Prolactin and estrogen up-regulate carboxypeptidase-d to promote nitric oxide production and survival of mcf-7 breast cancer cells.

Abstract

Carboxypeptidase-D (CPD) and carboxypeptidase-M (CPM) release C-terminal arginine (Arg) from polypeptides, and both are present in the plasma membrane. Cell-surface CPD increases intracellular Arg, which is converted to nitric oxide (NO). We have reported that prolactin (PRL) regulated CPD mRNA levels in MCF-7 breast cancer cells. This study examined PRL/17beta-estradiol (E2) regulation of CPD/CPM expression, and the role of CPD in NO production for survival of MCF-7 cells. We showed that PRL or E2 up-regulated CPD mRNA and protein expression. PRL/E2 increased CPD mRNA levels by 3- to 5-fold but had no effect on CPM. In Arg-free DMEM, exogenous L-Arg or substrate furylacryloyl-Ala-Arg (Fa-Ala-Arg) increased NO levels and cell survival, measured using 4,5-diaminofluorescein diacetate and the MTS assay, respectively. In the presence of Fa-Ala-Arg, NO production was enhanced by PRL and/or E2 but inhibited by CPD/CPM-specific inhibitor, 2-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MGTA). MGTA also decreased MCF-7 cell survival. In Arg-free medium, annexin-V staining showed that apoptotic MCF-7 cells (approximately 60%) were rescued by Fa-Ala-Arg (25%) or diethylamine/NO (10%). Finally, CPD or CPM gene expression was knocked down with small interfering (si) CPD or siCPM, respectively, with nontargeting siNT as controls. In Arg-free DMEM, the stimulatory effect of Fa-Ala-Arg on NO production was inhibited by siCPD only, showing that CPD depletion inhibited Fa-Ala-Arg cleavage. Furthermore, more than 60% of siCPD-transfectants were apoptotic, and L-Arg, not Fa-Ala-Arg, significantly decreased apoptosis to 32% (P<or=0.05). Thus, CPD gene knockdown did not affect L-Arg uptake, which protected cells from apoptosis. In summary, PRL/E2-induced cell-surface CPD released Arg from extracellular substrates, increased intracellular NO, promoted survival and inhibited apoptosis of MCF-7 cells.

• Authors: Salma A Abdelmagid, Catherine K L Too
• Journal: Endocrinology (Endocrinology) Vol. 149 Issue 10 Pg. 4821-8 (Oct 2008) ISSN: 0013-7227 [Print] United States
• PMID: 18535109 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Dipeptides
• Protease Inhibitors
• Proteins
• RNA, Messenger
• Nitric Oxide
• Estradiol
• furylacryloylalanylarginine
• 2-mercaptomethyl-3-guanidinoethylthiopropionic acid
• Prolactin
• Arginine
• 3-Mercaptopropionic Acid
• metallocarboxypeptidase D

Topics

• 3-Mercaptopropionic Acid (analogs & derivatives, pharmacology)
• Apoptosis (drug effects, physiology)
• Arginine (pharmacology)
• Breast Neoplasms (pathology, physiopathology)
• Cell Line, Tumor
• Cell Survival (drug effects, physiology)
• Dipeptides (pharmacology)
• Estradiol (pharmacology)
• Gene Expression Regulation, Enzymologic (drug effects, physiology)
• Gene Expression Regulation, Neoplastic (drug effects, physiology)
• Humans
• Nitric Oxide (metabolism)
• Prolactin (pharmacology)
• Protease Inhibitors (pharmacology)
• Proteins (genetics, metabolism)
• RNA, Messenger (metabolism)
• Transfection
• Up-Regulation (drug effects, physiology)