GYNECOLOGY

A processed metabolite of luteinizing hormone-releasinghormone has proliferative effects in endometrial cellsKathryn Walters, BS; Yue Pui Chin; T. John Wu, PhD

OBJECTIVE: The purpose of this study is to determine the possiblerole of the processed peptide of LHRH, LHRH-(1-5), in regulatinggrowth of endometrial cancer cells.

STUDY DESIGN: An endometrial cancer cell line, the Ishikawa cell line,was cultured under standard conditions and treated in a dose-depen-dent manner with 1 of 2 hormones, LHRH and LHRH-(1-5) to deter-mine the ability of these peptides to regulate cellular growth. A tetrazo-lium-based assay was used to determine the effect these peptides haveon cell proliferation. Furthermore, enzyme-linked immunosorbent as-say (ELISA)-based assays were used to determine the expression of

caspase-3/7 and pERK-1/2. Statistical analyses were conducted usingan analysis of variance followed by Fisher LSD as the post-hoc test.

RESULTS: The results show that LHRH is anti-proliferative whereasLHRH-(1-5) is proliferative on the cells. Furthermore, LHRH-(1-5) de-creased caspase-3/7 and pERK1/2 expression.

CONCLUSION: This is the first time LHRH-(1-5) is shown to have pro-liferative effects on cells.

Key words: caspase, GnRH, MAPKinase, metalloendopeptidase,proliferation.

Cite this article as: Walters K, Chin YP, Wu TJ. A processed metabolite of luteinizing hormone-releasing hormone has proliferative effects in endometrial cells.Am J Obstet Gynecol 2007;196:33.e1-33.e5.

L uteinizing hormone-releasing hor-mone (LHRH) is known primarily

for its role as the central regulator ofmammalian reproduction. Produced andreleased from the hypothalamus, this

neuropeptide acts via a specific G-pro-tein-coupled receptor, the LHRH recep-tor (LHRHR) on pituitary gonadotrophsto control the synthesis and release of lu-teinizing hormone and follicle stimulat-ing hormone.1 This hypophysiotropicform of the peptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) wasthe first discovered and has been desig-nated LHRH (LHRH-I, with the subse-quent discovery of multiple LHRH pep-tides). Studies have since found thatmost vertebrate species express at least 2forms of LHRH.2 Among these is a typeoriginally isolated from the chickenbrain (designated LHRH-II), which isnow discovered to be expressed in hu-mans.2 The presence of a specific recep-tor with a high affinity for LHRH-II(LHRHR-II) was first reported in am-phibians and more recently in marmo-set, African green monkey, and rhesusmonkey.3 However, only truncatedforms and not the fully functionalLHRHR-II are known to exist in the hu-man.4

Interestingly, a number of studieshave shown that LHRH-I and its ana-log have antiproliferative effects onhuman ovarian and endometrial can-cer cells in a cell line-dependent man-ner.5-7 While the mechanisms underly-

ing these observations are beingexamined, much remains to be eluci-dated. Our lab has been investigatingthe actions of a specific processed pep-tide of LHRH-I that comprises the first5 amino acids. This pentapeptide,LHRH-(1-5), is formed when the zincmetalloendopeptidase EC 3.4.24.15(EP24.15) cleaves the Tyr5-Gly6 pep-tide bond.8,9 We recently reported thatLHRH-(1-5) stimulates LHRH-I mRNAexpression within a mouse neuronal cellline and endometrial tissues, and oper-ates through a pathway that is distinctfrom that used by its parent mole-cule.10,11 Our data further suggest thatLHRH-(1-5) regulates certain aspects ofrodent behavior.12 Its apparent biologi-cal activity, along with the observed dif-ferences between LHRH-(1-5) andLHRH-I, lead us to hypothesize thatLHRH-(1-5) is involved in regulatingthe LHRH-I and/or LHRH-II systems inreproductive tissues. Thus, in context ofthe ability of LHRH-I and its analogs tofunction as a potential antiproliferativeagent, the purpose of this study was todetermine the proliferative/antiprolif-erative effects of the LHRH-I processedpeptide, the LHRH-(1-5), in reproduc-tive cancer cells.

From the Laboratory ofNeuroendocrinology and Women’s HealthResearch, Department of Obstetrics andGynecology, Uniformed Services Universityof the Health Sciences, Bethesda, MD.

Received April 4, 2006; revised May 17, 2006;accepted July 5, 2006.

Reprints: T. John Wu, PhD, Department ofObstetrics and Gynecology, Room B2020,Uniformed Services University of the HealthSciences, 4301 Jones Bridge Road,Bethesda, MD 20814; twu@usuhs.mil

This study was supported by the Henry M.Jackson Foundation for the Advancement ofMilitary Medicine, Gynecological CancerCenter, G185BZ-C3, and the NationalScience Foundation, IBN-0315923.

The opinions or assertions contained hereinare those of the authors and are not to beconstrued as official or as reflecting the viewsof the Department of Defense or of theUniformed Services University of the HealthSciences.

0002-9378/$32.00© 2007 Mosby, Inc. All rights reserved.doi: 10.1016/j.ajog.2006.07.054

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MATERIALS AND METHODSCell culture and treatmentIshikawa endometrial cancer cells13 weregrown in a 1:1 media consisting of Dul-becco’s Modified Eagle’s Medium andHam’s F12 (Gibco, Carlsbad, CA) sup-plemented with 10% fetal calf serum(HyClone Laboratories, Logan, UT) andantibiotics (100 U/mL penicillin and 100�g/mL streptomycin) and maintained at37°C with 5% CO2. Cells were passagedat 90-100% in a 1:5 ratio in 10-cm dishes(Nunc, Rochester, NY). Before the ex-periment, 1 10-cm dish of cells, at ap-proximately 90-100% confluency, waspassaged to 3 96-well dishes and grownto approximately 80% confluency before

treatment. The cells were changed to se-rum- and antibiotic-free medium 1 h be-fore treatment, and approximately 24 hafter plating the cells in 96-well plates.The Ishikawa cell line is a well-differen-tiated endometrial adenocarcinoma. Weused this cell line because it has been pre-viously characterized to respond toLHRH and its agonists, and is commonlyused as a model for endometrial can-cer.30-33

Proliferation assayThe proliferation experiments were con-ducted using a colorimetric method basedon a tetrazolium (3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

sulfophenyl)-2H-tetrazolium; MTS) com-pound and an electron coupling reagent(phenazine ethosulfate; PES) for deter-mining relative numbers of cells (CellTiter96 AQueous One Solution Cell ProliferationAssay; Promega Corporation, Madison,WI).14,15°The°MTS/PES°reagent°is°biore-duced by NADPH produced by dehydro-genase enzymes in metabolically activecells into a colored formazan product thatis soluble in culture medium. Thisformazan product may be recorded byreading the absorbance at 490 nm with aplate-reader (MRX Revelation, DYNEXCorporation, Chantilly, VA). Previousstudies have shown that the quantity offormazan product as measured by theamount of 490 nm absorbance is directlyproportional to the number of living cellsin°culture.16,17°The°experiments°for°eachassay were performed with 4-6 replicatesand each set of 4-6 replicates was repeatedat least 3 times.

Caspase-3/7 assaySpecific members of the cysteine asparticacid-specific protease (caspase) familyhave critical effector roles in apoptosisin°mammalian°cells.18,19° A°chemilumi-nescent-based assay that measurescaspase-3 and caspase-7 activities is usedto determine entry into the apoptosispathway (Caspase-Glo 3/7 Assay; Pro-mega). This assay is dependent on theability of a pro-luminescent caspase-3/7DEVD-aminoluciferin substrate to becleaved by caspase-3/7 to generate freeaminoluciferin which, in turn, reactswith luciferase to generate a luminescentsignal (relative luminescence unit) that ismeasured by a luminometer (Wallac1420 VICTOR Luminometer PerkinElmer Life and Analytical Sciences, Inc,Wellesley, MA).

pERK-1/2 assayA cell-based phospho-specific enzyme-linked immunosorbent assay (ELISA)assay was used to measure pERK-1/2(Fast Activated Cell-based ELISA; FACE,Active Motif, Carlsbad, CA). Detectionof specific activity is based on a chemilu-minescent°assay.20-22

FIGURE 1LHRH and LHRH-(1-5) effects on cellular proliferation

Dose effects of A, LHRH-(1-5) and B, LHRH Agonist (Des-Gly10) on cellular proliferation of Ishikawaendometrial cancer cells. Cells were incubated for 24 h without (0 nmol/L) or with increasingLHRH-(1-5) (A) or LHRH agonist (B) from 10�2 nmol/L to 102 nmol/L. Cell proliferation is givenas a percentage of control (0 nmol/L) (100%). The bars represent the mean � sem of eachexperiment. Statistical significance (P � .05) is represented by an asterisk (*). Statistical tendency(P � .10) is represented by the “^” symbol.

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Data analysesAll data were expressed as percentage ofcontrol to normalize the data for be-tween assay differences. Differences inexpression were determined by an anal-ysis of variance (ANOVA) followed by apost hoc comparison using Fisher LSD(significance at P � .05).

RESULTSLHRH agonist is antiproliferativewhereas LHRH-(1-5) isproliferativeTo determine the proliferative effects ofLHRH and its metabolite, LHRH-(1-5),Ishikawa cells were treated for 24 h in awide range of doses (0-, 10�2 to 102

nmol/L) (Figure 1). The LHRH agonist,LHRH-(Des-Gly10), decreased (P � .05)(ranging from 56-68% of controls) cel-lular proliferation at all doses testedwhereas the number of cells increasedwhen treated with 10�1, 100, and 101

nmol/L LHRH-(1-5). Significant in-creases (P � .05) were observed whenthe cells were treated with 10�1, 100, and101 nmol/L LHRH-(1-5), representing18%, 36%, and 28% increases, respec-tively, over the no treatment group.There was a tendency (P � .10) in anincrease (9%) in the number of cellswhen treated with 100 nmol/L LHRH-(1-5).

LHRH-(1-5) decreases caspase-3/7 activity and pERK-1/2 activityThere was an inverse relationship be-tween the proliferative effect of LHRH-(1-5) compared to the expression ofcaspase-3/7 and pERK-1/2 activity (Fig-ure 2). LHRH-(1-5) decreased (P � .05)caspase-3/7 expression between 20%and 35% at all doses tested (10�2 to 104

nmol/L). Likewise, LHRH-(1-5) also de-creased (P � .05) pERK-1/2 expressionbetween 17% and 35% at all doses exceptat the lowest dose tested (10�1 nmol/L)compared to controls.

COMMENTLocal effects of LHRH have been well es-tablished in both in vivo and in vitromodels. For example, it has been shownthat LHRH treatment can inhibit its se-cretion and gene expression in the GT1-7

FIGURE 2LHRH�(1-5) effect on caspase-3/7 and pERK�1/2

Dose effects of 10�2 to 104 nmol/L LHRH-(1-5) on A, cellular proliferation, B, caspase-3/7 activity,and C, pERK-1/2 activity. Cell proliferation and caspase-3/7 and pERK-1/2 activity are expressed asa percentage of control (0 nmol/L) (100%). The bars represent the mean � sem of each experiment.Statistical significance (P � .05) is represented by the asterisk (*).

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cell, a neuronal cell line that expressesLHRH, rodent hypothalamic explant tis-sues, and in the rat.10,23-27 In peripheraltissues, LHRH has been shown to exertantiproliferative effects3,5,7,28 as well asto stimulate its own gene expression.While the mechanism and circuitry isnot completely understood, the feedbackeffect of LHRH is mediated by the LHRHreceptor because LHRH receptor antag-onists block these LHRH effects.12,24 Incontrast to the effect of LHRH, we havepreviously shown that its processed me-tabolite, LHRH-(1-5), may stimulateLHRH gene expression in the LHRHneuronal cell line10 and mediate the abil-ity of LHRH to facilitate lordosis behav-ior in the rodent.12 The results from thepresent study show that the metabolite ofLHRH, LHRH-(1-5), has proliferativeeffects in the Ishikawa endometrial cellline. To our knowledge, this is the firsttime that the processed metabolite,LHRH-(1-5), has been shown to directlyeffect cell growth. Whether this observa-tion is unique to cancer cells or a charac-teristic of peripheral reproductive tissuesis the subject of present studies in thelaboratory. Furthermore, the link be-tween the ability of LHRH-(1-5) to stim-ulate cellular proliferation and to sup-press caspase-3/7 expression suggeststhat the cellular growth observed in thepresent study is linked to apoptosis. Fur-ther affirmation is shown by the positivecorrelation between caspase-3/7 activityand phosphor-ERK1/2 expression.29

Our laboratory previously showedthat LHRH and its processing metabo-lite, LHRH-(1-5), have different effectson regulating gene expression for theLHRH system, suggesting the possibilitythat these 2 peptides act through differ-ent receptors.11 We recently showed inan in vivo rat model, that the blockade ofthe LHRH receptor with a specific recep-tor antagonist, Antide, did not affect theability of LHRH-(1-5) to facilitate lordo-sis behavior in rats.12 Since the discoveryof multiple forms of LHRH (over 20 inmammals) and some of their cognate re-ceptors,3,4 it is possible that LHRH-(1-5)may act through 1 of the alternativeLHRH receptors. This is plausible sinceLHRH-(1-5) share the first 4 amino ac-ids with at least 9 LHRH forms that have

been found in various mammalian spe-cies. That LHRH-(1-5) does not appearto act through the LHRH receptor sug-gests that it may bind to a unique recep-tor or via 1 alternative form of the LHRHreceptor, such as receptor 2. Our find-ings show that LHRH-(1-5) may havecontrasting effects from its parent pep-tide, LHRH, could help explain the lackof correlation between the action ofLHRH analogs that behave as antago-nists at the pituitary level but result inagonist-like antiproliferative effects inmany reproductive system cancers.29

The identification of alternative LHRHreceptors or a unique LHRH-(1-5) re-ceptor will likely resolve some of thesequandaries.

In conclusion, our findings suggest thatthe metabolite of LHRH, LHRH-(1-5), incontrast to its parent peptide, LHRH, is aprocessed peptide that has proliferative ef-fects. That LHRH-(1-5) may have an effecton cellular growth through an apoptoticpathway suggests the complexities of localLHRH regulation. The LHRH-(1-5) bind-ing site and the inhibition of its processingby the zinc metalloendopeptidase, EC3.4.24.15, might serve as a novel target fortherapeutic approaches. The study alsosuggests that alternative designs are war-ranted for the next generation of LHRHagonists and antagonists. f

ACKNOWLEDGMENTSThe authors wish to thank Ms Cynthia Benningfor helpful discussions on the experimental de-sign and Mr Kevin Oakley for superb technicalassistance. We wish to thank Mr Michael Floraat the Biomedical Instrumentation Center, Uni-formed Services University, for synthesizing themetabolite, LHRH-(1-5), and Dr Andrew Satinfor critically reading the manuscript.

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