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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 6 3020-3026
Copyright © 2004 by The Endocrine Society

Type II Gonadotropin-Releasing Hormone Stimulates p38 Mitogen-Activated Protein Kinase and Apoptosis in Ovarian Cancer Cells

Ki-Yon Kim, Kyung-Chul Choi, Se-Hyung Park, Chun-Shan Chou, Nelly Auersperg and Peter C. K. Leung

Department of Obstetrics and Gynaecology, British Columbia Research Institute for Children’s and Women’s Health, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5

Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent results indicate that a novel second form of GnRH, GnRH-II, has an antiproliferative effect on ovarian and endometrial cancer cells and might be considered as a possible therapy for gynecological tumors. However, the mechanism of the GnRH-II-induced antiproliferative effect is not known. The p38 MAPK, one of the stress-activated protein kinases, is activated by diverse cellular stress and proinflammatory cytokines. In this study, the effect of GnRH-II on the activation of p38 MAPK was investigated, and its possible role in the regulation of cell proliferation and apoptosis was further examined in the human ovarian cancer cell line, OVCAR-3. Treatment with GnRH-II (100 nM) resulted in an activation of p38 MAPK in a time-dependent manner. A significant activation of p38 MAPK was observed at 2, 5, 10, and 15 min after GnRH-II treatment. The activation of p38 MAPK by GnRH-II was reversed in the presence of a specific inhibitor of p38 MAPK, SB203580 (1 µM). The transcription factor, activator protein-1, was activated (1.5-fold) by GnRH-II and attenuated in the presence of SB203580 (1 µM). Treatment with GnRH-II (1 nM, 100 nM, 10 µM) for 2, 4, and 6 d resulted in an inhibition of cell growth in OVCAR-3 cells as determined by thymidine incorporation assay. The effect of GnRH-II (100 nM) on cell proliferation was blocked by pretreatment with SB203580 (1 µM). Furthermore, a significant increase of apoptosis (1.6-fold) was observed after GnRH-II treatment, which was also reversed by pretreatment with SB203580 (1 µM). Taken together, these results indicate that p38 MAPK is involved in the GnRH-II-induced inhibition of cell growth through activator protein-1 activation, which may be related to induction of apoptosis in ovarian cancer cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVARIAN CANCER IS known as a major cause of cancer-related death in Western women (1). Because of the absence of symptoms in early stages and location in the pelvis, ovarian cancer is difficult to be detected in early stages (2) and has a high fatality and low 5-yr survival rate compared with other gynecological cancers. Hormonal factors have been implicated in the etiology of ovarian cancer, according to the incessant ovulation theory (3, 4). In addition to its well-established role as a neuroendocrine regulator at the level of the pituitary gland, GnRH has been proposed to be an autocrine regulator and exert an antiproliferative effect on gynecological cancers (1, 5, 6, 7). Treatment with GnRH has potential benefits for patients with ovarian cancer who do not respond to chemotherapy (8), and GnRH analogs produced favorable results when conventional therapy was supplemented for late-stage ovarian cancer treatment (9).

Recently, a second GnRH form (GnRH-II) was sequenced, and its gene expression was identified in humans (10). It appears that GnRH-II may exert a stronger antiproliferative effect than the classical mammalian GnRH (GnRH-I) in ovarian cancer cells (11). To date, the molecular mechanism underlying the antiproliferative effect of both GnRH-I and II is poorly understood. In the pituitary gonadotrope cells, MAPKs have been considered as important components of GnRH-induced signaling pathway (12, 13). The MAPK cascade is activated by mitogens and growth factors and plays an important role in the control of cell growth and differentiation. Of the 12 different MAPKs identified in mammalian cells to date, ERK1/ERK2, c-Jun N-terminal protein kinase/stress-activated protein kinase 1 (JNK/SAPK1), and p38/SAPK2 are the best characterized (14). ERK1/ERK2 are activated by mitogenic stimuli, whereas JNK/SAPK1 and p38/SAPK2 MAPK are activated by cellular stress, bacterial infection and proinflammatory cytokines, osmotic shock, heat shock, UV light, and DNA-damaging agents (15, 16). Together, these form a signal transduction cascade network that mediates cellular responses to diverse extracellular stimuli and play an important role in cell growth and differentiation. MAPKs are associated with transcription factors such as activator protein 1 (AP-1), c-Myc, Elk-1, c-Jun, and activating transcription factor-2 to induce biological changes in cells (15, 17, 18). ERK is proposed to contribute to the protective function in UV-induced damage, whereas JNK and p38 are involved in apoptosis via AP-1 (19, 20).

In the present study, the antiproliferative effect by GnRH-II was examined in the ovarian adenocarcinoma OVCAR-3 cell line. In addition to cell growth, the possible effect of GnRH-II in the activation of p38 MAPK and AP-1 transcriptional factor was investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatment

The human epithelial ovarian cancer cell line OVCAR-3, an ovarian adenocarcinoma derived from the ascites of ovarian cancer patients, was kindly provided by Dr. T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA). The cells were cultured in medium 199:MCDB 105 (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Life Technologies, Inc., Rockville, MD) at 37 C in a humidified atmosphere of 5% CO2-95% air as previously described (21, 22). Then, the cells were trypsinized with 0.06% trypsin (1:250)/0.01% EDTA (Life Technologies, Inc.) in Mg2+/Ca2+-free Hanks’ balanced salt solution and seeded at a density of 2 x 105 cells in 35-mm dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ) and cultured for 2 d. The cells were washed once with the medium and serum-starved for 6 h before GnRH-II treatment. To investigate the direct effect on p38 MAPK, SB203580 (1 µM), the inhibitor of p38 MAPK, was added for 20 min (23), and then GnRH-II (Bachem, Belmont, CA) was added in a time-dependent manner at 100 nM (24).

Immunoblot analysis

Immunoblot analysis was performed to investigate the effect of GnRH-II on the activation of MAPK as previously described (25). The cells treated with GnRH-II were washed once with ice-cold PBS and lysed in 100 µl of in ice-cold RIPA buffer [150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris (pH7.5), 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin]. The extracts were placed on ice for 10 min, collected into a 1.5-ml tube, and centrifuged for 10 min at 14,000 rpm. The supernatants were moved to new tubes, and the concentration of supernatants was determined using Bradford assay (Bio-Rad Laboratories, Mississauga, Ontario, Canada). Thirty-five micrograms of total protein were mixed with 6x sample buffer (75 mM Tri-HCl of pH 6.8, 15% SDS, 0.15% bromophenol blue, 15% glycerol, and 37.2% 2-mercapthoethanol) and boiled for 10 min. The sample mixture was run on 10% SDS-PAGE gels (acrylamide:bisacrylamide, 29:1) in 1x gel running buffer (25 mM Tris/250 mM glycine, pH 8.3/0.1% SDS) at 100 V for 2.5 h and electrotransferred to a nitrocellulose membrane (Hybond C, Amersham Pharmacia Biotech Inc., Oakville, Ontario, Canada) at 100 V for 1.5 h. The membrane was immunoblotted using a rabbit polyclonal antibody for phosphorylated p38 MAPK (Biosource International Inc., Camarillo, CA) with protein molecular marker (New England Biolabs, Inc., Ontario, Canada). After washing three times with TBS-T (0.1% Tween 20 in Tris-buffered saline) for 15 min, the signals were detected with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech Inc.) and visualized using the enhanced chemiluminescence system (Amersham Pharmacia Biotech Inc.). Alternatively, the membrane was probed with a mouse monoclonal p38 MAPK antibody (Biosource International Inc.), which detects total p38 MAPK levels. The intensity of signals was quantitated by densitometry (BioDocAnalyze, Biometra, Germany).

Proliferation assay

Proliferation assay was performed using [3H]thymidine incorporation assay as previously described (26, 27, 28). Briefly, 20,000 cells were seeded in 24-well plates and cultured in 0.5 ml medium. GnRH-II was diluted appropriately with medium, and the cells were treated for 2, 4, and 6 d every 12 h. The medium was changed after 24-h incubation. After treatment, the cells were then incubated with medium containing 1 µCi [3H]thymidine (0.5 Ci/mmol; Amersham Pharmacia Biotech Inc.) and collected after 6 h incubation. To block the effect of p38 MAPK, the cells were pretreated with SB203580 (1 µM), followed by treatment with GnRH-II. Control culture was treated with vehicle. The cells were washed three times with PBS and precipitated with 0.5 ml 10% trichloroacetic acid for 20 min at 4 C. The precipitate was washed in methanol twice and solubilized in 0.5 ml of 0.1 N sodium hydroxide. The radioactivity was measured in the Tri-Carb Liquid Scintillation Analyzer (model 2100TR, Packard Instrument Co., Meriden, CT).

DNA fragmentation assay

The amount of DNA fragmentation was measured using the cell death detection ELISA kit according to the manufacturer’s suggested procedure (Roche Applied Science, Laval, Québec, Canada). The cells were placed in each well of 24-well plates at 1 x 104 concentrations (27, 29). After treatment with GnRH-II (100 nM), the conditioned media were collected, cells were washed with PBS, and 0.1 ml lysis buffer was added. After 15 min incubation on ice, apoptotic cells in cell lysates and conditioned media were assayed for DNA fragments according to the manufacturer’s protocol using an ELISA kit. The same amount (1 µg) of cell lysate was used, and the amount of DNA fragmentation was measured at 405 nm. The control was treated with vehicle.

Transient transfection assay

AP-1-TA-Luc vector (Clontech, Palo Alto, CA) was designed to monitor the induction of the AP-1 signal transduction pathway and contained the firefly luciferase (luc) gene from Photinus pyralis. AP-1-TA-Luc contains an AP-1 response element located upstream of the minimal TA promoter: the TATA box from the herpes simplex virus thymidine kinase promoter (PTA). Located downstream of PTA is the firefly luciferase reporter gene (luc). The pTAL-Luc was used as a negative control to determine the background signals associated with the cell lysates. The enhancerless pTAL-Luc contains herpes simplex virus-thymidine kinase upstream of the luciferase coding sequence. Transient transfection was performed using Lipofetamine reagent (Invitrogen Life Technologies, Carlsbad, CA), following the manufacturer’s protocol. The OVCAR-3 cells were cotransfected using 1 µg plasmid with AP-1 per well. Five hours after transfection, the medium was changed to 10% FBS/DMEM, and 24 h after transfection, GnRH-II (100 nM) was treated for 6 h before harvest. Cellular lysates were collected with 200 µl cell lysis buffer and immediately assayed for luciferase activity with the Luciferase Assay kit (Promega, Madison, WI) using a Lumat LB9507 luminometer (EG&G, Berthold, Germany). ß-Galactosidase activity was also measured and normalized for varying transfection efficiencies. AP-1 activity was calculated as luciferase activity/ß-galactosidase activity. A pTA-basic vector was included as a control in the transfection experiments.

Statistical analysis

Data were subjected to ANOVA, and differences were determined by Tukey’s multiple comparison test. Each experiment was repeated three times in duplicate or triplicate. Data are shown as means of three individual experiments and presented as the mean ± SD. Expression level of MAPK are shown as fold changes compared with control levels. [3H]Thymidine incorporation assay was presented as the percentage of growth compared with control level and is the mean ± SD of three individual experiments with triple samples. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
p38 MAPK activation by GnRH-II in ovarian cancer cells

The phosphorylation of p38 MAPK by GnRH-II was examined in a time-dependent manner at a concentration of 100 nM (24). Treatment with GnRH-II induced a significant activation of p38 MAPK in 2, 5, 10, and 15 min, and the maximally increased level (3.5-fold vs. control) was observed at 10 min (Fig. 1Go). Total-p38 (nonphosphorylated form) activity was not changed by GnRH-II treatment in this time period. To investigate the direct effect of GnRH-II on the activation of p38 MAPK, SB203580 (1 µM), a specific inhibitor of p38 (23), was added 20 min before GnRH-II treatment. The pretreatment with SB203580 completely reversed an activation of p38 MAPK by GnRH-II as seen Fig. 2Go. Treatment with SB203580 alone had no effect on the activation of p38 MAPK.



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  		FIG. 1. Time-dependent effect of GnRH-II on p38 activation in ovarian cancer cell line. OVCAR-3 cells were cultured and treated with GnRH-II (100 nM) in a time-dependent manner. The total (T-p38) and activated p38 (P-p38) levels were analyzed by immunoblot assay, and the intensities of the signals were quantitated. p38 levels are expressed as relative fold change to basal levels. The data were analyzed by ANOVA followed by Tukey’s multiple comparison test. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control.

 


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  		FIG. 2. The effect of SB203580 pretreatment on GnRH-II-induced p38 activation. OVCAR-3 cells were pretreated with 1 µM SB203580 for 20 min, followed by stimulation with 100 nM GnRH-II for 5 min. Control culture was treated with vehicle. The total p38 and activated p38 levels were analyzed by immunoblot assay. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. GnRH-II treatment.

 
Activation of AP-1 by GnRH-II involved in p38 MAPK signal pathway

The AP-1-TA-Luc vector was transfected into OVCAR-3 cells to investigate the relevance of p38 MAPK signal pathway with AP-1 transcriptional factor by GnRH-II treatment. Treatment with GnRH-II (100 nM) resulted in activation of AP-1 transcription factor (1.5-fold vs. control level), as demonstrated in Fig. 3Go. The pretreatment with SB203580 (1 µM) completely reversed the activation of AP-1 induced by GnRH-II treatment (Fig. 3Go).



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  		FIG. 3. The effect of GnRH-II and SB203580 on AP-1 activation. Five hours after transfection, the medium was changed to 10% FBS/DMEM, and, 24 h after transfection, GnRH-II was added. Luc + ß-galactosidase assay was performed with 1 µM SB203580 for 20 min, followed by stimulation with 100 nM GnRH-II for 5 min. Control culture was treated with vehicle. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. GnRH-II treatment.

 
Antiproliferative effect on ovarian cancer cells by GnRH-II

To determine the effect of GnRH-II on cell proliferation, the OVCAR-3 cells were treated with increasing doses of GnRH-II (1 nM, 100 nM, 10 µM) for different time periods (2, 4, and 6 d). Treatment with GnRH-II resulted in a significant decrease of cell proliferation compared with control (Fig. 4Go). Cell proliferation was dose-dependently decreased at each concentration of GnRH-II. After 2 d of treatment with GnRH-II, cell proliferation was decreased only at the 10-µM concentration (72.0 ± 13.4%). After treatment with GnRH-II for 4 d, the inhibitory effect of proliferation was observed at all three concentrations of GnRH-II: 89.2 ± 1.4% at 1 nM, 84.0 ± 1.4% at 100 nM, and 76.9 ± 9.0% at 10 µM, respectively. The inhibitory effect on cell proliferation was also shown in the treatment groups of 1 nM (87.5 ± 4.7%) and 100 nM GnRH-II (75.6 ± 5.0%). The maximal antiproliferative effect was observed in 6-d treatment at the 10-µM concentration of GnRH-II (66.4 ± 9.2%).



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  		FIG. 4. The effect of GnRH-II on ovarian cancer cell proliferation. OVCAR-3 cells were cultured and treated with GnRH-II (1 nM, 100 nM, 10 µM). A [3H]thymidine incorporation assay was performed to quantify DNA synthesis. After 24-h culture to attach to the wells, the medium was changed, and 20 µl GnRH-II was added, resulting in final concentrations. Every 12 h, GnRH-II was added, and every 24 h, the medium was changed. After 2, 4, and 6 d, the cells were incubated with 1 µCi [3H]thymidine for 6 h. Each experiment was repeated three times. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control.

 
To investigate the relevance of the p38 signal pathway in the proliferation of OVCAR-3 cells, SB203580 (1 µM) was added 20 min before GnRH-II (100 nM). This pretreatment with the p38 inhibitor completely reversed the antiproliferative effect of GnRH-II (Fig. 5Go).



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  		FIG. 5. The effect of p38 MAPK activation by GnRH-II on ovarian cancer cell proliferation. OVCAR-3 cells were pretreated with SB203580 before treatment with GnRH-II (100 nM). To quantify the DNA synthesis, a [3H]thymidine incorporation assay was performed as previously described. Treatment with GnRH-II for 6 d induced a significant decrease of growth in OVCAR-3 cells, but pretreatment with SB203580 reversed the effect of GnRH-II. Each experiment was repeated three times. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.05 vs. GnRH-II treatment.

 
Induction of apoptosis by GnRH-II

The cells were treated with GnRH-II for 2, 4, and 6 d, and the DNA fragmentation assay was performed to quantify the induction of apoptosis. When the cells were treated with GnRH-II for 6 d, treatment with GnRH-II (100 nM) resulted in a significant increase in DNA fragmentation by ELISA (1.6-fold vs. control) (Fig. 6AGo). No significant difference was observed after 2- and 4-d treatments with GnRH-II. Furthermore, pretreatment of OVCAR-3 cells with 1 µM SB203580 completely reversed the effect of GnRH-II-induced apoptosis (Fig. 6BGo).



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  		FIG. 6. The effect of GnRH-II in the induction of apoptosis. To quantify the induction of apoptosis, OVCAR-3 cells were cultured and treated with GnRH-II (100 nM) for 2, 4, and 6 d (A). In addition, the cells were cultured for 6 d and pretreated with 1 µM SB203580 for 20 min before treatment with 100 nM GnRH-II (B). The attached and detached cells were collected, and DNA fragmentation was measured by cell death detection ELISA. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In our previous study (27), we demonstrated that, in addition to the GnRH receptor (GnRHR), GnRH-II mRNA is expressed in normal OSE, immortalized OSE (IOSE) cells, primary cultures of ovarian tumors, and ovarian cancer cell lines. In addition, treatments with increasing doses (10–9 to 10–7 M) of GnRH-II resulted in a growth inhibition in IOSE cell lines, suggesting that GnRH-II, similar to GnRH-I, may have a growth-regulatory effect in normal and neoplastic OSE cells (27). Although GnRH-II may have a lesser effect than GnRH-I on the secretion of human choriogonadotropin (hCG) in cytotrophoblastic cells (30), the mRNA expression of GnRH-II was detected at higher levels (up to 30 times more than GnRH-I) in kidney, bone marrow, and prostate than that of GnRH-I (31). GnRH-II receptors binding GnRH-II were found more widely than GnRH-I receptors in the body, including the brain (21, 24, 32), even though a full-length GnRH-II receptor has yet to be cloned in the human (11, 33, 34, 35). Recent evidence that GnRH-II has the high-affinity binding sites for mammalian ovary suggests that GnRH-II may be a potent regulator of ovarian function (36). In addition, it is of interest that treatment with GnRH-II analog induced a stimulation of hCG at the lowest concentration (250 nM), whereas the release of hCG from human placental explants was inhibited at higher concentrations in a placental explant perifusion system (37). This was the first report that the pulsatile release of GnRH-II from the early human placenta was shown, and binding affinity for GnRH-II was greater than that for GnRH-I. The different findings in the activity of GnRH-II compared with GnRH-I may be derived from the different culture systems used, the concentrations of GnRHs used, and differential degradation of GnRHs in different tissues. Furthermore, previous reports suggested that a specific receptor for GnRH-II (type II GnRHR) might exist in human ovarian cancer cells (11, 33). Neill et al. (35) observed that type II GnRHR gene is present in humans and suggested several possible explanations, including frame shift or mRNA editing or the elaborating of partial type II receptor.

As we previously reported (27), the expression of GnRH-II and its inhibitory action of cell growth have been demonstrated in normal OSE and immortalized OSE (IOSE) cells. Grundker et al. (11) further demonstrated that GnRH-II had a more potent antiproliferative effect than GnRH-I in ovarian and endometrial tumors, and GnRH-II receptors are present in OVCAR-3 cells, suggesting that the antiproliferative effect of GnRH-II might be mediated through GnRH-II receptor in these gynecological tumor cells. In the present study, the treatment with GnRH-II resulted in a significant antiproliferative effect in a dose-dependent manner in OVCAR-3 cells, which is a neoplastic OSE cell line derived from the ascites of ovarian cancer patients. Despite a role of GnRH-II in the inhibition of cell growth in ovarian cancer cells compared with control, it appears that no significant difference was observed in the antiproliferative effect of GnRH-II at various doses (1 nM to 10 µM), suggesting that the effect of GnRH-II is very specific at these concentrations, and type II GnRHR would be one of the factors to regulate a function of GnRH-II. It is noteworthy that the effect of GnRH-II on cell proliferation and apoptosis does not seem to be significantly different from that of GnRH-I. The discrepancy with a previous report (11) that GnRH-II has a more potent antiproliferative effect than GnRH-I might be a result of the use of different cell types and culture conditions between the two studies.

To investigate the signal pathway involved in the antiproliferative effect by GnRH-II, the activation of p38, one of the MAPKs, was measured by Western blot analysis using the phospho-specific MAPK antibody after treatment with GnRH-II. p38 MAPK is activated by the phosphorylation on threonine 180 and tyrosine 182 in the activation loop and modulates cell cycle, transcriptional activity, and programmed cell death for the response to environmental stress, hormones, ligands that bind to G protein-coupled receptors, and inflammatory cytokines (16). We observed that GnRH-II (100 nM) induced the activation of p38 MAPK (3.5-fold vs. control) in as early as 10 min, which is in agreement with a previous study performed in COS-7 cells (24). Furthermore, the activation of p38 MAPK was completely blocked by SB203580 (1 µM), a specific inhibitor of p38 MAPK (23). Previously, it has been shown that GnRH-I induced the activation of p38 as well as ERK1/ERK2 and JNK in pituitary cell lines (38, 39, 40, 41), which was reversed by chronic phorbol ester treatment. Further research is required to elucidate the relative effect of GnRH-I vs. GnRH-II on activation of the p38 MAPK pathway in ovarian cancer cells.

In this study, treatment with increasing doses of GnRH-II for 2, 4, and 6 d decreased the amount of [3H]thymidine incorporation in DNA synthesis in OVCAR-3 cells. This result is in agreement with the previous report using the cell counting method with the Neubauer hemocytometer (11). To elucidate the signal pathway involved in p38 MAPK in this antiproliferative effect by GnRH-II, SB203580 was pretreated, followed by treatment with GnRH-II (100 nM) for 2, 4, and 6 d. It is of interest that an antiproliferative effect of GnRH-II was reversed in the presence of SB203580, indicating that the activation of p38 MAPK may play a crucial role in the regulation of cell proliferation in ovarian cancer. It has been reported that the activation of ERK is also involved in the antiproliferative effect of GnRH-I in the Caov-3 human ovarian cancer cell line (42). Although our preliminary results indicated that GnRH-II activated the ERK1/ERK2 but not JNK/SAPK1 in OVCAR-3 cells (data not shown), whether or not other members of the MAPK pathways are involved in the antiproliferative action of GnRH-II remains to be elucidated.

The importance of apoptosis in tumor cells has been widely recognized as a critical phenomenon to regulate cell proliferation. It has been reported that GnRH-II induced apoptosis in goldfish testis (43), and GnRH-I has been reported to induce apoptosis by several groups. It is noteworthy that the effect of GnRH-I analogs in the induction of apoptosis is still controversial (44, 45, 46, 47, 48). Recent data from our laboratory reported that GnRH-II had an antiproliferative effect on IOSE cells (27). Interestingly, the results presented herein demonstrate that a significant increase of apoptosis (1.6-fold) was induced after GnRH-II treatment in OVCAR-3 cells, which is the first report of an induction of apoptosis by GnRH-II in ovarian cancer cells. The increase in DNA fragmentation induced by GnRH-II was completely inhibited by SB203580 pretreatment, suggesting that the p38 MAPK signaling cascade plays a role in the induction of apoptosis by GnRH-II in these cells.

To clarify the signaling cascade in the induction of apoptosis, the role of AP-1 by GnRH-II was further investigated. AP-1 is thought to regulate cell proliferation, differentiation, and apoptosis in different systems (49, 50). It is known that MAPKs contribute to the induction of AP-1 activity, which is involved in a protective function against the effects of UV-induced cell damage (18, 51, 52). It is interesting that GnRH-I not only stimulated the activation of Elk-1 through MAPK in {alpha}T3 cells, but its analog also activated AP-1 transcriptional factor in human endometrial cancer cells (53, 54), which is composed of c-Fos and c-Jun that induce a cellular response after binding to AP-1 response elements in gene promoter. Our observation of a GnRH-II effect on AP-1 activation is consistent with these findings. The transfection assay using AP-1-TA-Luc vector revealed that GnRH-II induced the activation of AP-1 (1.5-fold vs. control level). The pretreatment with SB203580 blocked the GnRH-II-induced effect on the activation of AP-1, suggesting that GnRH-II induces a sequential activation of AP-1 after activation of p38 MAPK.

In conclusion, we have identified the p38 MAPK signaling pathway as an integral component in the GnRH-II signaling cascade and demonstrated that the antiproliferative effect of GnRH-II in ovarian cancer cells may involve, at least in part, the activation of p38 MAPK. Furthermore, we have demonstrated for the first time the induction of apoptosis by GnRH-II in ovarian cancer cells and demonstrated that GnRH-II induced the activation of AP-1 transcription factor via p38 MAPK, implicating a potential role of AP-1 in ovarian cancer cell growth. Further study of the relationship between the GnRH-II and signaling pathways involved in cell proliferation and apoptosis will provide a better insight for the understanding of ovarian cancer and design of novel therapeutic approaches.


   Acknowledgments
 
The epithelial ovarian cancer cell line OVCAR-3, an ovarian adenocarcinoma derived from the ascites of ovarian cancer patients, was kindly provided by Dr. T. C. Hamilton (Fox Chase Cancer Center, Philadelphia, PA).


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research. P.C.K.L. is the recipient of a Distinguished Scholar Award from the Michael Smith Foundation for Health Research.

Abbreviations: AP-1, Activator protein-1; FBS, fetal bovine serum; GnRHR, GnRH receptor; hCG, human choriogonadotropin; JNK, c-Jun N-terminal protein kinase; SAPK, stress-activated protein kinase; SDS, sodium dodecyl sulfate.

Received October 27, 2003.

Accepted February 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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