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Endocrinology, doi:10.1210/en.2008-0145
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Endocrinology Vol. 149, No. 10 4821-4828
Copyright © 2008 by The Endocrine Society

Prolactin and Estrogen Up-Regulate Carboxypeptidase-D to Promote Nitric Oxide Production and Survival of MCF-7 Breast Cancer Cells

Salma A. Abdelmagid and Catherine K. L. Too

Department of Biochemistry & Molecular Biology (S.A.A., C.K.L.T.) and Department of Obstetrics & Gynecology (C.K.L.T.), Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5

Address all correspondence and requests for reprints to: Catherine K. L. Too, Ph.D., Department of Biochemistry and Molecular Biology, Sir Charles Tupper Medical Building, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5. E-mail: ctoo{at}dal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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/17β-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 (~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 ≤ 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CARBOXYPEPTIDASE-D (CPD) is a membrane-bound metalloproteinase that cleaves C-terminal arginine or lysine residues from its protein substrates (1). The 180-kDa CPD is found primarily in the trans-Golgi network where it processes polypeptides or prohormones that transit the secretory pathway (2, 3, 4, 5), but significant amounts of CPD (~10%) are present in the plasma membrane (6, 7). CPD expression in mouse RAW 264.7 macrophages is up-regulated by interferon-{gamma} and bacterial lipopolysaccharides (8). Cell surface CPD, by releasing arginine from extracellular substrates, has been associated with the production of intracellular nitric oxide (NO) in RAW macrophages (8), rat lungs, and microvascular endothelial cells (9).

CPD-released arginine is a substrate for nitric oxide synthase (NOS). The three NOS isoforms, neuronal (n) NOS/NOSI, inducible (i) NOS/NOSII, and endothelial (e) NOS/NOSIII, catalyze the production of NO from L-arginine (10). NO is a pleiotropic regulator of numerous cellular processes including vasodilation and neurotransmission (11). NO may also act as a pro-tumor molecule in tumor progression (12), tumor cell invasiveness (13), and tumor-induced angiogenesis (14). There is also evidence that NOS activity and NO production are critical in the proliferation and survival of breast tumor cells. About 61% of human breast tumors have been reported to stain positively for iNOS, and patients with iNOS-positive breast carcinoma had a significantly worse overall survival rate than those with negative stains (15). About 50% of the human MCF-7 breast cancer cell line has been shown to immunostain positively for either iNOS or eNOS (16), and treatment with 10–8 M E2 increased eNOS at 48 h (17).

We have previously reported that prolactin (PRL) stimulated CPD expression in lactogen-deprived human HepG2 hepatoma and MCF-7 breast cancer cells (18). Using CPD-specific antibodies in Western analysis, we detected a single, 180-kDa immunoreactive protein in MCF-7 cells (18). CPD activity in MCF-7 cells was found mainly in the postnuclear 10,000 x g fraction (~70%), but significant amounts were present in the 100,000 x g microsomal (15–20%) and 700 x g nuclear (~10%) fractions. PRL and the cytokine IL-2 also stimulated the expression of a CPD isoform, CPD-N (18), which was detected as a 160-kDa protein found exclusively in the nuclei of hematopoietic tumor cells of mouse, rat, and human origin (19). We have also reported that PRL stimulated eNOS expression, leading to NO production which promoted cell survival and inhibited apoptosis in PRL-dependent rat lymphoma cells (20). The role of hormone-inducible CPD in the production of NO in breast cancer cells is not known.

Pituitary PRL is essential for the proliferation and terminal differentiation of the mammary gland during puberty, pregnancy, and lactation (21, 22). However, both normal and malignant human breast tissues are significant sources of extrapituitary PRL, which exerts a proliferative action in an autocrine/paracrine manner (23, 24). The PRL receptor has been shown to be expressed in 98% of all human breast cancers tested (25) and higher plasma PRL levels are associated with an increased risk of breast cancer in postmenopausal women (26). All these findings have led to a recognition of PRL as an active participant in breast cancer and are an impetus to redefine the molecular targets of anti-PRL strategies in this disease.

Ovarian estrogen is also essential for the normal growth and development of the mammary gland, and has a critical role in the development and malignancy of breast tumors (27). Estrogen is also synthesized in the breast where it acts as a paracrine/intracrine growth factor (28). The presence of estrogen receptors in breast tumors has been used to determine tumor aggressiveness, the feasibility of certain therapies, and the prediction of relapse (29).

In this study, we examined if PRL and 17β-estradiol (E2) regulated CPD expression, and the role of CPD in NO production in MCF-7 breast cancer cells. We showed that PRL or E2 up-regulated CPD expression. The hormone-inducible CPD, acting on a synthetic extracellular substrate, increased intracellular NO production to increase survival of MCF-7 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Poly-L-lysine, furylacryloyl-Ala-Arg (Fa-Ala-Arg), furylacryloyl-Ala-Lys (Fa-Ala-Lys), 4,5-diaminofluorescein diacetate (DAF-2DA), and DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, Ontario, Canada). The NO-releasing complex diethylamine/nitric oxide (DEA/NO) was from RBI Research Biomedicals International (Natick, MA).

Cell culture
Human MCF-7 breast cancer cells were maintained in DMEM containing 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) and supplemented with 1x MEM nonessential amino acid solution, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Invitrogen Canada Inc., Burlington, Ontario, Canada) at 37 C in a humidified, 5%-CO2 atmosphere. Approximately 50–60% confluent MCF-7 cells were washed twice with PBS and growth arrested for 24–48 h in phenol red-free DMEM containing 1% lactogen-free horse serum (HS) before PRL treatment or 1% charcoal-stripped FBS before E2 treatment. In some experiments, MCF-7 cells were incubated in Arg-containing DMEM (0.398 mM L-Arg-HCl) or Arg-free DMEM (Invitrogen), with or without the exogenous addition of 1 mM L-Arg-HCl (Sigma-Aldrich).

Semiquantitative RT-PCR analysis
Total RNA was isolated using GenElute Mammalian Total RNA miniprep kit (Sigma-Aldrich) following the manufacturer’s instructions. Polyadenylated mRNA was isolated using MicroPoly(A)Pure kit (Ambion, Inc., Austin, TX). Reverse transcription of 1 µg total RNA, followed by PCR amplification, was performed as previously described (20). Primer pairs for PCR were as follows: human CPD, 5'-ATG-GCA-GGG-GTA-TAT-TAA-ATG-CCA-3' and 5'-GGA-TAC-CAG-CAA-CAA-AAC-GAA-TCT-3' (576-bp product) (18), human CPM, 5'-GGC-AAA-GAC-CCT-GAA-ATC-ACA-3' and 5'-TCC-CTT-CCG-ATG-CTG-TAG-TAA-C-3' (122-bp product) and human glyceraldehyde-3-phosphate-dehydrogenase (gapdh), 5'-TGA-TGA-CAT-CAA-GAA-GGT-GGT-GA-3' and 5'-TCC-TTG-GAG-GCC-ATG-TAG-GCC-AT-3' (240-bp product) (20). Quantum RNA 18S Internal Standards kit was used to amplify the 18S rRNA (Ambion, Inc). RT-PCR products were resolved in 1–2% agarose gels.

SDS-PAGE and Western analysis
MCF-7 cells were homogenized and sonicated in lysis buffer [150 mM NaCl, 50 mM Tris (pH 7.4), 50 mM sodium pyrophosphate, containing 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM sodium orthovanadate]. Cell lysates were centrifuged at 100,000 x g for 1 h, and the resulting pellet was resuspended in lysis buffer. SDS-PAGE was performed using 36 µg of protein/lane. Western blotting was performed with anti-CPD (18) and anti-actin antibodies (Sigma-Aldrich). Immunoreactive signals were detected using Immobilon Western Chemiluminescent horseradish peroxidase Substrate kit (Millipore, Billerica, MA).

Measurement of NO: DAF-2DA assay
MCF-7 cells (50,000 cells/well) in Arg-containing DMEM were seeded onto poly-L-lysine-coated chambered slides. After 24 h, the cells were washed twice with PBS, then given fresh Arg-free DMEM containing 1% HS for 16–24 h to make cells quiescent. These cells were treated with 100 ng/ml PRL for specific times (2–4 h), as cells without hormone treatment showed poor or undetectable fluorescence (data not shown). The cells were washed thrice with PBS, then incubated with 5 µM DAF-2DA (Sigma-Aldrich) for 30 min at 37 C. Nonfluorescent DAF-2DA, when taken into the cell, was hydrolyzed by intracellular esterases to form membrane-impermeable DAF-2, which reacted with intracellular NO to produce the fluorescent triazole derivative DAF-2T. After incubation, extracellular DAF-2DA was removed and the cells were treated with either 1 mM Arg for 15 min or 4 mM Fa-Ala-Arg for 45 min. In some experiments, the cells were also given 10 µM MGTA (inhibitor of CPD or CPM) for 12–16 h before, as well as during, Fa-Ala-Arg treatment. Fluorescence was examined using Zeiss Axiovert 200 fluorescent microscope (Zeiss, Jena, Germany), and images were captured using AxioCam HRc camera (Zeiss).

Measurement of cell survival: MTS assay
MCF-7 cells in DMEM containing 10% heat-inactivated FBS were seeded into 24-well plates (50,000 cells/well). After treatment, the MTS assay (CellTiter 96 AQueous Non-Radioactive Proliferation Assay; Fisher Scientific, Ltd., Nepean, Ontario, Canada) was performed following the manufacturer’s instruction.

Measurement of apoptosis: Annexin-V staining assay
MCF-7 cells in DMEM containing 10% heat-inactivated FBS were seeded onto chambered slides and allowed to grow for 24 h before the start of experiments. Annexin-V staining was performed following manufacturer’s instruction (Roche Applied Sciences, Laval, Quebec, Canada). Fluorescent cells were counted in fields with a total number of 300–900 cells/ treatment.

Small interfering (si) RNA transfections
siRNAs were used to knockdown CPD or CPM gene expression. siCPD (siRNA ID no. 103996), siCPM (siRNA ID no. 112813) and the nontargeting controls (siNT, catalog no. 4605) were purchased from Ambion/Applied Biosystems (Streetsville, Ontario, Canada). The siRNAs were transfected into MCF-7 cells using RNAiMAX reagent (Invitrogen) following manufacturer’s instructions. After 24 h, the cells were seeded onto chambered slides in medium containing PRL (100 ng/ml). The cells were allowed to grow for another 24 h before RNA extraction or the start of other treatments.

Statistical analyses
Statistical analyses were performed using StatView (Abacus Concepts, Berkeley, CA), and results were expressed as mean ± SEM. ANOVA and Fisher’s protected least significant difference test were used for comparison of means. P values ≤ 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PRL or E2 up-regulates CPD mRNA and protein levels
We have previously reported that CPD is a PRL-inducible gene in MCF-7 cells (18). The present study showed that PRL (10 ng/ml) up-regulated the CPD transcript by 3- to 5-fold after 4 h of treatment (Fig. 1Go, A and B). This is the first report that E2 (10 nM) also up-regulated CPD expression, the increase was over 4-fold at 2 h, and the transcript remained elevated up to 8 h after treatment (Fig. 1Go, A and B). CPM is also present in the plasma membrane and, like CPD, CPM cleaves C-terminal Arg (9). However, PRL or E2 had little effect on the expression of CPM in MCF-7 cells (Fig. 1Go, C and D).


Figure 1
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  		FIG. 1. PRL or E2 up-regulates CPD, not CPM, mRNA. MCF-7 cells were made quiescent in phenol red-free DMEM containing either 1% lactogen-free HS for 24–48 h before the addition of PRL (10 ng/ml), or 1% charcoal-stripped FBS before the addition of E2 (10 nM). After hormonal treatment for the indicated times, total RNA was isolated for semiquantitative RT-PCR analysis as described in Materials and Methods. A, RT-PCR of CPD transcript. B, Densitometric scans of CPD/18S rRNA. Each is a representative of 3 separate experiments. C, RT-PCR of CPM mRNA. Representative of 2 experiments. D, Densitometric scans of CPM/gapdh, showing mean ± range (n = 2).

 
Western analysis showed that PRL or E2 up-regulation of the CPD transcript was accompanied by an increase in CPD protein levels (Fig. 2Go). In E2-stimulated cells, the transient decrease in CPD mRNA levels at 4 h was reproducible (Fig. 1BGo), suggesting mRNA turnover, but the protein level remained elevated between 2 and 4 h (Fig. 2Go).


Figure 2
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  		FIG. 2. PRL or E2 up-regulates CPD protein. Quiescent MCF-7 cells were treated with (A) PRL or (B) E2 as in Fig. 1Go. Total cell lysates were prepared, and the 100,000 x g pellets were used for SDS-PAGE and Western analysis. The CPD-immunoreactive band was 180 kDa. Actin was used as a loading control.

 
Exogenous L-Arg promotes NO production and MCF-7 cell survival
CPD releases C-terminal Arg from polypeptides. L-Arg is a substrate of NOS for the production of NO. To establish a link between exogenous L-Arg and intracellular NO production, MCF-7 cells were cultured either in Arg-containing (0.398 mM L-Arg-HCl; Invitrogen) or Arg-free DMEM, for up to 24 h before the addition of 1 mM exogenous L-Arg. Under each culture condition, the addition of 1 mM L-Arg increased the production of intracellular NO (Fig. 3AGo). In cells cultured in Arg-free DMEM, the addition of increasing doses of L-Arg (0.1–1 mM) increased MCF-7 cell survival by over 2-fold, as measured using the MTS assay (Fig. 3BGo).


Figure 3
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  		FIG. 3. L-Arg promotes NO production and MCF-7 cell survival. A, MCF-7 cells were cultured in Arg-containing (Arg-plus) or Arg-free DMEM for 16 - 24 h. Cells were treated with PRL (100 ng/ml) for 2 h, then washed in PBS before loading with 5 µM DAF-2DA for 30 min at 37 C (see Materials and Methods). L-Arg (1 mM) was added and after a 15-min incubation, fluorescent microscopy was performed to detect intracellular NO. Arg-treated cells were noticeably more robust. Bar, 20 µm. B, MCF-7 cells, cultured in Arg-free DMEM for 16–24 h, were treated with increasing doses of L-Arg and after 3 d, the MTS assay was used to measure cell survival. Representative of at least three separate experiments, each in triplicate. Mean ± SE (SEM), triplicates of one experiment. *, P ≤ 0.0001 vs. control (0 mM Arg).

 
Fa-Ala-Arg increases NO production and survival of MCF-7 cells
The stimulatory effects of free L-Arg on MCF-7 cells were mimicked by the synthetic carboxypeptidase-specific substrate Fa-Ala-Arg (Fig. 4Go). When added to Arg-depleted MCF-7 cells, Fa-Ala-Arg increased both intracellular NO production (Fig. 4AGo) and cell survival (Fig. 4BGo), but the latter was abrogated by L-lysine (Fig. 4CGo), an Arg transport inhibitor (30). Unlike Fa-Ala-Arg, Fa-Ala-Lys had no effect on cell survival (Fig. 4CGo), indicating that Arg but not Lys contributed to the survival of the MCF-7 cells. Taken together, these results suggested that cell surface CPD was capable of catalyzing the cleavage of Arg from extracellular substrates, and the released Arg promoted NO production and survival of MCF-7 cells.


Figure 4
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  		FIG. 4. Fa-Ala-Arg increases NO production and survival of MCF-7 cells. A, MCF-7 cells were cultured in Arg-free DMEM, treated with PRL, then washed in PBS before loading with 5 µM DAF-2DA for 30 min at 37 C as in Fig. 3Go. Fa-Ala-Arg (4 mM) was added and after a 45-min incubation, fluorescent microscopy was performed. Bar, 20 µm. B and C, Arg-deprived MCF-7 cells were treated with increasing doses of Fa-Ala-Arg (±10 mM L-lysine) or Fa-Ala-Lys. The MTS assay was performed on d 3. Each graph is a representative of three separate experiments, each done in triplicate. Mean ± SEM. *, P ≤ 0.05 vs. control (0 mM Fa-Ala-Arg).

 
PRL/E2 increases NO production but MGTA decreases NO and cell survival
To further delineate the role of PRL/E2-inducible CPD on NO production, MCF-7 cells were made quiescent in serum-reduced, Arg-free DMEM. Fa-Ala-Arg was added as the exogenous substrate for the cell surface CPD. Control MCF-7 cells not given the CPD substrate showed undetectable NO but cells given the substrate produced a low, but detectable level of NO (Fig. 5AGo, panels 1 and 2). Cells treated with Fa-Ala-Arg, as well as PRL and/or E2, showed a further increase in NO production (Fig. 5AGo, panels 3–5). The PRL-stimulated increase in NO was inhibited by CPD-specific inhibitor, MGTA (Fig. 5BGo). MGTA treatment also resulted in a significant decrease in MCF-7 cell viability (Fig. 5CGo). Thus, PRL/E2-inducible CPD up-regulated NO production in the presence of the CPD substrate but inhibition of CPD decreased both NO production and cell survival.


Figure 5
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  		FIG. 5. PRL/E2 increases but MGTA decreases NO and cell survival. A, Quiescent MCF-7 cells, in Arg-free medium, were treated with PRL (100 ng/ml) and/or E2 (10 nM) for 4 h to up-regulate CPD, or had no hormone treatment. All the cells were preloaded with DAF-2DA, and all but controls were given Fa-Ala-Arg (4 mM) before fluorescent microscopy as in Fig. 4Go. B, MCF-7 cells, in Arg-free medium, were treated with 10 µM MGTA (+) or left untreated (–) for 16 h. This was followed by addition of PRL (2 h), then DAF-2DA and Fa-Ala-Arg, and fluorescent microscopy. Bar, 20 µm. C, PRL-stimulated MCF-7 cells, were left without further treatment (Control) or given Fa-Ala-Arg (1 mM; positive control) or Fa-Ala-Arg with increasing doses of MGTA. MTS assay was performed on d 2. Mean ± SEM (n = 3). **, P ≤ 0.0001; *, P ≤ 0.05 vs. positive control (Fa-Ala-Arg). All experiments were repeated two to three times.

 
Fa-Ala-Arg or DEA/NO decreases apoptosis in MCF-7 cells
MCF-7 cells in Arg-free DMEM were treated with PRL to up-regulate CPD expression. Annexin-V staining showed that about 60% of these cells (controls) were apoptotic under the culture conditions used (Fig. 6Go). However, the additional presence of Fa-Ala-Arg or DEA/NO significantly reduced the number of apoptotic cells to about 25% and 10%, respectively (Fig. 6BGo). These results implied that Arg, released by PRL-induced CPD, and the production of intracellular NO protected MCF-7 cells from undergoing apoptosis.


Figure 6
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  		FIG. 6. Fa-Ala-Arg or DEA/NO decreases apoptosis in MCF-7 cell. Quiescent MCF-7 cells in Arg-free DMEM for 24 h were treated with PRL (100 ng/mL; control), and further given Fa-Ala-Arg (4 mM) or DEA/NO (600 µM) for 3 d. A, Annexin-V staining was performed to measure apoptosis. B, The % of apoptotic cells was determined. Mean ± SEM (n = 7–10). *, P ≤ 0.0001 vs. control.

 
CPD gene knockdown decreases NO production
Using siRNAs, the effect of CPD gene knockdown on NO production in MCF-7 cells was next examined. CPD gene expression was successfully reduced by at least 90% in siCPD-transfected cells (Fig. 7AGo). As anticipated, the stimulatory effect of Fa-Ala-Arg on intracellular NO production (see also Fig. 5AGo) was unaffected by transfection of nontargeting siNT (Fig. 7BGo, upper panels) but was abrogated by siCPD (Fig. 7BGo, lower panels).


Figure 7
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  		FIG. 7. siCPD, not siCPM, decreases NO production. A and B, MCF-7 cells were transfected with 10 nM siCPD or siNT as described in Materials and Methods. Control (Con) cells were untransfected. After 24 h, cells were seeded in medium containing PRL for another 24 h. In A, total RNAs were isolated for semiquantitative RT-PCR 48 h after transfection (left panel). The CPD/gapdh ratio in transfected cells was analyzed by densitometry (right panel). B, Intracellular NO production in transfected cells was measured using DAF-2DA, ± Fa-Ala-Arg. C and D, MCF-7 cells were transfected with up to 30 nM siCPM or siNT, whereas controls (Con) were not transfected. C, RT-PCR for the CPM transcript (left panel) and densitometric analysis of the CPM/gapdh ratio (right panel). D, NO production in transfected cells, ± Fa-Ala-Arg. Bar, 20 µm. Each is a representative of two to three separate experiments.

 
Similarly, CPM gene expression was successfully knocked down using siCPM (Fig. 7CGo). However, NO production in the presence of Fa-Ala-Arg was equally high in cells transfected with siCPM or with siNT (Fig. 7DGo), that is, siCPM had little or no effect. Therefore, CPD, but not CPM, contributed significantly to NO production in MCF-7 cells.

L-Arg rescues apoptotic siCPD-transfected cells
MCF-7 cells in Arg-free DMEM, with PRL, for 24 h were approximately 60% apoptotic (see Fig. 6Go). We reasoned that Arg deprivation should not affect the ability of the cells to take up Arg and, if so, the inhibitory effects resulting from CPD-depletion (see Fig. 7Go) should be alleviated by exogenous Arg. To test this hypothesis, MCF-7 cells were transfected with siCPD or siNT, and cultured in DMEM that either contained Arg or was Arg-free. In Arg-containing DMEM (–PRL), the siCPD- or siNT-transfected cells were healthy, and only approximately 20% of the cells were apoptotic (Fig. 8Go; control). In Arg-free DMEM (but +PRL), the apoptotic rates of siCPD- or siNT cells increased to at least 60% or higher (Fig. 8Go). The addition of Fa-Ala-Arg did not rescue the apoptotic siCPD cells but significantly decreased the apoptotic rate of siNT cells to 32% (P < 0.05), suggesting that siNT, but not siCPD, transfectants were able to cleave Fa-Ala-Arg, and the released Arg decreased apoptosis. The protective effect of L-Arg was confirmed by the addition of exogenous L-Arg, which rescued both siCPD- and siNT-transfected cells. Therefore, CPD gene knockdown itself did not affect L-Arg uptake, and L-Arg protected cells from undergoing apoptosis.


Figure 8
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  		FIG. 8. L-Arg rescues apoptotic siCPD-transfected cells. Quiescent MCF-7 cells were transfected with 30 nM siCPD or siNT as in Fig. 7Go. One set of transfected cells were maintained for 2 d in Arg-containing DMEM (–PRL) (control). Three other sets were maintained in Arg-free DMEM (+PRL), either without further treatment or with Fa-Ala-Arg (4 mM) or L-Arg (1 mM). A, Annexin-V staining was performed to measure apoptosis in siCPD- and in siNT-transfected cells. Representative images of si-CPD-treated cells are shown in panel A. B, The percentage of apoptotic cells was determined in siCPD- or siNT-transfected cells. Mean ± SEM (n = 3). *, P ≤ 0.05 as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study showed that both PRL and E2 up-regulated CPD mRNA and protein levels in MCF-7 breast cancer cells. Exogenous L-Arg, either free or cleaved from Fa-Ala-Arg, increased NO production and cell survivability. Because CPD, but not CPM, was induced by PRL or E2, our studies implicate a key role for the PRL/E2-induced cell-surface CPD in intracellular NO production, which increased survival and inhibited apoptosis of the MCF-7 cells. Furthermore, the survival of PRL-treated cells was enhanced by Arg or Fa-Ala-Arg (Figs. 3Go and 4Go). PRL-stimulated NO production was inhibited by MGTA or siCPD (Fig. 5Go), resulting in decreased cell viability (Fig. 5Go) and, in siCPD-treated cells, increased apoptosis (Fig. 6Go). Therefore, our results implicate the involvement of CPD-L-Arg-NO in PRL action.

The mechanism by which PRL or E2 up-regulated CPD is not known. PRL exerts its effects through the PRL receptor, which signals through the Jak2/signal transducers and activators of transcription (STAT) pathway. In the mammary gland, activated STAT5 proteins bind to the {gamma}-activated sequence element (GAS sequence) on the promoter regions of PRL-responsive genes, some of which are involved in cell proliferation, differentiation, and/or motility (25, 31). Our analysis of the CPD promoter region has revealed several potential GAS sequences but no estrogen response elements (data not shown). However, E2 has been shown to elicit nongenomic effects through the rapid activation of protein kinase cascades such as MAPK (32), the Src/Ras/Erk pathway (33, 34, 35), and the phosphatidylinositol 3-kinase/Akt pathway (36). There is also the possibility of cross talk between the PRL and E2 receptors because STAT5a has been shown to interact with estrogen receptor {alpha} in transfected cells and in MCF-7 and T-47D breast cancer cell lines (37).

The specific inhibitor MGTA at 10 µM inhibited 97 ± 3.5% of CPM activity and 96.5 ± 4.7% of CPD activity in endothelial cells (38). Our study showed 10 µM MGTA inhibited, but did not abolish, NO production in PRL-stimulated MCF-7 cells cultured in Arg-free medium (Fig. 5BGo), suggesting the presence of residual CPD/CPM activity. MGTA is membrane impermeable and although it could inhibit cell surface CPD or CPM, it would have no effect on the CPD enzyme localized in the trans-Golgi network or nuclei of MCF-7 cells.

Our study showed that PRL/E2-induced, and CPD-mediated, NO production increased the survival and inhibited apoptosis of the MCF-7 cells. However, it is to be noted that NO, depending on its concentrations and the cell types in which it is produced, can either stimulate or suppress growth of tumor cells. For example, low levels of NO have been shown to promote growth of human colon adenocarcinoma (39), whereas high NO concentrations, produced by iNOS, were cytotoxic and abrogated tumorigenicity and metastasis of murine melanoma cells (40). There have also been contradictory reports on the effects of NO in mammary tumors and cell lines. On one hand, high NOS activity in human breast cancer is associated with low tumor grade and slow proliferation rate (41) and, likewise, stimulation of endogenous NO production by the ovarian hormone relaxin inhibited growth and promoted differentiation of MCF-7 cells (42). On the other hand, inhibition of eNOS activity or treatment with a NO scavenger increased apoptosis of MCF-7 cells. The effective concentrations of intracellular NO is critical to cellular outcome because low concentrations of the NO donor sodium nitroprusside inhibited, whereas high concentrations stimulated, apoptosis of these cells (43).

Exogenous L-Arg is known to increase intracellular NO levels even in cells cultured in Arg-rich medium (44). This phenomenon, referred to as the "Arginine Paradox," may be explained by the existence of a caveolar complex between the Arg transporter cationic amino acid transporter 1 (CAT-1) and eNOS that provided a mechanism for the direct delivery of Arg to eNOS. Thus, Arg being transported into the cell is in close proximity to eNOS compared with intracellular Arg that may be sequestered in distant pools (45). The role of PRL and E2 in the regulation of Arg transporters in breast cancer cells is under investigation.

In summary, PRL-/E2-inducible CPD at the cell surface plays a key role in cleaving Arg from extracellular substrates. L-Arg, in turn, increases intracellular NO production to promote survival and inhibit apoptosis of MCF-7 cells.


   Footnotes
 
S.A.A. is a recipient of graduate studentships from the Cancer Research Training Program (CRTP) at Dalhousie University and the Nova Scotia Health Research Foundation. This work was funded by the Canadian Institutes of Health Research (MOP12895, ROP84154) and the Canada Breast Cancer Foundation, Atlantic Chapter (to C.K.L.T.).

Disclosure Statement: S.A.A. and C.K.L.T. have nothing to declare.

First Published Online June 5, 2008

Abbreviations: CPD, Carboxypeptidase-D; CPM, carboxypeptidase-M; DAF-2DA, 4,5-diaminofluorescein diacetate; DEA/NO, diethylamine/nitric oxide; E2, 17β-estradiol; Fa-Ala-Arg, substrate furylacryloyl-Ala-Arg; Fa-Ala-Lys, furylacryloyl-Ala-Lys; eNOS, endothelial NOS; FBS, fetal bovine serum; gapdh, glyceraldehyde-3-phosphate-dehydrogenase; GAS, {gamma}-activated sequence element; HS, horse serum; iNOS, inducible NOS; MGTA, 2-mercaptomethyl-3-guanidinoethylthio-propanoic acid; nNOS, neuronal NOS; NO, nitric oxide; NOS, nitric oxide synthase; NT, nontargeting; PRL, prolactin; si, small interfering; STAT, signal tranducers and activators of transcription.

Received January 30, 2008.

Accepted for publication May 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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