RESUMO
Mitochondrial dysfunction is a direct target of hypoxic/ischemic stress in astrocytes, which results in the increased production of reactive oxygen species (ROS). Previous reports showed that ROS can activate NF-kB in spinal cord astrocytes, which occurs as a secondary injury during the pathological process of spinal cord injury (SCI). Protein kinase RNA (PKR)-like ER kinase (PERK) plays an important role in mitochondrial dysfunction. To elucidate the specific role of PERK in hypoxic/ischemic-induced NF-kB activation in spinal astrocytes, we utilized an in vitro oxygen-glucose deprivation (OGD) model, which showed an enhanced formation of ROS and NF-kB activation. Knockdown of PERK resulted in reduced activation of PERK and ROS generation in astrocytes under OGD conditions. Notably, the knockdown of PERK also induced NF-kB activation in astrocytes. These data suggest that PERK is required for the hypoxic/ischemic-induced-dependent regulation of ROS and that it is involved in NF-kB activation in the astrocytes.
Assuntos
Astrócitos/efeitos dos fármacos , Glucose/farmacologia , NF-kappa B/metabolismo , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , eIF-2 Quinase/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Hipóxia Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Vetores Genéticos , Glucose/deficiência , Lentivirus/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/genética , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/genéticaRESUMO
Colorectal cancer (CRC) remains the most common malignancy worldwide. TGF-ß1 is often overexpressed in late stages of colorectal carcinogenesis and promotes tumour growth and metastasis. Several reports have verified that the loss of functional TGFBRII expression contributed to escape the tumour suppressor activity of TGF-ß1 and that the epithelial-to-mesenchymal transition (EMT) responded to TGF-ß1 involved in tumour invasion and metastasis. However, the mechanisms by which TGF-ß1 confers a growth advantage to TGFBRII-null colorectal cancer cells have not been elucidated. MicroRNAs (miRNAs) are post-transcriptional inhibitory regulators of gene expression that act by directly binding complementary mRNA and are key determinants of cancer initiation and progression. In this study, we revealed a role for miR-200b in colorectal cancer. MiR-200b was highly expressed in TGFBRII-null tumour tissues and colorectal cancer cell lines and positively correlated with cell proliferation in tumour tissues and cell lines. In contrast, decreasing the miR-200b level in TGFBRII-null cells suppressed cell proliferation and cell cycle progression. Furthermore, in vivo studies also suggested a stimulating effect of miR-200b on TGFBRII-null cell-derived xenografts. CDKN1B (p27/kip1) and RND3 (RhoE) have miR-200b binding sequences within their 3' untranslated regions and were confirmed to be direct targets of miR-200b using fluorescent reporter assays. Meanwhile, CDKN1B (p27/kip1) played a role in miR-200b-stimulated TGFBR-null CRC. This study suggests that miR-200b plays a tumour-promoting role by targeting CDKN1B (p27/kip1) in CRCs.
Assuntos
Neoplasias Colorretais/patologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/genéticaRESUMO
Although excessive nitric oxide (NO) induced from iNOS is critical for dysfunction of vascular endothelial cells (ECs) in the diabetic retina, its role on ECs injury remains unknown. RPE (retinal pigment epithelium) is the pigmented cell layer just outside the neurosensory retina that constitutes the blood-retinal-barrier (BRB) with ECs, and also serves as the limiting transport factor that maintains the retinal environment. Dysfunction of the RPE is related to oxidative stress that contributes to the progression of diabetic retina. Using a co-cultural biosystem, we demonstrate that NO generation and iNOS expression was increased in both ECs and RPE cells after high glucose treatment. Increased NO in ECs cocultured with RPE activate the endoplasmic reticulum (ER) and protein kinase RNA (PKR)-like ER kinase (PERK) pathway and involved in ECs apoptosis. Blockade of the iNOS pathway, or depletion of PERK effectively, reverses NO-mediated apoptosis. Our study demonstrates that iNOS and subsequently excessive NO generation in RPE cells can have an unanticipated effect by activating PERK pathways in ECs, resulting in a novel mechanism for vascular endothelium to avoid injury from prolonged hyperglycemia.
Assuntos
Apoptose , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Glucose/farmacologia , Óxido Nítrico/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , eIF-2 Quinase/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo , Relação Estrutura-AtividadeRESUMO
c-Met is over-expressed in hepatocellular carcinoma(HCC) but is absent or expressed at low levels in normal tissues. Therefore we generated a novel conjugate of a human anti-c-Met Fab fragment (MetFab) with doxorubicin (DOX) and assessed whether it had targeted antitumor activity against HCC and reduced the side-effects of DOX. The MetFab was screened from human phage library, conjugated with DOX via chemical synthesis, and the conjugation MetFab-DOX was confirmed by HPLC. The drug release patterns, the binding efficacy, and cellular distribution of MetFab-DOX were assessed. MetFab-DOX was stable at pH7.2 PBS while release doxorubicin quickly at pH4.0, the binding efficacy of MetFab-DOX was similarly as MetFab, and the cellular distribution of the MetFab-DOX is distinct from free DOX. The cytotoxicity of MetFab-DOX was analyzed by the MTT method and the nude mouse HCC model. The MetFab-DOX demonstrated cytotoxic effects on c-Met expressing-tumor cells, but not on the cells without c-Met expression. MetFab-DOX exerted anti-tumor effect and significantly reduced the side effect of free DOX in mice model. Furthermore, the localization of conjugate was confirmed by immunofluorescence staining of tumor tissue sections and optical tumor imaging, respectively, and the tissue-distribution of drug was compared between free DOX and MetFab-DOX treatment by spectrofluorometer. MetFab-DOX can localize to the tumor tissue, and the concentration of doxorubicin in the tumor was higher after MetFab-DOX administration than after DOX administration. In summary, MetFab-DOX can target c-Met expressing HCC cells effectively and have obvious antitumor activity with decreased side-effects in preclinical models of HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/farmacologia , Imunoconjugados/farmacologia , Fragmentos Fab das Imunoglobulinas/genética , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Estabilidade de Medicamentos , Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Concentração de Íons de Hidrogênio , Imunoconjugados/química , Imunoconjugados/genética , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Biblioteca de Peptídeos , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/imunologiaRESUMO
BACKGROUND & OBJECTIVE: The mutation of von Hippel-Lindau (VHL) tumor suppressor gene is closely related with tumorigenesis of renal clear cell carcinoma (RCCC). Cyclin D1 gene plays an important role in progression of RCCC by stimulating cell proliferation. This study was to determine the mutation of VHL gene in RCCC, and explore its correlation to overexpression of Cyclin D1. METHODS: The specimens of RCCC and adjacent normal renal tissue from 50 patients were collected after surgery. Total RNA and genomic DNA were extracted from each sample. Variant exons in VHL gene were amplified by polymerase chain reaction (PCR) and sequenced, and DNA hypermethylation was detected by restriction analysis. Reverse transcription-PCR (RT-PCR) and Western blot were used to detect the expression of Cyclin D1. RESULTS: Of the 50 specimens, 42 (84.0%) had various VHL gene mutations, 12 (24.0%) had more than 1 kind of gene mutation. Of the 57 cases of exon mutation of VHL gene, 17 (29.8%) were located in exon 1, 26 (45.6%) in exon 2, and 14 (24.6%) in exon 3. The expression of Cyclin D1 in the 42 cases with VHL gene mutation was increased to 2-10 (3.91+/-1.54) times that of normal controls (P<0.01). Cyclin D1 expression in the other 8 cases was normal. CONCLUSION: There are variant mutations of VHL gene in RCCC, which may lead to overexpression of Cyclin D1.