RESUMO
The prognostic and therapeutic values of fibronectin have been reported in patients with renal cell carcinoma (RCC). However, the underlying mechanisms of malignancy in RCC are not completely understood. We found that silencing of fibronectin expression attenuated human RCC 786-O and Caki-1 cell growth and migration. Silencing of potential fibronectin receptor integrin α5 and integrin ß1 decreased 786-O cell ability in movement and chemotactic migration. Biochemical examination revealed a reduction of cyclin D1 and vimentin expression, transforming growth factor-ß1 (TGF-ß1) production, as well as Src and Smad phosphorylation in fibronectin-silenced 786-O and Caki-1 cells. Pharmacological inhibition of Src decreased 786-O cell growth and migration accompanied by a reduction of cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. In 786-O cells, higher activities in cell growth and migration than in Caki-1 cells were noted, along with elevated fibronectin and TGF-ß1 expression. The additions of exogenous fibronectin and TGF-ß1 promoted Caki-1 cell growth and migration, and increased cyclin D1, fibronectin, vimentin, and TGF-ß1 expression, as well as Src and Smad phosphorylation. These findings highlight the role of fibronectin in RCC cell growth and migration involving Src and TGF-ß1 signaling.
Assuntos
Carcinoma de Células Renais/metabolismo , Movimento Celular , Proliferação de Células , Fibronectinas/metabolismo , Neoplasias Renais/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Fibronectinas/genética , Humanos , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina beta1/genética , Integrina beta1/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Vimentina/genética , Vimentina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Aspirin is a novel chemopreventive agent against malignancy. However, outcomes of aspirin monotherapy of renal cell carcinoma (RCC) are inconsistent across studies. ABT-737, an BH3 mimetic inhibitor, is also a promising antitumor drug. Cancer cells including those from RCC, that have high levels of Mcl-1, are refractory to ABT-737-induced apoptosis. We here investigated how aspirin treatment modulates the ABT-737-induced apoptosis. Using the in vitro model of human 786-O cells, we showed that aspirin had sensitized cells to ABT-737 induced apoptosis. Such aspirin-induced changes of ABT-737 resistance was accompanied by a host of biochemical events like protein phosphatase 2A (PP2A) activation, AKT dephosphorylation, Mcl-1/FLICE inhibiting protein (FLIP)/XIAP downregulation, and Bax mitochondrial redistribution. The PP2A inhibitor, okadaic acid, was able to reverse the apirin-induced apoptotic changes. Apart from the aspirin treatment, Mcl-1 silencing also rendered cells vulnerable to ABT-737 induced apoptosis. Since PP2A, Akt, and Mcl-1 play critical roles in RCC malignancy and treatment resistance, our present study showed that aspirin, an alternative adjuvant agent, had recalled ABT-737 sensitivity in the RCC cells through processes involving the PP2A/Akt/Mcl-1 axis.
Assuntos
Aspirina/administração & dosagem , Compostos de Bifenilo/administração & dosagem , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Nitrofenóis/administração & dosagem , Sulfonamidas/administração & dosagem , Anticarcinógenos/administração & dosagem , Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Piperazinas/administração & dosagem , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Bone metastasis in metastatic breast cancer commonly results in osteolytic lesions due to osteoclast activity, promoting bone destruction and tumor progression. The bioactive fungal isolates, 4-acetyl-antroquinonol B (4-AAQB) and erinacine A, have diverse pharmacological and biological activities. However, their effects on breast cancer bone metastasis treatment remain unclear. Our study aimed to examine the impact of 4-AAQB or erinacine A on breast cancer metastases in bone. The effects of 4-AAQB and erinacine A on breast cancer-induced osteoclastogenesis, breast cancer migration, production of prometastatic cytokine (TGF-ß) and marker (MMP-9), as well as potential MAPK signaling transductions were assessed. The results revealed that 4-AAQB and erinacine A effectively suppressed breast cancer-induced osteoclastogenesis and migration, and reduced TGF-ß and MMP-9 production via Erk or JNK signaling transductions, specifically in breast cancer cells or in breast cancer cells-induced osteoclasts. Based on these findings, either 4-AAQB or erinacine A showed promise in preventing breast cancer metastases in bone.
Assuntos
Neoplasias da Mama , Metaloproteinase 9 da Matriz , Osteoclastos , Osteogênese , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Humanos , Feminino , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Animais , Fator de Crescimento Transformador beta/metabolismo , Neoplasias Ósseas/secundário , Neoplasias Ósseas/tratamento farmacológico , Camundongos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Cicloexanonas , 4-Butirolactona/análogos & derivadosRESUMO
Staphylococcus spp., including S. aureus, S. intermedius, S. hyicus, S. epidermidis, S. saprophyticus, S. haemolyticus, S. xylosus, and S. carnosus, are major bacterial species associated with food poisoning, and human and veterinary clinics. Traditional methods for the identification of these staphylococci are time-consuming, laborious, or inaccurate. Therefore, rapid and accurate diagnostic methods are needed. In this study, we designed the DNA probes and polymerase chain reaction (PCR) primers for the detection of the aforementioned Staphylococcus species. These primers were proved to be specific for the detection of their corresponding target strains. Furthermore, by using a consensus primer pair, we were able to co-amplify the intergenic region of groES-groEL for these staphylococci. Followed by a chromogenic macroarray system with the specific probes on the plastic chips, these staphylococci in milk products or clinical samples could be simultaneously detected. When the system was used for the inspection of milk or urine samples containing N × 10° target cells per milliliter of the sample, all these staphylococcal species could be identified after an 8-h pre-enrichment step. This system also allowed the adequate diagnosis of bacteremia, since N × 10° target cells per milliliter of the blood samples could be detected after a 12-h pre-enrichment. Compared to the multiplex PCR method, this approach has the additional advantage that it allowed the discrimination of more bacterial strains-even some bacterial strains that may generate PCR products with the same molecular sizes.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Primers do DNA/química , Expressão Gênica , Staphylococcus/classificação , Staphylococcus/isolamento & purificação , Animais , Bacteriemia/sangue , Bacteriemia/diagnóstico , Bacteriemia/microbiologia , Bacteriemia/urina , Proteínas de Bactérias/genética , Chaperonina 10/genética , Chaperonina 10/metabolismo , Chaperonina 60/genética , Chaperonina 60/metabolismo , Chaperoninas/genética , DNA Bacteriano/sangue , DNA Bacteriano/metabolismo , DNA Bacteriano/urina , DNA Intergênico/sangue , DNA Intergênico/metabolismo , DNA Intergênico/urina , Inspeção de Alimentos/métodos , Microbiologia de Alimentos , Humanos , Leite/microbiologia , Tipagem Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Sorotipagem/métodos , Intoxicação Alimentar Estafilocócica/sangue , Intoxicação Alimentar Estafilocócica/diagnóstico , Intoxicação Alimentar Estafilocócica/microbiologia , Intoxicação Alimentar Estafilocócica/urina , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/diagnóstico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/urina , Staphylococcus/genética , Staphylococcus/metabolismoRESUMO
Carbonic anhydrase VIII (CAVIII) is a member of the CA family, while CA8 is the oncogene. Here we observed increased expression of CAVIII with high expression in colorectal cancer tissues. CAVIII is also expressed in more aggressive types of human colorectal cancer cells. Upregulated CAVIII expression in SW480 cell lines increased vascular endothelial growth factor (VEGF) and reduced miRNA16-5p. Conversely, knockdown of the CAVIII results in VEGF decline by up-regulated miRNA16-5p. Moreover, the collection of different grades of CAVIII expression CRC cells supernatant co-culture with endothelial progenitor cells (EPCs) promotes the ability of tube formation in soft agar and migration in the Transwell experiment, indicating that CAVIII might facilitate cancer-cell-released VEGF via the inhibition of miRNA16-5p signaling. Furthermore, in the xenograft tumor angiogenesis model, knockdown of CAVIII significantly reduced tumor growth and tumor-associated angiogenesis. Taken together, our results prove that the CAVIII/miR-16-5p signaling pathway might function as a metastasis suppressor in CRC. Targeting CAVIII/miR-16-5p may provide a strategy for blocking its metastasis.
RESUMO
Oil bodies were observed in cells of both embryo and aleurone layers of mature adlay grains (Coix lachryma-jobi L. var. ma-yuen Stapf). Stable oil bodies were successfully isolated from the adlay grains. Thin-layer chromatography revealed that the contents stored in the adlay oil bodies were mainly neutral lipids (>90% triacylglycerols and about 5% diacylglycerols). The integrity of the isolated oil bodies was presumably maintained via electronegative repulsion and steric hindrance provided by their surface proteins. Immunological cross-recognition using antibodies against sesame oil-body proteins indicated that two oleosin isoforms (termed oleosin-H and oleosin-L) and one caleosin were present in the adlay oil bodies. Full-length cDNA fragments encoding these three unique oil-body proteins were obtained by PCR cloning. MALDI-MS analyses confirmed that the three full-length cDNA fragments encoded the two oleosin isoforms and one caleosin observed in the oil bodies isolated from the adlay grains.
Assuntos
Coix/ultraestrutura , Corpos de Inclusão/química , Óleos de Plantas/análise , Proteínas de Ligação ao Cálcio , Cromatografia em Camada Fina , Coix/química , DNA Complementar , Lipídeos/análise , Dados de Sequência Molecular , Proteínas de Plantas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
OBJECTIVE: Caveolin-1 (Cav-1) is expressed abundantly in adipose tissue and involved in many physiological processes. While Cav-1 has been reported to be secreted in pancreatic acinar cells and LNCaP prostate cancer cells, its secretion from adipose tissue awaits investigation. METHODS: Cav-1 secretion from 3T3-L1 adipocytes and fat tissues from normal chow diet- and high-fat diet (HFD)-fed mice was measured. Functions and uptake of secreted Cav-1 proteins were assessed by adding Cav-1 back to preadipocytes and LNCaP cells. RESULTS: Cav-1 secretion was evident in adipose tissues and were substantially promoted in HFD-fed mice. Cav-1 was detectable in the conditioned media of 3T3-L1 adipocytes but not preadipocytes. Hypertrophied adipocytes induced by glucose and fatty acids secreted more Cav-1, suggesting that hypertrophied adipocytes were responsible for enhanced Cav-1 secretion in obese mice. Secreted Cav-1 was taken up by preadipocytes and LNCaP cells. 3T3-L1 preadipocytes overexpressing Cav-1 were better differentiated, suggesting that secreted Cav-1 may promote adipogenesis. Hypertrophied 3T3-L1 adipocytes enhanced ERK1/2 activation, and the attenuation of ERK1/2 activity by PD98059 inhibited Cav-1 secretion. CONCLUSIONS: Cav-1 is actively secreted from adipocytes as a putative adipogenesis enhancer. Hypertrophied adipocytes secrete Cav-1 via ERK1/2-dependent mechanisms to promote adipogenesis, thus establishing a vicious cycle.
Assuntos
Adipócitos/metabolismo , Adipogenia/imunologia , Tecido Adiposo/metabolismo , Caveolina 1/metabolismo , Animais , Técnicas de Cultura de Células , Masculino , CamundongosRESUMO
Effects of leukemia inhibitory factor (LIF) and fibroblast growth factor 2 (FGF2) on establishment and maintenance of rabbit embryonic stem cell (rESC) lines were assessed. When grown on MEF feeders, rESC lines derived from fertilized embryos were established and maintained in medium containing paracrine factors LIF (via STAT3) and/or FGF2 (via MEK-ERK1/2 and PI3K-AKT). However, high levels of ERK1/2 and AKT activities in rESCs were crucial for maintaining their undifferentiated proliferation. Although rESCs under the influence of either LIF (500, 1,000, and 2,000 U/ml) or FGF2 (5, 10, and 20 ng/ml) alone had enhanced expression of pluripotency markers, peak expression occurred when both LIF (1,000 U/ml) and FGF2 (10 ng/ml) were applied. Induced dephosphorylation of STAT3, ERK1/2, and AKT by specific inhibitors limited growth of rESCs and caused remarkable losses of self-renewal capacity; therefore, we inferred that STAT3, ERK, and AKT had essential roles in maintaining rESC proliferation and self-renewal. We concluded that LIF and FGF2 jointly maintained the undifferentiated state and self-renewal of rESCs through an integrative signaling module.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator Inibidor de Leucemia/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Coelhos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Zigoto/citologiaRESUMO
Tetramethylpyrazine (TMP), which is widely used in the treatment of ischemic stroke by Chinese herbalists, is one of the most important active ingredients of the traditional Chinese herbal medicine, Ligusticum wallichii Franchat (Chung Xiong). However, the mechanism by which TMP protects the brain is still not clear. We examined neuroprotective effects of TMP after transient focal cerebral ischemia using common carotid artery and middle cerebral artery occlusion model in rats and evaluated the involvement of anti-inflammation. TMP administrated intraperitoneally significantly protected the brain against ischemic insult as evidenced by the reduction in infarction volume, preservation of neurons, and decrease in brain edema. TMP markedly reduced cerebral ischemia/reperfusion-induced inflammatory cell activation and proinflammatory mediator production. Moreover, TMP suppressed lipopolysaccharide/interferon-gamma-induced inflammation and prostaglandin E(2) production in cultured glial cells. Our findings suggest that one of neuroprotective effects of TMP against ischemic brain injury might involve its anti-inflammatory potential.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Pirazinas/uso terapêutico , Animais , Células Cultivadas , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be one of the most promising candidates in research on treatments for cancer, including renal cell carcinoma (RCC). However, many cells are resistant to TRAIL-induced apoptosis which limits the potential application of TRAIL in cancer therapy. Luteolin, a naturally occurring flavonoid, has been identified as a potential therapeutic and preventive agent for cancer because of its potent cancer cell-killing activity. In this study, we investigated whether luteolin treatment could modulate TRAIL-induced apoptosis in RCC. MAIN METHODS: The effect of luteolin on TRAIL sensitivity was assessed in human RCC 786-O, ACHN, and A498 cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS: We found that nontoxic concentration of luteolin alone had no effect on the level of apoptosis, but a combination treatment of TRAIL and luteolin caused significant extrinsic and intrinsic apoptosis. The sensitization was accompanied by Bid cleavage, Mcl-1 and FLIP down-regulation, DR4/DR5 protein expression and cell surface presentation, and Akt and signal transducer and activator of transcription-3 (STAT3) inactivation. Among these phenomena, changes in FLIP, Akt, and, STAT3 are more prone to the effects of luteolin treatment. Studies have further demonstrated that inactivation of Akt or STAT3 alone was sufficient to down-regulate FLIP expression and sensitized 786-O cells to TRAIL-induced apoptosis. SIGNIFICANCE: Data from this study thus provide in vitro evidence supporting the notion that luteolin is a potential sensitizer of TRAIL in anticancer therapy against human RCC involving Akt and STAT3 inactivation.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Renais/patologia , Luteolina/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Células Tumorais CultivadasRESUMO
The aim of this study was to purify and identify lipolysis-stimulating peptides derived from Flavourzyme®-soy protein isolate (SPI) hydrolysate (F-SPIH). Glycerol release was employed as a marker for lipolysis in 3T3-L1 adipocytes. A higher glycerol release represents a better lipolysis-stimulating activity. The peptide fraction with highest glycerol release obtained from F-SPIH fractionated by sequential ultrafiltration membranes was further purified using gel filtration chromatography and two steps of reverse-phase high-performance liquid chromatography. The peptides were identified using liquid chromatography-tandem mass spectrometry (LC/MS/MS). Three lipolysis-stimulating peptides were obtained, and the amino acid sequences were ILL, LLL and VHVV, respectively. The in vitro effect of gastrointestinal proteases on lipolysis-stimulating activity of synthetic ILL, LLL and VHVV, respectively, was also investigated. The result suggested that the gastrointestinal protease did not affect lipolysis-stimulating activity of the three novel peptides, which reveals their potential to act as anti-obesity ingredients.
Assuntos
Lipólise/efeitos dos fármacos , Peptídeo Hidrolases/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Hidrolisados de Proteína/química , Proteínas de Soja/química , Animais , Hidrólise , Camundongos , Células NIH 3T3 , Peptídeos/química , Hidrolisados de Proteína/farmacologiaRESUMO
A cDNA fragment (FaPR4) encoding a class I pathogenesis-related protein 4 (PR-4) from Ficus awkeotsang was obtained by PCR cloning. Plant PR-4s were grouped into class I and II, differing by the presence of ChtBD and hinge. The predicted mature FaPR4 comprises N-terminal chitin-binding domain (ChtBD), hinge, Barwin domain and C-terminal extension. FaPR4-C, an N-terminal truncated form of FaPR4, was designed to mimic the structural feature of class II PR-4s. FaPR4 and FaPR4-C were over-expressed in yeast Pichia pastoris, and both recombinants exhibited RNase and anti-fungal activities. To our knowledge, it is the first report that FaPR4, a member of class I PR-4s has RNase activity as class II. FaPR4 possesses better anti-fungal activities toward Fusarium oxysporum and Sclerotium rolfsii than FaPR4-C. Heat-treated FaPR4 remained RNase and anti-fungal activities; while heat-treated FaPR4-C lost those activities. Therefore, ChtBD of FaPR4 may not only contribute to its anti-fungal but also improve the thermal stability of protein. It also implied the correlation of RNase activity with anti-fungal activity of FaPR4-C. Furthermore, FaPR4 was detected to have weak but significant chitinase activity, and its chitinase activity was reduced after heat treatment. The chitinase activity by FaPR4-C was much lower than FaPR4.
Assuntos
Antifúngicos/farmacologia , Quitina/metabolismo , Ficus/química , Fungos/efeitos dos fármacos , Expressão Gênica , Proteínas de Plantas/farmacologia , Ribonucleases/farmacologia , Sequência de Aminoácidos , Antifúngicos/metabolismo , Quitinases/metabolismo , Clonagem Molecular , DNA Complementar , Fusarium/efeitos dos fármacos , Temperatura Alta , Dados de Sequência Molecular , Pichia/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Sementes/químicaRESUMO
Microglial activation plays a pivotal role in the pathogenesis of neurodegenerative disease by producing excessive proinflammatory cytokines and nitric oxide (NO). Luteolin, a naturally occurring polyphenolic flavonoid antioxidant, has potent anti-inflammatory and neuroprotective properties both in vitro and in vivo. However, the molecular mechanism of luteolin-mediated immune modulation in microglia is not fully understood. In the present study, we report the inhibitory effect of luteolin on lipopolysaccharide (LPS)/interferon γ (IFN-γ)-induced NO and proinflammatory cytokine production in rat primary microglia and BV-2 microglial cells. Luteolin concentration-dependently abolished LPS/IFN-γ-induced NO, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) production as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression. Luteolin exerted an inhibitory effect on transcription factor activity including nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF-1) in LPS/IFN-γ-activated BV-2 microglial cells. Biochemical and pharmacological studies revealed that the anti-inflammatory effect of luteolin was accompanied by down-regulation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), Akt and Src. Further studies have demonstrated that the inhibitory effect of luteolin on intracellular signaling execution and proinflammatory cytokine expression is associated with resolution of oxidative stress and promotion of protein phosphatase activity. Together, these results suggest that luteolin suppresses NF-κB, STAT1 and IRF-1 signaling, thus attenuating inflammatory response of brain microglial cells.
Assuntos
Luteolina/farmacologia , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Regulação para Baixo , Fator Regulador 1 de Interferon , Interleucina-1beta/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Camundongos , Óxido Nítrico/fisiologia , Fosfoproteínas Fosfatases/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/efeitos dos fármacos , Fator de Transcrição STAT1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Zinc overload is known to cause the death of neural cells. Although the activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A(2) (cPLA(2)) have been implicated in zinc-induced astrocyte death, the detailed mechanisms of their activation and upstream regulatory cascades are incompletely understood. Here, we report that protein kinase C (PKC)- and Src-related Ras/Raf/ERK cascades and ERK-associated cPLA(2) participate in astrocyte death caused by ZnCl(2). Sustained exposure to ZnCl(2) caused damage to astrocytes in a time- and concentration-dependent manner. The cell death caused by ZnCl(2) was accompanied by increased reactive oxygen species (ROS) generation, PKC-α membrane association, Src phosphorylation, Ras membrane association, Raf phosphorylation, ERK phosphorylation, and cPLA(2) activation, and decreased protein phosphatase activity. Pharmacological studies revealed that these activations/inactivations all contributed to ZnCl(2)-induced astrocyte death. ROS, such as superoxide, appear to be a key trigger in response to ZnCl(2) treatment in astrocytes because of the attenuations in protein phosphatase inhibition, signaling activation, and cell death by antioxidant treatments. Mechanistic studies had suggested that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK were potential signals linking zinc and cPLA(2). These observations indicated that ROS/PKC-α/Ras/Raf/ERK and ROS/Src/Ras/Raf/ERK signaling and cPLA(2) were actively involved in zinc-induced astrocyte damage.
Assuntos
Astrócitos/efeitos dos fármacos , Cloretos/toxicidade , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Transdução de Sinais/fisiologia , Compostos de Zinco/toxicidade , Animais , Astrócitos/citologia , Morte Celular , Células Cultivadas , Proteína Quinase C/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Quinases raf/fisiologia , Proteínas ras/fisiologia , Quinases da Família src/fisiologiaRESUMO
Zinc and cytosolic phospholipase A(2) (cPLA(2)) have been implicated in the death of neural cells and the pathogenesis of ischemia, and hyperglycemia is a potential augmenting factor. However, their potential crosstalk and/or interaction in mediating cell damage have not yet been fully elucidated. Here, we report that a potential link between cPLA(2) activation and zinc-induced astrocyte damage involving reactive oxygen species (ROS)/protein kinase C-α (PKC-α)/extracellular signal-regulated kinase (ERK) signaling and glucose is able to increase zinc uptake and potentiate zinc-induced alterations and astrocyte damage. The cell death caused by ZnCl(2) was accompanied by increased ROS generation, PKC-α membrane translocation, ERK phosphorylation, and cPLA(2) phosphorylation and activity. Pharmacological studies revealed that these activations contributed to ZnCl(2)-induced astrocyte death. Mechanistic studies had suggested that ROS/PKC-α/ERK was a potential signal linking zinc and cPLA(2). Glucose increased zinc uptake and potentiated ZnCl(2)-induced alterations and astrocyte death. These observations indicated that ROS/PKC-α/ERK signaling and cPLA(2) were actively involved in zinc-induced astrocyte damage, and suggested zinc was a potential downstream effector in hyperglycemia-aggravated astrocyte injury.
Assuntos
Astrócitos/efeitos dos fármacos , Cloretos/toxicidade , Glucose/farmacologia , Compostos de Zinco/toxicidade , Animais , Animais Recém-Nascidos , Astrócitos/patologia , Western Blotting , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citosol/efeitos dos fármacos , Citosol/enzimologia , Sinergismo Farmacológico , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , L-Lactato Desidrogenase/metabolismo , Fosfolipases A2/metabolismo , Proteína Quinase C-alfa/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
p73 is one of the family proteins that share structural and functional homologies with the tumor suppressor p53. To analyze the status of p73 in hepatocellular carcinoma (HCC), the allelic loss, allelic expression, mutation and methylation status of the p73 gene were examined in 18 paired HCC and normal tissues. No allelic loss was found. All heterozygous individuals contained RNA of both alleles, indicating that p73 was biallelically expressed in the liver. Notably, semiquantitative reverse transcriptase polymerase chain reaction analysis showed that p73 was consistently overexpressed in the cancerous tissues. Single-stranded conformation polymorphism and sequencing analysis revealed several polymorphisms, but no mutations were found in the entire coding sequence. Finally, the methylation patterns in the promoter and exon 1 regions of p73 were not altered in the cancerous tissues. These results do not support p73 as a tumor suppressor in HCC, but suggest that overexpression of p73 may in some way be associated with the pathogenesis of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hepáticas/genética , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Carcinoma Hepatocelular/metabolismo , Genes Supressores de Tumor , Humanos , Neoplasias Hepáticas/metabolismo , Perda de Heterozigosidade , Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , Polimorfismo Conformacional de Fita Simples , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
BACKGROUND: Races and Gm(23) allotypes can modify the serum levels of IgG subclasses. The average serum levels of IgG subclasses of African-Americans have been reported to be higher than those of Caucasians in both healthy young adults and patients with aggressive periodontitis; Gm(23)-positive subjects generally had higher IgG2 levels than Gm(23)-negative subjects. OBJECTIVE: We examined serum immunoglobulin G (IgG) concentrations in Taiwanese patients with different forms of periodontitis. METHODS: The serum levels of four IgG subclasses were determined by enzyme-linked immunosorbent assay and Gm(23) allotypes were verified by radial immunodiffusion tests in 50 patients with chronic periodontitis, 30 patients with aggressive periodontitis, and 74 healthy controls. RESULTS: There were no differences in the concentrations of four IgG subclasses in patients with chronic periodontitis compared with age-matched controls. However, in subjects younger than 35 years, levels of IgG2 were significantly elevated in patients with aggressive periodontitis compared with controls. We also found significant differences in IgG2 levels within the control group when stratified by age (< or = 35 years and > 35 years). Gm(23) allotypes were not correlated with the serum levels of IgG2 in either patient group. CONCLUSION: Microbial challenge might not provoke significant changes in systemic IgG response in patients with chronic periodontitis. However, in patients with aggressive periodontitis, IgG2 levels were increased when compared with age-matched controls. Gm(23) allotypes had no influence on IgG2 levels in well-established generalized chronic periodontitis or aggressive periodontitis.
Assuntos
Imunoglobulina G/sangue , Periodontite/imunologia , Adulto , Fatores Etários , Periodontite Agressiva/sangue , Periodontite Agressiva/imunologia , Povo Asiático , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Alótipos Gm de Imunoglobulina , Masculino , Pessoa de Meia-Idade , Periodontite/sangue , TaiwanRESUMO
OBJECTIVES: Given the diversity of the distribution of the Gm (23) allotypes and FcgammaR genotypes in different ethnic groups, it was our purpose to examine their clinical significance in periodontitis in Taiwan. MATERIAL & METHODS: Genomic DNA of 50 patients with chronic periodontitis (CP), 30 patients with generalized aggressive periodontitis (G-AP) and 74 healthy controls were harvested. The Gm (23) allotypes were determined by radial immunodiffusion test, and the FcgammaR IIa (CD32) and IlIb (CD16) genotypes were determined by polymerase chain reaction-based allele-specific oligonucleotide hybridization. RESULTS: The overall carrier rate of the Gm (23+) allotype was higher than 85%, and the Gm (23-) allotype was statistically over-represented in patients with CP compared to the controls. There were no differences in the distributions of the three genotypes of FcgammaR IIa and IIIb among the three tested groups. The frequency of the R131 allele of the FcgammaR IIa polymorphisms was higher in G-AP than in CP when R/H allelic frequencies (p = 0.01) were examined by the chi2 test. CONCLUSION: The Gm (23-) allotype might be a potential risk factor for CP. Although the R131 allele of FcgammaR IIa occurred more frequently in G-AP than in CP, its clinical significance could not be justified in this study.
Assuntos
Antígenos CD/genética , Povo Asiático/genética , Alótipos Gm de Imunoglobulina/genética , Periodontite/genética , Periodontite/imunologia , Receptores de IgG/genética , Adulto , Antígenos CD/sangue , Distribuição de Qui-Quadrado , Doença Crônica , Feminino , Proteínas Ligadas por GPI , Frequência do Gene/genética , Predisposição Genética para Doença , Genótipo , Heterozigoto , Humanos , Alótipos Gm de Imunoglobulina/sangue , Masculino , Pessoa de Meia-Idade , Periodontite/sangue , Periodontite/classificação , Polimorfismo Genético/genética , Receptores de IgG/sangue , Valores de Referência , Fatores de Risco , TaiwanRESUMO
Nasopharyngeal carcinoma (NPC) is a human cancer of epithelial cell origin. Infection by Epstein-Barr virus has been shown to be closely associated with this tumor. Recent studies have indicated that another common epitheliotropic virus, human papillomavirus (HPV), is also found in a significant number of NPC cases. In this study, we evaluated the feasibility of using the HPV regulatory long control region (LCR) to drive the expression of the thymidine kinase (tk) gene to achieve chemosensitivity for gene therapeutic treatment of NPC. Testing HPV-11-LCR-tk constructs in NPC cell lines in the presence of ganciclovir (GCV) led to 50-60% cell death of transfected cells. The therapeutic efficacy was further tested in an in vivo model using nude mice transplanted with tumors derived from transfected NPC cells. Injection of 50 mg/kg body weight GCV twice daily for 14 days resulted in visually complete regression of the transplanted NPC tumor loads within 20 days after GCV treatment. Taken together, results from this pilot study indicate the feasibility of the development of a gene therapeutic protocol based on the chemosensitive gene constructs described in this paper.