RESUMEN
AIM: To examine whether attenuated Salmonella typhimurium (S typhimurium) could be used as an anti-cancer agent or a tumor-targeting vehicle for delivering shRNA-expressing pDNA into cancer cells in a mouse tumor model. METHODS: Mouse bladder transitional cancer cell line (BTT-T739) expressing GFP was used, in which the GFP expression level served as an indicator of RNA interference (RNAi). BTT-T739-GFP tumor-bearing mice (4-6 weeks) were treated with S typhimurium carrying plasmids encoding shRNA against gfp or scrambled shRNA. The mRNA and protein expression levels of GFP were assessed 5 d after the bacteria administration, and the antitumor effects of S typhimurium were evaluated. RESULTS: In BTT-T739-GFP tumor-bearing mice, S typhimurium (1×10(9) cfu, po) preferentially accumulated within tumors for as long as 40 d, and formed a tumor-to-normal tissue ratio that exceeded 1000/1. S typhimurium carrying plasmids encoding shRNA against gfp inhibited the expression of GFP in tumor cells by 73.4%. Orally delivered S typhimurium significantly delayed tumor growth and prolonged the survival of tumor-bearing mice. CONCLUSION: This study demonstrates that attenuated S typhimurium can be used for both delivering shRNA-expressing vectors into tumor cells and eliciting RNAi, thus exerting anti-tumor activity, which may represent a new strategy for the treatment of solid tumors.
Asunto(s)
Vectores Genéticos , Interferencia de ARN , ARN Interferente Pequeño/genética , Salmonella typhimurium/genética , Neoplasias de la Vejiga Urinaria/terapia , Animales , Línea Celular Tumoral , Ratones , ARN Interferente Pequeño/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidadRESUMEN
Estrogen receptor alpha (ERalpha) signaling is paramount for normal mammary gland development and function and the repression of breast cancer. ERalpha function in gene regulation is mediated by a number of coactivators and corepressors, most of which are known to modify chromatin structure and/or influence the assembly of the regulatory complexes at the level of transcription initiation. Here we describe a novel mechanism of attenuating the ERalpha activity. We show that cofactor of BRCA1 (COBRA1), an integral subunit of the human negative elongation factor (NELF), directly binds to ERalpha and represses ERalpha-mediated transcription. Reduction of the endogenous NELF proteins in breast cancer cells using small interfering RNA results in elevated ERalpha-mediated transcription and enhanced cell proliferation. Chromatin immunoprecipitation reveals that recruitment of COBRA1 and the other NELF subunits to endogenous ERalpha-responsive promoters is greatly stimulated upon estrogen treatment. Interestingly, COBRA1 does not affect the estrogen-dependent assembly of transcription regulatory complexes at the ERalpha-regulated promoters. Rather, it causes RNA polymerase II (RNAPII) to pause at the promoter-proximal region, which is consistent with its in vitro biochemical activity. Therefore, our in vivo work defines the first corepressor of nuclear receptors that modulates ERalpha-dependent gene expression by stalling RNAPII. We suggest that this new level of regulation may be important to control the duration and magnitude of a rapid and reversible hormonal response.