RESUMO
Certain bacteria have emerged as biological gene vectors with natural tumor specificity, capable of specifically delivering genes or gene products to the tumor environment when intravenously (i.v.) administered to rodent models. We show for the first time that oral administration of bacteria to mice resulted in their translocation from the gastrointestinal tract (GIT) with subsequent homing to and replication specifically in tumors. The commensal, nonpathogenic Bifidobacterium breve UCC2003 harboring a plasmid expressing lux fed to mice bearing subcutaneous (s.c.) tumors were readily detected specifically in tumors, by live whole-body imaging, at levels similar to i.v. administration. Reporter gene expression was visible for >2 weeks in tumors. Mice remained healthy throughout experiments. Cytokine analyses indicated a significant upregulation of interferon-gamma (IFN-gamma) in the GIT of bifidobacteria-fed mice, which is associated with increases in epithelial permeability. However, B. breve feeding did not increase systemic levels of other commensal bacteria. The presence of tumor was not necessary for translocation to systemic organs to occur. These findings indicate potential for safe and efficient gene-based treatment and/or detection of tumors via ingestion of nonpathogenic bacteria expressing therapeutic or reporter genes.
Assuntos
Bifidobacterium/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Melanoma Experimental/metabolismo , Administração Oral , Animais , Bifidobacterium/imunologia , Bifidobacterium/isolamento & purificação , Citocinas/metabolismo , Ensaios Enzimáticos , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Trato Gastrointestinal/microbiologia , Vetores Genéticos/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Von Hippel-Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-α (HIF-α), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP)A2/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. METHODS: The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by Real-Time RT-PCR. Expression of pVHL's protein targets were determined by Western blotting in 17 paired patient samples. RESULTS: VHL genetic alterations were identified in 43.5% (10/23) of CCRCCs. HIF-1α, HIF-2α and CAIX were up-regulated in 88.2% (15/17), 100% (17/17) and 88.2% (15/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. CONCLUSION: As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), this data suggests that other pVHL targets may be more crucial in renal carcinogenesis.
Assuntos
Antígenos de Neoplasias/genética , Anidrases Carbônicas/genética , Carcinoma de Células Renais/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Renais/genética , Osteopontina/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Antígenos de Neoplasias/metabolismo , Anidrase Carbônica IX , Anidrases Carbônicas/metabolismo , Carcinoma de Células Renais/enzimologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/enzimologia , Neoplasias Renais/patologia , Mutação/genética , Osteopontina/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
BACKGROUND: von Hippel-Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-α (HIF-α), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. METHODS: The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by real-time RT-PCR. Expression of pVHL's protein targets were determined by Western blotting in 17 paired patient samples. RESULTS: VHL genetic alterations were identified in 43.5% (10/23) of CCRCCs. HIF-1α, HIF-2α and CAIX were up-regulated in 88.2% (15/17), 100% (17/17) and 88.2% (15/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. CONCLUSION: As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), these data suggests that other pVHL targets may be more crucial in renal carcinogenesis.