staining. RESULTS: CSE had an anti-proliferation effect on human gastric cancer BGC-823 cells in a dose- and time-dependent manner. After treatment, the apoptotic rate significantly increased, with morphological changes typical of apoptosis observed with LSCM by DAPI staining. Cell cycle and apoptosis related proteins, such as cyclin A, CDK2 and bcl-2 were all down-regulated, whereas bax was up-regulated. CONCLUSIONS: The molecular determinants of inhibition of cell proliferation as well as apoptosis of CSE may be associated with cycle arrest in the S phase.
Assuntos
Apoptose/efeitos dos fármacos , Extratos Vegetais/farmacologia , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Neoplasias Gástricas/patologia , Apium/química , Caspase 3/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sementes/química , Neoplasias Gástricas/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The purpose of the present study is to explore the effect of oxidized low density lipoprotein (ox-LDL) on the induction of endoplasmic reticulum stress (ERS) and the underlying mechanisms in ox-LDL-induced macrophage foam-forming process. RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum, and then treated with ox-LDL (25, 50 and 100 mg/L), anti-CD36 monoclonal antibody+ox-LDL and tunicamycin (TM), respectively. After incubation for 24 h, the cells were collected. The cellular lipid accumulation was showed by oil red O staining and the content of cellular total cholesterol was quantified by enzymatic colorimetry. The expression of glucose-regulated protein 94 (GRP94), a molecular marker of ERS, was determined by immunocytochemistry assay. The levels of GRP94 protein, phosphorylated inositol-requiring enzyme 1 (p-IRE1) and X box binding protein 1 (XBP1) in RAW264.7 cells were detected by Western blotting. The results indicated that after incubation with ox-LDL (25, 50 and 100 mg/L) for 24 h, a large amount of lipid droplets were found in the cytoplasm, and the contents of cellular total cholesterol were increased by 2.1, 2.8 and 3.1 folds compared with the control, respectively. Anti-CD36 antibody decreased markedly the cellular lipid accumulation induced by ox-LDL at 100 mg/L. Both ox-LDL and TM, a specific ERS inducer, could up-regulate the protein expression of GRP94 in a dose-dependent manner. Furthermore, p-IRE1 and XBP1, two key components of the unfolded protein response, were also significantly induced by the treatment with ox-LDL. The up-regulations of the three proteins induced by ox-LDL were inhibited significantly when the macrophages were pre-incubated with anti-CD36 antibody. These results suggest that ox-LDL may induce ERS in a dose-dependent way and subsequently activate the unfolded protein response signaling pathway in RAW264.7 macrophages, which is potentially mediated by scavenger receptor CD36.