title: Prolactin- and testosterone-induced carboxypeptidase-D correlates with increased nitrotyrosines and Ki67 in prostate cancer.
abstract: BACKGROUND
  Carboxypeptidase-D (CPD) cleaves C-terminal arginine for conversion to nitric oxide (NO) by nitric oxide synthase (NOS). Prolactin (PRL) and androgens stimulate CPD gene transcription and expression, which increases intracellular production of NO to promote viability of prostate cancer (PCa) cells in vitro. The current study evaluated whether hormonal upregulation of CPD and NO promote PCa cell viabilty in vivo, by correlating changes in expression of CPD and nitrotyrosine residues (products of NO action) with proliferation marker Ki67 and associated proteins during PCa development and progression.
  
  
  METHODS
  Fresh prostate tissues, obtained from 40 men with benign prostatic hyperplasia (BPH) or PCa, were flash-frozen at the time of surgery and used for RT-qPCR analysis of CPD, androgen receptor (AR), PRL receptor (PRLR), eNOS, and Ki67 levels. Archival paraffin-embedded tissues from 113 men with BPH or PCa were used for immunohistochemical (IHC) analysis of CPD, nitrotyrosines, phospho-Stat5 (for activated PRLR), AR, eNOS/iNOS, and Ki67.
  
  
  RESULTS
  RT-qPCR and IHC analyses showed strong AR and PRLR expression in benign and malignant prostates. CPD mRNA levels increased ∼threefold in PCa compared to BPH, which corresponded to a twofold increase in Ki67 mRNA levels. IHC analysis showed a progressive increase in CPD from 11.4 ± 2.1% in benign to 21.8 ± 3.2% in low-grade (P = 0.007), 40.7 ± 4.0% in high-grade (P < 0.0001) and 50.0 ± 9.5% in castration-recurrent PCa (P < 0.0001). Immunostaining for nitrotyrosines and Ki67 mirrored these increases during PCa progression. CPD, nitrotyrosines, and Ki67 tended to co-localize, as did phospho-Stat5.
  
  
  CONCLUSIONS
  CPD, nitrotyrosine, and Ki67 levels were higher in PCa than in benign and tended to co-localize, along with phospho-Stat5. The strong correlation in expression of these proteins in benign and malignant prostate tissues, combined with abundant AR and PRLR, supports in vitro evidence that the CPD-Arg-NO pathway is involved in the regulation of PCa cell proliferation. It further highlights a role for PRL in the development and progression of PCa.
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada.
authors.forename: Lynn N
authors.initials: LN
authors.lastname: Thomas
authors.affiliation: Department of Pathology, Dalhousie University, Halifax, NS, Canada.|Department of Urology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Merrimen
authors.affiliation: Department of Urology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada.
authors.forename: David G
authors.initials: DG
authors.lastname: Bell
authors.affiliation: Department of Urology, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Rendon
authors.affiliation: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada.
authors.forename: Catherine K L
authors.initials: CK
authors.lastname: Too
mesh_terms: D002268:Carboxypeptidases / Q000378:metabolism*; D006801:Humans; D019394:Ki-67 Antigen / Q000378:metabolism*; D008297:Male; D060787:Neoplasm Grading; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011388:Prolactin / Q000494:pharmacology*; D011467:Prostate / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011470:Prostatic Hyperplasia / Q000378:metabolism* / Q000473:pathology; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011944:Receptors, Androgen / Q000378:metabolism; D011981:Receptors, Prolactin / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013739:Testosterone / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D019394:Ki-67 Antigen; D011944:Receptors, Androgen; D011981:Receptors, Prolactin; D050799:STAT5 Transcription Factor; C002744:3-nitrotyrosine; D013739:Testosterone; D014443:Tyrosine; D011388:Prolactin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D002268:Carboxypeptidases; C078874:carboxypeptidase D
keywords: Ki67; carboxypeptidase-D; nitric oxide; prolactin; prostate cancer