Gene therapy approach utilizing adenovirus to deliver the pro-apoptotic gene, Bax, for tissue-specific treatment of prostate cancer.

摘要

In this study, we describe the development of a novel approach for treating prostate cancer with cytoreductive gene therapy. The necessity of limiting cytotoxic gene expression to specific cell populations can be achieved through the use of tissue-specific promoters. However, viral regulatory elements retained in the commonly used 1st and 2nd generation adenoviral vectors have been shown to interfere with promoter activity. Here we confirm the adenoviral E1A enhancer element can interfere with tissue-specific and regulatable promoter activities inserted directly proximal and describe an alternate transgene insertion site to retain promoter specificity.; Following these findings, we constructed a 2nd generation adenovirus vector that utilizes a prostate-specific promoter, ARR₂PB, to direct expression of the proapoptotic gene, Bax, for induction of apoptosis in prostate cancer. ARR₂PB promoter activity is prostate-specific and responsive to androgens end glucocorticoids. LNCaP PCa cells infected with ARR₂PB.Bax.GFP adenovirus showed high levels of Bax expression resulting in a significant reduction in cell viability. Importantly, LNCaP cells stably-transfected to overexpress anti-apoptosic Bcl-2 (as is found frequently in advanced prostate cancer) were not resistant, but showed similar patterns of cell death when infected with ARR₂PB.Bax.GFP adenovirus. Tissue-ppecificity of ARR₂PB promoter was evaluated using A549 (lung), SK-Hep-1 (liver), and Hela (cervical) cancer cells, which did not show detectable expression of virally delivered Bax protein or any increase in cell death.; To evaluate in vivo activity of the prostate-specific promoter and Bax-mediated apoptosis, LNCaP xenografts were established in male nude mice and treated with intratumoral injections of ARR₂PB.Bax.GFP adenovirus. Androgen receptor-positive LNCaP xenografts showed significant growth suppression with high levels of Bax expression and extensive apoptosis in tumor sections treated with ARR₂PB.Bax.GFP adenovirus. Evaluation of promoter activity in androgen receptor-negative PC3 cells and PC3-AR cells (stably-transfected to express wild type AR) showed that promoter activity was limited to AR-positive cells which could activate the ARR₂PB promoter with significant growth reduction in PC3-AR cells. Systemic administration of ARR₂PB.Bax.GFP Ad showed no toxicity in any of the vital organs examined including lung, liver, spleen, and kidney. Thus, results of this investigation represent an efficacious approach for the treatment of prostate cancer with cytoreductive gene therapy.