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[Cancer Research 60, 6590-6596, December 1, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Neutral Endopeptidase Promotes Phorbol Ester-induced Apoptosis in Prostate Cancer Cells by Inhibiting Neuropeptide-induced Protein Kinase C {delta} Degradation1

Makoto Sumitomo, Ruoqian Shen, Jonathan S. Goldberg, Jie Dai, Daniel Navarro and David M. Nanus2

Urologic Oncology Research Laboratory, Department of Urology [M. S., R. S., J. D, D. N., D. M. N.], and the Division of Hematology and Medical Oncology, Department of Medicine [J. S. G., D. M. N.], Joan and Stanford I. Weill Medical College of Cornell University, New York, New York 10021


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Phorbol esters induce apoptosis in androgen-sensitive LNCaP cells, which express neutral endopeptidase (NEP), but not in androgen-independent prostate cancer (PC) cells, which lack NEP expression. We investigated the role of NEP in PC cell susceptibility to 12-O-tetradecanoylphorbol-13-acetate (TPA). Western analysis showed that expression of NEP and protein kinase C{delta} (PKC{delta}) correlated with PC cell sensitivity to TPA-induced growth arrest and apoptosis in LNCaP cells and in TSU-Pr1 cells expressing an inducible wild-type NEP protein. Inhibition of NEP enzyme activity using the specific NEP inhibitor CGS24592, or inhibition of PKC{delta} using Rottlerin at concentrations that inhibit PKC{delta} but not PKC{alpha}, significantly inhibited TPA-induced growth inhibition and cell death. Furthermore, pulse-chase experiments showed PKC{delta} is stabilized in LNCaP cells and in TSU-Pr1 cells overexpressing wild-type NEP compared with PC cells lacking NEP expression. This results from NEP inactivation of its neuropeptide substrates (bombesin and endothelin-1), which in the absence of NEP stimulate cSrc kinase activity and induce rapid degradation of PKC{delta} protein. These results indicate that expression of enzymatically active NEP by PC cells is necessary for TPA-induced apoptosis, and that NEP inhibits neuropeptide-induced, cSrc-mediated PKC{delta} degradation.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
TPA3 exerts numerous effects on cells, including proliferation, malignant transformation, differentiation, and cell death (1 , 2) . These effects are mediated in part through modulation of PKC isoenzymes. Numerous investigators have shown that phorbol esters can induce apoptotic cell death in androgen-sensitive PC LNCaP cells but not in androgen-independent PC-3 or DU145 cells (3, 4, 5) . Powell et al. (6) reported that TPA-induced cell death in LNCaP cells correlated with increased expression of PKC{alpha} mRNA, but not other PKC isoforms, and with translocation of PKC{alpha} to non-nuclear membranes. However, Fujii et al. (7) recently reported that PKC{delta} mediates phorbol ester-mediated apoptosis in LNCaP cells, demonstrating that phorbol ester-induced cell death can be partially (~50%) blocked by a PKC{delta}-inhibitor or a dominant-negative PKC{delta} mutant. The biological effect of PKC{delta} is cell-type specific, and overexpression can both inhibit cell growth (8) or enhance anchorage-independent growth and metastatic potential (9) . PKC{delta} activity appears to be regulated in part through tyrosine phosphorylation by Src kinase, which results in degradation of PKC{delta} protein (10) .

LNCaP cells express neutral endopeptidase 24.11 (NEP, CD10, CALLA, EC 3.4.24.11), a Mr 90,000–110,000 zinc-dependent cell-surface metallopeptidase, whereas androgen-independent PC cell lines do not (11) . NEP can regulate through its enzymatic function access of neuropeptides such as bombesin, neurotensin, and ET-1 to their cell-surface G-protein-coupled receptors. Neuropeptide signaling involves activation of cSrc kinase activity, which in turn leads to phosphorylation of several downstream substrates such as focal adhesion kinase and p130Cas (12, 13, 14) . Mari et al. (15) reported previously that a catalytically active NEP protein is required for phorbol ester-induced growth arrest in Jurkatt T cells. In the present study, we considered whether NEP and its substrates were involved in regulating the expression of PKC{delta} in LNCaP cells and whether NEP and PKC{delta} expression were required for TPA-induced apoptosis. We report that ET-1 and bombesin induce PKC{delta} down-regulation caused by rapid PKC{delta} degradation in PC cells, which is mediated by cSrc kinase activation, and that PKC{delta} down-regulation is blocked by NEP in LNCaP cells, which facilitates TPA-induced apoptosis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Culture and Reagents.
Prostate cancer cell lines were maintained in RPMI 1640 media supplemented with 2 mM glutamine, 1% nonessential amino acids, 100 units/ml streptomycin and penicillin, and 10% FCS. WT-5, TN-12, and M-22 were derived and maintained as described previously (14) . TPA-resistant, LNCaP-derivative LN10H (6) was kindly provided by Dr. C. T. Powell (Cleveland Clinic Foundation) and maintained in the above media containing 10 nM TPA. Recombinant NEP was obtained from Arris Pharmaceutical Corp. CGS24592, a competitive inhibitor of NEP, was supplied by Novartis Pharmaceuticals. The specific Src inhibitor PP2 and the specific PKC inhibitors Rottlerin and Gö6976 were purchased from Calbiochem-Novabiochem, Ltd. (La Jolla, CA).

Cell Growth Assays.
PC cells (1 x 104 /well) were plated in 12-well tissue culture plates (Falcon Division, Becton Dickinson, Cockeysville, MD). After overnight culture in regular media (LNCaP, TSU-Pr1, DU145, and PC-3) or culture for 48 h in media with or without tetracycline (WT-5, TN-12, and M-22), cells were treated with various reagents for 48 h. Cells were harvested and counted using a Coulter Counter ZM (Coulter Electronics, Hialeah, FL). Each data point represents the average cell number of triplicate samples from a single experiment. Statistical analyses were performed using an unpaired t test. Ps less than 0.005 are reported as <0.005. All growth assays were performed on three separate occasions with similar results.

Apoptosis Assays.
Early apoptotic cells were detected using the Annexin V apoptosis detection kit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Briefly, cells evenly distributed in Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL) were treated with various reagents for 24 h. Cells were washed twice with cold PBS, washed once with 1 x Assay Buffer and with 1 µg of Annexin V-FITC with 500 µl of 1 x Assay Buffer added. Propidium iodide (0.5 µg ) was added to each well for nuclear counterstain. After incubation for 15 min at room temperature in the dark, positively stained cells were enumerated using a fluorescence microscope at x100–400. Each data point represented the average cell number in six independent microscopic fields of a single experiment. The statistical analysis was performed using an unpaired t test. Ps <0.05 were regarded as statistically significant. All assays were performed on three separate occasions with similar results.

For cell cycle analysis, cells were fixed in 70% ethanol and stained with 50 µg/ml propidium iodide. Cell cycle progression and apoptosis were analyzed by flow cytometry using a Becton Dickinson fluorescence-activated cell sorting system. Twenty thousand events were recorded for each treatment.

Immunoblotting.
Cells were lysed in 1 ml of RIPA buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, 1.2% aprotinin, 5 µM leupeptin, 4 µM antipain, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM Na3VO4), and lysates were separated on an 8% SDS-PAGE, transferred to nitrocellulose, and incubated in 1% BSA for 2 h. Membranes were immunoblotted with anti-NEP (NCL-CD10-270; Vector Laboratories, Inc., Burlingame, CA; 1:100), anti-PKC{alpha} (1:2000), anti-PKC{delta} (Santa Cruz Biotechnology, Inc.; 1:1000), or anti-actin (Chemicon International, Inc., Temecula, CA; 1:3000) and detected using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ).

Pulse-Chase Assay.
An equal number of cells were cultured in RPMI 1640 lacking methionine for 30 min, in the same media containing 300 µCi/ml [35S]methionine for 1 h, and washed with PBS and then in RPMI supplemented with 10% FCS and 0.15 mg/ml nonradioactive methionine for specific time periods. Cells were lysed in RIPA buffer. For immunoprecipitation, 300-µg lysates were incubated for 1 h with 1 µg of anti-PKC{delta} antibody and then for 1 h with 40 µl of protein G-Sepharose beads (Amersham Pharmacia) at 4°C. Immunoprecipitates were collected by centrifugation at 12,000 x g for 1 min, washed with RIPA buffer, and resuspended in 2 x Laemmli sample buffer. Samples were resolved on an 8% SDS-PAGE and transferred to nitrocellulose. Autoradiography and immunoblotting were performed using the same membrane. The relative intensity of each band obtained by autoradiography was measured by NIH image.

cSrc Kinase Assay.
Cell lysates (500 µg) were incubated with cSrc antibody (2 µg; Santa Cruz Biotechnology, Inc.) and immunoprecipitated as described above. cSrc kinase activity was measured using an Src kinase assay kit (Upstate Biotechnology, Inc.) as per the manufacturer’s recommendation. Briefly, 20 µl of kinase buffer (100 mM Tris-HCl (pH 7.2), 125 mM MgCl2, 25 mM MnCl2, 2 mM EGTA, 0.25 mM Na3VO4, and 2 mM DTT), 0.5 mg/ml of a specific substrate peptide (KVEKIGEGTYGVVYK), and 10 µl of [{gamma}-32P]ATP diluted to 1 µCi/µl with Mn/ATP mixture (75 mM MnCl2, 500 µM ATP) were added to 20 µl of washed protein G-Sepharose beads. The reaction mixture was incubated for 10 min at 30°C, the reaction was stopped by adding 20 µl of 40% trichloroacetic acid, and the phosphorylated substrate was separated from the residual [{gamma}-32P]ATP using P81 phosphocellulose paper and quantified with a scintillation counter. Each experiment was performed at least three times in triplicate. Results are expressed as fold increases compared with untreated controls.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
PKC{delta} Expression and TPA-induced Growth Inhibition in PC Cells Expressing Functional NEP.
Western blot analysis revealed high levels of NEP and PKC{delta} proteins in total cell lysates derived from LNCaP cells but not in lysates derived from androgen-independent TSU-Pr1, DU145, or PC-3 cells (Fig. 1ACitation , panel 1 and panel 2). In comparison, PKC{alpha} protein was expressed at lower levels in LNCaP cells relative to other PC cell lines (Fig. 1ACitation , panel 3), as reported previously (6) . In contrast to parental TPA-sensitive LNCaP cells, NEP and PKC{delta} proteins could not be detected in a TPA-resistant subclone of LNCaP, LN10H cells (6) , whereas PKC{alpha} protein was expressed at similar levels.



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  		Fig. 1. Increased PKC{delta} expression and TPA-induced growth inhibition in PC cells expressing functional NEP. A, total cell lysates (20 µg) from PC cells were analyzed for NEP protein by Western blot as described in "Materials and Methods" using the anti-NEP mAb NCL-CD10–270 (panel 1), anti-PKC{delta} Ab (panel 2), anti-PKC{alpha} Ab (panel 3), and anti-actin Ab (panel 4). B, cell growth assays were performed with the addition of the complex indicated (10 nM TPA, 10 µM Rottlerin, and 100 nM Gö6976) as described in "Materials and Methods." Bars, SD. C, LNCaP cells were treated with or without 100 nM CGS24592 for 16 h, and the expressions of PKC{delta} (panel 1) and actin (panel 2) were determined by Western blot. D, NEP-inducible TSU-Pr1 clones were treated with or without tetracycline (Tet.) for 48 h and analyzed for NEP protein by Western blot using anti-NEP mAb (panel 1), anti-PKC{delta} Ab (panel 2), anti-PKC{alpha} Ab (panel 3), and anti-actin Ab (panel 4). E, cell growth assays were performed in media containing FCS with the addition of 10 nM TPA with or without 50 µM Rottlerin. Bars, SD.

 
Incubation of PC cells in media containing FCS plus 10 nM TPA for 48 h induced ~80% growth inhibition in LNCaP cells compared with untreated control (P < 0.005) but not in TSU-Pr1, DU145, or PC-3 cells (Fig. 1B)Citation . Both PKC{alpha} and PKC{delta} have been implicated in TPA-induced growth inhibition of LNCaP cells (6 , 7) . We therefore assessed the effects on TPA-induced growth inhibition of the PKC-inhibitors Rottlerin (IC50 = 3–6 µM for PKC{delta}, IC50 = 40 µM for PKC{alpha}, PKCßI, and PKC{gamma}) at a concentration which selectively inhibits PKC{delta}, and Gö6976 (IC50 = 2–6 nM for PKC{alpha} and PKCßI; no inhibition at µM concentrations for PKC{delta}) as a PKC{alpha} inhibitor. As illustrated in Fig. 1BCitation , TPA-induced growth inhibition of LNCaP cells was reversed by pretreatment with 10 µM Rottlerin 2 h before TPA (P < 0.005) but not by 100 nM Gö6976. No significant effect of either inhibitor was observed in TSU-Pr1, DU145, and PC-3 cells. Taken together, these results show that TPA sensitivity correlates with NEP and PKC{delta} expression in PC cells and support previous studies which suggest that TPA-induced growth inhibition in LNCaP cells is mediated by PKC{delta}.

To assess whether NEP is needed for PKC{delta} expression and TPA sensitivity in PC cells, we cultured LNCaP cells in media containing FCS with the addition of the specific NEP enzyme inhibitor CGS24592 at a concentration of 100 nM, which completely inhibits NEP enzyme activity (14) , and found that PKC{delta} protein levels were significantly less than control-treated LNCaP cells (Fig. 1C)Citation . Next we examined PKC{delta} protein expression in TSU-Pr1 cells containing a tetracycline-repressed (tet-off) inducible wild-type NEP (WT-5 cells), catalytically inactive NEP (M-22 cells), which contain a point mutation in the zinc-binding domain required for NEP enzymatic function (14) , and control (empty vector; TN-12 cells). Western blot analysis (Fig. 1DCitation , panel 1) and enzyme studies confirmed NEP protein expression in both WT-5 and M-22 cells, but not in control TN-12 cells, 48 h after withdrawal of tetracycline from the media, whereas high levels of NEP-specific activity could be detected only in total cell lysates from WT-5 cells (not shown; see Ref. 14 ). High levels of PKC{delta} protein expression were present in cells expressing wild-type NEP proteins (WT-5), whereas barely detectable PKC{delta} protein was detected in cells which did not express NEP (tet-repressed WT5, TN-12) or which expressed catalytically inactive NEP proteins (M-22; Fig. 1DCitation , panel 2). In contrast, PKC{alpha} expression was not affected by NEP expression or tetracycline in these cell lines (Fig. 1DCitation , panel 3). Cell growth assays showed that culturing in media containing FCS with 10 nM TPA for 48 h after expression of wild-type cell-surface NEP resulted in a >60% decrease in cell number in WT-5 cells (P < 0.005) but did not alter cell growth in TN-12 cells or in M-22 cells expressing catalytically inactive NEP (Fig. 1E)Citation . Similar to experiments using LNCaP cells, pretreatment with 10 µM Rottlerin 2 h before 10 nM TPA treatment in NEP-expressing WT-5 cells reversed TPA-induced growth inhibition in TPA-treated WT-5 cells (P < 0.005; Fig. 1ECitation ). Rottlerin alone had no significant effect on these cells (data not shown). Taken together, these results suggest that the expression of catalytically active NEP protein is required for increase in PKC{delta} protein expression in PC cells, and that PKC{delta} mediates susceptibility of PC cells to TPA-induced growth inhibition.

TPA-induced Apoptosis Requires Expression of Functional NEP in PC Cells.
Previous studies show that TPA induces apoptosis in LNCaP cells (7) . Cell cycle analysis showed that 10 nM TPA treatment of LNCaP cells for 24 h resulted in 35.1% of cells with sub-G0-G1 DNA content (Fig. 2ACitation , lower left), which was blocked by incubation with 100 nM CGS24592 16 h before TPA treatment (Fig. 2ACitation , lower right). As illustrated in Fig. 2BCitation , determination of Annexin V-FITC staining as a marker of apoptosis in LNCaP cells revealed that TPA treatment for 24 h resulted in 53.7% (Lane 2; range 46.7–62.7%) stained cells compared with untreated control (Lane 1; 8.5%, range 6.2–10.8%; P < 0.005). Pretreatment for 16 h with 100 nM CGS24592 resulted in only 12% of cells staining positive with Annexin V-FITC (Lane 4; P < 0.005, compared with Lane 2). Pretreatment for 2 h with 10 µM Rottlerin also blocked TPA-induced apoptosis (Lane 6, 14.8%, P < 0.005, compared with Lane 2). Annexin V-FITC-positive staining cells did not increase after TPA treatment in TPA-resistant LN10H cells, which do not express NEP or PKC{delta} protein (Lane 7). Similar results to LNCaP cells were observed in WT-5 cells expressing NEP (following tetracycline removal; Fig. 2CCitation ). These results suggest that NEP enzyme activity is necessary for TPA-induced apoptotic cell death in LNCaP cells.



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  		Fig. 2. TPA-induced apoptotic cell death in PC cells expressing functional NEP. A, DNA flow cytometry histograms of LNCaP cells cultured in media containing 10% FCS treated with or without 10 nM TPA for 24 h. Note marked accumulation of sub-G0-G1 DNA content (area F in each histogram) in 10 nM TPA-treated LNCaP cells, which is blocked by pretreatment with 100 nM CGS24592 16 h before TPA treatment. B, LNCaP cells were cultured in media with the addition of the complex indicated [10 nM TPA, 100 nM CGS24592 (CGS), or10 µM Rottlerin] for 24 h. Cells were stained with Annexin V-FITC (Annexin V binds to membrane phosphatidylserine, which accumulates to the extracellular surface in early apoptotic cells). Annexin V-FITC positively staining cells were enumerated in six independent microscopic fields. Bars, SD. C, WT-5 cells cultured as indicated above with or without tetracycline for 48 h before TPA. NEP-expressing WT-5 cells [tetracycline- (Tet.) negative] treated with 10 nM TPA resulted in a significant increase in Annexin V-FITC positively staining cells [C, Lane 4; P < 0.005 compared with tetracycline-negative (Lane 3)]. Pretreatment with 100 nM CGS24592 16 h before treatment with TPA resulted in a significant decrease in Annexin V positively staining cells (C, Lane 6; P < 0.005, compared with Lane 4). Pretreatment with 10 µM Rottlerin 2 h before TPA also resulted in a significant decrease in positive-staining cells (C, Lane 8; P < 0.005, compared with Lane 4). Bars, SD.

 
NEP Increases PKC{delta} Expression by Blocking Neuropeptide-induced PKC{delta} Degradation.
PKC{delta} protein is highly expressed in PC cells expressing NEP. To assess the mechanisms by which NEP may regulate PKC{delta} expression, we first determined whether NEP increased protein production or prolonged turnover of PKC{delta} protein. As shown in Fig. 3ACitation (upper panel), the level of PKC{delta} protein production in TSU-Pr1 cells at time 0 was similar to that in LNCaP cells, but rapidly decreased within 2 h. Pulse-chase assays revealed that the half-life of PKC{delta} protein was longer in NEP-positive LNCaP cells (T1/2 = 6.9 h) compared with NEP-negative TSU-Pr1 cells (T1/2 = 1.3 h). Western blot analysis of immunoprecipitated PKC{delta} protein showed a marked decrease in total PKC{delta} protein expression in TSU-Pr1 cells compared with LNCaP cells (Fig. 3ACitation , lower panel). Similar results were obtained in WT-5 cells incubated with or without tetracycline for 48 h. Pulse-chase assays showed that the half-life of PKC{delta} protein was prolonged in NEP-expressing WT-5 cells (no tetracycline, T1/2 = 4.5 h) compared with NEP-negative WT-5 cells (with tetracycline, T1/2 =1.4 h; Fig. 3BCitation , upper panel). Western blot analysis of immunoprecipitated PKC{delta} protein showed a marked increase in PKC{delta} protein in NEP-expressing WT-5 cells compared with NEP-negative WT-5 cells (Fig. 3BCitation , lower panel).



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  		Fig. 3. NEP stabilizes PKC{delta} protein expression. A, B, and C, the turnover of PKC{delta} was evaluated by pulse-chase assay as described in "Materials and Methods." The half-life of PKC{delta} was prolonged in NEP-positive LNCaP cells (A), WT-5 cells under tetracycline- (Tet.) free media (B), and TSU-Pr1 cells cultured in serum-free media (C) compared with NEP-negative TSU-Pr1 cells (A), WT-5 cells with tetracycline (B), and TSU-Pr1 cells incubated with 10 nM ET-1 (C). IgGH, IgG heavy chain. D, TSU-Pr1 cells cultured in serum-free media were treated with 10% FCS, 10 nM ET-1, or 10 nM bombesin (Bomb.) for 16 h with or without 50 mg/ml recombinant NEP (rNEP) 2 h before each treatment, and the expressions of PKC{delta}, PKC{alpha}, and actin were determined by Western blot.

 
The effect of NEP expression in WT-5 cells on PKC{delta} protein levels occurs within 2 h, suggesting that NEP stabilizes PKC{delta} protein in PC cells, possibly by blocking PKC{delta} degradation (Fig. 3BCitation , bottom panel). Furthermore, as shown above, NEP catalytic activity is critical to the interaction between NEP and PKC{delta} proteins. NEP neuropeptide substrates such as ET-1 and bombesin can contribute to paracrine/autocrine PC cell growth and cell survival by activating various signal transduction pathways (16 , 17) . We therefore assessed whether these neuropeptides affected PKC{delta} protein expression. Pulse-chase assays revealed that incubation of TSU-Pr1 cells with 10 nM ET-1 in serum-free media resulted in a decrease in the half-life of PKC{delta} from 5.7 h to 1.2 h (Fig. 3CCitation , upper panel). In addition, Western blot of immunoprecipitated PKC{delta} protein showed that PKC{delta} protein levels decreased by 6 h after ET-1 treatment (Fig. 3CCitation , lower panel). Furthermore, as shown in Fig. 3DCitation , Western blot analysis showed that incubation of TSU-Pr1 cells in serum-free media containing either 10% FCS (Lane 3), 10 nM ET-1 (Lane 5), or 10 nM bombesin (Lane 7) for 16 h resulted in significantly lower levels of PKC{delta} protein compared with control. Pretreatment with 50 µg/ml recombinant NEP for 2 h before the addition of FCS, ET-1, or bombesin inhibited the neuropeptide-induced down-regulation of PKC{delta} protein (Fig. 3DCitation , Lanes 4, 6, and 8). These incubations had no effect on PKC{alpha} protein expression (Fig. 3D)Citation . Similarly, in LNCaP cells, pretreatment with 100 nM CGS24592 for 2 h before treatment with 10 nM ET-1 resulted in decreased PKC{delta} expression, whereas ET-1 alone did not alter PKC{delta} expression (data not shown). Taken together, these results suggest that NEP neuropeptide substrates such as ET-1 and bombesin stimulate PKC{delta} degradation, and that this effect is inhibited by NEP.

NEP Inhibits Neuropeptide-mediated cSrc Kinase Activation, which Induces Rapid PKC{delta} Degradation and TPA-resistance in PC Cells.
Recent studies show that cSrc kinase activation regulates PKC{delta} tyrosine phosphorylation leading to a decrease in PKC{delta} protein expression (10 , 18) . Furthermore, neuropeptides can activate cSrc kinase activity, which contributes to neuropeptides-mediated action (12) . We therefore considered that neuropeptides stimulate cSrc kinase activity, which in turn phosphorylates PKC{delta}, inducing rapid PKC{delta} protein degradation and inactivation. As shown in Fig. 4ACitation , cSrc kinase activity of TSU-Pr1 cells incubated with 10% FCS for 16 h was 6.1-fold higher than that of LNCaP cells. Incubation of TSU-Pr1 cells in serum-free media containing 10% FCS (Fig. 4BCitation , Lane 3), 10 nM ET-1 (Fig. 4BCitation , Lane 5), or 10 nM bombesin (Fig. 4BCitation , Lane 7) for 20 min resulted in 4.4, 6.9, or 5.6-fold increase in cSrc kinase activity, respectively, compared with serum-free control (Fig. 4BCitation , Lane 1; P < 0.005). Pretreatment with 50 µg/ml recombinant NEP for 2 h partially blocked cSrc kinase activation (Lanes 4, 6, and 8). In addition, the expression of wild-type cell-surface NEP in WT-5 cells for 48 h resulted in a 75% decrease in the cSrc kinase activity compared with untreated control, but did not alter cSrc kinase activity in control TN-12 cells (data not shown). To confirm the requirement of cSrc activity in neuropeptide-mediated down-regulation of PKC{delta} expression, we incubated TSU-Pr1 cells with the specific Src kinase inhibitor PP2 (10 µM) for 2 h before treatment with ET-1 or 10% FCS for 16 h, and we showed that PP2 completely blocked ET-1-induced (Fig. 4CCitation , Lane 5, compared with Lane 2) or 10% FCS-induced (Fig. 4CCitation , Lane 6, compared with Lane 3) down-regulation of PKC{delta} protein expression.



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  		Fig. 4. NEP inhibits neuropeptide-mediated cSrc kinase activation. A, LNCaP and TSU-Pr1 cells were cultured in media with 10% FCS for 16 h, and cSrc kinase activity was measured as described in "Materials and Methods." Bars, SD. B, TSU-Pr1 cells cultured in serum-free media overnight were treated with 10% FCS, 10 nM ET-1, or 10 nM bombesin (Bomb.) for 20 min, and cSrc kinase activity was measured. Fifty µg/ml recombinant NEP (rNEP) was added 2 h before each treatment. Bars, SD. C, TSU-Pr1 cells cultured in serum-free media were treated with 10 nM ET-1 or 10% FCS for 16 h with (Lanes 5 and 6) or without (Lanes 2 and 3) 10 µM PP2 2 h before each treatment, and the expressions of PKC{delta} and actin were determined by Western blot. D, TSU-Pr1 cells cultured in media with 10% FCS were treated with 10 nM TPA for 24 h with or without 10 µM PP2 and/or 10 µM Rottlerin. Apoptotic cells were enumerated by Annexin V detection assay. PP2 and Rottlerin were added 16 h and 2 h, respectively, before the treatment with TPA. Bars, SD.

 
Finally, to confirm that the inhibition of cSrc kinase activity is required for TPA-induced apoptotic cell death, we incubated TSU-Pr1 cells with TPA, PP2, plus Rottlerin and performed Annexin V detection. As illustrated in Fig. 4DCitation , whereas 10 nM TPA alone (Lane 2) did not induce apoptosis, pretreatment with 10 µM PP2 resulted in 64.3% (range 57.5–71.1%) positively stained cells (Lane 4, P < 0.005; compared with Lane 2). Moreover, pretreatment with the PKC{delta} inhibitor Rottlerin (10 µM) before TPA plus PP2 (Lane 6) partially reversed the TPA-plus-PP2 induction of positive-staining cells (20.3%, P < 0.005; compared with Lane 4). These data suggest that, in TSU-Pr1 cells, inhibition of cSrc kinase activity permits TPA-induced apoptotic cell death and that PKC{delta} activation is needed for apoptosis to occur.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The results presented here help clarify previous reports on phorbol ester-induced cell death in androgen-sensitive LNCaP cells but not in androgen-independent PC cells. Our data suggest that neuropeptides such as ET-1 and bombesin stimulate cSrc kinase activity, which in turn phosphorylates PKC{delta} and leads to rapid degradation of PKC{delta} protein. TPA-induced apoptosis is mediated through PKC{delta}. Thus, androgen-independent PC cells, which do not express NEP, are resistant to TPA treatment because they express low levels of PKC{delta} protein. In contrast, LNCaP cells constitutively express NEP. NEP inactivates through hydrolysis neuropeptides such as ET-1 and bombesin, leading to diminished cSrc kinase activity and stable expression of PKC{delta} protein. Consequently, PKC{delta}-expressing LNCaP cells are extremely sensitive to TPA-induced apoptosis. Although a previous study reported that NEP is required for TPA-induced growth arrest in Jurkatt T cells (15) , the mechanism of NEP in allowing susceptibility to TPA had not been elucidated. Our results highlight the involvement of NEP in TPA-induced apoptosis mediated by PKC{delta} in PC cells.

Our studies indicate that TPA-induced apoptosis and growth inhibition in LNCaP cells is predominantly mediated by PKC{delta} rather than by PKC{alpha}. Henttu et al. (5) have reported that calcium-independent PKC isoenzymes such as PKC{delta}, rather than PKC{alpha}, are predominantly activated in TPA-treated LNCaP cells, which supports our studies. Recent reports also implicate PKC{delta} as a proapoptotic kinase (19, 20, 21) . A proteolytic cleavage site for caspase-3 has been identified at the V3 (hinge) region of PKC{delta} with cleavage resulting in the release of an active Mr 40,000 fragment corresponding to the PKC{delta} COOH-terminal kinase domain (19) . However, similar to a previous report (7) , pretreatment with the selective caspase-3 inhibitor DEVD-CHO showed little inhibitory effect on TPA-induced cell death in LNCaP or WT-5 cells (data not shown). These results suggest that the TPA-induced apoptotic pathway mediated by PKC{delta} in PC cells is independent of caspase-3 activity or caspase-3-mediated PKC{delta} cleavage, and that PKC{delta} can act as a primary effector or is involved in other pathways for apoptosis via its allosteric activation (7) . Recent studies show that translocation of PKC{delta} holoenzyme, and not its catalytic fragment, onto mitochondria induces cytochrome c release and apoptosis, and that this translocation precedes the activation of caspases (22 , 23) . This suggests that proteolytic cleavage may not be required for PKC{delta} kinase activation and apoptosis induction. Others have suggested that various cell cycle regulators (3) or ceramide (4) mediate TPA-induced apoptosis in LNCaP cells. We have found NEP expression in WT-5 cells up-regulates p21WAF/CIP1 expression and inactivates the retinoblastoma protein, which induces G0-G1 arrest,4 leading us to speculate on the possibility that PKC{delta} stabilized by NEP can affect these cell cycle regulators via its allosteric activation.

NEP neuropeptide substrates such as ET-1 and bombesin can act as survival and antiapoptotic factors (24 , 25) , transactivators of epidermal growth factor receptor (26) , and activators of Akt/protein kinase B cell survival pathway (27 , 28) . As a regulator of these peptides to their cell surface receptors, NEP is involved in various critical signaling pathways. Our data for the first time define one mechanism of NEP function as an inducer of apoptosis through stabilization of PKC{delta} expression. PKC{delta} activity has been implicated in mediating apoptosis in response to various antitumor reagents such as etoposide (20) and cis-platinum (29) as well as TPA. Thus, through its ability to inhibit various cell survival pathways by inactivating mitogenic neuropeptides, NEP may be a potential therapeutic modality to use in combination with various agents to treat prostate cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Thomas Powell for useful discussions and for supplying the LN10H PC cell line, and Catherine Kearney and Lana Winter for secretarial assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant CA 80240, the Association for the Cure of Cancer of the Prostate (CaP CURE), and the Dorothy Rodbell Foundation for Sarcoma Research. J. D. is a recipient of a Department of Defense Prostate Cancer Research Program Post-doctoral Traineeship Award. Back

2 To whom requests for reprints should be addressed New York Presbyterian Hospital-Weill Medical College of Cornell University, 525 E. 68th Street, New York, NY 10021. Phone: (212) 746-2920; Fax: (212) 746-6645; E-mail: dnanus{at}med.cornell.edu Back

3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; PC, prostate cancer; ET-1, endothelin-1; NEP, neutral endopeptidase; RIPA, radioimmunoprecipitation assay; Ab, antibody. Back

4 Unpublished data. Back

Received 8/ 8/00. Accepted 10/17/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science, 258: 607-614, 1992.[Abstract/Free Full Text]
  2. Blumberg P. M. Protein kinase C as the receptor for the phorbol ester tumor promoters: sixth Rhoads memorial award lecture. Cancer Res., 48: 1-8, 1988.[Free Full Text]
  3. Blagosklonny M. V., Prabhu N. S., El-Deiry W. S. Defects in p21WAF1/CIP1, Rb, and c-myc signaling in phorbol ester-resistant cancer cells. Cancer Res., 57: 320-325, 1997.[Abstract/Free Full Text]
  4. Garzotto M., White-Jones M., Jiang Y., Ehleiter D., Liao W. C., Haimovitz-Friedman A., Fuks Z., Kolesnick R. 12-O-tetradecanoylphorbol-13-acetate-induced apoptosis in LNCaP cells is mediated through ceramide synthase. Cancer Res., 58: 2260-2264, 1998.[Abstract/Free Full Text]
  5. Henttu P., Vihko P. The protein kinase C activator, phorbol ester, elicits disparate functional responses in androgen-sensitive and androgen-independent human prostatic cancer cells. Biochem. Biophys. Res. Commun., 244: 167-171, 1998.[Medline]
  6. Powell C. T., Brittis N. J., Stec D., Hug H., Heston W. D., Fair W. R. Persistent membrane translocation of protein kinase C {alpha} during 12-O- tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells. Cell Growth Differ., 7: 419-428, 1996.[Abstract]
  7. Fujii T., Garcia-Bermejo M. L., Bernabo J. L., Caamano J., Ohba M., Kuroki T., Li L., Yuspa S. H., Kazanietz M. G. Involvement of protein kinase C {delta} (PKC{delta}) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKC{delta}. J. Biol. Chem., 275: 7574-7582, 2000.[Abstract/Free Full Text]
  8. Watanabe, T., Ono, Y., Taniyama, Y., Hazama, K., Igarashi, K., Ogita, K., Kikkawa, U., and Nishizuka, Y. Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-{delta} subspecies. Proc. Natl. Acad. Sci. USA 89: 10159–10163, 1992.
  9. Kiley S. C., Clark K. J., Goodnough M., Welch D. R., Jaken S. Protein kinase C {delta} involvement in mammary tumor cell metastasis. Cancer Res., 59: 3230-3238, 1999.[Abstract/Free Full Text]
  10. Blake R. A., Garcia-Paramio P., Parker P. J., Courtneidge S. A. Src promotes PKC{delta} degradation. Cell Growth Differ., 10: 231-241, 1999.[Abstract/Free Full Text]
  11. Papandreou C. N., Usmani B., Geng Y., Bogenrieder T., Freeman R., Wilk S., Finstad C. L., Reuter V. E., Powell C. T., Scheinberg D., Magill C., Scher H. I., Albino A. P., Nanus D. M. Neutral endopeptidase 24. 11 loss in metastatic human prostate cancer contributes to androgen-independent progression. Nat. Med., 4: 50-57, 1998.[Medline]
  12. Zachary I., Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell, 71: 891-894, 1992.[Medline]
  13. Okuda M., Takahashi M., Suero J., Murry C. E., Traub O., Kawakatsu H., Berk B. C. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J. Biol. Chem., 274: 26803-26809, 1999.[Abstract/Free Full Text]
  14. Sumitomo, M., Shen, R., Walburg, M., Dai, J., Geng, Y., Navarro, D., Boileau, G., Papandreou, C. N., Giancotti, F. G., Knudsen, B., and Nanus, D. M. Neutral endopeptidase Inhibits Prostate Cancer Cell Migration by Blocking Focal Adhesion Kinase (FAK) Signaling. J. Clin. Invest., in press, 2000.
  15. Mari B., Guerin S., Maulon L., Belhacene N., Farahi Far D., Imbert V., Rossi B., Peyron J. F., Auberger P. Endopeptidase 24. 11 (CD10/NEP) is required for phorbol ester-induced growth arrest in Jurkat T cells. FASEB J., 11: 869-879, 1997.[Abstract]
  16. Nelson J. B., Chan-Tack K., Hedican S. P., Magnuson S. R., Opgenorth T. J., Bova G. S., Simons J. W. Endothelin-1 production and decreased endothelin B receptor expression in advanced prostate cancer. Cancer Res., 56: 663-668, 1996.[Abstract/Free Full Text]
  17. Markwalder R., Reubi J. C. Gastrin-releasing peptide receptors in the human prostate: relation to neoplastic transformation. Cancer Res., 59: 1152-1159, 1999.[Abstract/Free Full Text]
  18. Lu Z., Hornia A., Jiang Y. W., Zang Q., Ohno S., Foster D. A. Tumor promotion by depleting cells of protein kinase C {delta}. Mol. Cell. Biol., 17: 3418-3428, 1997.[Abstract]
  19. Emoto Y., Manome Y., Meinhardt G., Kisaki H., Kharbanda S., Robertson M., Ghayur T., Wong W. W., Kamen R., Weichselbaum R., et al Proteolytic activation of protein kinase C {delta} by an ICE-like protease in apoptotic cells. EMBO J., 14: 6148-6156, 1995.[Medline]
  20. Reyland M. E., Anderson S. M., Matassa A. A., Barzen K. A., Quissell D. O. Protein kinase C {delta} is essential for etoposide-induced apoptosis in salivary gland acinar cells. J. Biol. Chem., 274: 19115-21913, 1999.[Abstract/Free Full Text]
  21. Cross T., Griffiths G., Deacon E., Sallis R., Gough M., Watters D., Lord J. M. PKC-{delta} is an apoptotic lamin kinase. Oncogene, 19: 2331-2337, 2000.[Medline]
  22. Dal Pra I., Whitfield J. F., Chiarini A., Armato U. Increased activity of the protein kinase C-{delta} holoenzyme in the cytoplasmic particulate fraction precedes the activation of caspases in polyomavirus-transformed pyF111 rat fibroblasts exposed to calphostin C or topoisomerase-II inhibitors. Exp. Cell Res., 255: 171-183, 2000.[Medline]
  23. Majumder P. K., Pandey P., Sun X., Cheng K., Datta R., Saxena S., Kharbanda S., Kufe D. Mitochondrial translocation of protein kinase C {delta} in phorbol ester-induced cytochrome C release and apoptosis. J. Biol. Chem., 275: 21793-21796, 2000.[Abstract/Free Full Text]
  24. Shichiri M., Sedivy J. M., Marumo F., Hirata Y. Endothelin-1 is a potent survival factor for c-Myc-dependent apoptosis. Mol. Endocrinol., 12: 172-180, 1998.[Abstract/Free Full Text]
  25. Eberl, L. P., Valdenaire, O., Saintgiorgio, V., Jeannin, J. F., and Juillerat-Jeanneret, L. Endothelin receptor blockade potentiates FasL-induced apoptosis in rat colon carcinoma cells. Int. J. Cancer 86: 182–187, 2000.
  26. Prenzel N., Zwick E., Daub H., Leserer M., Abraham R., Wallasch C., Ullrich A. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature, 402: 884-888, 1999.[Medline]
  27. Clerk A., Sugden P. H. Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists. Am. J. Cardiol., 83: 64H-69H, 1999.[Medline]
  28. Murga C., Laguinge L., Wetzker R., Cuadrado A., Gutkind J. S. Activation of Akt/protein kinase B by G protein-coupled receptors. A role for {alpha} and ß {gamma} subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinase {gamma}. J. Biol. Chem., 273: 19080-19085, 1998.[Abstract/Free Full Text]
  29. Basu A., Akkaraju G. R. Regulation of caspase activation and cis-diamminedichloroplatinum(II)-induced cell death by protein kinase C. Biochemistry, 38: 4245-4251, 1999.[Medline]



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