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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-1894
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4287-4298
Copyright © 2005 by The Endocrine Society

Differential Effects of Gonadotropin-Releasing Hormone (GnRH)-I and GnRH-II on Prostate Cancer Cell Signaling and Death

Kaushik Maiti, Da Young Oh, Jung Sun Moon, Sujata Acharjee, Jian Hua Li, Dong Gyu Bai, Hee-Sae Park, Keesook Lee, Young Chul Lee, Neon Chul Jung, Kyungjin Kim, Hubert Vaudry, Hyuk Bang Kwon and Jae Young Seong

Hormone Research Center (K.M., D.Y.O., J.S.M., S.A., J.H.L., D.G.B., H.-S.P., K.L., Y.C.L., H.B.K., J.Y.S.) and School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea; Neurogenex Co. Ltd. (N.C.J.), Biotechnology Incubating Center Golden Helix, Seoul National University, Seoul 151-744, Republic of Korea; School of Biological Sciences (K.K.), Seoul National University, Seoul 151-742, Republic of Korea; Institut National de la Santé et de la Recherche Médicale U413, Laboratory of Cellular and Molecular Neuroendocrinology (H.V.), European Institute for Peptide Research, University of Rouen, 76821 Mont-Saint-Aignan, France; and Laboratory of G Protein Coupled Receptors (J.Y.S.), Korea University College of Medicine, Seoul 136-705, Republic of Korea

Address all correspondence and requests for reprints to: Jae Young Seong, Ph.D., Laboratory of G Protein Coupled Receptors, Korea University College of Medicine, Seoul 136-705, Republic of Korea. E-mail: jyseong{at}korea.ac.kr; or Hyuk Bang Kwon, Ph.D., Hormone Research Center and School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: kwnohb{at}chonnam.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Context: GnRH is known to directly regulate prostate cancer cell proliferation, but the precise mechanism of action of the peptide is still under investigation.

Objective: This study demonstrates differential effects of GnRH-I and GnRH-II on androgen-independent human prostate cancer cells.

Results: Both GnRH-I and GnRH-II increased the intracellular Ca2+ concentration ([Ca2+]i) either through Ca2+ influx from external Ca2+ source or via mobilization of Ca2+ from internal Ca2+ stores. Interestingly, the [Ca2+]i increase was mediated by activation of the ryanodine receptor but not the inositol trisphosphate receptor. Trptorelix-1, a novel GnRH-II antagonist but not cetrorelix, a classical GnRH-I antagonist, completely inhibited the GnRH-II-induced [Ca2+]i increase. Concurrently at high concentrations, trptorelix-1 and cetrorelix inhibited GnRH-I-induced [Ca2+]i increase, whereas at low concentrations they exerted an agonistic action, inducing Ca2+ influx. High concentrations of trptorelix-1 but not cetrorelix-induced prostate cancer cell death, probably through an apoptotic process. Using photoaffinity labeling with 125I-[azidobenzoyl-D-Lys6]GnRH-II, we observed that an 80-kDa protein specifically bound to GnRH-II.

Conclusions: This study suggests the existence of a novel GnRH-II binding protein, in addition to a conventional GnRH-I receptor, in prostate cancer cells. These data may facilitate the development of innovatory therapeutic drugs for the treatment of prostate cancer.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTATE CANCER IS the most common malignancy in men and the second leading cause of cancer-related deaths in Western countries (1). Initial treatment of prostate cancer is hormonal therapy to remove or decrease serum androgen, a potential growth stimulant for prostate cancer. However, after an initial regression, most prostate cancers become resistant to this loss of androgens and progress further with eventual death (2). Thus, development of effective therapeutic drugs for androgen-independent prostate cancer is of utmost concern to oncologists.

Hormonal therapies to treat benign prostatic hyperplasia or androgen-dependent prostate cancers include drugs that target the GnRH receptor (3, 4, 5). The GnRH isoform synthesized in the hypothalamus is termed GnRH-I as opposed to a second form of GnRH (GnRH-II) that is widely expressed in the brain as well as in peripheral reproductive and immune tissues (6, 7, 8, 9, 10, 11, 12). GnRH-I stimulates the secretion of pituitary gonadotropins, LH, and FSH, which in turn modulates the synthesis and secretion of androgens from the testis. Chronic administration of a GnRH-I superagonist was found to down-regulate the GnRH receptor, resulting in a marked reduction in circulating levels of androgens (3). Alternatively, inactivation of the GnRH receptor by a GnRH-I antagonist is also effective in reducing androgen levels (4, 5).

Recently a series of studies demonstrated the direct action of GnRH-I on prostate cancer cells. A GnRH-I superagonist exerted a direct inhibitory action on epidermal growth factor- or IGF-induced prostate cancer cell proliferation (13, 14). The authors suggested that this effect may be meditated by the pituitary type GnRH receptor (the mammalian type 1 GnRH receptor distinct from the type 2 GnRH receptor found in the monkey) because they observed expression of the mammalian type 1 GnRH receptors by RT-PCR and Western blot analysis (15). The mammalian type 1 GnRH receptor in the pituitary and other cells is known to be coupled to Gq/11, which in turn activates phospholipase C (PLC), producing inositol trisphosphate (IP3) and diacylglycerol (16). However, it has been suggested that, in prostate cancer cells, GnRH activates a Gi-linked signaling cascade (15). The exact reason for such a discrepancy of the signaling pathways from the same receptor is unclear. Alternatively, it may be ascribed to the presence of uncharacterized GnRH receptors in prostate cancer cells that may trigger a different signaling cascade.

Another question raised from these studies is whether GnRH-II could be involved in the regulation of prostate cancer cell growth because it is likely to have diverse functions in reproductive and immune tissues (6, 7, 8, 9, 10, 11, 12). It is noteworthy that, in human endometrial and ovarian cancer cells, GnRH-II appears to be a more potent inhibitor of cell proliferation than GnRH-I (17). GnRH-II exhibits a differential hormonal regulation pattern in human granulosa-luteal cells (11). Recently it was found that GnRH-II, produced by human T cells, triggers laminin receptor gene expression and cell migration (12). Interestingly, GnRH-II-induced laminin receptor gene expression is not blocked by the GnRH-I antagonist cetrorelix (12), suggesting the existence of an uncharacterized receptor for GnRH-II in human. However, involvement of GnRH-II and its novel receptor in the regulation of prostate cancer cell growth has not been carefully studied.

Recently we and others identified the receptors having a higher sensitivity/affinity for GnRH-II than GnRH-I in nonmammalian species, referred to as nonmammalian GnRH receptors (18, 19). Furthermore, the functional receptor for GnRH-II was cloned from the monkey and called mammalian type 2 GnRH receptor (20, 21, 22). In humans, the genes for the mammalian type 2 GnRH receptor are localized in chromosomes 1 and 14 and are expressed in a variety of tissues including brain (23). However, these genes are thought to be nonfunctional pseudogenes due to the introduction of a premature stop codon (23, 24). Although alternative splicing patterns to circumvent the frame-shift that allows the production of a two-membrane domain protein has been suggested, the functionality of such a protein is still in question (25).

Although genetics-based studies suggest the absence of a functional type 2 GnRH receptor in humans, many physiological and pharmacological findings raise the possibility of novel human receptors for GnRH-II (11, 12, 17). Based on the characterization of GnRH-II receptors in both nonmammalian and mammalian species (26, 27, 28), we designed specific GnRH-II analogs (22, 29, 30). In the present study, using these GnRH-II analogs, we provide evidence for the possible involvement of a novel GnRH-II-binding protein in cell signaling and death of androgen-independent prostate cancer cells. We also demonstrate that a novel GnRH-II antagonist, trptorelix-1, induces prostate cancer cell death probably via an apoptotic process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
GnRH and its analog

GnRH-I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and [des-Gly10, D-Trp6]-GnRH-I (GnRH-Ia) were purchased from Sigma (St. Louis, MO). Cetrorelix (Ac-D-Nal (2)-D-Phe(4Cl)-D-Pal (3)-Tyr-D-Cit-Leu-Arg-Pro-D-Ala-NH2) was obtained from ASTA Medica AG (Frankfurt am Mein, Germany). GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), [His5]-GnRH-I, [Trp8]-GnRH-I, [D-Trp6]-GnRH-I, [Tyr5]GnRH-II, [Leu5, D-Ala6]-GnRH-II, [D-Ala6, Leu7]-GnRH-II, [D-Ala6, Tyr7, Trp8]-GnRH-II salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2), trptorelix-1 (Ac-D-Nal (2)-D-Phe(4Cl)-D-Pal (3)-Ser-Tyr-D-Cit-Trp-Tyr-Pro-D-Ala-NH2), trptorelix-2 (Ac-D-Nal (2)-D-Phe(4Cl)-D-Pal (3)-Ser-His-D-Cit-Trp-Tyr-Pro-D-Ala-NH2), [azidobenzoyl-D-Lys6]-GnRH-I, and [azidobenzoyl-D-Lys6]-GnRH-II were synthesized by AnyGen (Gwangju, Korea).

Cell culture

DU-145 and LNCaP cells were obtained from Korea Cell Bank (Seoul, Korea) and American Type Culture Collection (Manassas, VA), respectively. PPC-1 (31), ALVA-41 (32), and TSU-Pr1 (33) cells were obtained from Dr. J. Y. Bahk (Gyeongsang National University, Chinju, Korea) with the permissions of their original developers. DU-145, ALVA-41, TSU-Pr1, PPC-1, MCF-7, CV-1, HEK-293, and GH3 cells were cultured at 37 C in DMEM containing 10% heat-inactivated fetal bovine serum (FBS), 1 mM glutamate, 100 U penicillin, and 100 µg/ml streptomycin. LNCaP cells were grown in RPMI 1640 medium containing 10% FBS at 37 C.

Establishment of stable cell lines using retroviral expression vectors

The establishment of stable cell lines expressing bullfrog GnRHR (bfGnRHR)-3 or rat GnRHR has been previously described (22, 34). Briefly, the entire open reading frames of bfGnRHR-3 and rat GnRH receptor (GnRHR) were cloned into the retrovirus expression vector, pLXSN (Clontech, Palo Alto, CA) at the EcoRI and XhoI sites. The pLXSN retroviral vectors containing either bfGnRHR-3 or rat GnRHR were transfected in the dual-tropic packaging cell line PT67 using the Superfect transfection kit (QIAGEN, Chatsworth, CA). The cells were treated with 25 mM chloroquinine (Sigma) 1 h before transfection, and the media were replenished (DMEM, 10% FBS). After 24 h, the cells were treated with 800 µg/µl G418 (Clontech) for 2 wk. Large healthy colonies were selected and expanded. The presence of the stably expressed genes was confirmed by RT-PCR and inositol phosphate (IP) assay. The culture media soup was used for infection of GH3 cells with retroviruses containing the GnRH receptors. Two days after infection, GH3 cells were selected with G418 for 2 wk, and selected clones were expanded. GH3 cell lines expressing rat GnRHR or bfGnRHR-3 with the highest IP fold induction were chosen and maintained in DMEM containing 10% FBS until use.

Generation of recombinant adenovirus

Ad-bf2-SDY, an E1-deleted adenovirus expressing bfGnRHR-2-SDY mutant, was generated by Neurogenex (Seoul, Korea). Briefly, DNA encoding bfGnRHR-2-SDY (30) was inserted into the pShuttle-cytomegalovirus vector (Clontech). The pShuttle-cytomegalovirus with bfGnRHR-2-SDY was linearized with PmeI and cotransformed into BJ 5183 Escherichia coli along with the pAdEasy-1 (Clontech) adenoviral vector. Transformed cells were overlaid on kanamycin-containing agarose plates, and individual colonies were checked for the presence of the proper recombinant. After sequence confirmation, recombinant adenoviral stocks were expanded by infection of HEK-293A cells, followed by extraction and CsCl gradient purification. Viral titers were determined using plaque assays and reported as multiplicity of infection (MOI), in which one MOI represents one plaque-forming unit/plate. Viruses expressing enhanced green fluorescent protein (Ad-EGFP) were used as controls.

Cell binding assay

GnRH-II and GnRH-Ia were iodinated with the chloramine-T (Sigma) method and purified by chromatography on a Sephadex G-25 (Sigma) column in 0.01% acetic acid and 0.1% BSA. Cells (3 x 105 cells/well) were grown in 12-well plates for 24 h before assay. The cells were washed with warm PBS and incubated at 4 C for 60 min with the iodinated ligand (60,000–80,000 cpm/well) in DMEM containing 0.1% BSA in the absence or presence of cold GnRH (10 µM). The cells were then washed twice with PBS. After solubilizing the cells in 1% sodium dodecyl sulfate (SDS) and 0.2 M NaOH, radioactivity was determined.

RT-PCR analysis

Total RNA was isolated from ALVA-41, DU-145, PPC-1, TSU-Pr1, HEK-293, and HeLa cells using Tri-reagent (Sigma). Five micrograms of total RNA were reverse transcribed with Superscript II Ribonuclease H– reverse transcriptase (Life Technologies, Inc., Rockville, MD), according to a provided protocol. The first-strand cDNA was amplified by PCR using the following primers: hGnRHR1-F (5'-CTTGAAGCTCTGTCCTGGG-3'; located at –6 to –25 from the translation start point); hGnRHR1-R (5'-AAGCTAGGGGCCTCGTGATA-3'; 426–445); hGnRHR2e1-F (5'-ATGTCTGCAGGCAACGG-3'; 380–396); hGnRHR2e1-R (5'-CTGGGGCAAGGCAAGCAGGAAACT-3'; 865–888); hGnRHR2e2-F (5'-CTGTTCCTGTTCCACACGG-3'; 880–907); hGnRHR2e2-R (5'-CATGGCTCCCCTTCCTT-3'; 1083–1099); F-ß-actin (5'-CTGAAGTCACCCATTGAACATGGC-3'); and R-ß-actin (5'-CAGAGCAGTAATCTCCTTCTGCAT-3'). The PCR cycling parameters were: denaturation at 94 C for 5 min followed by 40 cycles of denaturation at 94 C for 30 sec, annealing at 56 C for 30 sec, and extension at 72 C for 45 sec. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and photographed under a UV light source.

IP assay

Prostate cancer cells (1 x 105 cells/well) were seeded in 12-well plates, grown for 1 d, and then incubated in inositol-free DMEM (Life Technologies) containing 2% dialyzed FBS and 1 µCi (1 Ci = 37 GBq) [3H] myo-inositol (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 24 h. The medium was removed and 0.5 ml buffer A (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM CaCl2, 1 mM MgCl2, and 1 mg/ml fatty acid-free BSA) containing 10 mM LiCl was applied to cells for 30 min. The cells were then treated with 1 µM GnRH-II for 45 min. Reactions were terminated by aspirating the medium and adding 1 ml 10 mM formic acid. After 30 min, the formic acid extracts were transferred to a column containing Dowex anion-exchange resin (Bio-Rad Laboratories, Hercules, CA). Total IP was eluted with 1 M ammonium formate and 0.1 M formic acid, and radioactivity was determined using a liquid scintillation counter. Adenoviral infections were performed 48 h before the IP assays. The cells were infected with 100 MOI adenoviral soup in serum-free DMEM for 3 h and incubated in normal DMEM with 10% FBS.

Calcium signaling

Cells were grown on poly-L-lysine-coated glass cover slips for 24–48 h before experiment and incubated in a physiological solution (138 mM NaCl, 6 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 1 mM Na2HPO4, 5 mM NaHCO3, 5 mM glucose, 10 mM HEPES, and 0.1% BSA) with 5 µM fura-2/AM (Molecular Probes, Eugene, OR) at 25 C for 45 min. In the bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA-AM) experiments, 1 µM of BAPTA-AM (Calbiochem, La Jolla, CA) was coincubated with fura-2/AM. For pharmacological inhibitor experiments, the cells were preincubated with 100 µM dantrolene (Sigma) or 1 µM U73122 (Sigma) for 20 min before calcium imaging. To determine the Ca2+ concentration in the internal Ca2+ pool, 5 µM thapsigargin (Tg) (Sigma), a sarcoendoplasmic Ca2+-ATP inhibitor was treated. The cells were washed twice with the dye-free physiological solution and mounted in an acrylamide chamber that allows perfusion of incubation medium. Fura-2/AM fluorescence was measured at excitation wavelengths 340 and 380 nm and at an emission wavelength 510 nm using an IX70 fluorescence microscope (Olympus, Tokyo, Japan) coupled to a digital Cool CCD camera (CoolSNAP fx CCD Camera, Roper Scientific, Tucson, AZ). All calcium data were analyzed by MetaFluor (version 5; MetaFluor, West Chester, PA).

Thymidine incorporation assay

Cells (5 x 104 cells/well) were seeded in 12-well plates. After a 24-h preincubation, cells were treated with GnRHs or GnRH antagonists for 48 h and then further incubated with 1 µCi/ml 3H-thymidine (NEN Life Science Products, Inc., Boston, MA) for 24 h. The cells were washed twice with cold PBS and treated with 1 ml 5% trichloroacetic acid at 4 C for 30 min. Cells were washed with PBS and solubilized in 0.5 N NaOH and 0.5% SDS. Radioactivity was counted with a liquid scintillation counter.

4',6-Diamidino-2-phenylindole (DAPI) staining

The morphology of nuclei was analyzed by DAPI staining. Cells (8 x 104 cells) were seeded on poly-L-lysine-coated glass cover slips in 6-well plates. After treatment with GnRH antagonists for 72 h, the cells were washed twice with PBS and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. After rinsing and permeabilization with 0.2% Triton X-100, the cells were washed and stained with the DAPI solution [0.1% Triton X-100, 2 mM MgCl2, 0.1 M NaCl, 100 mM 1,4-piperazine diethane sulfonic acid, 1 µg/ml DAPI (pH 6.8)] for 5 min. The cells were washed and analyzed with a fluorescence microscope.

DNA fragmentation assay

Cells were seeded into 100-mm dishes at a density of 1 x 106 per plate in 2% FBS-containing DMEM and treated with GnRH antagonists for 72 h. The cells were then harvested by scraping and incubated in lysis buffer [20 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% Triton X-100] for 30 min on ice. The homogenates were centrifuged at 15,000 rpm for 15 min. The supernatants were collected and extracted with equal volume of phenol-chloroform-isoamylalcohol. DNA was precipitated overnight with 70% ethanol at –70 C, and the pellets were washed with 70% ethanol, air dried, and dissolved in distilled water. The DNA was treated with RNase at 37 C for 15 min. The DNA was electrophoresed on a 1.5% agarose gel.

Terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL)

To analyze DNA fragmentation histologically, TUNEL was performed using an in situ cell death detection kit, POD (Roche Diagonastics Corp., Indianapolis, IN). Cells were seeded onto poly-L-lysine-coated glass cover slips in 6-well plates at a density of 105 cells/well. After 24 h, the cells were treated with GnRH antagonists for 72 h. The cells were rinsed with PBS at 25 C and fixed with 4% paraformaldehyde in PBS for 1 h at room temperature. Peroxidase activity in cells was blocked with 3% H2O2 in methanol for 15 min at room temperature. The cells were then permeabilized with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. The cells were incubated in 50 µl of TUNEL reaction mixture (Roche Diagonastics) at 37 C for 1 h in a humidified atmosphere in the dark, then rinsed three times with PBS for 5 min, and dried. The cover slips were incubated in 50 µl converter-POD in a humidified chamber at 37 C for 30 min, washed three times with PBS, and incubated in 50 µl of diaminobenzidine tetrahydrochloride at 25 C for 10 min. Finally, the cover slips were washed three times and analyzed on a light microscope.

Photoaffinity labeling

[Azidobenzoyl-D-Lys6]-GnRH-I and [azidobenzoyl-D-Lys6]-GnRH-II were iodinated using the chloramine-T method and purified on a Sephadex G-25 (Sigma) column with 0.01% acetic acid under a minimal light source. Prostate cancer cells were grown to maximum confluence in 100-mm dishes. The cells were washed twice by PBS and incubated in 2 ml buffer A containing 125I-[azidobenzoyl-D-Lys6]-GnRH-I or 125I-[azidobenzoyl-D-Lys6]-GnRH-II (100,000 cpm) in the presence or absence of graded concentrations of cold GnRHs at 20 C for 30 min. Dishes were irradiated under an auto-cross-linking mode three times at a distance of 2 cm from the UV source in Stratalinker 2400 (Stratagene, La Jolla, CA). The cells were harvested in ice-cold binding buffer [10 mM HEPES, 1 mM EDTA (pH 7.4)], homogenized with 15 strokes of a glass Dounce homogenizer, and centrifuged for 10 min at 400 x g to remove nuclei (35). The supernatants were centrifuged at 10,000 x g for 30 min at 4 C and membrane pellets were resuspended in binding buffer. Labeled receptors were denatured by adding 0.5% SDS and 1% Triton X-100 and heated at 60 C for 15 min. Samples were mixed with SDS sample buffer containing 5% (vol/vol) 2-mercaptoethanol and electrophoresed at room temperature for 18 h on three-phase polyacrylamide gels (4, 10, and 16.5% polyacrylamide) using a tricine buffer system. The gels were fixed and subjected to autoradiography.

Data analysis

Results were expressed as mean ± SEM. Plots were produced using PRISM2 software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen-independent prostate cancer cells bind to GnRH-II with a higher affinity than GnRH-I

To examine whether prostate cancer cells bind to GnRH-I or GnRH-II, we employed three different androgen-independent prostate cancer cell lines, DU-145, ALVA-41, and PPC1, a bladder carcinoma cell line TSU-Pr1 (36), and an androgen-sensitive cell line LNCaP. Cancer cells were incubated with 125I-GnRH-Ia or 125I-GnRH-II in the presence or absence of cold GnRHs. 125I-GnRH-Ia efficiently bound to GH3 cells expressing the rat GnRHR and the binding was inhibited by excess cold GnRH-I and GnRH-II. DU-145 cells did not bind 125I-GnRH-Ia (Fig. 1AGo), indicating that DU-145 cells do not or only marginally express the conventional mammalian type 1 GnRH receptor. In contrast, DU-145 cells bound 125I-GnRH-II as efficiently as GH3 cells stably expressing bfGnRHR-3. Both cold GnRH-I and GnRH-II were able to inhibit the binding of 125I-GnRH-II to GH3 cells expressing bfGnRHR-3, whereas only cold GnRH-II efficiently decreased binding of 125I-GnRH-II to DU-145 cells (Fig. 1BGo). The binding affinity of ALVA-41, PPC-1, and TSU-Pr1 cells for GnRH-I and GnRH-II was examined. All three cell types showed a higher affinity for GnRH-II than GnRH-I as observed in DU-145 cells (Fig. 1CGo). The binding affinity of LNCaP cells for GnRH-I and GnRH-II was examined. LNCaP cells did not specifically bind to 125I-GnRH-I. For GnRH-II, however, the LNCaP cells have a low-affinity binding because a GnRH-II analog [Tyr5]GnRH-II significantly inhibited the binding, although native and other GnRH-II analogs did not (Fig. 1DGo). To further define the binding specificity of DU-145 cells, GnRH-II analogs, Tyr5-GnRH-II, Leu5-D-Ala6-GnRH-II, D-Ala6-Leu7-GnRH-II, and D-Ala6-Tyr7-Trp8-GnRH-II were employed. Except for Tyr5-GnRH-II, GnRH-II analogs, having considerable hydrophobicity, failed to displace 125I-GnRH-II binding (Fig. 1EGo), suggesting specific binding of 125I-GnRH-II. Binding of DU-145 cells to 125I-GnRH-II was inhibited by unlabeled GnRH-II and salmon GnRH in a dose-dependent manner (Fig. 1FGo). These results indicate that GnRH-II specifically binds to prostate cancer cells, although it has a relatively low affinity.



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  		FIG. 1. Cell binding assay with radioiodinated GnRH-I and GnRH-II. Binding affinities of GH3 cells stably expressing rat GnRHR (GH3-rat-GnRHR) or prostate cancer cells for GnRH-I and GnRH-II were examined using 125I-GnRH-Ia and 125I-GnRH-II in the presence or absence of 1 µM unlabeled GnRH-I or GnRH-II and their analogs. A and B, Binding of 125I-GnRH-Ia (A) or 125I-GnRH-II (B) on GH3-rat-GnRHR and DU-145 cells. C, Binding of 125I-GnRH-Ia or 125I-GnRH-II to ALVA-41, PPC-1, and TSU-Pr1 cells. D, Binding of 125I-GnRH-Ia or 125I-GnRH-II to LNCaP cells. Competition binding was performed in the presence of a variety of GnRH analogs. Note that Tyr5-GnRH-II significantly reduced 125I-GnRH-II binding. E, Displacement of 125I-GnRH-II binding to DU-145 cells by GnRH-II analogs. F, Inhibition of 125I-GnRH-II binding by graded concentrations of GnRH-II ({blacksquare}), salmon GnRH (sGnRH, {triangledown}), and GnRH-I ({triangleup}).

 
RT-PCR analysis of GnRH receptors in prostate cancer cells

Because the binding assay indicates the presence of a receptor with a higher affinity to GnRH-II than GnRH-I, the presence of mRNAs for already known human mammalian type 1 and type 2 GnRH receptors was sought using RT-PCR. It should be noted that 35 rounds of PCR amplification brought about very faint signals for each GnRH receptor, whereas 40 rounds of PCR amplification produced intense signals for all receptors (Fig. 2Go). ALVA-41 and DU-145 cells expressed the mRNA for type 1 GnRH receptor. Interestingly, the mRNAs for individual exon 1 or exon 2 of the human type 2 GnRH receptor were amplified in all cell lines. However, we failed to obtain PCR products consisting of exon 1 and exon 2 together (Fig. 2Go). This result is consistent with a previous observation that mRNA specific for human type 2 GnRH receptor is expressed ubiquitously, but the full-length mRNA may be truncated due to inappropriate posttranscriptional processing (25). This result indicated that high-affinity binding of GnRH-II to prostate cancer cells is not due to expression of the conventional type 2 GnRH receptor.



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  		FIG. 2. RT-PCR analysis of human GnRH-I and GnRH-II receptor mRNAs in prostate cancer cells. To exclude possible DNA contamination, PCR was performed using samples with or without reverse transcription (RT). Human type 1 GnRH receptor (GnRHR-1), exon 1 of human type 2 GnRH receptor (GnRHR-2, e1), exon 2 of human type 2 GnRH receptor (GnRHR-2, e2), and exons 1 and 2 of human type 2 GnRH receptor (GnRHR-2, e1-e2) cDNAs were amplified by 40 rounds of PCR. ß-Actin cDNA was amplified by 30 rounds of PCR.

 
GnRHs failed to induce IP production in prostate cancer cells

It is well known that both mammalian type 1 and type 2 GnRH receptors are coupled to Gq/11, producing IP through PLC activation. To examine the presence of functional conventional GnRH receptors, we determined IP production in response to GnRH stimulation. Treatment of prostate cancer cells with GnRHs failed to increase IP formation (Fig. 3Go). One may suggest that the GnRH receptor in prostate cancer cells does not couple with Gq/11 or that prostate cancer cells lack the Gq/11/PLC machinery. To test these possibilities, we infected prostate cancer cells with adenovirus containing a mutant bfGnRHR-2 (bfGnRHR-2-SDY) that has a high affinity for GnRH-II (30). The cells infected with the adenovirus containing bfGnRHR-2-SDY significantly induced IP production in response to GnRH (Fig. 3Go). However, the cells infected with adenovirus with EGFP did not respond to GnRH stimulation. Similar results were observed with MCF-7, a breast cancer cell-line and CV-1, a monkey kidney cell line (Fig. 3Go). These results indicate that prostate cancer cells contain the functional Gq/11 and PLC system and thus may express an uncharacterized GnRH receptor that does not link to the Gq/11/PLC system. Because GnRH did not induce IP production in prostate cancer cells, we examined other signaling systems, such as cAMP: GnRH did not alter basal or forskolin-induced cAMP levels (data not shown).



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  		FIG. 3. GnRH-induced IP production in prostate cancer cells. Prostate or nonprostate cells infected with mock (open bar), Ad-GFP (hatched bar), or Ad-bfGnRHR2-SDY (closed bar) were treated with GnRH-II (100 nM) or vehicle. Each bar represents the percentage of IP production over basal. Data are the mean ± SE of three independent experiments.

 
GnRHs induce calcium mobilization in prostate cancer cells

Although GnRH failed to influence IP or cAMP production, both GnRH-I and GnRH-II provoked an increase in intracellular Ca2+ concentration ([Ca2+]i) in prostate cancer cells (Fig. 4Go). All androgen-independent prostate cancer cells, ALVA-41, DU-145, and PPC-1, responded to both GnRH-I and GnRH-II. TSU-Pr1 cells responded to only GnRH-II. Concurrently androgen-sensitive LNCaP cells and nonprostate MCF-7 and HEK-293 cells did not respond to either GnRH-I or GnRH-II (Fig. 4Go). BAPTA-AM is known to effectively chelate Ca2+ release from internal stores but is not as effective at chelating Ca2+ entering through plasma membrane channels (37). To determine the Ca2+ source responsible for GnRH-induced [Ca2+]i increase, GnRHs were applied to DU-145 cells in the absence of extracellular Ca2+ or under a BAPTA-AM-pretreated condition. Removal of Ca2+ in the incubation medium abrogated the [Ca2+]i response to GnRH-I but did not affect the response to GnRH-II (Fig. 5AGo). Replacement of Ca2+-free with Ca2+-containing medium restored the [Ca2+]i response to GnRH-I (data not shown). In contrast, in BAPTA-AM-pretreated cells, GnRH-I but not GnRH-II induced an increase in [Ca2+]i (Fig. 5BGo). These observations indicate that GnRH-II causes [Ca2+]i increase via mobilization of intracellular stores, whereas GnRH-I provokes Ca2+ influx probably through activation of a certain Ca2+ channel. Furthermore, graded concentrations of GnRH-I and GnRH-II were applied to DU-145 cells. In Ca2+-free medium, the log EC50 value of GnRH-II was –8.21 ± 0.08, whereas the log EC50 value of GnRH-I was very high (–5.25 ± 0.10), such that GnRH-II appeared to be 1000 times more potent than GnRH-I in this condition (Fig. 5CGo). In the BAPTA-AM-pretreated cells, however, GnRH-I (log EC50 –7.96 ± 0.10) was more potent than GnRH-II (log EC50 –6.57 ± 0.07) (Fig. 5DGo). Thus, it is likely that GnRH-I may activate a membrane Ca2+ channel, causing Ca2+ entry into the cells, whereas GnRH-II provokes Ca2+ mobilization from internal stores. It is also noteworthy that the GnRH-II-evoked [Ca2+]i increase rapidly declined in Ca2+-free medium but induced a sustained response in the presence of extracellular Ca2+, indicating a capacitative Ca2+ entry.



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  		FIG. 4. Ca2+ mobilization by GnRH-I and GnRH-II in prostate or nonprostate cancer cells. Changes in [Ca2+]i by GnRH-I and GnRH-II were examined in fura-2/AM-preloaded cells. The fura-2/AM 340:380 ratio of individual cells was determined. Arrows indicate exposure to GnRH-I or GnRH-II (10 nM) each. The cells that did not respond to GnRH were subsequently exposed to 1 µM Tg or 1 nM ionomycin.

 


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  		FIG. 5. Identification of the source of Ca2+ mobilized by GnRH. Changes in [Ca2+]i by GnRHs were examined in Ca2+-free medium (A and C) or BAPTA-AM-pretreated DU-145 cells (B and D). The effects of graded concentrations of GnRH-I and GnRH-II on the amplitude of the Ca2+ responses were examined in calcium-free medium (C) or BAPTA-AM-pretreated cells (D).

 
GnRH-II activates the ryanodine receptor to release Ca2+

The release of Ca2+ from internal stores may be mediated by either the IP3 receptor or ryanodine receptor. To determine which mechanism was responsible for GnRH-II-induced Ca2+ mobilization, cells were treated with U73122, a PLC inhibitor, or dantrolene, a ryanodine receptor inhibitor. In Ca2+-free medium, GnRH-I induced an increase of [Ca2+]i in GH3 cells stably expressing rat GnRHR. This increase was inhibited by U73122 but not by dantrolene, indicating that the [Ca2+]i increase induced by rat GnRHR can be accounted for by Ca2+ mobilization from IP3-sensitive intracellular sources (Fig. 6AGo). In contrast, in DU-145 cells, the GnRH-II-induced [Ca2+]i increase was completely blocked by dantrolene but not U73122 (Fig. 6BGo). Involvement of Gi coupling or receptor tyrosine kinase in GnRH-induced Ca2+ mobilization was determined using pertussis toxin, an inhibitor of Gi, and genistein, an inhibitor of tyrosine protein kinase. Neither of these inhibitors affected Ca2+ mobilization by GnRH-I and GnRH-II in DU-145 cell (data not shown). These data, together with the observation that neither GnRH-I nor GnRH-II-induced IP3 production in prostate cancer cells, strongly suggest that the GnRH-II-induced [Ca2+]i increase is mediated by the ryanodine receptor rather than the IP3 receptor.



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  		FIG. 6. Ryanodine receptor-mediated Ca2+ mobilization by GnRH-II. The Ca2+ responses to GnRH-II were determined in Ca2+-free medium to exclude the possible contribution of external Ca2+ sources. A, GH3 cells expressing rat GnRHR (GH3-rat-GnRHR) were exposed to GnRH-I (1 µM) in the presence of 1 µM U73122, 100 µM dantrolene, or vehicle. B, DU-145 cells were exposed to GnRH-II (1 µM) in the presence of 1 µM U73122, 100 µM dantrolene, or vehicle. Each point is the mean ± SE from six individual cells.

 
Effect of GnRH antagonists on [Ca2+]i

Two types of GnRH antagonists were used to study the pharmacological characteristics of GnRH-induced [Ca2+]i transients: cetrorelix, a potent GnRH-I antagonist, and trptorelix-1 and trptorelix-2, potent GnRH-II antagonists (22, 29). It should be noted that none of these antagonists induced an increase in [Ca2+]i in GH3 cells expressing conventional GnRH receptors, such as rat GnRHR, bfGnRHR-2, or monkey type 2 GnRH receptor, and that these antagonists efficiently blocked the GnRH-induced [Ca2+]i increase in these cells (data not shown). In prostate cancer cells, however, all three antagonists induced a bell-shaped [Ca2+]i response in the presence of extracellular Ca2+: low concentrations of antagonists induced an increase in [Ca2+]i, whereas high concentrations of antagonists did not (Fig. 7AGo). Trptorelix-1 was the most potent analog because it could induce a massive increase in [Ca2+]i in DU-145 cells at a concentration as low as 10 pM. In the absence of extracellular Ca2+, neither of the antagonists induced a [Ca2+]i increase (data not shown). Pretreatment of DU-145 cells with cetrorelix blocked the [Ca2+]i increase induced by low doses of GnRH-I (1 and 10 nM) but did not block the GnRH-II-induced [Ca2+]i increase. Thus, trptorelix-1 can inhibit both GnRH-I- and GnRH-II-induced [Ca2+]i increase (Fig. 7BGo).



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  		FIG. 7. Effect of GnRH antagonists on Ca2+ mobilization. A, DU-145 cells were treated with various concentrations of GnRH antagonists, cetrorelix (Cet), trptorelix-1 (Trp-1), or trptorelix-2 (Trp-2) in a Ca2+-containing media. B, DU-145 cells were treated with various concentrations of GnRH-I or GnRH-II in the presence of 100 nM cetrorelix or 100 nM trptorelix-1.

 
A GnRH-II antagonist induces prostate cancer cell death

Because GnRHs are known to influence prostate cancer cell proliferation, we examined the effects of GnRHs and their antagonists on thymidine incorporation in DU-145 and ALVA-41 cells. GnRH-II and cetrorelix did not affect thymidine incorporation, whereas trptorelix-1 reduced thymidine incorporation in a dose-dependent manner (Fig. 8Go). Despite the failure of [Ca2+]i induction in LNCaP cells by GnRH-I and GnRH-II, LNCaP cells may have a low-affinity GnRH-II binding protein (Fig. 1DGo). In addition, trptorelix-1 has a growth-inhibitory effect on LNCaP cells (data not shown). This observation suggests that trptorelix-1 could be useful for the treatment of prostate cancer cells that retain some sensitivity to androgens as well as androgen-independent cancer cells. To determine whether the trptorelix-1-induced decrease in thymidine incorporation was due to a decreased proliferation rate or an increased cell death, cellular morphology and DNA fragmentation were examined in cetrorelix- or trptorelix-1-treated DU-145 and ALVA-41 cells. Marked changes in cellular morphology were seen in trptorelix-1- but not cetrorelix-treated cells. When cells were stained with DAPI, fragmented nuclei and cells lacking nuclei were observed in trptorelix-1-treated cells (Fig. 9AGo). To further confirm that trptorelix-1 induced apoptosis of prostate cancer cells, DNA fragmentation was visualized by electrophoresis and TUNEL. DNA laddering, and TUNEL-positive cells were observed only in the trptorelix-1-treated group (Fig. 9Go, B and C).



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  		FIG. 8. Effect of GnRH antagonists on thymidine incorporation. ALVA-41 (A) and DU-145 (B) cells were incubated with graded concentrations of GnRH-II, cetrorelix (Cet), or trptorelix-1 (Trp-1) for 3 d. At the end of the second day, cells were treated with 1 µCi/ml 3H-thymidine. Each point is the mean ± SE of three independent experiments performed in quadruplicate.

 


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  		FIG. 9. Apoptosis of prostate cancer cells after trptorelix-1 treatment. ALVA-41 and DU-145 cells were incubated with vehicle (Veh), 10 µM cetrorelix (Cet), or trptorelix-1 (Trp-1) for 3 d. A, After GnRH antagonist treatment, DAPI staining was performed. Arrowheads show the division of nuclei and arrows indicate a cell lacking a nucleus in the Trp-1-treated group. B, DNA was isolated from antagonist-treated DU-145 cells and separated on 1.5% agarose. DNA laddering is seen in the Trp-1-treated group. C, To analyze DNA fragmentation histologically, TUNEL was performed in ALVA-41. DNase-treated cells were used as positive controls. TUNEL-positive cells were seen in the Trp-1-treated group.

 
GnRH-II antagonist induces a decrease in Ca2+ concentration of internal stores

It is well known that depletion of internal Ca2+ pools induces apoptosis of cells (38, 39). To elucidate the possible mechanism for trptorelix-1-induced cell death, we examined Ca2+ concentration in internal stores of cells treated with 10 µM trptorelix-1 or cetrorelix for 3 d in DU-145 and ALVA-41 cells. Ca2+ concentration in internal stores was measured by treating Tg, a sarcoendoplasmic Ca2+-ATPase inhibitor that generally results in emptying Ca2+ from the internal Ca2+ pool (40). Tg-releasable Ca2+ was greatly reduced in trptorelix-1-treated cells, compared with control and the cetrorelix-treated group (Fig. 10Go). This finding indicates that trptorelix-1 may affect Ca2+ homeostasis in prostate cancer cells.



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  		FIG. 10. Trptorelix-1-induced a decrease in Ca2+ concentration of internal stores. DU-145 (A) and ALVA-41 (B) cells were treated with 10 µM trptorelix-1 (TRP-1) or cetrorelix (Cet) for 3 d. Temporal changes in [Ca2+]i by 5 µM Tg were examined in fura-2/AM-preloaded cells. Fura-2/AM 340:380 nm ratio of an individual cell was examined. Each line indicates mean ± SEM of data observed in at least five cells.

 
Photoaffinity labeling of GnRH-II binding protein in prostate cancer cells

The presence of a novel GnRH-II binding protein in prostate cancer cells was investigated by photoaffinity labeling using an azidobenzoyl-conjugated GnRH-II. 125I-[azidobenzoyl-D-Lys6]-GnRH-II cross-linked with an approximately 80-kDa protein in all prostate cancer cells but not in HEK-293 and CV-1 cells (Fig. 11AGo). This band was abolished in the presence of an excess of unlabeled [azidobenzoyl-D-Lys6]-GnRH-II. 125I-[azidobenzoyl-D-Lys6]-GnRH-I exhibited a different binding pattern from that of [azidobenzoyl-D-Lys6]-GnRH-II. It bound to an approximately 35- to 40-kDa protein in ALVA-41 and DU-145 cells. The intensity of the 80-kDa band that bound to [azidobenzoyl-D-Lys6]-GnRH-II was dose-dependently decreased by cold natural GnRH-II but not GnRH-I (Fig. 11CGo). It should be noted that there were many other bands whose intensities were decreased by cold azidobenzoyl-D-Lys6-GnRH-II, but the 80-kDa band was the only one whose intensity was decreased by cold native GnRH-II.



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  		FIG. 11. Photoaffinity labeling with 125I-azidobenzoyl-D-Lys6-GnRH-II (AZ2) and 125I-azidobenzoyl-D-Lys6-GnRH-I (AZ1). Proteins were obtained from ALVA-41 (ALVA), DU-145, PPC-1, TSU-Pr1, HEK-293 (HEK), and CV-1 cells. Autoradiography shows the protein that binds 125I-azidobenzoyl-D-Lys6-GnRH-II (A) and 125I-azidobenzoyl-D-Lys6-GnRH-I (B). Binding and UV cross-linking were performed in the absence (–) or presence (+) of 1 µM unlabeled azidobenzoyl-D-Lys6-GnRH-II (cold comp). The arrows indicate the 80-kDa and 40-kDa proteins band whose intensities were decreased in the presence of cold competitor. C, Binding of the 80-kDa protein from DU-145 cells was visualized in the presence of graded concentrations of native GnRH-I or GnRH-II.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated differential effects of GnRH-I and GnRH-II on signaling and proliferation of androgen-independent prostate cancer cells (Table 1Go). Although these cells express transcripts for either conventional mammalian type 1 and/or type 2 GnRH receptors, however, the amount of protein translated from the transcript for the type 1 receptor must be very low because we did not observe specific binding of GnRH-Ia to these cells. Protein translated from the type 2 receptor transcript may be truncated or abnormal because we failed to obtain the transcript consisting of exon 1 and exon 2 together. Furthermore, the responses to GnRHs in prostate cancer cells were completely different from those found in cells expressing conventional mammalian type 1 or type 2 GnRH receptors.


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  		TABLE 1. Differential effects of GnRH-I and GnRH-II on DU-145 cells

 
Conventional GnRH receptors are known to induce [Ca2+]i transients through an increased formation of IP3 leading to mobilization of IP3-sensitive intracellular Ca2+ stores (16). In contrast, GnRHs failed to induce IP3 formation in prostate cancer cell. This failure of GnRH to activate polyphosphoinositide turnover is not due to a defect of the signaling machinery, such as a lack of Gq/11 or PLC, in prostate cancer cells because we observed GnRH-induced IP3 production when a conventional GnRH receptor was introduced into these cells. Interestingly, the GnRH-II-induced increase in [Ca2+]i was completely blocked by a ryanodine receptor antagonist but not by a PLC inhibitor, indicating that GnRH-II in prostate cancer cells causes Ca2+ mobilization through activation of the ryanodine receptor. However, the signaling cascade that is responsible for activation of the ryanodine receptor remains to be elucidated.

The effect of GnRH antagonists on [Ca2+]i in prostate cancer cells was also different from that observed in cells expressing conventional GnRH receptors. Neither trptorelix-1 nor cetrorelix induced a [Ca2+]i increase in cells expressing conventional GnRH receptors, yet low concentrations of both antagonists did induce a [Ca2+]i increase in prostate cancer cells incubated in Ca2+-containing medium. It is of interest to note that the effects of GnRH antagonists on [Ca2+]i increase is comparable with that of ryanodine. Ryanodine stimulates Ca2+ release from the endoplasmic vesicle at nanomolar to micromolar concentrations, whereas it inhibits Ca2+ release at higher concentrations (41, 42). GnRH-II antagonists, however, may not directly affect the ryanodine receptor activation because trptorelix-1 and cetrorelix failed to induce the [Ca2+]i in the absence of Ca2+ source. Thus, it is likely that, in prostate cancer cells, the GnRH antagonists can act as agonists for the GnRH-I receptor under a certain condition. In support of this notion, we found that TSU-Pr1 cells, which responded to only GnRH-II but not GnRH-I, were not affected by trptorelix-1 and cetrorelix. Furthermore, the molecular mass of the protein that binds to GnRH-II in prostate cancer cells is approximately 80 kDa, whereas that of the conventional GnRH receptors is 40–50 kDa (15, 35, 43). Altogether these results strongly suggest that GnRH-II exerts its action through a novel GnRH-II binding protein in androgen-independent prostate cancer cells. However, we do not exclude the possibility that GnRH-II could act through the mammalian GnRH-I receptor because the ligand selectivity and intracellular responses of a single receptor can be vastly altered by receptor conformation and the cellular context.

Several lines of evidence also support the idea of a functional receptor for GnRH-II in humans. In human T cells, either GnRH-I or GnRH-II can induce laminin receptor gene expression and cell migration. Interestingly, the effect of GnRH-I is completely blocked by cetrorelix, whereas the effect of GnRH-II is not (12). In human ovarian cancer cells, cetrorelix also exerts an antiproliferative effect in very much the same as GnRH-I agonists, indicating that the distinction between GnRH-I agonists and antagonists may not apply to the GnRH-I system (17). When GnRH-I receptors are depleted in human ovarian cancer cells, the antiproliferative effect of a GnRH-I agonist is abolished, whereas the effects of a GnRH-I antagonist and GnRH-II are still observed (44), indicating that the effect of GnRH-I antagonists and GnRH-II in these cells are not mediated by the conventional GnRH-I receptor.

Prostate cancer develops from an androgen-dependent stage to an androgen-independent stage. It is likely that during the androgen-dependent stage, the main effect of GnRH analogs as prostate cancer therapy is to block or to desensitize the pituitary type 1 GnRH receptor, thereby reducing serum androgen levels (3, 4, 5), whereas during the androgen-independent stage, GnRH-I and GnRH-II may act directly on prostate cancer cells. According to our findings, GnRH-I and GnRH-II themselves are not likely to affect proliferation of prostate cancer cells as neither peptide altered thymidine incorporation. However, the [Ca2+]i increase induced by GnRHs indicates a possible role of GnRHs in cancer cell activity. Indeed, cytosolic Ca2+ is known to control numerous cell functions, including contraction, proliferation, differentiation, and migration (40, 45, 46). Although the exact regulatory role of GnRHs in prostate cancer cells is currently unknown, it is conceivable that GnRH-I and/or GnRH-II may affect gene expression and/or cell migration in androgen-independent prostate cancer cells. In particular, ryanodine receptor activation by GnRH-II in prostate cancer cells is likely important because activation of these receptors is involved in the control of cell migration (46).

To elucidate the role of GnRHs in prostate cancer cell regulation, it is also of interest to determine the sources of endogenous GnRHs. The GnRH-I and/or GnRH-II genes are expressed in a number of peripheral tissues such as placenta (8), T cells (12), breast (10), endometrium (9), and ovarian surface cells (47). However, the GnRH peptides produced from these tissues are generally thought to play an autocrine or paracrine role rather than endocrine activities. Considering the extremely short half-life of GnRHs, an endocrine action of the peptides would not be expected. Because the GnRH genes are expressed in prostate cancer cells (6, 48), it seems likely that GnRHs can act as paracrine or autocrine factors in prostate cancer development. Therefore, regulation of the GnRH-I and GnRH-II genes during prostate cancer differentiation, and biosynthesis of GnRH from these transcripts deserve further investigation.

An interesting finding in this study is that trptorelix-1 induced prostate cancer cell death, probably through apoptosis. Although there are numerous reports indicating that GnRH analogs decrease proliferation of a variety of reproductive tissue cancer cells (14, 17, 43, 44, 49, 50, 51), few of them have shown that a GnRH analog can induce cancer cell death. The mechanism involved in trptorelix-1-induced apoptosis of prostate cancer cells is currently a matter of speculation. A possible explanation is that trptorelix-1 may disturb Ca2+ homeostasis because high concentrations of trptorelix-1 completely blocked either GnRH-I- or GnRH-II-induced [Ca2+]i increase. It is well established that depletion of internal Ca2+ pools induces apoptosis of cells (38, 39). Moreover, Ca2+ mobilization via the ryanodine receptor is known to play a role in apoptosis of androgen-dependent prostate cancer cells (52). Indeed, we observed that trptorelix-1, but not cetrorelix, decreased Ca2+ concentration in internal store. Alternatively, it is possible that trptorelix-1 can induce apoptosis through a yet-unknown mechanism.

In conclusion, GnRH-I agonists and antagonists are already widely used in various reproductive tissue cancer therapies. However, the direct effects of GnRH-II agonists and antagonists have been barely examined in these cancer cells, although many of them are responsive to GnRH-II (38). Whether these cancer cells express novel GnRH-II receptors, as observed in prostate cancer cells, and whether GnRH-II antagonists can induce their death needs to be elucidated. This study provides evidence that androgen-independent prostate cancer cells possess novel receptors for GnRH-I and GnRH-II. Further information on these receptors will facilitate development of new drugs for the treatment of reproductive tissue cancers.


   Footnotes
 
This work was supported by the Korea Research Foundation (2002-CP0337); a grant (M103KV010004 03K2201 00410) from Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, Republic of Korea; and the Science and Technology Amicable Research exchange program between Korea and France.

First Published Online May 3, 2005

Abbreviations: BAPTA-AM, Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester; bfGnRHR, bullfrog GnRHR; [Ca2+]i, intracellular calcium concentration; DAPI, 4',6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; GnRHR, GnRH receptor; IP, inositol phosphate; IP3, inositol trisphosphate; MOI, multiplicity of infection; PLC, phospholipase C; SDS, sodium dodecyl sulfate; Tg, thapsigargin; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine 5-triphosphate nick-end labeling.

Received September 24, 2004.

Accepted April 15, 2005.


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 Introduction
 Materials and Methods
 Results
 Discussion
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