RESUMO
Prostate cancer is one of the most common cancers in men. The overactivation of IL-6/JAK/STAT3 signaling and silencing of SOCS3 are frequently observed in prostate cancer. In the present study we undertook to develop Ad-SOCS3 gene therapy for the treatment of prostate cancer and also investigated whether Ad-SOCS3 increased sensitivity to NK cells. We demonstrated that Ad-SOCS3 could significantly inhibit growth of castration-resistant prostate cancer (CRPC) cell lines expressing pSTAT3, DU-145 (at 10, 20, and 40 MOI), and TRAMP-C2 (at 40 MOI), but not the PC-3 CRPC cell line with the STAT3 gene deleted. Ad-SOCS3 (40 MOI) could suppress IL-6 production in DU-145 cells and PD-L1 expression induced by IFN-γ in TRAMP-C2 cells, and increased the NK cell sensitivity of both TRAMP-C2 and DU-145 cells. In the DU-145 mouse xenograft tumor model, intratumoral injections (twice/week for 3 weeks) of 1 × 108 pfu of Ad-SOCS3 significantly inhibited tumor growth and combining the Ad-SOCS3 treatment with intratumoral injections (once/week for 2 weeks) of 1 × 107 human NK cells showed the highest tumor growth inhibitory effect. These results suggested that a combination of Ad-SOCS3 gene therapy and NK cell immunotherapy could be a powerful treatment option for advanced CRPC overexpressing pSTAT3.
Assuntos
Adenoviridae/genética , Expressão Gênica , Vetores Genéticos/genética , Células Matadoras Naturais/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Animais , Apoptose/genética , Biomarcadores Tumorais , Ciclo Celular/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Humanos , Interleucina-6/metabolismo , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Neoplasias de Próstata Resistentes à Castração/metabolismo , Transdução de Sinais , Transdução Genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Plants contain potentially toxic compounds for animals and animals have developed physiological strategies to detoxify the ingested toxins during evolution. Feeding mice with various plant seeds and grains showed unexpected result that only sesame killed PPARα-null mice but not wild-type mice at all. A detailed analysis of this observation revealed that PPARα is involved in the metabolism of toxic compounds from plants as well as endobiotic substrates by inducing phase I and phase II detoxification enzymes. PPARα plays a vital role in direct or indirect activation of the relevant genes via the complex network among other xenobiotic nuclear receptors. Thus, PPARα plays its wider and more extensive role in energy metabolism from natural food intake to fat storage than previously thought.
RESUMO
A systematic analysis to examine the effects of peroxisome proliferator-activated receptor (PPAR)alpha agonists on the expression levels of all the nutrient/drug plasma-membrane transporters in the mouse small intestine was performed. Transporter mRNAs that were induced or repressed by two independent PPARalpha-specific agonists were identified by a genome-wide microarray method, and the changes were confirmed by real-time PCR using RNA isolated from the intestines and livers of wild-type and PPARalpha-null mice. Expression levels of seven nutrient/drug transporters (Abcd3, Octn2/Slc22a5, FATP2/Slc27a2, Slc22a21, Mct13/Slc16a13, Slc23a1 and Bcrp/Abcg2) in the intestine were up-regulated and the expression level of one (Mrp1/Abcc1) was down-regulated by PPARalpha; although the previously report that the H(+)/peptide co-transporter 1 (Pept1) is up-regulated by PPARalpha was not replicated in our study. We propose that the transport processes can be coordinately regulated with intracellular metabolism by nutrient nuclear receptors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Coenzima A Ligases/genética , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Proteínas de Membrana/genética , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Proteínas de Transporte de Cátions Orgânicos/genética , PPAR alfa/agonistas , Simportadores/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transportadores de Sódio Acoplados à Vitamina C , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
Peroxisome proliferator (PPAR)alpha ligand Wy14,643 induces liver-fatty acid binding protein (FABP) spontaneously and heart-FABP gradually, but not intestine-FABP mRNA expression in the mouse liver. These strict regulations have not been reproduced in cultured cell systems. We applied a DNA electroporation method to directly introduce reporter gene constructs into the livers of mice. This system reproduced the in vivo responses of the above three FABP gene promoters to the PPARalpha ligand but not that of a promoter containing the typical three PPAR binding sites in tandem. Deletion and mutation analyses of the mouse L-FABP gene suggested that, in addition to the binding site for PPARalpha, a far upstream sequence is required for PPAR-dependent transactivation in the liver. In contrast to the cultured cell systems, our in vivo DNA electroporation method showed that PPARalpha binding to the promoter is necessary but not sufficient for PPARalpha ligand-dependent transcriptional activation of the L-FABP gene in vivo.
Assuntos
Proteínas de Transporte/genética , Eletroporação/métodos , Regulação da Expressão Gênica , Fígado/fisiologia , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , DNA/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Genes Reporter , Masculino , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Distribuição TecidualRESUMO
FAT/CD36 is involved in various processes including uptake of fatty acid into the heart and of oxidized low density lipoprotein (LDL) into macrophages. Expression of the FAT/CD36 gene is regulated in a tissue-specific manner, and loss or inadequately regulated expression of FAT/CD36 is thought to be one of the causes of some diseases such as cardiomyopathy and atherosclerosis. We recently found that the mouse and human FAT/CD36 genes have two independent promoters. To elucidate the physiological significance of the two promoters, we characterized the peroxisome proliferator-activated receptor ligand-responsive new promoter that is located 14 kb upstream of the previously reported promoter of the human gene. We found several SNPs in this region some of which were found only when analyzing DNA samples from the patients lacking FAT/CD36 totally or in a cell-type-specific manner. However, we could not detect any negative effect of these SNPs on the transcription by transient transfection analysis, suggesting that the identified SNPs alone are not directly linked to low transcriptional activities.
Assuntos
Regiões 5' não Traduzidas/genética , Antígenos CD36/genética , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Transportadores de Ânions Orgânicos/química , Transportadores de Ânions Orgânicos/genética , Regiões Promotoras Genéticas/genética , Regiões 5' não Traduzidas/química , Regulação da Expressão Gênica/genética , Humanos , Glicoproteínas de Membrana/deficiência , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos/deficiência , Polimorfismo Genético/genética , Células Tumorais CultivadasRESUMO
Fatty acid translocase (FAT)/CD36 is a glycoprotein involved in multiple membrane functions including uptake of long-chain fatty acids and oxidized low density lipoprotein. In mice, expression of the gene is regulated by peroxisome proliferator-activated receptor (PPAR) alpha in the liver and by PPAR gamma in the adipose tissues (Motojima, K., Passilly, P. P., Peters, J. M., Gonzalez, F. J., and Latruffe, N. (1998) J. Biol. Chem. 273, 16710-16714). However, the time course of PPAR alpha ligand-induced expression of FAT/CD36 in the liver, and also in the cultured hepatoma cells, is significantly slower than those of other PPAR alpha target genes. To study the molecular mechanism of the slow transcriptional activation of the gene by a PPAR ligand, we first cloned the 5' ends of the mRNA and then the mouse gene promoter region from a genomic bacterial artificial chromosome library. Sequencing analyses showed that transcription of the gene starts at two initiation sites 16 kb apart and splicing occurs alternatively, producing at least three mRNA species with different 5'-noncoding regions. The PPAR alpha ligand-responsive promoter in the liver was identified as the new upstream promoter where we found several possible binding sites for lipid metabolism-related transcriptional factors but not for PPAR. Neither promoter responded to a PPAR alpha ligand in the in vitro or in vivo reporter assays using cultured hepatoma cells and the liver of living mice. We also have cloned the human FAT/CD36 gene from a bacterial artificial chromosome library and identified a new independent promoter that is located 13 kb upstream of the previously reported promoter. Only the upstream promoter responded to PPAR alpha and PPAR gamma ligands in a cell type-specific manner. The absence of PPRE in the responding upstream promoter region, the delayed activation by the ligand, and the results of the reporter assays all suggested that transcriptional activation of the FAT/CD36 gene by PPAR ligands is indirectly dependent on PPAR.