RESUMO
BACKGROUND: Previous studies demonstrated the EGF-targeted phagemid particles carrying siRNA against Akt could be expressed efficiently in the presence of hydroxycamptothecin (HCPT). However, no significant cell growth inhibition was obtained. This study was to further investigate whether the EGF-targeted phagemid particles carrying siRNA would be a promising tool for anti-cancer siRNA delivery. RESULTS: We found that pSi4.1-siFAK phagemid particles could significantly inhibit the expression of focal adhesion kinase in the HCPT-treated cells. Moreover, we also observed that the particles could potently suppress cell growth and cell invasion. CONCLUSION: These results indicated that EGF-targeted phagemid particles might be a promising tool for anti-cancer siRNA delivery in the presence of HCPT.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Neoplasias/terapia , Interferência de RNA , Bacteriófagos/genética , Sequência de Bases , Camptotecina/análogos & derivados , Camptotecina/química , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Terapia Combinada , Fator de Crescimento Epidérmico/genética , Vetores Genéticos , Humanos , Dados de Sequência Molecular , Invasividade Neoplásica , Neoplasias/patologia , RNA Interferente Pequeno/genéticaRESUMO
AIM: To develop an efficient RNA interference system using phagemid particles displaying the epidermal growth factor (EGF) ligand. METHODS: pSilencer1.0-siEGFP and pSilencer4.1-siAkt plasmids were constructed by gene clone technology. The modified helper phage genome (plasmid) M13KO7EGFCT was used to package phagemids, such as pSilencer1.0-siEGFP and pSilencer4.1-siAkt. ELISA was used to quantify the titer of the progeny virus particles. Single-strand DNA was extracted and analyzed by agarose gel electrophoresis to evaluate the percentage of the phagemid particles. The expression level of the reporter gene enhanced green fluorescence protein (EGFP) was determined by transducing phagemid particles packaging pSilencer1.0-siEGFP into cells. The level of Akt gene expression in cells transduced phagemid particles packaging pSilencer4.1-siAkt was examined by Western blotting. Hydroxycamptothecin (HCPT) was used to enhance the gene transduction efficiency. RESULTS: RNAi vectors pSilencer1.0-siEGFP and pSilencer4.1-siAkt were successfully constructed. Phagemid-encoding siRNA can be packaged efficiently. After the cells were infected by EGF displaying phagemid particles in the presence of HCPT, the expression of the target gene EGFP or Akt was substantially downregulated. CONCLUSION: Cell-targeted phagemid particles are efficient siRNA delivery vectors in the presence of HCPT.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Plasmídeos , Interferência de RNA , Antineoplásicos Fitogênicos/farmacologia , Bacteriófagos/genética , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/genética , Escherichia coli/genética , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução GenéticaRESUMO
Previous studies have shown that all-trans retinoic acid (ATRA) suppresses growth of hepatocarcinoma cell in vitro. To understand the underlying mechanisms, we investigated the protein expression profiles by 2-DE in hepatocarcinoma cell line SMMC-7721 treated with ATRA. Our results reveal that six proteins were differently expressed in response to ATRA. Using MS and database searching, they were identified as profilin 1, phosphoglycerate kinase 1, RuvB-like 1, alpha-enolase, pyridoxal kinase and F-actin capping protein. We selected the up-regulated protein, profilin 1 (PFN1), for further studies. The PFN1 expression was increased in response to ATRA in a dose- and time-dependent manner. The PFN1 expression was reduced dramatically in four hepatoma cell lines compared to L02 cell line of non-tumor origin. The PFN1 expression was also examined in 4 cases of primary hepatocarcinoma tissues by Western blot and 30 cases by tissues microarray. It was found that the protein level of PFN1 was lower in hepatocarcinoma tissues compared to that in the adjacent tissues. Similar to ATRA, overexpression of PFN1 led to inhibition of cell proliferation and migration. Furthermore, RNAi-based PFN1 knockdown could rescue the inhibitory effect of ATRA on cell proliferation and migration. In conclusion, ATRA inhibited cell proliferation and migration through up-regulation of PFN1.
Assuntos
Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Profilinas/metabolismo , Proteômica/métodos , Tretinoína/farmacologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Relação Dose-Resposta a Droga , Eletroforese em Gel Bidimensional , Humanos , Neoplasias Hepáticas/genética , Profilinas/genética , Profilinas/fisiologia , Interferência de RNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
PTEN is a major tumor suppressor gene that has been shown to inhibit cell invasion. Its mutation has been found in 20-40% of malignant gliomas. Meanwhile, the type III EGFR mutation (EGFRvIII), which was frequently found in gliomas, promoted cell invasion. In the present study, the effects of PTEN on cell invasion were investigated in U87DeltaEGFR glioblastoma cells with EGFRvIII expression but missing PTEN. The cell invasion was downregulated by transfection of phosphatase-active forms of PTEN (wild-type and G129E) but not by PTEN (C124A) with an inactive phosphatase domain; the effects were correlated with decreased tyrosine phosphatase levels of FAK at Tyr397, which was increased by EGFRvIII. Overexpression of FAK mutant (Y397F) could partially mimic the effect of PTEN on cell invasion. Although EGFRvIII increased the levels of P-Akt and PTEN eliminated it, PI-3K inhibitors, wortmannin or Ly294002, could not decrease the cell invasion. In conclusion, PTEN could inhibit cell invasion even in the presence of the constitutively active EGFR; this inhibition depended on its protein phosphatase activity, partially by dephosphorylating FAK, but not depended on its lipid phosphatase activity.
Assuntos
Receptores ErbB/genética , Glioma/genética , Glioma/patologia , Mutação , Invasividade Neoplásica/prevenção & controle , PTEN Fosfo-Hidrolase/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Receptores ErbB/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Expressão Gênica , Glioblastoma , Humanos , Invasividade Neoplásica/genética , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas/metabolismo , Transfecção , CicatrizaçãoRESUMO
Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin beta1 imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21(CIP1). Furthermore, our findings suggested that transcription activity of the p21(CIP1) gene could be upregulated in the integrin beta1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21(CIP1) promoter.
