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1 Department of Medicine and Sydney Cancer Centre, 2 Institute of Biomedical Research, School of Biomedical Sciences and Pathology, The University of Sydney, Australia; 3 St Vincent’s Hospital Clinical School, School of Medical Sciences and Department of Clinical Medicine, The University of New South Wales, Australia; 4 Department of Anatomical Pathology, Royal Prince Alfred Hospital, Sydney, Australia; 5 Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington; 6 Oncology Research Centre, Prince of Wales Hospital, Sydney, Australia; and 7 Therapeutics Centre, St. Vincent’s Hospital, Sydney, Australia
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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(cPLA2-) levels are unchanged. sPLA2-IIA mRNA expression is detectable and inducible by androgen (0.01–10 nmol/L) in the androgen-sensitive cell line LNCaP, and exogenous addition of sPLA2-IIA (1–100 nmol/L), but not an inactive sPLA2-IIA mutant (H48Q), results in a dose-dependent increase in cell numbers or the fraction of cells in G2-M phase, which is inhibited by sPLA2-IIA-selective inhibitors. The effect of exogenous sPLA2-IIA can also be blocked by inhibition of cPLA2-, suggesting a role for cPLA2- in mediating sPLA2-IIA action. sPLA2-IIA inhibitors suppressed basal proliferation in LNCaP cells and in the androgen-independent, sPLA2-positive cell line PC3 but not in the sPLA2-IIA-negative androgen-independent cell line DU145. Established PC3 xenograft tumors grew more slowly in mice treated with sPLA2-IIA inhibitors than those treated with saline only. The PLA2 enzymes, and sPLA2-IIA in particular, thus represent important targets for the treatment of sPLA2-IIA-positive androgen-independent prostate cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Phospholipase A2 (PLA2), catalyzes the hydrolysis of membrane glycerophospholipids, leading to production of free fatty acids and lysophospholipids. If the esterified fatty acid is arachidonic acid, this is converted in the prostate to prostaglandins by COX or hydroxyeicosatetraenoic acids by LOX. PLA2 is a growing family of proteins, and in human, there are 10 secreted PLA2 forms (sPLA2), at least three isoforms of cytosolic PLA2 (cPLA2), and multiple isoforms of calcium-independent intracellular PLA2 (5, 6, 7, 8, 9) .
The aim of this study was, first, to determine the expression levels of sPLA2 and cPLA2 in patients with prostate cancer treated with androgen ablation therapy; second, to determine oncogenic potential of sPLA2 and cPLA2; and last, to determine the effect of specific sPLA2-IIA inhibitors on prostate cancer cell growth. We have now obtained evidence that sPLA2-IIA is constitutively expressed in remaining prostate cancer cells after 3 months of androgen ablation therapy. The increased sPLA2-IIA has an oncogenic action that requires the activity of sPLA2-IIA and of cPLA2-. Our newly designed sPLA2-IIA inhibitors can block basal and sPLA2-IIA-induced prostate cancer cell proliferation in vitro and in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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inhibitor pyrrolidine-1 was synthesized as described previously (13)
. Polyclonal anti-sPLA2-IIA (160502; Cayman Chemicals, Sydney, Australia) and anti-cPLA2- (SC-438; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were purchased. The monoclonal anti-sPLA2-IIA antibody 4A1 was raised and purified (Bioquest, Ltd., Sydney, Australia) as described previously (11)
.
Immunohistochemistry.
Prostate tissues were all fixed in buffered 10% formaldehyde solution for approximately 24 hours and paraffin-embedded. Immunohistochemistry was performed on tissue sections (5 µm) using an avidin-biotin complex method with diaminobenzidine as a chromogen as described previously (14)
. The sections were subjected to microwave epitope retrieval before staining. Immunostaining was considered positive and specific when the intensity of staining with the post-immune IgG exceeded that observed with the preimmune IgG (isotype control) and with no IgG (method control). Specific staining was graded as described previously (15)
. In brief, the staining intensity was graded as low, intermediate, and high. The percentage of cells with the highest intensity staining was stratified into three scores: 1, 0 to 33%; 2, 34 to 66%; and 3, 67 to 100%. Zero represented no cells with high intensity staining.
Cell Lines and Culture.
The LNCaP, DU145, and PC3 cell lines were purchased from American Type Culture Collection (Rockville, MD). All cell cultures were maintained in RPMI 1640 (Sigma-Aldrich, Sydney, Australia), supplemented with 5% fetal bovine serum (FBS; ICN Biomedical, Sydney, Australia) for LNCaP and 10% FBS for DU145 and PC3 at 37°C in a humidified environment of 5% CO2. For experiments with the addition of androgens, LNCaP cells were cultured in the absence or presence of methyltrienolone (R1881; New England Nuclear Life Science, Wilmington, DE) for 72 hours in phenol red-free RPMI containing 5% charcoal-stripped FBS. The passage numbers of cells described in this article were between 30 and 45 for LNCaP, 65 and 80 for DU145, and 25 and 40 for PC3.
Charcoal-Stripped Fetal Bovine Serum.
Activated charcoal (40 g; 250–350 mesh; Sigma) was mixed with distilled water (1 L) and soaked for 1 hour at room temperature, and floating charcoal was removed. The soaking process was repeated until all floating charcoal was removed. Charcoal was then soaked in a minimal volume of acetone and allowed to dry by evaporation overnight in a fume hood. Dried charcoal was suspended in FBS to 0.4 mg/mL, incubated at 55°C for 10 minutes, and centrifuged for 5 minutes at 2000 x g; and the supernatant was filtered through a 0.2-µm filter. Because FBS has a low concentration of sex steroids, which makes it difficult to determine the efficiency of charcoal stripping, pooled human serum was used for validation of our protocol for charcoal stripping. Relative to unstripped serum, the concentration of testosterone, estradiol, and progesterone in charcoal-stripped serum was under the detection limit by competitive immunoassay (IMMULITE 2000; DPC, Los Angeles, CA), whereas the concentration of luteinizing hormone and follicular stimulating hormone, representatives of larger Mr proteins, were unchanged by microparticle enzyme immunoassay (Abbott Laboratories, Abbott Park, IL).
Reverse Transcription-Polymerase Chain Reaction.
The effect of added androgen on the steady-state level of sPLA2-IIA mRNA in LNCaP cells was measured by quantitative real-time reverse transcription-PCR. After cell treatments, total RNA was isolated using Trizol reagent. The first-strand cDNA was synthesized from 2 µg of total RNA using a combination of random hexamers and oligo(dT) as described previously (16)
. Primers were designed based on the human sPLA2-IIA mRNA (NM_000300). Forward, 5'-TTTGTCACCCAAGAACTCTTAC-3'. Reverse, 5'-GGGAGGGAGGGTATGAGA-3'. Hypoxanthine phosphoribosyltransferase was used as the house-keeping gene, and its primer sequences were published previously (14)
. The PCR reaction (15 µL) contained 5.5 µL of water, 7.5 µL of 2x platinum quantitative PCR superMix-UDG (Invitrogen, Melbourne, Australia), 0.25 µL each of 0.01 mmol/L primers, 0.5 µL of 25x SYBR Green 1 dye (Molecular Probe, Sydney, Australia), and 1 µL of cDNA. The following protocol was used on ABI Prism 7000 sequence detection system: 50°C for 2 minutes, 95°C for 2 minutes followed by 45 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. After verification of parallelism in amplification efficiency (<10% variation in slopes on plots of crossing point cycles versus cDNA concentration) between sPLA2-IIA and hypoxanthine phosphoribosyltransferase, the 
- method was used to calculate relative changes in mRNA levels of sPLA2-IIA corrected for hypoxanthine phosphoribosyltransferase (HPRT) (17)
. For endogenous sPLA2-IIA mRNA level, total RNA from LNCaP, DU145, and PC3 cells was isolated and reverse transcribed as above. Conventional end-time PCR was performed at an annealing temperature of 55°C.
Cell Proliferation Assay.
Cells were plated at 1 x 104 per well in 96-well plates with 0.1 mL of media. Treatments were performed on cells at 70 to 80% confluence, in medium containing 5% FBS for 72 hours. After treatment, the number of viable cells was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Sydney, Australia). In brief, 20 µL of solution were added to each well and incubated for 1 hour. The absorbance at 490 nm was measured with a microplate reader (Multiscan EX; Labsystems, Helsinki, Finland). Each experiment was performed in quadruplicate and repeated at least three times.
Flow Cytometric Analysis.
LNCaP cells were seeded in 50-mL flasks and grown to 70 to 80% confluence before treatment for 72 hours. After trypsinization and cell counting, the treated or untreated cells (1 x 106) were suspended in PBS (1 mL) and incubated after the addition of 200 µL of Triton X-100 (0.4% stock) for 5 minutes at room temperature in the presence of 50 µL of propidium iodide solution (1 mg/mL stock) and 20 µL of RNase A Type I-A (50 mg/mL, stock; Sigma-Aldrich). DNA content per cell was measured by flow cytometry using a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ). Analysis was performed on 10,000 events per sample.
PC3 Xenograft Study in Nude Mice.
Animal studies were performed according to the guidelines for the ethical use of animals published by the National Health and Medical Research Council of Australia and with the approval of the University of New South Wales Animal Ethics Committee. Four- to 6-week-old athymic male nude mice (BALB/c) were inoculated subcutaneously with 1 x 107 PC3 cells. The cells were suspended in 0.1 mL of Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum before injection and injected via a 26-gauge needle. When the size of the xenograft reached 5 x 5 mm (tumor volume, 65.5 mm3 calculated as V = 
/6(d1.d2)3/2; ref. 18
), mice were randomly assigned into four groups (n = 15 per group), i.e., cFLSYR (1 mg/kg), cFLSYR (10 mg/kg), 2-naphthylalanine [c(2NapA)LS(2NapA)R; 1 mg/kg], and saline. cFLSYR was dissolved directly in sterile saline, whereas c(2NapA)LS(2NapA)R was diluted into sterile saline from a stock solution (40 mg/mL in dimethylformamide) immediately before use. The saline group received sterile saline containing 0.75% dimethylformamide. Subcutaneous injections were given three times weekly on the opposite flank to the xenograft site, and treatments were blinded to experimentalists throughout the study. Tumor growth was normalized for differing starting sizes by calculating relative tumor volume, i.e., the ratio of each tumor volume on a given day to its volume on day 1 of treatment. The mice were treated for a period of 8 weeks unless culled due to tumor volume reaching 15 x 15 mm (1768 mm3).
Statistical Analysis.
The Number Cruncher Statistical System (NCSS, Kaysville, UT) was used for statistical analysis. Data were analyzed by 
2 or analysis of variance (ANOVA) as appropriate. A two-tailed P value <0.05 was considered significant.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, -ß, and -
), intracellular PLA2 (-A), and platelet-activating factor acetylhydrolase families were either unchanged or not present. There are three published reports describing an increased sPLA2-IIA mRNA (19)
and protein levels (20
, 21)
in prostate cancer tissue and the enhanced sPLA2-IIA expression was inversely related to 5-year patient survival (21)
. The chromosomal location of sPLA2-IIA (1p35-ter) was found to overlap with a prostate cancer susceptibility locus CAPB (22)
. To verify the SAGE results and to extend them to cancer cells in the absence of androgens, we examined, immunohistochemically, sPLA2-IIA expression in prostate cancer tissues from patients treated with androgen-ablation therapy for 3 months (100 mg of cyproterone acetate, twice daily) before radical prostatectomy. An undetectable serum prostate-specific antigen was confirmed in each patient before radical prostatectomy. Although the remaining cancer cells in these specimens are regarded qualitatively as being androgen independent, it is likely that the degree of androgen independence varies among these cells. Two antibodies were used for immunohistochemistry, and both showed the same staining pattern on consecutive sections. In the presence of androgens (n = 50), there was patchy cytoplasmic staining in benign glands (Fig. 1A)
adjacent to cancer cells and extensive cytoplasmic staining in cancer cells (Fig. 1B)
. With androgen ablation therapy (n = 26), benign glands showed substantially decreased staining, whereas cancer cells maintained sPLA2-IIA expression (Fig. 1C)
. The percentage of stained cancer and of adjacent benign glands was then stratified into three scores (Fig. 2)
. No difference was found in immunohistochemical staining of cPLA2- between normal and cancer cells irrespective of androgen status (data not shown).
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suggests that expression of sPLA2-IIA gene requires androgens. To verify that, we first searched the 5'-flanking region of the sPLA2-IIA gene using MatInspector Release 5.3 (Genomatix, München, Germany) and found an androgen response element GAGGTAAATGGTATTCTC from –546 to –527. Second, we treated the androgen-responsive cell line, LNCaP, with metabolically stable synthetic androgen (methyltrienolone; R1881; 0.01–10 nmol/L) for 4, 8, and 24 hours and measured sPLA2-IIA mRNA by real-time PCR. Indeed, there was a significant increase in the steady-state mRNA level of sPLA2-IIA at 8 hours (Fig. 3)
. However, we could not find an androgen response element within 3 kb of the 5-flanking region of the cPLA2 genomic DNA. Androgen treatment had no effect on cPLA2 mRNA by real-time PCR (data not shown). Together, these findings suggest that expression of sPLA2-IIA but not cPLA2 is likely to be dependent on androgens in normal prostate. In cancer cells remaining after androgen ablation therapy, sPLA2-IIA expression is constitutively active via an as yet unknown mechanism.
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. This potent effect was blocked by the sPLA2-IIA inhibitor, cFLSYR, at a 1:1 molar ratio of inhibitor to added enzyme (Fig. 4B)
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. In the presence of both sPLA2 and its inhibitor (cFLSYR), the proportion of cells in G1 and G2 phase returned to basal levels (Fig. 4D)
. These results suggest the biological importance of the highly expressed sPLA2-IIA in prostate cancer and demonstrate that the sPLA2-IIA-induced cell proliferation can be attributed at least partly to an increased proportion of cells entering the G2-M phase from the G1 phase.
Oncogenic Action of Exogenous Secreted Phospholipase A2-IIA Requires the Activity of Both Secreted Phospholipase A2-IIA and Cytosolic Phospholipase A2-.
In contrast to the essential in vivo role of cPLA2- in eicosanoid production under most physiologic conditions and in response to inflammatory stimuli, whether and if so, how sPLA2-IIA increases eicosanoid production in vivo is unclear. Currently, two models are proposed to explain the action of sPLA2 on eicosanoid production in cellular systems (23)
. In the first model, sPLA2 binds directly to membrane phospholipids, and the released arachidonic acid serves as a substrate to produce eicosanoids. Lysophospholipids mediate indirect activation of endogenous cPLA2- via mobilization of calcium. The enhanced cPLA2- activity can in turn cause an increased production of eicosanoids. The second model is indirect modulation of intracellular eicosanoid pathways via cell surface glycosyl phosphatidyl inositol (GPI)-linked heparan sulfate proteoglycan receptors. The internalized sPLA2 supplies arachidonic acid to downstream enzymes either directly or indirectly via activation of cPLA2- through mitogen-activated protein kinase-mediated phosphorylation. Enzyme activity of sPLA2 is not obligatory for activation of the mitogen-activated protein kinase pathway.
To determine whether sPLA2-IIA-induced prostate cancer cell proliferation depends on its enzyme activity, H48Q purified by immunoaffinity chromatography was used to treat LNCaP cells. Over the same dose range as the wild-type sPLA2-IIA, H48Q, which has no more than 2 to 4% of wild-type activity (12)
, had no growth-promoting effect (Fig. 5A)
, demonstrating that sPLA2-IIA enzyme activity and thus its products are required for the proliferative effect of sPLA2-IIA.
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mediates exogenously added sPLA2-IIA action, we treated LNCaP cells with various doses of the selective cPLA2- inhibitor pyrrolidine-1 (13)
with or without a constant effective dose of sPLA2-IIA (1 nmol/L). Blockade of cPLA2 abolishes sPLA2-induced cell growth completely, demonstrating that cPLA2- activity is necessary for sPLA2-IIA-dependent prostate cancer cell proliferation (Fig. 5B)
. Furthermore, the dose response at sub-micromolar to low micromolar range of pyrrolidine-1 is consistent with the dose required to block arachidonate release in a number of cPLA2--expressing cells (13)
. Considering the common loss of annexins 1 (24)
and 2 (14)
in prostate cancer and the known inhibitory effect of the two annexins on cPLA2- activity, the activity of cPLA2- could be increased significantly in prostate cancer cells.
Cyclic Peptide Inhibitors of Secreted Phospholipase A2-IIA Suppress Endogenous Secreted Phospholipase A2-IIA-Dependent Proliferation In vitro and In vivo.
Based on our finding that the growth-promoting sPLA2-IIA is constitutively expressed in prostate cancer cells and the knowledge that production of arachidonic acid and lysophospholipids by PLA2 is, in most cases, the rate-limiting step in eicosanoid synthesis, we have considered the potential of sPLA2-IIA as a target for treatment of prostate cancer. We reason that a better outcome can be achieved with the PLA2 inhibitor than with a COX inhibitor alone because the latter suppresses the production of prostaglandins only. The first sPLA2-IIA inhibitor, LY311727, was rationally designed by Eli Lilly. Studies examining structure-activity relationships show that the Lilly inhibitor is relatively nonselective (25)
. We have previously shown that human sPLA2-IIA is dose-dependently inhibited by a pentapeptide sequence comprising residues 70 to 74 of the native sPLA2-IIA protein (70FLSYK74; ref. 26
). Because of the inherent flexibility of the linear peptide sequence, inhibition was weak in in vitro activity assays. We have recently designed two novel cyclic peptides (10)
, cFLSYR and a cyclic peptide in which F and Y are substituted with c(2NapA)LS(2NapA)R. Both have shown significant improvement in potency over linear peptides in sPLA2-IIA inhibition assays and do not bind to a structurally related enzyme sPLA2-IB.
To test the effect of blocking endogenous sPLA2-IIA on cell growth, we first determined the basal mRNA levels of sPLA2-IIA in three human prostate cancer cell lines. mRNA encoding sPLA2-IIA was undetectable in DU145 compared with LNCaP and PC3 cells (Fig. 6A)
. We then tested the effect of individual inhibitors cFLSYR and c(2Nap)LS(2Nap)R on cell growth over a range of doses (1–100 nmol/L). LNCaP and PC-3 cell numbers were significantly decreased in the presence of inhibitor, and the smallest effective dose was 1 nmol/L (Fig. 6B)
. In contrast, neither of the inhibitors had an effect on DU145, presumably due to the lack of endogenous sPLA2-IIA.
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and 6
. In the presence of exogenous sPLA2-IIA in Fig. 4
, cFLSYR did not reduce cell number below that of untreated cells at concentrations up to 100 nmol/L. However, in Fig. 6
, c(2Nap)LS(2Nap)R at 1 nmol/L results in 
40% fewer cells than in control. Recent studies have shown that the effects of exogenous sPLA2-IIA are mechanistically separable from those of endogenous intracellular sPLA2-IIA in model cell systems (27)
. It is thus possible that sPLA2-IIA may modulate cell growth by separate mechanisms depending on its cellular location. Also, the difference in potency between the two inhibitors (see Materials and Methods) may also contribute to the observation. Additional work will examine the detailed mechanism underlying the effect of both inhibitors in the presence or absence of exogenous sPLA2-IIA.
In vivo, cFLSYR, at either 1 mg/kg or 10 mg/kg, and c2(NapA)LS2(NapA)R at 1 mg/kg were injected three times a week subcutaneously to the flank opposite to the site of the PC3 xenograft. The treatment commenced only when the xenograft has reached the size of 5 x 5 mm. We found that inhibitor c(2NapA)LS(2NapA)R at 1 mg/kg and inhibitor cFLSYR at 10 mg/kg slowed the rate of growth of tumors from 5 weeks post-treatment commencement resulting in significant suppression of PC3 xenograft volume by 8 weeks of treatment compared with saline-treated mice (Fig. 7)
. cFLSYR had no effect at 1 mg/kg (data not shown). This in vivo order of potency of the two peptides correlates well with their relative potencies in in vitro enzyme activity assays (10)
. There were no visible side effects of either treatment including eating, drinking, and general behavior and no evidence of toxicity at the injection site. Although the inhibitory effect of both peptides is clear at 8 weeks after treatment-commencement, it is worth noting that, because pharmacokinetic data on these peptide inhibitors are not available at present, additional efficacy studies with optimized delivery route, dose, and dosing regimen are needed. It is nonetheless remarkable that blockade of xenograft-derived sPLA2-IIA alone, as host-derived sPLA2-IIA expression is largely confined to secretory cells in the gut in BALB/c mice and does not respond to agonist stimulation (28)
, seems to be sufficient to slow xenograft growth. In addition, evaluation of this effect relative to a COX-2-selective inhibitor would be valuable to corroborate the hypothesis that suppression of phospholipase A2 is more beneficial than suppression of COX alone.
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. The loss of Annexin 1 and 2 in prostate cancer could enhance sPLA2-IIA action by increasing the activity of cPLA2-. A better therapeutic outcome might be achieved through the use of PLA2 inhibitors in the treatment of prostate cancer, particularly in the hormone refractory form.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
Note: P. Sved and K. Scott contributed equally to this work.
Requests for reprints: Qihan Dong, Department of Medicine, D06, University of Sydney, NSW 2006, Australia. Phone: 612-95155186; Fax: 612-95161273; E-mail: qhd{at}med.usyd.edu.au
8 K. J. Bryant, P. W. Lei, M. Bidgood, M. Taberner, A. Cunningham, K. F. Scott, manuscript in preparation. 
9 Internet address: www.ncbi.nlm.nih.gov/SAGE.
Received 9/24/03. Revised 6/ 7/04. Accepted 7/19/04.
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. J Biol Chem 2004;279:25024-38.This article has been cited by other articles:
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  J. Saegusa, N. Akakura, C.-Y. Wu, C. Hoogland, Z. Ma, K. S. Lam, F.-T. Liu, Y. K. Takada, and Y. Takada Pro-inflammatory Secretory Phospholipase A2 Type IIA Binds to Integrins {alpha}v{beta}3 and {alpha}4{beta}1 and Induces Proliferation of Monocytic Cells in an Integrin-dependent Manner J. Biol. Chem., September 19, 2008; 283(38): 26107 - 26115. [Abstract] [Full Text] [PDF]
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 L. Linderoth, T. L. Andresen, K. Jorgensen, R. Madsen, and G. H. Peters Molecular Basis of Phospholipase A2 Activity toward Phospholipids with sn-1 Substitutions Biophys. J., January 1, 2008; 94(1): 14 - 26. [Abstract] [Full Text] [PDF]
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T. T. C. Yang, P. M. U. Ung, M. Rincon, and C.-W. Chow Role of the CCAAT/Enhancer-binding Protein NFATc2 Transcription Factor Cascade in the Induction of Secretory Phospholipase A2 J. Biol. Chem., April 28, 2006; 281(17): 11541 - 11552. [Abstract] [Full Text] [PDF]
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) or cancer cells (
) from patients with (+, n = 26) or without (–, n = 50) androgen ablation therapy was graded on a three-point score, (1, 0–33% cells positive; 2, 33–66% cells positive; and 3, > 66% cells positive). Data are presented as the percentage of cases at each score. *, P < 0.05 versus benign glands without androgen ablation therapy by 
) and absence (
