title: Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells.
abstract: In prostate carcinoma, overexpression of the anti-apoptotic gene Bcl-2 has been found to be associated with resistance to therapies including radiation and androgen ablation. Restoring the balance of Bcl-2 family members may result in the induction of apoptosis in prostate cancer cells previously resistant to treatment. To accomplish this, a strategy involving overexpression of the pro-apoptotic gene Bax was executed. The use of cytotoxic genes such as Bax require selective expression of the gene. In this study, we examined the ability of selective expression of Bax protein directed by a prostate-specific promoter to induce apoptosis in human prostate carcinoma. A second-generation adenoviral vector was constructed with the modified prostate-specific probasin promoter, ARR2PB, directing expression of an HA-tagged Bax gene and a green fluorescent protein reporter translated from an internal ribosome entry site (ARR2PB.Bax.GFP). ARR2PB promoter activity is tightly regulated and highly prostate specific and is responsive to androgens and glucocorticoids. The prostate-specific promoter-Bax-GFP transgene cassette was inserted into a cloning site near the right inverted terminal repeat 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 expression 48 h after infection resulting in an 85% reduction in cell viability. Importantly, LNCaP cells stably transfected to overexpress Bcl-2 showed similar patterns of cell death when infected with Ad/ARR(2)PB.Bax.GFP, an 82% reduction in cell viability seen 48 h after infection. Apoptosis was confirmed by measuring caspase activation and using the TUNEL assay. Tissue specificity was evaluated using A549 cells (lung adenocarcinoma), SK-Hep-1 (liver cancer) cells, and Hela (cervical cancer) cells which did not show detectable expression of virally delivered Bax protein or any increase in cell death. Systemic administration of Ad/ARR2PB. Bax.GFP in nude mice revealed no toxicity in liver, lung, kidney, or spleen. This study shows that infection with the second-generation adenovirus, ARR2PB.Bax.GFP, results in highly specific cytotoxicity in LNCaP cells, and that consequent overexpression of Bax in prostate carcinoma, even in the context of high levels of Bcl-2 protein, resulted in apoptosis. These results suggest that a second-generation adenovirus-mediated, prostate-specific Bax gene therapy is a promising approach for the treatment of prostate cancer.
authors.affiliation: Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, 29425, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Lowe
authors.forename: S
authors.initials: S
authors.lastname: Rubinchik
authors.forename: T
authors.initials: T
authors.lastname: Honda
authors.forename: T J
authors.initials: TJ
authors.lastname: McDonnell
authors.forename: J Y
authors.initials: JY
authors.lastname: Dong
authors.forename: J S
authors.initials: JS
authors.lastname: Norris
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004789:Enzyme Activation; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000633:toxicity; D006801:Humans; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008819:Mice, Nude; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D013997:Time Factors; D014407:Tumor Cells, Cultured; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; C494827:Bax protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases