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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Coulter
Jonathan A
JA
School of Pharmacy, McClay Research Centre, Queen's University, Belfast, Ireland, UK.
Page
Natalie L
NL
Worthington
Jenny
J
Robson
Tracy
T
Hirst
David G
DG
McCarthy
Helen O
HO
D000818:Animals; D000954:Antigens, Surface / Q000235:genetics; D017209:Apoptosis; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D003110:Colonic Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D043425:Glutamate Carboxypeptidase II / Q000235:genetics; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D009367:Neoplasm Staging; D009376:Neoplasms, Hormone-Dependent / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D019076:Transgenes / Q000502:physiology; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay; D023041:Xenograft Model Antitumor Assays
D000954:Antigens, Surface; D009573:Nitrites; D015675:Osteocalcin; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; C473848:FOLH1 protein, human; D043425:Glutamate Carboxypeptidase II