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Extracellular Signal-Regulated Protein Kinase, But Not c-Jun N-Terminal Kinase, Is Activated by Type II Gonadotropin-Releasing Hormone Involved in the Inhibition of Ovarian Cancer Cell Proliferation

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, Room 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

 
Although a novel second form of GnRH (GnRH-II) has been reported to have an antiproliferative effect on gynecologic cancer cells, its biological mechanism remains to be elucidated. We have previously demonstrated that GnRH-II activates p38 MAPK. There is accumulating evidence that activation of MAPKs by GnRH-I and -II is important for cell proliferation, differentiation, and apoptosis. In the present study, we further investigated the involvement of GnRH-II in the inhibition of cell proliferation and activation of ERK1/2 and c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK) in ovarian cancer cells, OVCAR-3. The [³H]thymidine incorporation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that treatment with GnRH-II suppresses cell proliferation of ovarian cancer cells. Western blot analysis demonstrated that ERK1/2 was activated by GnRH-II (100 nM). Moreover, PD98059 (10 µM), an inhibitor of a MAPK/ERK kinase, reversed the activation of ERK1/2 induced by GnRH-II. The activation of ERK1/2 by GnRH-II subsequently phosphorylated Elk-1 as a downstream pathway, which was blocked by PD98059. On the other hand, it is not likely that GnRH-II activates the JNK/SAPK pathway. Taken together, these results indicate that the ERK1/2 pathway is involved in the effect of GnRH-II on antiproliferation and may be an important target for ovarian cancer therapy.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
GnRH, ALSO KNOWN as LHRH, is a key molecule of the hypothalamus-pituitary-gonadal axis in the reproduction of vertebrates. In addition to its well known physiological roles in the regulation of gonadotropins in the brain, GnRH has also been used for the therapy of reproductive cancers (1). It has been reported that agonists of a classical form of GnRH (GnRH-I) suppressed various tumors by blocking the secretion of FSH and LH from pituitary gland, called chemical castration. In addition, direct application is considered important for the induction of apoptosis or suppression of proliferation (2, 3). Because a second form of GnRH (GnRH-II) was identified in mammals, the role and possible mechanism of action of GnRH-II has been investigated in normal physiology and cancer cells (4). Although it has been noted that GnRH-II induced the secretion of human chorionic gonadotropin (hCG) in cytotrophoblastic cells, the normal physiological function of GnRH-II is poorly understood (5). In mammals, GnRH-II receptors have been found to be more widely expressed than GnRH-I receptors in the body, suggesting that GnRH-II may have additional various functions (6). The expression and potential antiproliferative effect of GnRH-II indicate that GnRH-II, similar to GnRH-I, may have a growth-regulatory effect in normal and neoplastic ovarian surface epithelial cells (7). Furthermore, a recent study demonstrated that GnRH-II has an antiproliferative effect in gynecologic tumors and may exert a stronger antiproliferative effect than GnRH-I in ovarian cancer cells, suggesting that GnRH-II could be considered as a novel target for antiproliferative therapeutic approaches (4, 8). However, in contrast to GnRH-I, the biological mechanism of GnRH-II remains obscure.

MAPK cascades are activated via two distinct classes of cell surface receptors, receptor tyrosine kinases and G protein-coupled receptors. Signals transmitted through these cascades induce the activation of diverse molecules that regulate cell growth, survival, and differentiation (9, 10, 11). ERK1 (p44 MAPK) and ERK2 (p42 MAPK) are activated by mitogenic stimuli and represent a group of the most extensively studied members. In contrast, c-Jun N-terminal protein kinase/stress-activated protein kinase (JNK/SAPK1) and p38 MAPK are activated in response to stress such as heat shock, osmotic shock, cytokines, protein synthesis inhibitors, antioxidants, UV light, and DNA-damaging agents (12, 13, 14, 15). MAPK family members are directly regulated by kinases known as MAPK kinases, which activate MAPKs by phosphorylation of tyrosine and threonine residues (15, 16). It has been reported that ERK1/2 is involved in cell cycle arrest and the inhibition of growth (17, 18, 19) as well as cell survival and differentiation (9). The role of the MAPK family in the antiproliferative effect of GnRH in the CaOV-3 ovarian cancer cell line has been demonstrated (20). In our previous reports, we demonstrated that FSH stimulated activation of the ERK1/2 cascade and phosphorylated Elk-1 in neoplastic ovarian surface epithelial cells (21) and that the p38 MAPK pathway is involved in the antiproliferative effect of GnRH-II in ovarian cancer cells (4).

Although it is difficult to define each of the mechanisms involved in the regulation of MAPKs in response to external stimuli, it is important to clarify the specific signaling pathways used by GnRH-II. Increasing our understanding of GnRH-II signaling pathways may improve the efficacy of chemotherapy using these agonists in ovarian cancer treatment. Thus, in the present study, we examined the effect of GnRH-II on the activation of ERK1/2 and JNK/SAPK1 in ovarian cancer cells. In addition, the possible involvement of the ERK1/2 pathway in mediating the antiproliferative effects of GnRH-II was investigated in ovarian cancer cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

A GnRH-II analog, D-Arg(6)-Azagly(10)-NH₂, was purchased from Peninsula Laboratories (Belmont, CA). PD98059, a MAPK/ERK kinase (MEK) inhibitor, was purchased from New England Biolabs Inc. (Beverly, MA) and was dissolved in dimethylsulfoxide (DMSO).

Cell culture and treatment

Human ovarian adenocarcinoma cell lines, OVCAR-3 and SKOV-3, were cultured as previously described (4, 22, 23). The nontumorigenic SV40 Tag-immortalized ovarian surface epithelium (OSE)-derived cell line, and IOSE-80 post crisis (IOSE-80PC), were cultured in the above mentioned culture conditions and used in the present study. Briefly, the cells were cultured in medium 199:MCDB 105 (Sigma-Aldrich Corp., St. Louis, MO) supplemented with 10% 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 and 95% air. Cells were trypsinized with 0.06% trypsin (1:250)/0.01% EDTA (Life Technologies) in Mg²⁺/Ca²⁺-free Hank’s 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. Cells were washed once with medium and serum starved for 6 h before GnRH-II treatment. To investigate the direct effect on ERK1/2, the cells were pretreated with 10 µM PD98059 for 1 h and then treated with GnRH-II (100 nM).

Immunoblot analysis

Immunoblot analysis was carried out as previously described (4, 24). The extracts were run on a 10% SDS-PAGE gel and transferred to nitrocellulose membrane. The membrane was immunoblotted using a rabbit polyclonal antibody for phosphorylated ERK1/2 and JNK/SAPK (Biosource International Inc., Camarillo, CA) (25, 26) with protein molecular marker (New England Biolabs, Inc., Mississauga, Ontario, Canada). Alternatively, the membrane was probed with pan ERK1/2 and pan JNK/SAPK1 antibodies (Biosource International), which detect total ERK1/2 and JNK/SAPK1 level, respectively. The intensity of signals was quantitated by densitometry (BioDocAnalyze, Biometra, Germany). The activity of ERK1/2 and JNK/SAPK was represented as a ratio of phosphorylated MAPKs (P-MAPKs) to total MAPKs (T-MAPKs).

In vitro MAPK assay

OVCAR-3 cells were seeded at a density of 4 x 10⁵ cells in 60-mm dishes (Corning, Corning Laboratory Sciences Co., Corning, NY) and cultured for 2 d. After treatment with GnRH-II in the presence or absence of PD98059, protein extracts were prepared under the conditions described above. Cellular protein (200 µg) was immunoprecipitated with an immobilized phospho-ERK1/2 MAPK monoclonal antibody. The in vitro MAPK assay was performed using an Elk-1 fusion protein as a substrate for activated MAPK, according to the manufacturer’s suggested procedure (New England Biolabs).

Proliferation assay

The proliferation assay was performed using [³H]thymidine incorporation as previously described (7, 27, 28). Briefly, 2 x 10⁴ OVCAR-3 cells were plated in 24-well dishes in 0.5 ml medium as described above. GnRH-II was appropriately diluted with medium, and the cells were treated with a final concentration of GnRH-II (100 nM) after 24 h of incubation. Cells were treated for 4 d with medium changes every 24 h. After treatment with GnRH-II, the cells were then incubated with medium containing 1 µCi [³H]thymidine (0.5 Ci/mmol; Amersham Pharmacia Biotech Inc., Piscataway, NJ) and collected after 6 h incubation. To block the activation of ERK1/2, the cells were pretreated with PD98059 (10 µM) for 1 h, followed by the addition of GnRH-II (100 nM final concentration) or vehicle.

MTT assay

Cell viability was estimated by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] (MTT) (Sigma-Aldrich Corp.) assay. OVCAR-3 cells were seeded onto 96-well dishes. The MTT colorimetric assay was performed to detect tumor cell viability after 96 h of incubation (29). The cells were incubated at 37 C with 50 µl MTT solution (2 mg/ml in PBS) for 4 h. The supernatants were removed and the cells were solubilized in DMSO (200 µl) for 30 min. The OD at 570 nm was determined using an ELISA reader (Fisher Scientific Ltd., Ottawa, Canada).

Statistical analysis

The results of three separate experiments are presented as the mean ± SD. Each individual experiment was performed in duplicate or triplicate. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of GnRH-II on the activation of ERK1/2 in ovarian cancer cells

To investigate the activation of ERK1/2 in ovarian cancer cell lines and immortalized OSE-derived cell lines, the cells were treated with GnRH-II (100 nM) in a time-dependent manner. The phosphorylation of ERK1/2 was examined using the antibodies targeting ERK1/2 and T-ERK1/2 (activated plus inactivated forms). Treatment with GnRH-II (100 nM) activated ERK1/2 in OVCAR-3, SKOV-3, IOSE-80PC, and IOSE-80 cell lines in a time-dependent manner (Fig. 1A). As seen in Fig. 1B, GnRH-II activated ERK1/2 significantly in OVCAR-3 cells, and the maximal level of ERK1/2 activation was observed (2-fold increase) at 10 min. These results indicate that GnRH-II activates ERK1/2 in both ovarian carcinomas and IOSE cell lines.

View larger version (40K): [in this window] [in a new window]   FIG. 1. Effect of GnRH-II on ERK1/2 in ovarian adenocarcinomas and IOSE cell lines. The cells were treated with GnRH-II (100 nM) in a time-dependent manner. The T-ERK1/2 and P-ERK1/2 levels were analyzed by immunoblot assay. EKR1/2 level is expressed as a fold change relative to basal level. 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.

To determine the activation of JNK/SAPK1 by GnRH-II, we treated OVCAR-3 cells with GnRH-II (100 nM) in a time-dependent manner. In contrast to the activation of ERK1/2, no difference was observed in P-JNK/SAPK1 levels after treatment with GnRH-II (Fig. 2). In addition, GnRH-II had no effect on the activation of JNK/SAPK1 in SKOV-3, IOSE-80, and IOSE-80PC cells (data not shown).

View larger version (44K): [in this window] [in a new window]   FIG. 2. Effect of GnRH-II on the activation of JNK/SAPK1. The T-JNK/SAPK1 and P-JNK/SAPK1 levels were analyzed by immunoblot assay. JNK/SAPK1 level is expressed as a fold change relative to basal level. Values are represented as the mean ± SD of three individual experiments.

View larger version (43K): [in this window] [in a new window]   FIG. 3. Effect of GnRH-II on ERK1/2 activation in the presence or absence of PD98059. OVCAR-3 cells were pretreated with PD98059 (10 µM) for 1 h, followed by treatment with GnRH-II (100 nM) for 10 min. A control was treated with vehicle. The T-ERK1/2 and P-ERK1/2 levels were analyzed by immunoblot assay. EKR1/2 level is expressed as a fold change relative to basal level. Values are represented as the mean ± SD of three individual experiments. a, P < 0.01 vs. control.

To investigate whether GnRH-II-induced activation of ERK1/2 leads to phosphorylation of Elk-1 in vitro as a downstream pathway of MAPK, the cells were treated with GnRH-II (100 nM) for 10 min in the presence or absence of PD98059 (10 µM) 9 for 1 h. As shown in Fig. 4, the treatment with GnRH-II resulted in a significant increase in Elk-1 phosphorylation, whereas pretreatment with PD98059 significantly reduced GnRH-II-induced Elk-1 phosphorylation in OVCAR-3 cells.

View larger version (32K): [in this window] [in a new window]   FIG. 4. Effect of GnRH-II in the presence or absence of PD98059 on Elk-1 phosphorylation. To evaluate whether GnRH-II induced ERK1/2 activation leading to the activation of Elk-1 in vitro, the cells were pretreated with PD98059 (10 µM) for 1 h, followed by treatment with GnRH-II (100 nM) for 10 min. A control was treated with vehicle. Values are represented as the mean ± SD of three individual experiments. a, P < 0.05 vs. control; b, P < 0.01 vs. GnRH-II treatment.

To determine the role of GnRH-II in ovarian cancer, we further examined the effect of GnRH-II on proliferation by thymidine incorporation and MTT assays in OVCAR-3 cells. The cells were treated for 4 d with 100 nM GnRH-II followed by 24 h culture. As illustrated in Fig. 5, treatment with GnRH-II (100 nM) induced a decrease in cell proliferation in OVCAR-3 cells as assessed by thymidine incorporation assay. To confirm the antiproliferative effect of GnRH-II, cellular viability was measured by MTT assay. Treatment with GnRH-II (100 nM) resulted in a significant reduction in cell viability after 4 d of treatment (Fig. 6). Furthermore, to elucidate the relevance of ERK1/2 activation in the proliferation of ovarian cancer cells, we challenged OVCAR-3 cells with 10 µM PD98059. Pretreatment with PD98059 (10 µM) completely abolished the antiproliferative effect induced by GnRH-II (Fig. 5), suggesting that the MEK/MAPK pathway mediates the antiproliferative effect of GnRH-II in ovarian cancer cells.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effect of PD98059 on GnRH-II-induced growth inhibition. OVCAR-3 cells were treated with GnRH-II (100 nM) in the presence or absence of the MEK inhibitor PD98059 (10 µM). A [3H]thymidine incorporation assay was performed to quantify s-phase growing cells. After 24-h culture, the medium was changed and 20 µl of GnRH-II (100 nM) was added. Then, the radioactivity of thymidine-incorporated cells was measured by ß-counter as mentioned in Materials and Methods. Data are shown as the means of three individual experiments and are presented as the mean ± SD. a, P < 0.05 vs. control.

View larger version (10K): [in this window] [in a new window]   FIG. 6. Effect of GnRH-II on cell viability. The cells were seeded into 96-well plates at a density of 104 cells per well. The cells were treated with GnRH-II (100 nM) for 4 d, and cell viability was measured by MTT assay. 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, we demonstrated that the antiproliferative effect of GnRH-II in ovarian cancer cells involve p38 MAPK, which led us to investigate the role of other MAPK family members. Over 12 different types of MAPKs have been identified in mammalian cells. ERK1/2, JNK/SAPK1, and p38/SAPK2 are three of the best-characterized MAPK family members that exert their effects via the activation of transcription factors resulting in cellular responses such as cell proliferation or apoptosis (13, 14, 15, 16).

In this study, we demonstrated that treatment with GnRH-II activated ERK1/2 in immortalized OSE and ovarian cancer cell lines in a diverse pattern. It is of interest to note that GnRH-II appears to activate ERK1/2 in a different time manner in these cell lines, indicating that different signal pathways may be involved in the GnRH-II-induced ERK1/2 activation in different ovarian cell types. Moreover, PD98059, an inhibitor of MEK, markedly attenuated the activation of ERK1/2 by GnRH-II in OVCAR-3 cells. This is in agreement with previous studies that demonstrated that GnRH-I activates ERK1/2 in normal and ovarian cancer cells (20, 22). The present results indicate that the ERK1/2 pathway might be an important signaling pathway mediating the effects of GnRH-II in ovarian cancer cells. It has been shown that treatment of CaOV-3 cells with GnRH results in an activation of ERK at 5 min with maximal activation occurring at 3 h and sustained until 24 h, whereas GnRH had no effect on the activation of the JNK (20). In addition, ERK1/2 kinase was also activated, and an increase in phosphorylation of son of sevenless (Sos) and Shc was observed after GnRH treatment. Treatment with a MEK inhibitor, PD98059, reduced the antiproliferative effect of GnRH analog and the GnRH-induced dephosphorylation of the retinoblastoma protein, indicating that the activation of ERK may play an important role in the antiproliferative effect of GnRH (20). In parallel with the previous study, ERK may play a critical role in GnRH-II-induced antiproliferation in ovarian cancer cells. Furthermore, GnRH-I agonist activated the JNK pathway in endometrial cancer cells (38) and the T3-1 gonadotroph cell line (39) but not in ovarian cancer cells (20). In this study, JNK/SAPK1 was not activated by GnRH-II in OVCAR-3 cells. This is in agreement with a previous report using GnRH-I agonist in CaOV-3 cells (20). In addition, JNK/SAPK1 was not activated by GnRH-II in SKOV-3 cells, an ovarian cancer cell line (data not shown), suggesting that the JNK/SAPK1 pathway may not be involved in GnRH-I and -II signaling to induce cellular responses. We have also examined the effect of GnRH-II on TE671, a human brain tumor cell line, using an antibody to phospho-JNK/SAPK1, and demonstrated that GnRH-II phosphorylates JNK/SAPK1 in this cell line until 60 min of treatment (unpublished data). Taken together, these results indicate that the effects of GnRH-I and GnRH-II on the activation of ERK1/2 but not JNK/SAPK1 may be identical in ovarian cancer cells.

Activated MAPKs translocate from the cytoplasm to the nucleus and activate transcription factors. ERK1/2 induces gene expression by the activation of transcription factors including the phosphorylation of ternary complex factors such as Elk-1 and SAP-1 (40, 41, 42). Elk-1, an Ets family transcription factor, is a physiological substrate for ERK1/2 and mediates the c-fos and other coregulated gene activity through the serum response element. Therefore, the ability of GnRH-II to activate a downstream pathway of ERK1/2 was examined using the immunoprecipitation method. In this study, the treatment with GnRH-II resulted in substantial phosphorylation of Elk-1 fusion protein in vitro. Furthermore, PD98059, an inhibitor of MEK, abolished the effect of GnRH-II on the phosphorylation of Elk-1, suggesting that GnRH-II-induced ERK1/2 activation resulted in the phosphorylation of Elk-1, possibly mediating cellular response in ovarian cancer cells. In our previous study, we demonstrated the in vitro effect of GnRH-II in regard to inhibited cell growth and induced apoptosis in the ovarian cancer cell line OVCAR-3 (4). To confirm its effect on the inhibition of tumor growth, [³H]thymidine incorporation and MTT assays were performed. After a 4-d treatment, GnRH-II (100 nM) inhibited cell growth and cell viability. The importance of MEK-MAPK in cell proliferation and apoptosis is now widely recognized (43). It has been reported that inhibition of the ERK1/2 signaling pathway with PD98059 may affect the growth of prostate and breast tumors (44, 45), and PD98059 restored cell proliferation inhibition (46). In addition, it has been observed that PD98059 reduced the antiproliferative effect GnRH-I, suggesting that the MEK-MAPK pathway has a critical role in the effect of GnRH-I (20). Therefore, we investigated the involvement of the MEK-MAPK pathway in the antiproliferative effect of GnRH-II on ovarian cancer cells. In the present study, GnRH-II-induced growth inhibition in ovarian cancer cells was completely abolished by PD98059, suggesting that ERK1/2 mediate the antiproliferative effect of GnRH-II and that the MEK-MAPK pathway may be an integral mediator of GnRH-induced functions such as cell growth and/or apoptosis. In the previous study, p38 MAPK is involved in the GnRH-II-induced inhibition of cell growth through activator protein-1 (AP-1) activation, which may be related to induction of apoptosis in ovarian cancer cells (4). It has been known that PD98059 is a specific inhibitor for MEK1 and does not block p38 MAPK (47), and SB203580 is also a specific inhibitor of p38 MAPK (48). Furthermore, we have tested the effect of PD98059 or SB203580 to examine the specificity of these inhibitors and found that PD98059 could not block an activation of p38 in the preliminary experiment. It can be hypothesized that p38 MAPK is involved in the GnRH-II-induced apoptotic pathway via AP-1 transcriptional factor and that ERK1/2 MAPK is involved in GnRH-II-induced cell growth inhibition via Elk-1 transcriptional factor in ovarian cancer cells as proposed in Fig. 7. However, we cannot rule out the possibility that the ERK1/2 pathway activated by GnRH-II may induce subsequent AP-1 transcriptional activation or that the p38 MAPK pathway by GnRH-II may result in Elk-1 phosphorylation in this cell type. It has been reported that the MAPK pathways are regulated by other signaling pathways (49); thus, further study is necessary to investigate a possible involvement of other pathways in this cellular response.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Proposed GnRH-II-induced signaling pathways in ovarian cancer. GnRH-II activates p38 MAPK (4 ) and ERK1/2 but not JNK in our previous and present studies. The activation of p38 and ERK activates transcriptional factors such as AP-1 and Elk-1, respectively. Furthermore, the antiproliferative effect of GnRH-II appears to be mediated by the activation of these MAPKs.

In conclusion, the present study demonstrates that treatment with GnRH-II induces the phosphorylation of ERK1/2 in OVCAR-3 cells, which was reduced by a MEK inhibitor, PD98059. In an in vitro kinase assay, treatment with GnRH-II resulted in the phosphorylation of Elk-1, and this effect was also blocked by PD98059. This suggests that Elk-1 may mediate cellular responses to GnRH-II-induced ERK1/2 activation. Furthermore, the growth inhibitory effect of GnRH-II was attenuated by PD98059. These results, taken together with our previous study, strongly suggest that GnRH-II-induced MAPK activation, including ERK1/2 and p38 but not JNK/SAPK1, mediate cellular responses such as growth inhibition and induction of apoptosis in ovarian cancer cells and may be a potential target in the treatment of ovarian cancer.

	Acknowledgments

 
We are grateful to Dr. Catherine J. Pallen at the British Columbia Research Institute for Children’s and Women’s Health, University of British Columbia, for critical review of the manuscript.

	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. K.-Y.K. is the recipient of a travel award from the Society for the Study of Reproduction 2004 Annual Meeting (Vancouver, British Columbia, Canada) to present this paper and graduate studentship awarded from the British Columbia Research Institute for Children’s and Women’s Health. The epithelial ovarian cell lines, OVCAR-3 and IOSE-80PC, were kindly provided by Drs. T. C. Hamilton and A. Godwin (Fox Chase Cancer Center, Philadelphia, PA), respectively.

First Published Online December 14, 2004

Abbreviations: AP-1, Activator protein 1; DMSO, dimethylsulfoxide; JNK, c-Jun N-terminal protein kinase; MEK, MAPK kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OSE, ovarian surface epithelium; P, phosphorylated; SAPK, stress-activated protein kinase; T, total.

Received August 16, 2004.

Accepted December 7, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kiesel L, Rody A, Greb R, Szilagyi A 2002 Clinical use of GnRH analogues. Clin Endocrinol (Oxf) 56:677–687[CrossRef][Medline]
2. Kang S, Cheng K, Nathwani P, Choi K, Leung P 2000 Autocrine role of gonadotropin-releasing hormone and its receptor in ovarian cancer cell growth. Endocrine 13:297–304[CrossRef][Medline]
3. Florio S, Pagnini U, Crispino A, Pacilio C, Crispino L, Giordano A 2002 GnRH and steroids in cancer. Front Biosci 7:d1590–d1608
4. Kim KY, Choi KC, Park SH, Chou CS, Auersperg N, Leung PC 2004 Type II gonadotropin-releasing hormone stimulates p38 mitogen-activated protein kinase and apoptosis in ovarian cancer cells. J Clin Endocrinol Metab 89:3020–3026[Abstract/Free Full Text]
5. Islami D, Chardonnens D, Campana A, Bischof P 2001 Comparison of the effects of GnRH-I and GnRH-II on HCG synthesis and secretion by first trimester trophoblast. Mol Hum Reprod 7:3–9[Abstract/Free Full Text]
6. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641[Abstract/Free Full Text]
7. Choi KC, Auersperg N, Leung PC 2001 Expression and antiproliferative effect of a second form of gonadotropin-releasing hormone in normal and neoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab 86:5075–5078[Abstract/Free Full Text]
8. Grundker C, Gunthert AR, Millar RP, Emons G 2002 Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. J Clin Endocrinol Metab 87:1427–1430[Abstract/Free Full Text]
9. Mansour S, Matten W, Hermann A, Candia J, Rong S, Fukasawa K, Vande Woude G, Ahn N 1994 Transformation of mammalian cells by constitutively active MAP kinase kinase. Science 265:966–970[Abstract/Free Full Text]
10. Smalley K 2003 A pivotal role for ERK in the oncogenic behaviour of malignant melanoma? Int J Cancer 104:527–532[CrossRef][Medline]
11. Choi KC, Auersperg N, Leung PC 2003 Mitogen-activated protein kinases in normal and (pre)neoplastic ovarian surface epithelium. Reprod Biol Endocrinol 1:71[CrossRef][Medline]
12. Kennedy N, Davis R 2003 Role of JNK in tumor development. Cell Cycle 2:199–201[Medline]
13. Lin A 2003 Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays 25:17–24[CrossRef][Medline]
14. Garrington TP, Johnson GL 1999 Organization and regulation of mitogen-activated protein kinase signaling pathways. Curr Opin Cell Biol 11:211–218[CrossRef][Medline]
15. Cobb MH, Goldsmith EJ 1995 How MAP kinases are regulated. J Biol Chem 270:14843–14846[Free Full Text]
16. Robinson MJ, Cobb MH 1997 Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9:180–186[CrossRef][Medline]
17. Herskowitz I 1995 MAP kinase pathways in yeast: for mating and more. Cell 80:187–197[CrossRef][Medline]
18. Alblas J, Slager-Davidov R, Steenbergh P, Sussenbach J, van der Burg B 1998 The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells. Oncogene 16:131–139[CrossRef][Medline]
19. Yen A, Roberson M, Varvayanis S, Lee A 1998 Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrest. Cancer Res 58:3163–3172[Abstract/Free Full Text]
20. Kimura A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N, Murata Y 1999 Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line. Cancer Res 59:5133–5142[Abstract/Free Full Text]
21. Choi KC, Kang SK, Tai CJ, Auersperg N, Leung PC 2002 Follicle-stimulating hormone activates mitogen-activated protein kinase in preneoplastic and neoplastic ovarian surface epithelial cells. J Clin Endocrinol Metab 87:2245–2253[Abstract/Free Full Text]
22. Kang SK, Tai CJ, Cheng KW, Leung PC 2000 Gonadotropin-releasing hormone activates mitogen-activated protein kinase in human ovarian and placental cells. Mol Cell Endocrinol 170:143–151[CrossRef][Medline]
23. Choi KC, Kang SK, Nathwani PS, Cheng KW, Auersperg N, Leung PC 2001 Differential expression of activin/inhibin subunit and activin receptor mRNAs in normal and neoplastic ovarian surface epithelium (OSE). Mol Cell Endocrinol 174:99–110[CrossRef][Medline]
24. Choi KC, Kang SK, Tai CJ, Auersperg N, Leung PC 2001 The regulation of apoptosis by activin and transforming growth factor-ß in early neoplastic and tumorigenic ovarian surface epithelium. J Clin Endocrinol Metab 86:2125–2135[Abstract/Free Full Text]
25. Akagi T, Shishido T, Murata K, Hanafusa H 2000 v-Crk activates the phosphoinositide 3-kinase/AKT pathway in transformation. Proc Natl Acad Sci USA 97:7290–7295[Abstract/Free Full Text]
26. Lee SA, Dritschilo A, Jung M 2001 Role of ATM in oxidative stress-mediated c-Jun phosphorylation in response to ionizing radiation and CdCl2. J Biol Chem 276:11783–11790[Abstract/Free Full Text]
27. Choi KC, Kang SK, Tai CJ, Auersperg N, Leung PC 2001 Estradiol up-regulates antiapoptotic Bcl-2 messenger ribonucleic acid and protein in tumorigenic ovarian surface epithelium cells. Endocrinology 142:2351–2360[Abstract/Free Full Text]
28. Kang SK, Choi KC, Tai CJ, Auersperg N, Leung PC 2001 Estradiol regulates gonadotropin-releasing hormone (GnRH) and its receptor gene expression and antagonizes the growth inhibitory effects of GnRH in human ovarian surface epithelial and ovarian cancer cells. Endocrinology 142:580–588[Abstract/Free Full Text]
29. Mohseni H, Zaslau S, McFadden D, Riggs DR, Jackson BJ, Kandzari S 2004 COX-2 inhibition demonstrates potent anti-proliferative effects on bladder cancer in vitro. J Surg Res 119:138–142[CrossRef][Medline]
30. Schally AV, Comaru-Schally AM, Nagy A, Kovacs M, Szepeshazi K, Plonowski A, Varga JL, Halmos G 2001 Hypothalamic hormones and cancer. Front Neuroendocrinol 22:248–291[CrossRef][Medline]
31. Zidan J, Zohar S, Mijiritzky I, Kral S, Bilenca B 2002 Treating relapsed epithelial ovarian cancer with luteinizing hormone-releasing agonist (goserelin) after failure of chemotherapy. Isr Med Assoc J 4:597–599[Medline]
32. Schally AV 1999 Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis. Peptides 20:1247–1262[CrossRef][Medline]
33. Savino L, Baldini B, Susini T, Pulli F, Antignani L, Massi GB 1992 GnRH analogs in gynecological oncology: a review. J Chemother 4:312–320[Medline]
34. Santen R, Manni A, Harvey H, Redmond C 1990 Endocrine treatment of breast cancer in women. Endocr Rev 11:221–265[Abstract/Free Full Text]
35. Siler-Khodr TM, Grayson M, Eddy CA 2003 Action of gonadotropin-releasing hormone II on the baboon ovary. Biol Reprod 68:1150–1156[Abstract/Free Full Text]
36. Ling K, Wang P, Zhao J, Wu YL, Cheng ZJ, Wu GX, Hu W, Ma L, Pei G 1999 Five-transmembrane domains appear sufficient for a G protein-coupled receptor: functional five-transmembrane domain chemokine receptors. Proc Natl Acad Sci USA 96:7922–7927[Abstract/Free Full Text]
37. Neill JD 2002 GnRH and GnRH receptor genes in the human genome. Endocrinology 143:737–743[Abstract/Free Full Text]
38. Grundker C, Emons G 2003 Role of gonadotropin-releasing hormone (GnRH) in ovarian cancer. Reprod Biol Endocrinol 1:65[CrossRef][Medline]
39. Levi NL, Hanoch T, Benard O, Rozenblat M, Harris D, Reiss N, Naor Z, Seger R 1998 Stimulation of Jun N-terminal kinase (JNK) by gonadotropin-releasing hormone in pituitary T3–1 cell line is mediated by protein kinase C, c-Src, and CDC42. Mol Endocrinol 12:815–824[Abstract/Free Full Text]
40. Babu PS, Krishnamurthy H, Chedrese PJ, Sairam MR 2000 Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor: role in hormone signaling and cell proliferation. J Biol Chem 275:27615–27626[Abstract/Free Full Text]
41. Gille H, Kortenjann M, Thomae O, Moomaw C, Slaughter C, Cobb MH, Shaw PE 1995 ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation. EMBO J 14:951–962[Medline]
42. Janknecht R, Ernst WH, Pingoud V, Nordheim A 1993 Activation of ternary complex factor Elk-1 by MAP kinases. EMBO J 12:5097–5104[Medline]
43. Lewis T, Shapiro P, Ahn N 1998 Signal transduction through MAP kinase cascades. Adv Cancer Res 74:49–139[Medline]
44. Reddy KB, Krueger JS, Kondapaka SB, Diglio CA 1999 Mitogen-activated protein kinase (MAPK) regulates the expression of progelatinase B (MMP-9) in breast epithelial cells. Int J Cancer 82:168–173
45. Price DT, Rocca GD, Guo C, Ballo MS, Schwinn DA, Luttrell LM 1999 Activation of extracellular signal-regulated kinase in human prostate cancer. J Urol 162:1537–1542[CrossRef][Medline]
46. Tsukada Y, Miyazawa K, Kitamura N 2001 High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2. J Biol Chem 276:40968–40976[Abstract/Free Full Text]
47. Davies SP, Reddy H, Caivano M, Cohen P 2000 Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351:95–105[CrossRef][Medline]
48. Harris D, Chuderland D, Bonfil D, Kraus S, Seger R, Naor Z 2003 Extracellular signal-regulated kinase and c-Src, but not Jun N-terminal kinase, are involved in basal and gonadotropin-releasing hormone-stimulated activity of the glycoprotein hormone -subunit promoter. Endocrinology 144:612–622[Abstract/Free Full Text]
49. Zhang W, Liu HT 2002 MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 12:9–18[CrossRef][Medline]
50. Sugden PH, Clerk A 1997 Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell Signal 9:337–351[CrossRef][Medline]
51. van Biljon W, Wykes S, Scherer S, Krawetz S, Hapgood J 2002 Type II gonadotropin-releasing hormone receptor transcripts in human sperm. Biol Reprod 67:1741–1749[Abstract/Free Full Text]
52. Millar RP, Lu Z-L, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR 2004 Gonadotropin-releasing hormone receptors. Endocr Rev 25:235–275[Abstract/Free Full Text]

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