Transcriptional regulation of inducible nitric oxide synthase gene therapy: targeting early stage and advanced prostate cancer

Abstract

Using the tumour type specific human osteocalcin (hOC) promoter, we have previously reported strong promoter activation in hormone independent prostate cancer cells in vitro. In the present study, we present a comparative study of the tissue specific promoter prostate specific membrane antigen (PSMA), and the tumour-type specific hOC promoter driving the inducible nitric oxide synthase (iNOS) transgene using both in vitro and in vivo models. In vitro cytotoxicity was assessed by clonogenic assay. Quantification of nitric oxide expression was determined by the Griess test. In vivo anti-tumour efficacy was determined by tumour growth delay following direct intra-tumoural injection of the constructs into PC3 xenografts. In addition, tumours were dissected post mortem and examined for morphological differences as well as changes in apoptotic protein expression. PSMA/iNOS produced cytotoxicity in both androgen dependant and independent cell lines. Nitric oxide quantification confirmed that increased cytotoxicity was directly associated with nitric oxide production. Tumour growth delays were observed in all groups treated with the iNOS-expressing constructs ranging from 10.7 days for the hOC/iNOS single dose treatment group to a maximum of 52.2 days for the hOC/iNOS multiple dose group. Intra-tumoural assessment of iNOS and cleaved poly (ADP-ribose) polymerase protein expression demonstrated a significant up-regulation of both proteins, indicating cytotoxicity mediated through the intrinsic apoptotic pathway. Highly significant tumour growth delay coupled with no detrimental side-effects were observed following treatment with the PSMA/iNOS and hOC/iNOS constructs. We consider that these findings provide a basis for the development of systemically delivered PSMA/iNOS or hOC/iNOS targeting early stage and advanced prostate cancer.