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Endocrinology Vol. 147, No. 9 4245-4251
Copyright © 2006 by The Endocrine Society

The Endocrine-Gland-Derived Vascular Endothelial Growth Factor (EG-VEGF)/Prokineticin 1 and 2 and Receptor Expression in Human Prostate: Up-Regulation of EG-VEGF/Prokineticin 1 with Malignancy

Daniela Pasquali, Valentina Rossi, Stefania Staibano, Gaetano De Rosa, Paolo Chieffi, Domenico Prezioso, Vincenzo Mirone, Massimo Mascolo, Donatella Tramontano, Antonio Bellastella and Antonio Agostino Sinisi

Department of Clinical and Experimental Medicine and Surgery (D.Pa., V.R., A. B., A.A.S), Endocrine Unit, and Department of Experimental Medicine (P.C.), Second University of Naples, 80131 Naples, Italy; Department of Biomorphological and Functional Sciences (S.S, G.D.R, M.M.), Pathology Section, Urologic Clinic (D.Pr., V.M.), and Department of Biology and Molecular Pathology (D.T.), University of Naples Federico II, 80131 Naples, Italy

Address all correspondence and requests for reprints to: Prof. Antonio A. Sinisi, Department of Clinical and Experimental Medicine and Surgery, Endocrine Unit, Second University of Napoli, Via Pansini 5, 80131 Napoli, Italy. E-mail antonio.sinisi{at}unina2.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A new family of angiogenic factors named endocrine-gland-derived vascular endothelial growth factors (EG-VEGF)/prokineticins (PK) have been recently described as predominantly expressed in steroidogenic tissues. Whether the normal and malignant epithelial prostate cells and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. We studied the expression of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in human prostate and their involvement in cancer. Using immunohistochemistry, Western blot, and RT-PCR, we determined the expression of EG-VEGF/PK1 in normal prostate (NP) and malignant prostate tissues (PCa), in epithelial cell primary cultures from normal prostate (NPEC) and malignant prostate (CPEC) and in a panel of prostate cell lines. In NPEC, CPEC, and in EPN, a nontransformed human prostate epithelial cell line, EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels were evaluated by quantitative RT-PCR. EG-VEGF/PK1 transcript was found in PCa, in CPEC, in EPN, and in LNCaP, whereas it was detected at low level in NP and in NPEC. EG-VEGF/PK1 was absent in androgen-independent PC3 and DU-145 cell lines. Immunochemistry confirmed that EG-VEGF/PK1 protein expression was restricted to hyperplastic and malignant prostate tissues, localized in the glandular epithelial cells, and progressively increased with the prostate cancer Gleason score advancement. EG-VEGF/PK1 and PK2 were weakly expressed in NPEC and EPN. On the other hand, their transcripts were highly detected in CPEC. PK-R1 and PK-R2 were found in NPEC, EPN, and CPEC. Interestingly, CPEC showed a significantly (P < 0.05) higher expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 compared with NPEC and EPN. We demonstrated that PKs and their receptors are expressed in human prostate and that their levels increased with prostate malignancy. It may imply that EG-VEGF/PK1 could be involved in prostate carcinogenesis, probably regulating angiogenesis. Thus, the level of EG-VEGF/PK1 could be useful for prostate cancer outcome evaluation and as a target for prostate cancer treatment in the future.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANGIOGENESIS PLAYS A pivotal role in the growth, invasion, and metastasis of several malignant tumors including prostate cancer (1, 2). It has been demonstrated that formation of new microvascular endothelium is also important for normal prostate growth (1, 3). A link between hormonal regulation of prostate tissue and angiogenesis from paracrine effects of endothelial cells has been suggested (1, 3). Prostate cells have the ability to produce several angiogenic factors, including the vascular endothelial growth factor (VEGF), a key regulator of angiogenesis. Increased microvessel density and VEGF levels have been described in most prostate cancers and associated with poorer prognosis (2, 4, 5, 6, 7). Recently, two new angiogenic factors that selectively act on the endothelium of endocrine gland cells have been described and named endocrine-gland-derived VEGF (EG-VEGF)/prokineticin 1 (PK1) and PK2 (8). EG-VEGF is identical to PK1, which was previously cloned as a mammalian homolog of mamba intestinal toxin-1 (9). EG-VEGF/PK1 and -2 act by inducing proliferation, migration, and fenestration of endothelium derived from adrenal capillaries but not of other endothelium types such as those derived from aorta, umbilical vein, and dermis (8). These peptides, with 10 cysteine residues in identical positions in all members of the family, are structurally unrelated to VEGF and regulate diverse biological functions in steroidogenic and nonsteroidogenic tissues, such as smooth muscle contraction and in particular angiogenesis. EG-VEGF/PK1 and -2 exert their physiological functions through G-protein-coupled receptors recently identified and named PK receptor type 1 (PK-R1) and PK-R2 (10, 11, 12). These receptors bind to and are activated by nanomolar concentrations of recombinant PK. Activation of PK-R induces calcium mobilization, induction of phosphoinositide turnover, and activation of MAPK signaling pathways that are consistent with the effects of PK on smooth muscle contraction and angiogenesis (10, 11, 12). EG-VEGF seems to play different roles in different tissues. It has recently been found that EG-VEGF/PK1 overexpression in a colorectal cancer line induces the angiogenesis and tumor when implanted into nude mice (13). However, in other tissues, such as ovary, EG-VEGF expression was detected in the early stage of the disease and reduced in advanced-stage ovarian carcinoma (14), whereas its expression was often lost in endometrial carcinoma (15). Whether the normal prostate (NP) and malignant prostate (CP) epithelial cells (EC) and tissues express EG-VEGF/PK1 and PK2 and their receptors is still unknown. EG-VEGF/PK1 mRNA is barely detectable by Northern blot in human normal prostate tissue (8). To evaluate the potential role of these new angiogenic factors in human prostate growth, tumorigenesis, and cancer progression, we analyzed EG-VEGF/PK1 expression in normal, benign prostate hyperplasia, and prostate cancer tissues in cultured normal (NPEC) and malignant (CPEC) prostate EC and in EPN, LNCaP, PC3, and DU-145 cell lines. Moreover, we studied by quantitative RT-PCR EG-VEGF/PK1 and -2 and their receptor expression levels in NPEC, CPEC, and EPN. We demonstrated the presence and differential expression levels of the PKs and their receptors in NPEC, EPN, and CPEC. Furthermore, EG-VEGF/PK1 protein was localized by Western blot and immunohistochemistry in hyperplastic and malignant prostate tissues.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Prostate tissue specimens, prostate cell primary cultures, and prostate cancer (PCa) cell lines
NP tissues were collected from patients who had undergone radical cystectomy for bladder cancer. PCa tissues were obtained from patients who had undergone radical prostatectomy (Gleason score 4–9). After prostatectomy, a wedge-shaped specimen of fresh prostate was removed and divided in two equally representative parts; one was immediately frozen in liquid nitrogen until RNA extraction was performed, and the other was used to set primary cultures. Some slices were cut from each tissue specimen, fixed in 4% paraformaldehyde, and processed for histological examination to confirm the prostatic origin, the diagnosis, and the absence of other diseases. Only specimens containing 100% normal or cancer prostate cells were used to establish primary cultures according to a previously described method (16). EC were separated by different centrifugations of minced and collagenase-digested tissues (Collagenase IV, 10 mg/ml; Life Technologies, Inc.-BRL, Milan, Italy). The EC were plated on keratinocyte-SFM medium (Life Technologies) supplemented with bovine pituitary extract (10 mg/ml), epidermal growth factor (10 ng/ml), cholera toxin (10 ng/ml), 5% fetal calf serum (FCS), and antibiotics (fungizon and penicillin-streptomycin). At confluence, cultures were then split after EDTA-trypsin treatment and used at first passage. The epithelial nature was established if cytokeratin immunostaining was positive in nearly 100% of cells according to previously described methods (16). The malignant nature of cells derived from prostate carcinomas was confirmed by a high expression of proliferative antigen Ki67 and, particularly, by a high expression of mutated p53 protein, demonstrated by immunoreactivity with monoclonal antibody clone Ki-67 (Dako, Milan, Italy) and Pab 240 (Serotec, Delta Biological, Milan, Italy), respectively (16). Four cell strains from normal prostates (NPEC) and four from PCa specimens (CPEC) were used in the experimental protocols that were repeated at least three times. EPN cells, a nontransformed cell line isolated and characterized by our laboratory (17), were cultured in HAM-F12 supplemented with 3% FCS and antibiotics (fungizon and penicillin-streptomycin). LNCaP cells (American Type Culture Collection, Rockville, MD) were maintained in RPMI 1640 supplemented with 10% FCS. PC3 cells were maintained in DMEM with heat-inactivated FCS. DU-145 cells were maintained in MEM supplemented with 10% FCS, 1.5 g/liter sodium bicarbonate, 1 mM sodium pyruvate, 2 nM L-glutamine, and 0.1 mM nonessential amino acids. All cultures were maintained at 37 C in a humidified 5% CO2 atmosphere.

RNA isolation
RNA was extracted from frozen tissue specimens or from the cell cultures. Total RNA was recovered with TRIZOL kit (Invitrogen, Milan, Italy). Residual DNA was removed by RNase-free DNase I treatment (Promega, Florence, Italy).

RT-PCR
RNAs were reverse transcribed using 5 µg total RNA as previously described (16). To obtain a negative control for the amplification reactions, we carried out an RNA transcription without adding reverse transcriptase. cDNA (400 ng) obtained by RT of RNAs was amplified in a total volume of 50 ml containing 10 mmol Tris-HCl, 1.5 mmol MgCl2, 50 mmol KCl (pH 8.3), and 100 ng 5'-3' end primers. PCR conditions were 35 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 90 sec. To evaluate variability in the expression of EG-VEGF/PK1, PK2, PK-R1, and PK-R2, a semiquantitative PCR was performed in which these genes were amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (22 cycles) as previously described (16). The number of cycles was chosen in the middle of the exponential phase of the reaction, separately for each type. To establish the number of cycles, GAPDH was amplified at 15, 22, 32, and 40 PCR cycles, and EG-VEGF/PK1 and -2 and PK-R1 and -R2 were subjected to 25, 32, and 40 amplification cycles (data not shown). The levels of mRNAs, quantified by densitometry scanning of the amplification products electrophoresed on agarose gels, are expressed as the ratio between the density of each gene product and that of coamplified GAPDH. We used oligonucleotide sequences for GAPDH (16). We also used the following primers: EG-VEGF/PK1 sense (5'-CGC GAG TCT CAA TCA TGC TCC T-3') and antisense (5'-GGC AAG GCG CTA AAA ATT GAT G-3'); PK-R1 sense (5'-GCG GCA TTG GAA ACT TCA-3') and antisense (5'-GGC CCA CGA ATT CTA TGC C-3'); PK2 sense (5'-TTG GCC TGT TTA CGG ACT TC-3') and antisense (5'-TGC AAG AGG AGG GAA GAG AA-3'); and PK-R2 sense (5'-ATA TCT CGA CCA TCG T TC ACC-3') and antisense (5'-AGC ACA TAG GCC GTG AGA AAT-3') (Table 1Go). PCR products were then separated on a 1.2% agarose gel containing ethidium bromide using a 100-bp DNA ladder (Life Technologies) as size marker.


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  		TABLE 1. Oligonucleotide sequences used for RT-PCR

 
Real-time quantitative RT-PCR
Real-time quantitative PCR was used to determine the amounts of EG-VEGF/PK1 in NP and PCa, and EG-VEGF/PK1, PK2, PK-R1 and PK-R2 mRNA in EPN, NPEC, and CPEC as previously described (18). In these experiments, the amount of specific amplicon present was related to ß2-microglobulin and subsequently to an internal control. RNA was extracted from NP, PCa, and epithelial prostate cells as described above. Total RNA (100 ng) was reverse transcribed to first cDNA as previously described (16). Real-time PCR was repeated three times for each sample. Oligonucleotides were as follows: EG-VEGF/PK1 [5'-CCACATGTATCCCTCGGTCT-3' (sense) and 5'-ACCTGGGACTCTGAGCAATG-3' (antisense)]; PK2 [5'-CTTGCCTCTTCCACCTCAAA-3' (sense) and 5'-TGCAAGAGGAGGGAAGAGAA-3' (antisense)]; PK-R1 [5'-CGGCATTGGAAACTTCATCT-3' (sense) and 5'-GATGAGCAGGTTGGTGAGGT-3' (antisense)]; and PK-R2 [5'-CACCATGACCCTGTGCTATG-3' (sense) and 5'-GAATCTGCTCCGTCTGGAAC-3' (antisense)]. Real-time PCR was also repeated three times for a housekeeping gene, ß2-microglobulin. The following primers were used: sense, 5'-CCAGCAGAGAATGGAAAGTC-3', and antisense, 5'-GATGCTGCTTACATGTCTCG-3'. The iQ SYBR Green Supermix kit (Bio-Rad Laboratories, Milan, Italy) was used in an iCycler iQ Real-Time PCR Detection System (Bio-Rad Laboratories). Data are expressed as the amount of specific PCR products from each gene in cells treated with fibrate (500 µM) over untreated cells after normalization based on the housekeeping gene product ß2-microglobulin (which showed no significant difference in any treatment).

Immunohistochemistry
Formalin-fixed, paraffin-embedded blocks of 30 prostatic cancers (surgical specimens from radical prostatectomy) and 30 normal-benign/hyperplastic prostatic tissues (as a control population), retrieved from the archives of the Department of Biomorphological and Functional Sciences, Pathology Section, University Federico II of Naples, Italy, were used for the immunohistochemical determination of EG-VEGF/PK1 expression. We studied prostate tissue samples from four patients with low-grade (Gleason score 4–5), 17 patients with medium-grade (Gleason score 6–7), and nine patients with high-grade (Gleason score 8–9) PCa malignancy. Age, Gleason score, tumor-node-metastasis stage, and EG-VEGF/PK1 expression is summarized in Table 2Go. A paraffin block holding a representative area of the prostate gland or tumor was cut in serial sections of 4 µm in thickness. A colored section with hematoxylin-eosin was examined using a Leitz Laborlux K microscope (Leica Imaging System, Inc., Cambridge, UK) to confirm the original diagnosis. The 4-µm serial sections were cut for each case and mounted on poly-L-lysine-coated glass slides. Deparaffinized sections were boiled three times for 3 min in a 10–3 M sodium citrate buffer (pH 6.0) as antigen retrieval method. To prevent the nonspecific binding of the antibodies, sections were preincubated with nonimmune mouse serum (1:20; Dakopatts, Hamburg, Germany) diluted in PBS/BSA (1%) for 25 min at room temperature. After quenching of endogenous peroxidases with 0.3% hydrogen peroxide in methanol, followed by two rinses with Tris-HCl buffer, the sections were incubated with the anti-EG-VEGF primary antibody (EG-VEGF, no. MAB1209; R&D Systems Inc., Milan, Italy) diluted 1:150 overnight at 4 C. The standard streptavidin-biotin-peroxidase complex technique, using sequential 20-min incubation with biotinylated linking antibody and peroxidase-labeled streptavidin (Dako LSAB kit HRP; Dako, Carpinteria, CA) was performed. 3,3'-Diaminobenzidine (Vector Laboratories, Burlingame, CA) was used as a substrate chromogen solution for the development of the peroxidase activity. Hematoxylin was used for nuclear counterstaining, and then the sections were mounted and coverslipped with a synthetic mounting medium (Entellan; Merck, Darmstadt, Germany).


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  		TABLE 2. Clinical, histological data, and immunohistochemical score for EG-VEGF/PK1 in prostate cancer tissue samples

 
As positive control for both the antibodies, the immunoreactivity of normal ovary tissue was evaluated. Negative controls were performed using an antibody with irrelevant specificity but of the same isotype as the primary antibody and included in each staining run. The immunostaining was evaluated semiquantitatively according to an arbitrary scale and expressed in arbitrary units as follows: 0 (no positive cells), 1 (≤5% positive cells), 2 (5–10%), 3 (10–30%), or 4 (30%) (for each sample in 10 fields, at x400 magnification). At least three distinct preparations from each patient were checked, and in all the cases, sections were examined and immunostaining was graded without previous knowledge of the clinical data of the patients.

Protein extraction and Western blot analysis
Prostate tissue samples were homogenized directly into lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg aprotinin, 0.5 mM sodium orthovanadate, 20 mM sodium pyrophosphate). The lysates were clarified by centrifugation at 14,000 x g for 10 min. Protein concentrations were estimated by a Bio-Rad assay (Bio-Rad, Munich, Germany) and boiled in Laemmli buffer [0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.002% bromophenol blue] for 5 min before electrophoresis. Proteins were subjected to SDS-PAGE (15% polyacrylamide) under reducing conditions. After electrophoresis, proteins were transferred to nitrocellulose membranes (Immobilon Millipore Corp., Milan, Italy); complete transfer was assessed using prestained protein standards (Bio-Rad, Hercules, CA). After blocking with Tris-buffered saline/BSA [25 mM Tris (pH 7.4), 200 mM NaCL, 5% BSA], the membrane was incubated with the primary antibody against EG-VEGF (1:400, no. MAB1209; R&D Systems) and ERK1 (1:1000, no. sc-94-G; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 1 h (at room temperature). Membranes were then incubated with the horseradish-peroxidase-conjugated secondary antibody (1:4000) for 45 min (at room temperature), and the reaction was detected with an enhanced chemiluminescence system (Amersham Life Science, Milan, Italy).

Statistical analysis
The data were reported as mean ± SEM obtained from at least three separate experiments in which each point was performed in triplicate. The means were compared by ANOVA and paired t test. P < 0.05 was used to define statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presence of EGVEGF/PK1 transcript in NP and in PCa tissues and in androgen-dependent prostate cell lines EPN and LNCaP
Although a low level of expression of EG-VEGF/PK1 mRNA in human prostate tissue was shown by Northern blot (8), whether PCa express EG-VEGF/PK1 was unknown. We therefore examined the expression of EG-VEGF/PK1 in 10 NP and 10 PCa samples (Figs. 1Go and 2Go) and in a panel of prostate cell lines (EPN, LNCaP, PC3, and DU-145) by RT-PCR (Fig. 2Go). We found EG-VEGF/PK1 mRNA in all prostate tissue specimens with a weaker expression in normal tissue samples. Real-time PCR analysis of EG-VEGF/PK1 mRNA in NP and PCa showed a significant higher level of expression in PCa compared with NP (P < 0.01). EG-VEGF/PK1 transcript was found in the androgen-dependent nontransformed human prostate epithelial cell line EPN and LNCaP cancer cell line but not in the androgen-independent PC3 and DU-145 cell lines (Fig. 2Go).


Figure 1
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  		FIG. 1. EG-VEGF/PK1 mRNA level in NP and in PCa. A, EG-VEGF/PK1 transcript was detected in 10 NP and in 10 PCa tissues. Total RNA was reverse transcribed and amplified with GAPDH and EG-VEGF/PK1 primers. PCR products were electrophoresed on 1.2% agarose gel, stained with ethidium bromide, and photographed. B, Real-time PCR products of EG-VEGF/PK1 were normalized to a housekeeping gene product in each experiment (ß2-microglobulin), showing a significant higher level in PCa compared with NP. Data are the mean ± SD (n = 3); **, P < 0.01.

 

Figure 2
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  		FIG. 2. EG-VEGF/PK1 mRNA level in PCa and NP and in EPN, LNCaP, PC3, and DU-145 cell lines. EG-VEGF/PK1 transcript was detected in normal and PCa tissue and in nontransformed human prostate epithelial cell lines EPN and LNCaP and was absent in PC3 and DU-145 cell lines. Total RNA was reverse transcribed and amplified with GAPDH and EG-VEGF/PK1 primer. PCR products were electrophoresed on 1.2% agarose gel, stained with ethidium bromide, and photographed. Data are the mean ± SD (n = 3); *, P < 0.05.

 
EG-VEGF/PK1 protein expression was localized in the prostate glandular compartment and was up-regulated with PCa grade progression
To determine the cellular distribution and the level of EG-VEGF/PK1 expression, immunohistochemical studies were performed on sections of NP, benign prostatic hyperplasia, and low-medium-grade and high-grade PCa (Fig. 3Go, A–D). Interestingly, in NP, EG-VEGF/PK1 was absent (Fig. 3AGo). Immunoreactivity was present in the cytoplasm of glandular EC and increased progressively from benign prostatic hyperplasia with a weak signal, according to an arbitrary scale, between 0 and 1 (Fig. 3BGo), low-medium grade PCa (Fig. 3CGo) showing an intensity of staining between 2 and 3, and high-grade PCa (Fig. 3DGo) in which was detected the greater intensity of staining (from 3–4). The EG-VEGF/PK1 expression score was significantly higher in high-grade compared with low- and medium-grade PCa (P < 0.01 and P < 0.02, respectively). The progressive significant up-regulation of EG-VEGF/PK1 protein in PCa suggested that EG-VEGF/PK1 could be a marker of PCa progression and outcome, even if it is necessary to follow the patient’s outcome together with the levels of that protein expression to draw more reliable conclusions.


Figure 3
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  		FIG. 3. Expression of EG-VEGF/PK1 in normal, hyperplastic, and malignant prostate epithelia. Paraffin-embedded sections of human normal and hyperplastic prostate were stained with a polyclonal antibody, anti-EGF-VEGF/PK1. Specific cytoplasmic EGF-VEGF/PK1 expression is absent in normal prostate (A), barely detectable in hyperplastic benign prostate (B), present in low-medium-grade PCa (C), and highly expressed in the periglandular epithelium of high-grade PCa (D). Magnification, x250.

 
Western blot analysis of total protein extracted from archive prostate tissues comprising NP and cancer tissues scoring Gleason 4–9 confirmed the morphological results. A band of the appropriate molecular mass (9.5 kDa) was observed in all the samples tested, but EG-VEGF/PK1 expression dramatically increased with increasing Gleason score being high in Gleason 8/9 (Fig. 4AGo, K5 and K6) compared with the samples K1, K2, K3, and K4, classified as Gleason score 4, 5, 6, and 7, respectively. In addition, ERK1/2 Western blot was used to assess equal amounts of protein (Fig. 4BGo).


Figure 4
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  		FIG. 4. Western blot analysis of EG-VEGF/PK1 expression in prostate human tissues. The expression of EG-VEGF/PK-1 protein increases with increasing Gleason score. EG-VEGF/PK-1 was detected in samples K1, K2, K3, and K4, classified as Gleason’score 4, 5, 6, and 7, respectively, being maximal in Gleason score 7/8 (K5, K6) carcinomas. ERK1/2 (Santa Cruz Biotech. Santa Cruz, CA) was used to assess the equal amounts of protein. The blots are representative of three separate assays.

 
EG-VGF/PK1 and PK2 and their receptors mRNA levels in EC primary cultures from normal and malignant prostates and in EPN
Whether the normal and malignant epithelial prostate cells and tissues express PK2 and PK receptor was unknown. We therefore examined the expression pattern of these genes in NPEC and CPEC primary cultures and in the EPN cell line by semiquantitative RT-PCR. PK1 and PK2 transcripts were detected at low levels in NPEC and in EPN, whereas the level of expression was significantly increased (P < 0.05) in CPEC (Fig. 5Go, A and B). NPEC, EPN, and CPEC expressed PK-R1 and PK-R2 (Fig. 5AGo). Expression of PK-R1 did not differ significantly from normal and malignant prostate cells, whereas PK-R2 mRNA showed a significant increase in CPEC (Fig. 5Go, A and B). Moreover, PK2 and PK-R2 transcripts were more expressed (P < 0.05) than PK1 and PK-R1 in all cells studied. The real-time PCR results confirmed the semiquantitative analysis data. (Fig. 6Go)


Figure 5
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  		FIG. 5. A, Presence of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 mRNA levels in NPEC and CPEC and in EPN. Total RNA was reverse transcribed and amplified with GAPDH and EG-VEGF/PK1, PK2, PK-R1, and PK-R2 primers, respectively. PCR products were electrophoresed on 1.2% agarose gel, stained with ethidium bromide, and photographed. B, The mRNA levels of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 were determined by semiquantitative RT-PCR using GAPDH as internal control. PCR products were electrophoresed on 1.2% agarose gel, stained with ethidium bromide, and photographed. Data are the mean ± SEM (n = 3) of the densitometric analysis of PK1, PK2, PK-R1, and PK-R2 relative to GAPDH expression. *, Significant difference between PK1, PK2, PK-R1, and PK-R2; *, P < 0.05.

 

Figure 6
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  		FIG. 6. Evaluation of the expression level of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 in NPEC and CPEC and in EPN. Real-time PCR products of EG-VEGF/PK1, PK2, PK-R1, and PK-R2 were normalized to a housekeeping gene product in each experiment (ß2-microglobulin). Data are the mean ± SD (n = 3); *, P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that EG-VEGF/PK1 expression is barely detectable in the normal human prostate and is up-regulated in PCa. Both RNA and protein (Western blotting and immunohistochemistry) analyses show that prostate EC express EG-VEGF/PK1, and its expression level increases with Gleason score advancement. RT-PCR data show that PK2 also has a similar pattern of expression. Moreover, the presence of EG-VEGF receptor (PK-R1 and PK-R2) transcripts in epithelial cell primary cultures of normal (NPEC) and malignant (CPEC) prostates and in EPN suggest that PKs may directly act on epithelial cells. The significance of increased PK-R2 expression in cancer cells is as yet unknown. The molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. Altered blood vessel formation is related to aberrant pathways regulating apoptosis, androgen receptor signaling, signal transduction, and cytokine and cell adhesion molecules (19, 20, 21, 22, 23). Recent studies suggested that the VEGF pathway is one of the key regulators of this process. This factor, widely expressed in normal and malignant tissues, is the main one responsible for the development and maintenance of the aberrant tumor vascular network. These observations have led to current preclinical and clinical studies in oncology on the inhibition of the VEGF pathway. Although anti-VEGF therapy may have antiangiogenic effects, and seems to be promising, it has other systemic effects as well, leading to the induction of endothelial cell apoptosis and altering the functionality of the vascular bed. The cloning of the EG-VEGF gene by LeCouter et al. (8), a new angiogenic factor expressed mainly in endocrine cells that selectively acts on the endothelium of endocrine gland cells, has opened a new perspective for the pathophysiology and management of endocrine-related cancers. In fact, the identification of tissue-selective angiogenic factors may pave the way for tissue-specific angiogenic therapeutics with reduced systemic effects. EG-VEGF has low homology with VEGF, and differs in various respects, especially for the selectivity of area of expression. In fact EG-VEGF/PK1 is found only in hormone-producing cells such as adrenal gland, ovaries, testis, and placenta (8). Very little is known about the expression of PKs in malignant tissues. A recent study demonstrated EG-VEGF/PK1 expression in colorectal cancer cells that express estrogen receptor ß and are affected by endocrine hormones (13). EG-VEGF/PK1 overexpression induces angiogenesis and promotes cell proliferation and liver metastasis in colorectal cancer cells. Antisense EG-VEGF/PK1 injected into mice induces inhibition of angiogenesis and tumor growth in mice (13). There are no data on PKs and PCa, and very little is known about PK-R expression in human normal and malignant tissues (14, 15). Because a better understanding of EG-VEGF/PK1 and PK2 and their receptors (PK-R1 and PK-R2) in PCa development and progression is important, these data could represent the first step toward a future clinical application. Three spontaneously established cell lines, PC3, DU-145, and LNCaP, are by far the most widely used cell culture model to study PCa (24). Our data showed a barely detectable EG-VEGF/PK1 mRNA in the the androgen-dependent LNCaP and no expression in the androgen-independent PC3 and DU-145. The absence of EG-VEGF/PK1 in the androgen-independent PC3 and DU-145 cell lines, although we found an increased expression in the higher-Gleason-score PCa tissues, could be explained with the current knowledge that the most commonly used cell culture models of PCa do not span the range of PCa phenotypes and are not necessarily representative of the biological behavior of primary adenocarcinomas of the prostate (25). Furthermore, the question of how extensively long-term cultures alter the biological properties of the cell lines is always under debate. Because it is known that the most informative studies are performed with donor-matched pairs of normal and malignant primary cultures, grown under identical conditions (25), we decided to study the PK and PK-R expression using as an in vitro model primary cultures of normal (NPEC) and malignant (CPEC) epithelial prostate cells and EPN, a nontransformed human prostate EC line (17). Moreover, we searched for EG-VEGF/PK1 protein expression in human normal, benign hyperplasia, and low-medium- and high-grade malignant prostate tissues.

Our data suggest the association between PK expression and increased angiogenesis in tumor cells, leading to an expansion in the tumor volume as a result of vascular network formation by new blood vessels. In fact, tumor growth is angiogenesis dependent. Moreover, the higher level of PK expression in cancer cells compared with normal cells could suggest that PKs are cancer specific, and possibly tissue specific. Interestingly, EG-VEGF/PK1 protein was absent in normal prostate tissues, whereas a low level of EG-VEGF/PK1 transcript expression was found in both NPEC and EPN. These data could indicate a functional block of the transduction signal pathway of EG-VEGF/PK1 or its inactivation in normal tissues. The progressive activation of EG-VEG/PK1 protein expression from benign to malignant prostate gland seems to be a signal for the initiation of prostate carcinoma. This is probably because of its effect on vascular permeability, fenestration of endothelial cells, and the creation of a vascular network in the tumor tissues with consequent increase in the tumor volume. We also found a significant and progressive increase of EG-VEGF/PK1 protein level with PCa progression from low-medium grade to high grade, indicating that PKs are important and specific prognostic biomarkers for PCa progression.

In conclusion, our data showed that PK and PK-R expression could be useful for PCa outcome. Moreover, PKs may be a target for PCa treatment in the future.


   Acknowledgments
 
We are grateful to Dr. Joseph Sepe for editing this manuscript.


   Footnotes
 
This work was supported in part by grants from Prin 2003, Associazione Italiana per la Ricerca sul Cancro (AIRC) Regionale 2005 to A.A.S.

Author disclosure summary: All of the authors of this paper have nothing to declare.

First Published Online June 8, 2006

Abbreviations: CPEC, Malignant prostate epithelial cells; EC, epithelial cells; EG, endocrine gland; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NP, normal prostate; NPEC, normal prostate epithelial cells; PCa, prostate cancer; PK, prokineticin; PK-R, PK receptor; VEGF, vascular endothelial growth factor.

Received May 8, 2006.

Accepted for publication May 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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