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Endocrinology Vol. 141, No. 6 2120-2128
Copyright © 2000 by The Endocrine Society

ARTICLES

Antagonists of Growth Hormone-Releasing Hormone and Vasoactive Intestinal Peptide Inhibit Tumor Proliferation by Different Mechanisms: Evidence from in Vitro Studies on Human Prostatic and Pancreatic Cancers1

Zoltan Rekasi2, Jozsef L. Varga, Andrew V. Schally, Gabor Halmos, Patricia Armatis, Kate Groot and Tamas Czompoly2

Endocrine, Polypeptide and Cancer Institute, Veterans Affairs Medical Center (Z.R., J.L.V., A.V.S., G.H., P.A., K.G., T.C.), and Department of Medicine, Tulane University School of Medicine (Z.R., J.L.V., A.V.S., G.H., T.C.), New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: Dr. Andrew V. Schally (151), Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, Louisiana 70112-1262.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antagonists of GH-releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) inhibit the proliferation of various tumors in vitro and in vivo, but a comparison of their antitumor effects and mechanisms of action has not been reported to date. We recently synthesized and characterized a series of analogs, some of which are primarily GHRH antagonists (JV-1–36, JV-1–38, and JV-1–42), whereas others are more selective for VIP receptors (VPAC-R; JV-1–50, JV-1–51, JV-1–52, and JV-1–53). LNCaP human prostatic cancer cells express VPAC-R, with predominant subtype 1 determined by RT-PCR. Our studies show that GHRH antagonists significantly inhibit the proliferation of both VPAC-R positive LNCaP cells (P < 0.001) and VPAC-R negative MiaPaCa-2 human pancreatic cancer cells cultured in vitro (P < 0.05 to P < 0.001). Growth inhibition of LNCaP cells is accompanied by a proportional reduction in prostate-specific antigen (PSA) secretion (P < 0.001). In a superfusion system, the inhibitory activities of the analogs on the rate of VIP and GHRH-induced PSA secretion correlate well with their VPAC-R binding affinities to LNCaP cell membranes. Antagonists more selective for VPAC-R display a stronger inhibition of inducible PSA release than GHRH antagonists, but have smaller effects or no effects on proliferation and PSA secretion in culture. Collectively, our findings demonstrate that the antiproliferative activity of the analogs on cancer cells is not correlated to their VPAC-R antagonistic potencies. Because GHRH antagonists inhibit the proliferation of LNCaP cells more powerfully than VPAC-R antagonists and also suppress the growth of VPAC-R-negative MiaPaCa-2 cells, it can be concluded that their antiproliferative effect is exerted through a mechanism independent of VPAC-R.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANTAGONISTIC ANALOGS of GH-releasing hormone (GHRH) could be useful for the treatment of endocrine disorders such as acromegaly, diabetic retinopathy, or diabetic nephropathy, but their main applications are likely to be in cancer therapy (1, 2, 3, 4, 5). GHRH antagonists synthesized in this laboratory (6, 7, 8) inhibit the growth of human osteosarcomas, small cell and nonsmall cell lung carcinomas, prostate cancers, renal cell carcinomas, malignant gliomas, and pancreatic, colorectal and breast cancers in vitro and in vivo in nude mice (5, 9, 10, 11, 12, 13, 14, 15, 16, 17). In vivo, these analogs may inhibit tumor progression, acting indirectly through the suppression of the pituitary GH/hepatic insulin-like growth factor I axis (5, 9, 10, 11, 12, 15) or by direct actions on cancer cells (5, 13, 14, 15, 16, 17). The antiproliferative activity of GHRH antagonists in vitro in cell cultures of various cancers under conditions that clearly exclude endocrine effects through the pituitary GH/insulin-like growth factor I axis demonstrate that these compounds act directly on cancer cells (9, 10, 11, 12, 13, 14, 16, 17, 18, 19). The receptors that mediate these direct antiproliferative effects of GHRH antagonists are still not identified, as the classic pituitary type GHRH receptors (GHRH-R) are not present on tumor cells (5, 14).

Receptors for VIP (VPAC-R), with two subtypes, VPAC1-R and VPAC2-R, are abundant in many types of cancers (20, 21, 22, 23, 24). It was also reported that VIP (22, 25) and its antagonists (26, 27) can inhibit the growth of various human carcinomas. Because of the structural similarity between VIP and GHRH as well as considerable homology between their receptor proteins, GHRH analogs may interact with VPAC-R (28, 29). This might contribute to their antiproliferative effect on human tumors. This prompted us to study the possible interactions of GHRH antagonists with VPAC-R on cancer cells.

We recently synthesized three new GHRH analogs, JV-1–50, JV-1–51, and JV-1–52, which are derivatives of the potent GHRH antagonists JV-1–36, JV-1–38, and JV-1–42 (8), but contain D-Phe2 instead of D-Arg2 (30). Analogs JV-1–50 and JV-1–51 are structurally related to JV-1–42 and JV-1–36, whereas JV-1–52 is derived from JV-1–38. Analogs JV-1–50, JV-1–51, and JV-1–52 have reduced antagonistic activities on GHRH-R and increased antagonistic properties on VPAC1-R and VPAC2-R compared with their parent compounds. We also synthesized a potent VPAC1-R and VPAC2-R antagonist, JV-1–53, that is devoid of detectable antagonistic activity on GHRH-R (30). The inhibitory activities of these peptides on GHRH-R, VPAC1-R, and VPAC2-R were evaluated on rat pituitary and pineal cells (30) and compared with that of a selective VPAC1-R antagonist, PG 97–269 (31).

According to earlier reports, human prostate has relatively rich VIP-like immunopositive innervation (32, 33), and VPAC-R have been identified in both normal and malignant human prostate cells and tissue (20, 34). VIP stimulates prostatic secretion and increases the proliferation of prostatic epithelial cells in culture (35, 36). VIP is also known to potentiate the invasive capacity of the androgen-responsive human carcinoma cell line LNCaP (37). In addition, VIP increases the secretion of human prostate-specific antigen (PSA) and the production of cAMP in LNCaP cancer cells (38). PSA is produced primarily by prostatic epithelial cells (39) and is established as a marker for the early detection of prostate cancer (40).

In this study we compared the inhibitory effects of GHRH antagonists and VPAC-R antagonists on the proliferation and PSA secretion of VPAC-R-positive LNCaP human prostatic cancer cells in static culture. In addition, the inhibitory effects of the analogs on the growth of the MiaPaCa-2 human pancreatic cancer cell line, which served as a VPAC-R- negative (41) control, were evaluated. The expression of VPAC1-R, VPAC2-R, and GHRH-R on LNCaP and MiaPaCa-2 cancer cells was studied by RT-PCR and radioligand competition assay. We also measured, in a superfusion system, the inhibitory activity of the antagonists on the rate of inducible PSA release from LNCaP cells and evaluated their VPAC-R binding affinity. The overall aim of this project was comparison of the specificity and antitumor activity of GHRH antagonists and VIP antagonists.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptides
The synthesis of human GHRH-(1–29)NH2, and GHRH analogs JV-1–36, JV-1–38, JV-1–42, JV-1–50, JV-1–51, JV-1–52, and JV-1–53 was previously described (8, 30). VIP was obtained from California Peptide Research (Napa, CA). A selective VPAC1-R antagonist (PG 97–269) was provided by Drs. P. Gourlet and P. Robberecht (Université Libre de Bruxelles, Brussels, Belgium) (31).

Tissue cultures
Human prostatic (LNCaP) and pancreatic (MiaPaCa-2) cancer cell lines were obtained from American Type Culture Collection (Manassas, VA). The cell culture media and reagents were purchased from Life Technologies, Inc. (Grand Island, NY). The LNCaP cell line was grown in RPMI 1640 medium supplemented with 10% FBS and a mixture of antibiotics and antimycotics (100 U/ml penicillin G, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin). The MiaPaCa-2 cell line was routinely maintained in DMEM containing 1 mM pyruvate, 10% FBS, 2.5% horse serum, and a mixture of antibiotics and antimycotics as described above. The cultures were maintained in a humidified atmosphere containing 95% air-5% CO2 at 37 C. The cells were passaged weekly and were routinely monitored for the presence of mycoplasma using a test kit from Roche Molecular Biochemicals (Indianapolis, IN).

Cell proliferation assay
For all experiments, cells were grown to 80–90% confluence, harvested by use of trypsin-EDTA solution, and seeded at low concentration in 96-well plates. After 24 h, the culture medium was removed and replaced with test medium (for LNCaP: RPMI 1640 and 2% heat-inactivated dextran-coated charcoal-treated FBS; for MiaPaCa-2: DMEM, 1 mM pyruvate, 1.6% FBS, and 0.4% horse serum) containing the test compounds (in octuplicate wells each) at 3- and 10-µM concentrations or the vehicle (0.1% dimethylsulfoxide) as a control for 72 h. Cell growth was determined by a microculture tetrazolium assay based on a method described by Plumb et al. (42). This assay measures the reduction of substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) to a dark blue formazan product by mitochondrial dehydrogenases in living cells. Briefly, after 72-h treatment with various compounds, 40 µl 2 mg/ml MTT (Sigma, St. Louis, MO) were added to each well, and the cells were incubated for 4 h at 37 C in darkness. The medium was replaced by 200 µl DMSO and then with 25 µl Sorensen’s glycine buffer (0.1 M glycine plus 0.1 M NaCl, pH 10.5). After shaking to dissolve the formazan, absorbance was read at 540 nm in the plate reader (Beckman Coulter, Inc., Palo Alto, CA). Results were calculated as a percentage of the control, where the OD540nm of treated cultures (medium plus compounds) was related to the OD540nm of the control culture (medium plus vehicle) x 100. Aliquots of medium were kept frozen for PSA immunoradiometric assay (IRMA) at -20 C.

Superfusion
Superfusion analysis of cultured LNCaP cells was performed in a system similar to that described previously for studying dispersed anterior pituitary cells (43, 44). Tissue culture medium and reagents for the superfusion technique were obtained from Sigma. In brief, cultured tumor cells were harvested and resuspended in 1.5 ml medium, and the cells were transferred onto superfusion columns (4.5–5 million cells each) and allowed to sediment simultaneously with 0.8 ml Sephadex G. The dead volume of the system was set at 1 ml. Medium 199 containing BSA (1 g/liter), NaHCO3 (2.2 g/liter), penicillin G (50 mg/liter), and gentamicin sulfate (87 mg/liter) was equilibrated with a mixture of 95% air-5% CO2 and used as the culture medium. The medium was pumped at a flow rate of 1 ml/3 min. After an overnight recovery period, in which the baseline stabilized and the cells regained their full responsiveness, 2-ml fractions of the effluent medium were collected every 6 min. The cells were exposed periodically to VIP (1 nM) or GHRH (10 nM), which was dissolved in fresh medium immediately before application, for 12 min at 60-min intervals. The antagonists were infused at 100 or 300 nM for 12 min (two fractions), immediately followed by the mixture of an antagonist and 1 nM VIP or 10 nM GHRH for an additional 12 min. Fractions of effluent medium were kept frozen for PSA IRMA at -20 C. Each experiment was performed in three superfusion columns simultaneously.

Receptor binding
Preparation of membrane fractions of human prostatic (LNCaP) and pancreatic (MiaPaCa-2) cancer cells and receptor binding of VIP and GHRH were performed as previously reported (45, 46). Radioiodinated VIP was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL), and 125I-labeled [His1,Nle27]hGHRH-(1–32)NH2 was prepared as previously described (46). The protein concentration was determined using a Bio-Rad Laboratories, Inc., protein assay kit. The LIGAND PC computerized curve-fitting program developed by Munson and Rodbard (47) was used to determine the type of receptor binding, the dissociation constant (Kd), and the maximal binding capacity of receptors. The receptor binding affinity of GHRH-related peptides to tumor membranes of LNCaP cells was measured in displacement experiments based on competitive inhibition of radiolabeled VIP binding using various concentrations (10-12-10-6 M) of GHRH analogs. IC50, defined as the dose causing 50% inhibition of specific binding of [125I]VIP, was calculated by a computerized curve-fitting program (48). Relative affinities of GHRH analogs for VPAC-R compared with VIP were calculated as the ratio of the IC50 of the tested compounds to the IC50 of VIP (1.07 nM).

RT-PCR analysis
Total RNA of cultured LNCaP and MiaPaCa-2 cancer cells was extracted according to the Tri-Reagent protocol (Sigma). The concentration of RNA was determined by spectrophotometric analysis at A260/280nm. One microgram of total RNA was reverse transcribed and then amplified using the reagents and protocol of the GeneAmp RNA PCR Core kit (Perkin-Elmer Corp., Norwalk, CT). The RT reaction was performed in a final volume of 20 µl containing 2.5 µM random hexamers, 1 mM each of deoxynucleoside triphosphate, 1 x PCR buffer, 5 mM MgCl2, 1 U/µl ribonuclease inhibitor, and 2.5 U/µl Moloney leukemia virus reverse transcriptase. One fourth (5 µl) of the RT reaction was used for each PCR amplification with a primer set that would amplify complementary DNA (cDNAs) for human VPAC1-R and VPAC2-R, GHRH-R, or ß-actin (Table 1Go). The PCR reaction included 1 x PCR buffer, 2 mM MgCl2, 0.15 µM (for VPAC1-R and ß-actin), or 1.0 µM (for VPAC2-R and GHRH-R) of each primer and 2.5 U/100 µl AmpliTaq DNA polymerase in a 25-µl volume. The PCR amplification was performed in a GeneAmp PCR System 2400 (Perkin-Elmer Corp.) with the following cycle profile: 95 C for 180 sec, followed by 45 (receptors) or 25 (ß-actin) cycles of 95 C for 30 sec, 60 C for 30 sec, and 72 C for 45 sec. After the last cycle, there was a final extension for 7 min at 72 C. PCR products were electrophoresed on a 1.5% agarose gel stained with 0.5 µg/ml ethidium bromide and visualized under UV light, followed by scanning and quantification of gel (GDS 7500 Gel Documentation System, UVP, Upland, CA; and GS-700 Imaging Densitometer, Bio-Rad Laboratories, Inc., Hercules, CA). The quality of RNA extracted was tested by PCR amplification of human ß-actin cDNA (49) from the same RT reaction as that used for various receptor cDNA amplifications.


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  		Table 1. Oligonucleotide primers used in RT-PCR analysis for GHRH-R, VPAC1-R, VPAC2-R, and ß-actin expression in LNCaP human prostatic cancer cells and MiaPaCa-2 human pancreatic cancer cells

 
PSA quantitation
Quantitative measurement of total PSA of collected medium was performed using the reagents and protocol provided with the DSL-9700 Active PSA Coated-Tube IRMA kit (Diagnostics Systems Laboratories, Inc., Webster, TX).

Statistical analysis
IRMA results from superfusion system were further analyzed with computer software developed in our institute (43). In brief, the program first separates the fractions containing a basal level of the secreted PSA from the response fraction by using several iterations. The statistical parameters of the baseline are determined from the values of the basal fractions. A fraction is considered as a response if it is larger or smaller than the 95% confidence limits of the baseline. Further statistical analysis is based on the net integral values (area of the response curve above the baseline) of the responses (NET INT). Results of IRMA and MTT test from static cell culture were subjected to one-way ANOVA, and differences between groups were determined by Tukey’s test. P < 0.05 was considered significant. Data are expressed as a percentage of the control values (set at 100%) and represent the mean ± SEM PSA content or the formazan level of octuplicates of wells with the same treatment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of GHRH, VIP, and their antagonists on proliferation and PSA secretion of LNCaP prostatic cancer cells in static culture
Cell proliferation, as measured by the MTT test, was slightly stimulated by GHRH, but was not influenced by VIP (Fig. 1AGo). Both peptides caused a significant increase in the rate of PSA secretion (total PSA secretion per cell number) in cell culture [3 µM GHRH, 1.9-fold; 10 µM GHRH, 2.2-fold (P < 0.001); 3 µM VIP, 1.7-fold (P < 0.05); 10 µM VIP, 2.0-fold (P < 0.001); Fig. 1Go, B and C]. GHRH antagonists JV-1–38, JV-1–36, and JV-1–42 at 10-µM concentrations significantly inhibited cell proliferation by 73%, 54%, and 44%, respectively (P < 0.001); JV-1–38 was the most potent inhibitor of the peptides tested (Fig. 1AGo). Their structurally related analogs JV-1–52, JV-1–51, and JV-1–50, with decreased GHRH-R antagonistic potency and increased VPAC-R antagonistic activity, produced a weaker inhibition of cell proliferation than the parent peptides (Fig. 1AGo). JV-1–52 was the most potent inhibitor among these nonselective antagonistic analogs of VPAC-R/GHRH-R (Fig. 1AGo). However, its antiproliferative effect was weaker than that of its parent compound, JV-1–38 (53% vs. 73%). JV-1–53, the most potent antagonist of VPAC1-R and VPAC2-R and devoid of measurable antagonistic activity on GHRH-R, caused only a marginal, statistically insignificant inhibition (P > 0.05). The selective VPAC1-R antagonist PG 97–269 also had a weak antiproliferative effect. The specific GHRH antagonists JV-1–38, JV-1–36, and JV-1–42 caused the strongest inhibition of total PSA levels (69%, 45%, and 46%, respectively; P < 0.001; Fig. 1BGo). The nonspecific VPAC-R/GHRH-R antagonist JV-1–52 also markedly reduced total PSA secretion. These changes in total PSA secretion from LNCaP cells after exposure to various antagonists correlated well with the alteration in cell number, indicating that the rate of PSA secretion was not reduced (Fig. 1CGo).



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  		Figure 1. Effects of GHRH, VIP, and their antagonists on cell proliferation (A), total PSA secretion (B), and rate of cellular PSA secretion (ratio of total PSA level and cell number) (C) from LNCaP human prostatic cancer cells in static culture. The cells were treated for 72 h with various compounds at 3 or 10 µM. Medium was assayed for PSA secretion, and the growth of human prostatic cancer cells was determined by MTT test. Data represent the mean ± SEM cell number and total PSA secretion of eight wells, expressed as a percentage of untreated control cell numbers (A) and total PSA secretion (B) or as the rate of cellular PSA secretion (ratio of total PSA production and cell number) expressed as a percentage of the rate of untreated control PSA secretion (C). *, P < 0.05; **, P < 0.001 (vs. control).

 
Effects of GHRH, VIP, and their antagonists on proliferation of MiaPaCa-2 pancreatic cancer cells in static culture
In agreement with the results in LNCaP cells, the three potent GHRH antagonists JV-1–38, JV-1–36, and JV-1–42 at 10-µM concentrations significantly reduced the growth of MiaPaCa-2 pancreatic cancer cells by 63%, 42%, and 40%, respectively (P < 0.05 to P < 0.001; Fig. 2Go). Among their derivatives with decreased GHRH-R antagonistic potency and increased VPAC-R antagonistic activity, only JV-1–52 inhibited the proliferation of this VPAC-R-negative cell line (55% inhibition; P < 0.001). VPAC1-R and VPAC2-R antagonist JV-1–53, devoid of GHRH-R antagonistic activity, caused only a marginal, statistically insignificant inhibition (P > 0.05), and the selective VPAC1-R antagonist PG 97–269 proved ineffective (Fig. 2Go). VIP and GHRH also did not influence the proliferation of MiaPaCa-2 pancreatic cancer cells.



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  		Figure 2. Effects of GHRH, VIP, and their antagonists on cell proliferation of MiaPaCa-2 human pancreatic cancer cells in static culture. The cells were treated for 72 h with various compounds at 3 or 10 µM. Growth of human pancreatic cancer cells was determined by MTT test. Data represent the mean ± SEM cell number of eight wells, expressed as a percentage of the untreated control cell number. *, P < 0.05; **, P < 0.001 (vs. control).

 
Binding characteristics and messenger RNA (mRNA) expression of VPAC1-R, VPAC2-R, and GHRH-R in LNCaP and MiaPaCa-2 cancer cells
RT-PCR analyses were performed to detect the expression of mRNA for VPAC1-R, VPAC2-R, and GHRH-R in human prostate (LNCaP) and pancreatic (MiaPaCa-2) cancer cells (Fig. 3Go). Using primers for human GHRH-R mRNA, we could not find the expression of GHRH-R (387 bp) in either LNCaP or MiaPaCa-2 cells. In contrast, the amplification with specific primers for human VPAC1-R produced a large amount of a single product of 324 bp in LNCaP cells. Using specific primers for human VPAC2-R, only a low expression of VPAC2-R mRNA (586 bp) could be detected in LNCaP cells. MiaPaCa-2 pancreatic cancer cells proved to be negative for both VPAC1-R and VPAC2-R mRNA.



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  		Figure 3. RT-PCR analysis of VPAC1-R, VPAC2-R, and GHRH-R mRNA in human prostatic (LNCaP) and pancreatic (MiaPaCa-2) cancer cells. Total RNA was reverse transcribed and PCR amplified with primers for VPAC1-R, VPAC2-R, GHRH-R, and ß-actin. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were of the expected sizes of 324 bp (VPAC1-R), 586 bp (VPAC2-R), 387 bp (GHRH-R), and 459 bp (ß-actin). Lane M, 100-bp DNA mol wt marker; lane 1, LNCaP; lane 2, MiaPaCa-2; lane +, positive control from human pituitary adenoma.

 
A single class of specific, high affinity, and low capacity binding sites for VIP was found on LNCaP cells, with a mean Kd of 4.97 ± 0.41 nM and a mean binding capacity of 234.3 ± 18.2 fmol/mg membrane protein (Fig. 4Go). We could not identify VPAC-R on membranes of MiaPaCa-2 pancreatic cancer cells. Similarly, using [125I]His1,Nle27-hGHRH-(1–32)NH2 as radioligand, no binding sites for GHRH were detected on either the LNCaP prostatic or MiaPaCa-2 pancreatic cancer cells examined.



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  		Figure 4. Representative example of Scatchard plots of [125I]VIP binding to the membrane fraction isolated from LNCaP human prostate cancer cells. Specific binding was determined as described. Each point represents the mean of triplicate determinations.

 
Receptor binding affinities of antagonists
To assess the potency of our GHRH-related antagonists to displace radiolabeled VIP from receptors on LNCaP cancer cell membranes, heterologous displacement experiments were performed (Fig. 5Go). The binding of radioligand was completely displaced by increasing concentrations (10-12–10-6 M) of unlabeled VIP (IC50 = 1.07 nM; Table 2Go). The selective VPAC-R antagonist JV-1–53 had the highest binding affinity to VPAC-R on prostate cancer cells, even slightly higher than that of VIP itself (Fig. 5Go). Other peptides (JV-1–50, JV-1–51, and JV-1–52), also more selective for VPAC-R, displayed lower receptor binding affinity than VIP, but this affinity was still 1 order of magnitude higher than that of GHRH antagonists JV-1–36, JV-1–38, and JV-1–42 (Fig. 5Go). The selective VPAC1-R antagonist (PG 97–269) also showed a very strong binding affinity to VPAC-R (Table 2Go). GHRH antagonists containing D-Arg in position 2 (JV-1–36, JV-1–38, and JV-1–42) displayed the lowest binding affinity to VPAC-R on LNCaP cell membranes (Fig. 5Go). In our study hGHRH-(1–29)NH2 had an almost negligible binding affinity for VPAC-R.



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  		Figure 5. Ability of unlabeled peptides to compete with [125I]VIP for binding to membrane fraction isolated from LNCaP human prostate cancer cells. One hundred percent specific binding is defined as the difference between binding in the absence and presence of 10-5 M VIP. Each point represents the mean of duplicate or triplicate determinations. The IC50 values for VIP ({triangledown}), human GHRH-(1–29)NH2 ({triangleup}), JV-1–36 ({blacktriangledown}), JV-1–38 (•), JV-1–42 ({circ}), JV-1–50 ({diamondsuit}), JV-1–51 ({blacktriangleup}), JV-1–52 ({diamond}), JV-1–53 ({square}), and PG 97–269 ({blacksquare}) were calculated using a computerized curve-fitting program (48 ).

 

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  		Table 2. Relative affinities of GHRH, VIP, and their antagonists to VPAC-R on LNCaP cells, the relative intrinsic activity, and inhibitory effect of antagonists (300 nM) on VIP- or GHRH-induced PSA release in the superfusion system

 
Effects of antagonists on PSA release from LNCaP prostatic cancer cells induced by VIP or GHRH in the superfusion system
The antagonistic activities of these analogs on VPAC-R were tested in human LNCaP prostate cancer cells in a superfusion system. A pulsatile stimulation of LNCaP cells with 1 nM VIP for 12 min at 60-min intervals caused a sharp increase in PSA secretion (Fig. 6Go). PSA release reached the maximum value in 3–9 min and then rapidly returned to basal values (337.2 ± 17.8 pg/ml) The areas of the peaks of PSA responses caused by VIP stimulation gradually declined during the experiment (after first VIP, 2356.5 ± 49.5 pg; after third VIP, 1985.1 ± 54.6 pg). GHRH infused at 10 nM for 12 min at 60-min intervals was also able to evoke PSA release from LNCaP prostatic cancer cells, with similar kinetics, but its effects were approximately 25-fold smaller. In the inhibitory tests, the cells were preincubated with 100 or 300 nM antagonistic peptides for 12 min and then immediately exposed to a mixture of the analogs and 1 nM VIP or 10 nM GHRH for an additional 12 min (Fig. 6Go, A and B). The results obtained from superfusion system indicate that the order of inhibitory potencies of these analogs on PSA release was similar to their VPAC-R binding affinities: JV-1–53{cong}PG 97–269>JV-1–51>JV-1–52{cong}JV-1–50>>JV-1–36{cong}JV-1–38{cong}JV-1–42 (Table 2Go). The analogs with D-Phe2 substitution, which had been designed to possess enhanced VIP antagonistic characteristics (JV-1–50, JV-1–51, JV-1–52, and JV-1–53), were able to block more powerfully the VIP- or GHRH-induced PSA release than D-Arg2-containing peptides, JV-1–36, JV-1–38, and JV-1–42. JV-1–53 (Fig. 6BGo), JV-1–51, and the selective VPAC1-R antagonist (PG 97–269) at 300 nM concentrations completely blocked VIP- or GHRH-stimulated PSA response, whereas JV-1–50 and JV-1–52 inhibited VIP- or GHRH-evoked PSA release to a lesser extent. In contrast, GHRH antagonists JV-1–36, JV-1–42, and JV-1–38 (Fig. 6AGo) proved to be practically ineffective at concentration tested. None of the GHRH-R and VPAC-R antagonists tested was able to influence basal PSA secretion.



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  		Figure 6. Effects of JV-1–38 (300 nM; A) and JV-1–53 (300 nM; B) on basal and VIP-induced PSA secretion from LNCaP human prostatic cancer cells in the superfusion system. After two 12-min infusions of 1 nM VIP (filled, black bars), the cells were exposed to JV-1–38 or JV-1–53 for 12 min (checkered bars), followed by the simultaneous infusion of these analogs and 1 nM VIP for an additional 12 min (filled, black bars). The second VIP-induced PSA response before antagonist exposure was used as a reference.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we compared in vitro the antiproliferative activities of the selective and high affinity VPAC-R antagonists JV-1–53 and PG 97–269 to those of the most potent GHRH antagonists available today. Although GHRH antagonists are efficacious antitumor agents inhibiting the growth of a wide variety of cancers (9, 10, 11, 12, 13, 14, 15, 16, 17), their antiproliferative mechanism of action is still not fully elucidated (5). In previous studies we could not detect classic pituitary-type GHRH receptors on various cancer cells, but VPAC-R were present in many tumor samples and cancer cell lines (5, 14, 17). Nevertheless, in our present in vitro experiments, GHRH antagonists JV-1–38, JV-1–36, and JV-1–42 strongly inhibited the proliferation as well as the total PSA secretion of cultured LNCaP cells. In contrast, VPAC-R antagonists JV-1–53 and PG 97–269, devoid of antagonistic activity on GHRH-R, did not significantly inhibit the growth of LNCaP cells under the concentrations tested. Similarly, peptides with the highest antagonistic activity on GHRH-R proved to be the most effective in inhibiting cell proliferation of the VPAC-R-negative MiaPaCa-2 human pancreatic cell line, whereas the antagonists more selective for VPAC-R were less effective or were inactive.

Consequently, in this study we also characterized VPAC1-R, VPAC2-R, and GHRH-R on LNCaP human prostatic cancer cells, which could be involved in the antiproliferative effect of the antagonists. Our findings, based on RT-PCR, radioligand competition assay, and superfusion system, clearly indicate that LNCaP cells have functional VPAC-R and are devoid of classic pituitary-type GHRH-R. The expression of mRNA for both types of VPAC-R was detected in LNCaP prostatic cancer cells, with VPAC1-R predominating in accordance with recent observations of rat prostate membranes (50). We also demonstrated in a superfusion system that a pulsatile stimulation of LNCaP cells with GHRH leads to a transient increase in the rate of PSA secretion, however, to a 25-fold lesser extent than with VIP. Because the release of this marker is not related to cell proliferation in this dynamic system, the superfusion method allowed characterization of the receptor-mediated, short-term endocrine effects of GHRH analogs. Antagonistic analogs of GHRH, JV-1–36, JV-1–38, and JV-1–42, inhibited VIP- or GHRH-evoked PSA release much less effectively than the selective and potent VPAC-R antagonists JV-1–53 and PG 97–269. These GHRH antagonists were also able to bind to VPAC-R on LNCaP cells, however, with a lesser affinity than VPAC-R antagonists. These findings suggest that GHRH and its antagonists may influence inducible PSA release through tumoral VPAC-R by acting as pharmacological analogs of VIP with lower affinity. A similar rank order of potency for these GHRH analogs was recently observed by us in tests on pinealocytes (blockade of VIP-evoked cAMP efflux) (30), where VPAC1-R were found to be predominant (51). These results indicate that the same receptor subtype may play a role in the induction of PSA secretion from LNCaP cells by both VIP and GHRH. A joint use of superfusion method and ligand binding assay proved to be suitable for screening GHRH analogs for their VPAC-R inhibitory activity on LNCaP cells.

In contrast, the antiproliferative effects of analogs were not correlated with their VPAC-R antagonistic potencies. Instead, a clear association was observed between tumor inhibition and antagonistic activity of analogs on GHRH-R, suggesting that VPAC-R are not the main receptors that mediate the antiproliferative effect of GHRH antagonists.

Although the potent GHRH antagonists JV-1–36, JV-1–38, and JV-1–42 have more than 1000-fold higher affinity for GHRH-R than for VPAC1-R and VPAC2-R on rat pituitary and pineal cells (8, 30), their antagonistic activity on VPAC-R is not negligible. These GHRH antagonists displaced radiolabeled VIP from VPAC-R on LNCaP cell membranes with IC50 values of 27–82 nM. This affinity is higher than that of a previously reported VPAC-R antagonist, VIPhyb, (IC50 = 500–700 nM), which inhibited the growth of nonsmall cell lung cancers and breast cancers in vitro and in vivo (26, 27). Despite this relatively high VPAC-R binding affinity of analogs JV-1–36, JV-1–38, and JV-1–42, their antagonistic properties on VPAC-R appear to be less involved in the antiproliferative mechanism than their GHRH-R inhibitory potency. This is supported by the findings that D-Phe2-containing analogs JV-1–50, JV-1–51, and JV-1–52, with decreased GHRH-R antagonistic potency and increased VPAC-R antagonistic activity, caused less inhibition of cell proliferation than their parent compounds. A possible nonspecific inhibitory effect of these analogs due to their high concentration (10 µM) can be also excluded, because GHRH, VIP, and a potent GHRH agonistic analog (JI-38) (52), structurally similar to the antagonists, either did not inhibit or slightly stimulated the proliferation of LNCaP and MiaPaCa-2 cells (data not shown).

These results provide, for the first time, evidence that the antiproliferative effects of GHRH antagonists in vitro are mainly exerted through a VPAC-R-independent mechanism. This conclusion is supported by the ability of GHRH antagonists to inhibit proliferation of VPAC-R-negative human pancreatic cancer cell line (MiaPaCa-2).

Therefore, it is likely that unknown receptors, which are different from the well known VPAC-R and the classic pituitary GHRH-R, might be involved in the antiproliferative mechanism of our GHRH antagonists. Using primers for human GHRH-R mRNA, a single band of a 387-bp product was amplified in the human pituitary cells, but no GHRH-R mRNA expression was found in LNCaP and MiaPaCa-2 human cancer cells, in accordance with an earlier report on ovarian tumors (53). The presence of splicing variants of GHRH-R was detected in human pituitary adenomas (53, 54), and thus, in extrapituitary tumor cells other GHRH-R variants or structurally related receptors may also exist, the cDNA of which was not recognized by our primers. The radiolabeled [His1,Nle27]human GHRH-(1–32)NH2, previously used for the characterization of pituitary GHRH-R (46, 55) might have very low affinity to these variant forms of receptors. Consequently, we could not find specific binding sites for this ligand on cancer cells. In contrast, some of the GHRH analogs with structures such as JV-1–36, JV-1–38, and JV-1–42 might bind to these variant forms of receptors with higher affinity and thus inhibit tumor proliferation. The identification of these unknown receptors involved in the antiproliferative action of our GHRH antagonists is in progress. In conclusion, although much additional work is required, the studies in vitro described herein and other findings suggest that potent antitumor effects of GHRH antagonists (5) are exerted by mechanisms independent of receptors for VIP.


   Acknowledgments
 
The selective VPAC1-R antagonist (PG 97–269) kindly provided by Drs. P. Gourlet and P. Robberecht (Université Libre de Bruxelles, Brussels, Belgium) is greatly appreciated. The authors also thank Dr. R. Kineman (University of Illinois, Chicago, IL) for providing us with the sequence of primers for human GHRH-R. The excellent experimental assistance of Ms. Elena Glotser is gratefully acknowledged.


   Footnotes
 
1 This work was supported by the Medical Research Service of the V.A. Department, a CaPCURE Foundation Research Award, and a grant from ASTA Medica AG (Frankfurt am Main, Germany) to Tulane University School of Medicine (all to A.V.S.). Back

2 On leave from Department of Human Anatomy, University Medical School, H-7643 Pécs, Hungary. Back

Received December 20, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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