Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells

In prostate carcinoma, overexpression of the anti-apoptotic gone Bcl-2 has been found to be associated with resistance to therapies including radiatio n and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously re sistant to treatment. To accomplish this, a strategy involving overexpressi on of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes s uch as Bax require selective expression of the gene. In this study, we exam ined the ability of selective expression of Bax protein directed by a prost ate-specific promoter to induce apoptosis in human prostate carcinoma. A se cond-generation adenoviral vector was constructed with the modified prostat e-specific probasin promoter, ARR(2)PB, directing expression of an HA-tagge d Bax gene and a green fluorescent protein reporter translated from an inte rnal ribosome entry site (ARR(2)PB.Bax.GFP). ARR(2)PB promoter activity is tightly regulated and highly prostate specific and is responsive to androge ns and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene ca ssette was inserted into a cloning site near the right inverted terminal re peat of the adenoviral vector to retain specificity of the promoter. LNCaP cells infected with Ad/ARR(2)PB.Bax.GFP showed high levels of Bax expressio n 48 h after infection resulting in an 85% reduction in cell viability. Imp ortantly, LNCaP cells stably transfected to overexpress Bcl-2 showed simila r patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% red uction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specific ity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver c ancer) cells, and Hela (cervical cancer) cells which did not show detectabl e expression of virally delivered Bax protein or any increase in cell death . Systemic administration of Ad/ARR(2)PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR(2)PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression o f Bax in prostate carcinoma, even in the context of high levels of Bcl-2 pr otein, resulted in apoptosis. These results suggest that a second-generatio n adenovirus-mediated, prostate-specific Bax gene therapy is a promising ap proach for the treatment of prostate cancer.