Báo cáo khoa học: Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview docx

MINIREVIEW
Gonadotropin-releasing hormone and ovarian cancer: a
functional and mechanistic overview
Wai-Kin So, Jung-Chien Cheng, Song-Ling Poon and Peter C. K. Leung
Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada
Gonadotropin theory of ovarian cancer
Ovarian cancer is the most lethal gynecological malig-
nancy. Although epithelial ovarian carcinomas account
for approximately 90% of all human ovarian cancers,
the etiology of this disease is poorly understood. Fat-
halla proposed the ‘incessant ovulation theory’ in 1971,
suggesting that continuous ovulation, associated with
successive rounds of surface rupture and repair,
increases the chance of accumulating genetic aberra-
tions and therefore malignant transformation [1]. The
hypothesis is supported by substantive epidemiological
data. For example, one case–control study of 150 ovar-
ian cancer patients under the age of 50 years demon-
strated that the risk of ovarian cancer decreased with
increasing numbers of live births, increasing numbers
Keywords
apoptosis; G protein; GnRH; gonadotropin-
releasing hormone; growth factor; invasion;
MAPK; migration; ovarian cancer;
proliferation
Correspondence
P. C. K. Leung, Department of Obstetrics
and Gynecology, University of British
Columbia, 2H30, 4490 Oak Street,
Vancouver, BC, Canada V6H 3V5
Fax: +1 604 875 2717

Tel: +1 604 875 2718
E-mail:
(Received 7 May 2008, revised 5 August
2008, accepted 15 August 2008)
doi:10.1111/j.1742-4658.2008.06679.x
The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is
well known for its role in the control of pituitary gonadotropin secretion,
but the hormone and receptor are also expressed in extrapituitary tissues
and tumor cells, including epithelial ovarian cancers. It is hypothesized that
they may function as a local autocrine regulatory system in nonpituitary
contexts. Numerous studies have demonstrated a direct antiproliferative
effect on ovarian cancer cell lines of GnRH and its synthetic analogs. This
effect appears to be attributable to multiple steps in the GnRH signaling
cascade, such as cell cycle arrest at G
0
⁄ G
1
. In contrast to GnRH signaling
in pituitary gonadotropes, the involvement of G
aq
, protein kinase C and
mitogen-activated protein kinases is less apparent in neoplastic cells.
Instead, in ovarian cancer cells, GnRH receptors appear to couple to the
pertussis toxin-sensitive protein G
ai
, leading to the activation of protein
phosphatase, which in turn interferes with growth factor-induced mitogenic
signals. Apoptotic involvement is still controversial, although GnRH ana-
logs have been shown to protect cancer cells from doxorubicin-induced
apoptosis. Recently, data supporting a regulatory role of GnRH analogs in

ovarian cancer cell migration ⁄ invasion have started to emerge. In this mini-
review, we summarize the current understanding of the antiproliferative
actions of GnRH analogs, as well as the recent observations of GnRH
effects on ovarian cancer cell apoptosis and motogenesis. The molecular
mechanisms that mediate GnRH actions and the clinical applications of
GnRH analogs in ovarian cancer patients are also discussed.
Abbreviations
AP-1, activator protein-1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase;
GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; IGF-I, insulin-like growth factor-I; JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MMP, matrix metalloproteinase; NF-jB, nuclear
factor kappa B; OSE, ovarian surface epithelium; PKC, protein kinase C; PLC, phospholipase C; PP2A, protein phosphatase 2A; PTP,
phosphotyrosine phosphatase; PTX, pertussis toxin; TIMP, tissue inhibitor of metalloproteinases.
5496 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS
of incomplete pregnancies, and the use of oral contra-
ceptives [2]. Another prevailing hypothesis addressing
the development of ovarian cancer was proposed by
Cramer and Welch in 1983. Their ‘gonadotropin the-
ory’ proposed that excessive gonadotropin stimulation
contributes to ovarian carcinogenesis [3]. The risk of
ovarian cancer increases during the perimenopausal
period, when serum gonadotropin levels peak and
thereafter remain elevated [4,5]. Moreover, only 10–
15% of tumors appear in premenopausal women [6].
Likewise, polycystic ovary syndrome patients (with
high luteinizing hormone levels) are more prone to
ovarian cancer [7]. Epidemiologic evidence supports the
idea that pregnancies, breast feeding, and oral contra-
ceptive use, which suppress pituitary gonadotropin
secretion, reduce the risk of ovarian cancer [8–11].
Experimentally, expression of gonadotropin receptors

has been detected in ovarian cancer tissue and in the
precursor ovarian surface epithelium (OSE) cells [12–
14]. Gonadotropins activate mitogenic pathways,
including the extracellular signal-regulated kinase
(ERK) pathway [14–16], and promote ovarian cancer
cell proliferation [12–14] and invasion [17].
Desensitization or downregulation of the gonadotro-
pin-releasing hormone (GnRH) receptors on pituitary
gonadotropes by chronic administration of GnRH
agonists or competitive binding of the GnRH receptors
by GnRH antagonists can block gonadotropin secre-
tion and subsequently suppress gonadotropin-depen-
dent functions in the ovary [18]. GnRH agonists have
also been shown to inhibit the growth of heterotrans-
planted ovarian cancer in nude mice, presumably via
altering circulating gonadotropin or steroid levels [19],
but a direct effect of GnRH on the cancer cells cannot
be excluded.
GnRH
⁄
GnRH receptor autocrine system
in ovarian cancer cells
Expression of GnRH receptors and specific GnRH-
binding sites have been detected in primary cultures of
ovarian carcinomas [20] and ovarian carcinoma biopsy
specimens [21,22], including mucinous and serous sub-
types [23]. The widespread presence (> 80%) of
GnRH-binding sites in biopsy samples [24,25] supports
the involvement of a GnRH regulatory system in ovar-
ian cancers. We and others have also demonstrated the

presence of GnRH receptors in various established
ovarian cancer cell lines, including BG-1, OVCAR-3,
SKOV-3, EFO-21 and EFO-27 (Table 1) [20,26–31].
The level of GnRH receptor expression in the ovarian
cancer cell lines was about 10-fold lower than that in
pituitary aT3 cells [20].
The extremely short half-life of hypothalamic GnRH
makes it an unlikely candidate to act on the ovary via
the systemic circulation and suggests the existence of a
local source of GnRH in ovarian cancer cells. Indeed,
our group and others have detected GnRH-I mRNA
in normal OSE and immortalized OSE cells, as well as
in primary cultures of ovarian tumors and ovarian car-
cinoma cell lines such as EFO-21, EFO-27, CaOV-3,
OVACR-3 and SKOV-3 [32,33]. Similarly, GnRH-II
mRNA has been detected in normal and neoplastic
OSE cell lines and primary cultures of ovarian carcino-
mas [28]. GnRH-like immunoreactivity was detected in
conditioned media [34] and cell lysates [21] from ovar-
ian cancer cell lines. The latter possessed bioactivity
comparable to that of authentic GnRH, as it stimu-
lated luteinizing hormone release from rat pituitary
[21]. Incubation of ES-2 ovarian cancer cells in vitro
with a GnRH-I antibody inhibited cell proliferation in
a time- and dose-dependent manner [34], whereas
Emons reported a significant increase in EFO-21 and
EFO-27 ovarian cancer cell proliferation after GnRH-I
antiserum treatment [35]. Despite this discrepancy,
these studies provide direct evidence for the endo-
genous secretion of bioactive GnRH as an autocrine

growth-regulatory loop in ovarian cancer cells.
Our laboratory demonstrated the existence of an
autocrine loop involving GnRH and the GnRH recep-
tor in primary cultures of human OSE cells (scraped
from the ovarian surface during laparoscopies for
nonmalignant disorders) [32]. The GnRH agonist
[D-Trp6]GnRH had a direct inhibitory effect on
growth of OSE cells in a time- and dose-dependent
manner. This inhibitory effect was reversed by cotreat-
ment with the GnRH receptor antagonist antide [32].
Moreover, the GnRH agonist has a homologous
regulatory effect on the expression of GnRH and the
GnRH receptor in OSE cells, which further supports
the presence of an autocrine regulatory GnRH system
that is operational in the ovary.
Antiproliferative effect of GnRH
analogs on ovarian cancer
Numerous in vitro studies have reported a growth-
modulating effect of GnRH-I ⁄ GnRH-II and their
synthetic analogs in various GnRH receptor-bearing
ovarian cancer cell lines (Table 1). In most cases,
GnRH-I and its agonists were reported to inhibit ovar-
ian cancer cell proliferation, as judged by decreased
cell number or DNA synthesis. For example, our labo-
ratory has reported that treatment with the agonist
[D-Ala6]GnRH caused a time- and dose-dependent
inhibition of cell proliferation in the ovarian cancer
W K. So et al. GnRH and ovarian cancer
FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5497
Table 1. In vitro effects of GnRH-I and GnRH-II analogs on OSE and ovarian cancer cell lines. R-I, GnRH-I receptor; R-II, GnRH-II receptor; ND, not determined; DOX, doxorubicin;

+, positive; ), negative. Expression of GnRH receptors is based on reports published by individual groups.
Reference GnRH analogs Dose Cell line R-I R-II Action
[31] GnRH-I antagonist cetrorelix 10
)8
–10
)5
M OV-1063 + ND Antiproliferation
GnRH-I agonist triptorelin 10
)8
–10
)5
M
[44] GnRH-I agonist triptorelin 10
)7
–10
)5
M OVCAR-3 + ND Antiproliferation and cell cycle
arrest
SKOV-3 + ND
[30] GnRH-I agonist triptorelin 10
)5
M EFO-21 + + Antiproliferation
GnRH-I antagonist cetrorelix, Hoe-013 10
)11
–10
)5
M EFO-27 + ) Antiproliferation for EFO-21 but not
EFO-27
[32] GnRH-I agonist [
D-Ala6]GnRH 10

)11
–10
)7
M Human OSE + ND Antiproliferation
[36] GnRH-I agonist [
D-Ala6]GnRH 10
)11
–10
)7
M OVCAR-3 + ND Antiproliferation and DNA
fragmentation
[35] Native GnRH-I 10
)12
–10
)5
M EFO-21 + + Antiproliferation
EFO-27 + )
[53] GnRH-I agonist triptorelin 10
)7
M EFO-21 + + Protection against DOX-induced
apoptosis
EFO-27 + )
[34] GnRH-I agonist triptorelin 10, 1000 ngÆmL
)1
ES-2 + ND Increase ⁄ decrease cell proliferation
(10 ngÆmL
)1
), antiproliferation
(1000 ngÆmL
)1

)
[28] GnRH-I agonist [
D-Ala6]GnRH 10
)9
–10
)7
M IOSE29, IOSE28-EC + ND Antiproliferation
[46] GnRH-I antagonist cetrorelix 10
)9
–10
)5
M HTOA + ND Antiproliferation and cell cycle
arrest (10
)9
,10
)5
M), apoptosis
(10
)5
M)
[43] GnRH-I agonist triptorelin 10
)9
–10
)7
M EFO-21 + + Antiproliferation and cell
cycle arrest
EFO-27 + )
[37] GnRH-I agonist triptorelin 10
)5
M EFO-21 + + Triptorelin: antiproliferation for

EFO-21, but not for SKOV-3.
GnRH-II: antiproliferation for all cell
lines
Native GnRH-II 10
)11
–10
)5
M OVCAR-3 + +
SKOV-3 ) +
[27] GnRH-I agonist triptorelin 10
)11
–10
)5
M EFO-21 + + Triptorelin: antiproliferation
GnRH-I antagonist cetrorelix 10
)11
–10
)5
M BG-1 + + Cetrorelix: antiproliferation
GnRH-II 10
)11
–10
)5
M OVCAR-3 + + GnRH-II: antiproliferation
EFO-27 + )
SKOV-3 ) +
GnRH and ovarian cancer W K. So et al.
5498 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS
Table 1. (Continued)
Reference GnRH analogs Dose Cell line R-I R-II Action

[127] GnRH-I agonist triptorelin 10
)11
–10
)5
M EFO-21 + + Suppression of
17b-estradiol-induced EFO-21 and
OVCAR-3 cell proliferation, but no
effect on SKOV-3
OVCAR-3 + +
SKOV-3 ) +
[57] GnRH-II agonist
D-Arg(6)-Azagly(10)-NH
2
10
)7
M OVCAR-3 + ND Antiproliferation and apoptosis
[54] GnRH-I agonist leuprolide 10
)6
M CaOV-3 + ND Protection against DOX-induced
apoptosis
SKOV-3 + ND
[50] GnRH-I antagonist cetrorelix 10
)8
–10
)6
M CaOV-3 + ND Apoptosis
SKOV-3 + ND
[42] GnRH-II agonist
D-Arg(6)-Azagly(10)-NH
2

10
)7
M OVCAR-3 + + Antiproliferation
[41] GnRH-I agonist triptorelin 10
)7
M OVCAR-3 + ND Antiproliferation
GnRH-II agonist
D-Arg(6)-Azagly(10)-NH
2
10
)7
M SKOV-3 + ND
[63] GnRH-I agonist [
D-Ala6]GnRH 10
)10
–10
)8
M CaOV-3 + ND Cell migration and invasion
OVCAR-3 + ND
[58] GnRH-II antagonists: [Ac-
D2Nal1, D-4Cpa2,
D-3Pal3, D-Lys6, D-Ala10]GnRH-II, [Ac-D2Nal1,
D-4Cpa2, D-3Pal3, D-Lys6, Leu8, D-Ala10]GnRH-II,
[Ac-
D2Nal1, D-4Cpa2, D-3Pal3,6, Leu8, D-Ala10]GnRH-II
10
)5
M EFO-21 + + Apoptosis
OVCAR-3 + +
SKOV-3 ) +

[64] GnRH-I agonist triptorelin 10
)10
–10
)7
M OVCAR-3 + ND Induction of OVCAR-3 invasion
(10
)10
–10
)8
M), suppression of
SKOV-3 invasion at 10
)8
and
10
)7
M
GnRH-II agonist D-Arg(6)-Azagly(10)-NH
2
SKOV-3 + ND
W K. So et al. GnRH and ovarian cancer
FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5499
cell line OVCAR-3. Significant inhibition was detected
as early as 2 days after treatment [36]. Interestingly,
GnRH-I antagonists consistently act like agonists and
inhibit cell proliferation in various cell lines (Table 2).
GnRH-I antagonists were reported to be more potent
than equimolar concentrations of agonists in inhibiting
ovarian cancer cell growth [29,37]. This phenomenon
was also observed in endometrial [38], prostate [39]
and breast cancers [40], suggesting that the dichotomy

between GnRH-I agonists and antagonists in the
pituitary might not be applicable to cancer cells. The
exact mechanism underlying the difference between
the pituitary and extrapituitary tissues remains to be
elucidated.
In contrast to the situation with GnRH-I, functional
studies on GnRH-II are rather limited. We and others
have reported that, like GnRH-I agonists, GnRH-II
and its agonists, such as d -Arg(6)-Azagly(10)-NH
2
,
exert an antiproliferative effect on ovarian cancer cell
lines [41,42]. The antiproliferative effect of GnRH-II is
more potent than that of GnRH-I [37]. Interestingly,
it has been reported that a GnRH-II agonist inhibits
the growth of SKOV-3 cells, which are GnRH-I recep-
tor-negative and unresponsive to GnRH-I [27]. The
antiproliferative effect of GnRH-I is associated with
an induction of cell cycle arrest at G
0
⁄ G
1
[43–46], cou-
pled with a downregulation of Cdk expression [44]
and cyclin A–Cdk2 complex levels [46], or inhibition
of telomerase activity without alteration of RNA
expression [47].
In addition to cell cycle arrest, apoptosis may also
be involved in the antiproliferative action of GnRH.
GnRH-I agonists have been reported to induce pros-

tate cancer apoptosis [48]. In ovarian cancer cells, a
high concentration (10
)5
m) of GnRH agonist has
been reported to induce tumor necrosis factor-a secre-
tion, interchromosomal DNA fragmentation, and a
marginal apoptotic effect [49]. An equimolar concen-
tration of the GnRH-I antagonist cetrorelix induced
apoptosis by upregulating p53 and p21 protein levels,
whereas concentrations as low as 10
)9
m resulted in
antiproliferative effects [46]. Recently, apoptosis was
shown to be induced by a low concentration of cetror-
elix in ovarian cancers [50]. We also observed DNA
fragmentation after prolonged (6 days) low-dose
GnRH-I agonist treatment [36]. In most studies, how-
ever, apoptosis was induced only when ovarian cancer
cells were treated with GnRH-I analogs at relatively
high concentrations or for a prolonged time. Although
Fas and FasL were detected in the majority of ovarian
carcinomas and ovarian cancer cell lines [51,52], and
GnRH agonists such as buserelin dose-dependently
induced FasL expression in ovarian cancer cells [52], a
causative linkage between Fas ⁄ FasL and the antipro-
liferative action of GnRH has not been established.
Indeed, there is no consensus about the proapoptotic
role of GnRH. The antiproliferative effect of GnRH
has been mainly attributed to the cytostatic action of
GnRH rather than induction of apoptosis. GnRH-I

agonists, including triptorelin [44,53] and leuprolide
[54], were marginally effective or ineffective in induc-
ing ovarian cancer cell apoptosis. In contrast, these
agonists exerted a protective effect against the cyto-
toxic action of the chemotherapy drug doxorubicin
[53,54]. Abolition of GnRH action by GnRH receptor
knock-down increased doxorubicin-induced apoptosis
[55]. A GnRH-generated protective effect against
doxorubicin-induced apoptosis was also observed in
human granulosa, breast cancer and endometrial
cancer cells [55,56]. The underlying mechanism of this
protective effect is unknown, although activation of
nuclear factor kappa B (NF-jB) may be involved.
Triptorelin was shown to activate NF-jB in ovarian
cancer cells, and blockage of NF-jB translocation into
the nucleus reversed GnRH-induced protection against
doxorubicin [53] (Fig. 1F). In this case, the antitumor
(antiproliferative) and antiapoptotic effects of GnRH
would appear to be paradoxical, but in fact doxorubi-
cin and most chemotherapy drugs are more efficacious
towards rapidly dividing cells, and thus the cell cycle
arrest induced by GnRH can protect the tumor cell
from doxorubicin. Regarding GnRH-II, we and others
showed that GnRH-II and its antagonist induced
ovarian cancer cell apoptosis [57,58], which was medi-
ated by p38 mitogen-activated protein kinase (MAPK)
and caspase-3 activation [57,58]. Furthermore, an
antitumor effect of GnRH-II antagonists was
Table 2. Overview of trials using GnRH agonists in ovarian cancer.
CR, complete response; PR, partial response; SD, stable disease.

Reference Drug Patients CR PR SD
[107] Leuprolide acetate 18 0 4 2
[108] Leuprolide acetate 5 1 3 1
[109] Leuprolide acetate 25 0 1 15
[110] Leuprolide acetate 32 0 4 5
[111] Leuprolide acetate 32 1 2 4
[112] Leuprolide acetate 37 0 0 4
[113] Leuprolide acetate 12 0 1 3
[114] Triptorelin 41 0 6 5
[115] Triptorelin 19 0 11 0
[116] Triptorelin 20 0 0 14
[117] Triptorelin 40 0 0 1
[118] Triptorelin 14 0 0 8
[106] Triptorelin 68 0 0 11
[119] Goserelin 23 0 4 7
[120] Goserelin 30 0 2 5
GnRH and ovarian cancer W K. So et al.
5500 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS
demonstrated in nude mice bearing ovarian cancer cell
xenografts [58].
In contrast to the relatively large number of studies
on GnRH actions such as antiproliferative and apop-
totic ⁄ antiapoptotic effects, reports of GnRH influences
on other parameters of ovarian cancer progression,
such as tumorigenic or metastatic processes, are lim-
ited. Spread of ovarian cancer beyond the ovaries to
the peritoneal cavity leads to later staging of the dis-
ease and poor prognosis. The fact that a high propor-
tion of advanced-stage (stages III and IV) ovarian
carcinomas express GnRH receptor mRNA and

protein, as compared to early-stage carcinomas [59],
has prompted us to investigate the participation of
GnRH in regulating migration and invasion in ovarian
cancer. Previously, we have demonstrated the potency
of GnRH-I and GnRH-II regulation of the urokinase-
type plasminogen activator ⁄ plasminogen inhibitor
system and matrix metalloproteinase (MMP)-2, MMP-
9, and tissue inhibitor of metalloproteinases (TIMP)-1
in other gynecological tissues, including extravillous
cytotrophoblasts and decidual stromal cells [60–62].
These proteolytic enzymes are involved in the degrada-
tion and remodeling of extracellular matrix, which has
been implicated in the multistep process of metastasis
formation. By activating MMP-2 and MMP-9 promot-
ers to increase gene expression, GnRH agonists
stimulated the migration and invasion potential of
CaOV-3 and OVCAR-3 ovarian cancer cells. The
GnRH-induced increase in invasiveness and migratory
activity was blocked by neutralizing antibodies against
MMP-2 and MMP-9 [63]. This motogenic action of
GnRH was mediated by GnRH receptor and c-Jun
N-terminal kinase (JNK), but not by ERK or p38
MAPK [63] (Fig. 2C). We further investigated
the effect of GnRH-II in ovarian cancer cells. The
GnRH-II agonist d-Arg(6)-Azagly(10)-GnRH-II, like
GnRH-I agonists, stimulated OVCAR-3 cell invasion.
Interestingly, high doses of GnRH-I and GnRH-II
agonists were observed to reduce the invasive potential
of SKOV-3 cells by altering the balance between MMP
and TIMP [64]. In this regard, it is noteworthy that

GnRH-I agonists and antagonists have been reported
to inhibit the migration and invasion of prostate
cancer, breast cancer and epidermoid carcinoma cells
[65–67]. Also, breast cancer cell invasiveness was sup-
pressed in vitro by both GnRH-I and GnRH-II [67].
Signaling and mechanism of GnRH
action in ovarian cancer cells
As a member of the serpentine receptor family, the
GnRH receptor transmits its signals mainly through
heterotrimeric G-proteins (GTP-binding proteins).
GnRH-I agonist/
antagonist
PTP
GnRH-I
receptor
β
β
γ
γ
EGFR
Expression
JNK
G α
α
i
Apoptosis
NF κ
κ
B
NF κ

κ
B
n o i t
a c o l s
n
a r T
PP2A
EGFR
P
G α
α
i
Proliferation
ERK1/2
MEK
Sos
c-fos expression
PKC
P
P
AP-1
GnRH-I
antagonist
GnRH-II
receptor ?
JunD
cAMP
FK
AC
EGF

Cell cycle
A
D C
B
E
F
G
Shc
Fig. 1. GnRH-I signaling in ovarian cancer
cells. (A) Through G
ai
, GnRH-I analogs acti-
vate PTP to dephosphorylate EGFR and
abolish EGF-induced ERK activation, c-fos
expression and proliferation. (B) G
bc
subunit
activates ERK and mediates GnRH-I-induced
growth inhibition. (C) GnRH-I activates ERK
through a PKC-dependent or PKC-indepen-
dent pathway to inhibit proliferation. (D)
GnRH-I activates JNK, which increases AP-1
activity and JunD–DNA binding to extend
the cell cycle. (E) GnRH-I suppresses apop-
tosis through activation of PP2A. (F) GnRH-I
stimulates NF-jB activity and nuclear trans-
location to protect ovarian cancer cells from
apoptosis. (G) GnRH-I acts through Gaito
counteract forskolin (FK)-induced cAMP. The
presence of a functional GnRH-II receptor

has yet to be evaluated. Dashed arrows rep-
resent the EGF-stimulated mitogenic signal-
ing pathway; FS, forskolin; AC; adenylate
cyclase.
W K. So et al. GnRH and ovarian cancer
FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5501
Upon stimulation, G
a
dissociates from the G
bc
dimer
and changes to its active GTP-bound form. According
to the subtype of their a-subunits, G-proteins can be
categorized into four groups: G
s
,G
i
,G
q ⁄ 11
, and
G
12 ⁄ 13
.G
as
and G
ai
mainly exert their effects via stim-
ulating or inhibiting, respectively, adenylate cyclase to
modulate the production of cAMP. G
aq

activates
membrane-associated phospholipase C (PLC), which
hydrolyzes phosphoinositides to generate the second
messengers inositol 1,4,5-triphosphate and diacylglyc-
erol, resulting in intracellular Ca
2+
mobilization and
protein kinase C (PKC) activation. Moreover, G
aq
-
activated PKC can activate MAPKs, including JNK,
ERK and p38 MAPK [68,69]. It is well established
that the GnRH receptor interacts with multiple G-pro-
teins, and that specificity is cell context dependent [68].
In hypothalamic neurons, the GnRH receptor interacts
with G
aq
,G
as
and G
ai
[70]. In pituitary gonadotropes,
GnRH preferentially or exclusively stimulates G
aq
[71].
However, G
ai
has been shown to mediate GnRH
receptor signaling in tumor cells such as ovarian
cancer [50,72,73], endometrial cancer [72,73] and pros-

tate cancer [74] cells. Consequently, the downstream
signaling pathways and the physiological outcomes of
GnRH action may be quite different in the gonado-
tropes and extrapituitary tissues.
The mechanism that leads to the inconsistency of
GnRH signaling and biological outcome in different
tissues is unknown. It is unlikely that divergent signal-
ing is due to alternatively spliced variants or mutant
receptors in cancer cells, as expression of the wild-type
GnRH receptor has been confirmed in OVCAR-3,
EFO-21 and EFO-27 ovarian cancer cell lines and
human OSE cells [32,72]. On the other hand, the
receptor may oscillate, in a cell context-dependent
manner, between multiple conformations, each with
specific ligand and intracellular signaling complex
selectivity. According to this hypothesis, the receptor
can adopt a conformation that preferentially binds cer-
tain ligand(s), in response to different ligand concen-
trations. The intracellular signaling complex could in
turn stabilize the receptor conformation and favor the
binding of the ligand [75]. There is direct evidence sup-
porting the existence of multiple conformations of a
G-protein-coupled receptor [76]. Thus, the GnRH
receptor in gonadotropes may preferentially bind
GnRH-I and be coupled to the G
aq
–PLC pathway in
order to modulate gonadotropin expression and secre-
tion, whereas in cancer cells, the GnRH receptor cou-
pling to G

ai
is selectively recognized and activated by
GnRH-II in order to regulate cell proliferation and
apoptosis. This kind of conformational preference may
be a result of cell context, including cell type and prior
exposure to other hormones [75]. Concrete evidence
for this specificity has been generated for the Xenopus
GnRH receptor: activation of PKC, which phosphory-
lates the C-terminus of the receptor, led to a marked
increase in GnRH-II binding to the Xenopus GnRH-I
receptor, but had no effect on GnRH-I binding [75].
This theory could resolve questions regarding the
Proliferation
Elk-1
p38
Apoptosis
Migration/
invasion
GnRHII
GnRH-I receptor
G α
α
i
PKC
EGFR
P
ERK1/2
MEK
c-fos expression
EGF

Sos
PTP
GnRH-II
receptor ?
JNK
AP-1
A
D
C
B
Shc
P
P
Fig. 2. GnRH-II signaling in ovarian cancer
cells. (A) Similar to GnRH-I, GnRH-II acti-
vates PTP to inhibit EGFinduced ERK activa-
tion, c-fos expression and proliferation. (B)
Through PKC, GnRH-II activates ERK and
Elk-1 to suppress proliferation. (C) GnRH-II
activates JNK to induce migration ⁄ invasion.
(D) GnRH-II activates G
ai
or p38 and AP-1 to
induce apoptosis. Dashed arrows represent
the EGF-stimulated mitogenic signaling
pathway.
GnRH and ovarian cancer W K. So et al.
5502 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS
GnRH system in different tissues, including why a
lower binding affinity of GnRH agonists has been

observed in tumor cells than in pituitary gonadotropes
[75], why GnRH antagonists act like agonists in cancer
cells (Table 2), and why GnRH-II is observed to be
more potent than GnRH-I in inhibiting cancer cell
proliferation through G
ai
, but less potent in stimulat-
ing G
aq
-mediated gonadotropin secretion in pituitary
gonadotropes [37,77].
At present, the identity of the GnRH receptor(s)
that mediate the antiproliferative actions of GnRH in
tumor cells and the agonistic effects of GnRH antago-
nists in cancer cells is still controversial. Grundker and
co-workers have compared the GnRH responsiveness
of SKOV-3 and EFO-27 ovarian cancer cells, and pro-
posed that their responsiveness correlates with the
expression of GnRH receptors. Accordingly, EFO-27
cells expressing GnRH-I receptor but not GnRH-II
receptor responded to the GnRH-I agonist triptorelin
but not to antagonists, even at high concentrations
(10
)5
m) [27,30,78]. By contrast, in SKOV-3 cells,
which are reportedly GnRH-I receptor-negative but
GnRH-II receptor-positive, both the GnRH-I antago-
nist cetrorelix and GnRH-II, but not triptorelin, inhib-
ited cell growth [27,37] or EGF-induced c-fos
expression [78]. Cell lines expressing both GnRH-I

and GnRH-II receptors were responsive to all of the
treatments. These findings were in accordance with
other reports that growth of GnRH-II receptor-
expressing Ishikawa and Hec-IA endometrial cancer
cell lines was inhibited by a GnRH-I antagonist
[27,38]. Moreover, the GnRH-I agonist-induced anti-
proliferative effect was abolished by GnRH-I receptor
knock-out, whereas the antiproliferative effects
induced by the antagonist cetrorelix or GnRH-II
agonist persisted [27,79]. These results support the
hypothesis that the GnRH-I receptor mediates the
actions of GnRH agonists, whereas GnRH antagonists
and GnRH-II may act through an additional receptor,
i.e. the putative GnRH-II receptor. Although a func-
tional GnRH-II receptor has not yet been identified in
humans, the presence and ⁄ or functionality of a type II
receptor has not been ruled out. The human GnRH-I
antagonist 135-18 and GnRH-II are capable of acti-
vating the marmoset GnRH-II receptor, which is over-
expressed in COS-7 cells [80]. However, the presence
of only one class of specific, high-affinity ⁄ low-capacity
receptors in humans has not been sufficiently demon-
strated. Binding of the agonist [D-Trp6]GnRH was
displaced by the antagonist SB-75, suggesting that
both analogs bind to the same receptor on OV-1063
cells [29]. Thus, the precise mechanisms underlying the
divergence of signaling pathways and biological
actions in pituitary gonadotropes and tumor cells
remain to be determined.
G

ai
and ⁄ or G
aq
have been detected in GnRH-I
receptor-expressing ovarian cancer cell lines and surgi-
cally removed ovarian carcinomas [72,73]. GnRH-I
receptor has been shown, by disuccinimidyl suberate
cross-linking experiments, to interact physically with
G
ai
and G
aq
[72]. Functionally, it has been suggested
that GnRH-I receptor couples with G
ai
, which is com-
mon in tumor cells [50,72–74]. G
ai
is pertussis toxin
(PTX)-sensitive and is not affected by cholera toxin.
PTX induced ADP-ribosylation of the a-subunit in the
GnRH-I receptor-positive tumor cell membrane
[50,72–74] and thus impaired GnRH-I receptor-linked
cellular events, including GnRH-induced phosphatase
activity [50,72,73], apoptosis [50], and antiproliferative
actions [72,74]. Conversely, incubation with GnRH
agonists substantially antagonized the PTX-catalyzed
ADP-ribosylation of G
ai
[72–74]. Furthermore, G

ai
sig-
nificantly counteracted the forskolin-induced increase
in intracellular cAMP levels [74] (Fig. 1G). These find-
ings strongly indicate that G
ai
is the major G-protein
mediating GnRH actions in tumor cells. In addition to
the PTX-sensitive G
ai
, G-protein bc-subunits also
mediated GnRH agonist-induced antiproliferative
effects and ERK activation in ovarian cancer cell lines,
and such ERK activation was blocked by ectopic
expression of the C-terminus of a-adrenergic receptor
kinase I, an antagonist of G-protein beta gamma-
subunits [81] (Fig. 1B).
In pituitary gonadotropes, PKC and PLC act down-
stream of G
aq
to relay the GnRH receptor signals.
However, the roles of PKC and PLC in GnRH recep-
tor signal transduction in tumor cells are less clear.
There is evidence that the signaling pathways induced
by GnRH-I in pituitary gonadotropes, including PLC
and PKC, are not activated by the GnRH-I agonist
triptorelin in ovarian, endometrial and breast cancer
cell lines [35,36]. Similarly, GnRH-I-stimulated MAPK
activation in pituitary aT3 cells was abolished by
4b-phorbol 12-myristate 13-acetate pretreatment (to

deplete PKC) or by depletion of Ca
2+
, whereas the
GnRH-I agonist activated MAPK via a PKC-indepen-
dent mechanism to inhibit growth of CaOV-3 ovarian
cancer cells [81]. However, evidence supporting PKC
involvement in GnRH actions in tumor cells or
extrapituitary tissues is available. Cetrorelix stimulated
PKC activity in DU-145 prostate cancer cells, resulting
in an increase in phosphorylation of the PKC substrate
MARCKS [82]. By activating PKC, GnRH analogs
inhibited the EGF receptor (EGFR) signal and the
growth of prostate cancer xenografts in athymic mice
[83] and cell invasion in vitro [82]. Essentially, prostate
W K. So et al. GnRH and ovarian cancer
FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5503
cancer cells carrying a mutated EGFR that lacks the
target site for PKC are resistant to GnRH-induced
in vivo and in vitro growth inhibition [82,83]. Our labo-
ratory has recently demonstrated that 12-O-tetradeca-
noyl phorbol-13-acetate (a PKC-activating phorbol
ester) can mimic the effects of GnRH-I and GnRH-II
in stimulating ERK1 ⁄ 2 phosphorylation and antipro-
liferation in ovarian cancer cells. Furthermore, the
effects of GnRH were abolished by pretreatment with
the PKC inhibitor GF109203X [41] (Figs 1C and 2B).
By analogy, the PKC inhibitor calphostin C and the
activator 12-O-tetradecanoyl phorbol-13-acetate can
block and mimic, respectively, the antiproliferative
action of the GnRH agonist buserelin on surgically

removed uterine leiomyoma [84] cells. In human gran-
ulosa-luteal cells, a GnRH agonist stimulates MAPK
activation through a PKC-dependent pathway [85].
However, the direct effect on PKC activation and the
identity of the PKC isoform activated by GnRH are
still under investigation.
In pituitary gonadotrope cells, there is ample evidence
that GnRH-I activates MAPK members, including
ERK, JNK and p38, in a PKC-dependent pathway to
control gonadotropin secretion [68]. MAPK members
mediate distinct roles in GnRH-induced gonadotropin
subunit gene transcription [86–88]. In sharp contrast to
the situation with pituitary gonadotropes, our under-
standing of MAPK activation by GnRH analogs in can-
cer cells is rather limited. In ovarian cancer OVCAR-3
cells and placental cancer JEG-3 cells, we have demon-
strated biphasic activation of ERK by [D-Ala6]GnRH.
High doses of GnRH agonist (10
)7
and 10
)6
m) signifi-
cantly activated ERK, whereas a low dose (10
)10
m)
resulted in decreased ERK activation [89]. In another
study, phosphorylation of ERK, Sos and Shc was
induced by the GnRH-I agonist leuprolide [81]. Leupro-
lide-induced ERK activation was rapid (within 5 min)
and long-lasting (sustained up to 24 h). ERK appeared

to mediate the antiproliferative action of GnRH and
such growth inhibition could be reversed by the mito-
gen-activated protein kinase kinase (MEK) inhibitor
PD98059 [81]. The dual roles of ERK in mediating
mitogenic effects (by growth factors) and antiprolifera-
tive effects (by GnRH) seem contradictory. Paradoxical
actions of ERK have also been reported in PC-12 cells:
transient activation induced by EGF led to prolifera-
tion, and nerve growth factor-induced prolonged ERK
activation caused differentiation and cessation of prolif-
eration [90]. It has been suggested that the duration of
ERK activation is important in determining its actions
and thus the resultant cell fate [90].
In addition to ERK, JNK was activated by triptore-
lin through induction of c-jun mRNA expression and
protein phosphorylation [91], in a manner that was
independent of PLC and PKC [92]. The same group
further demonstrated that triptorelin treatment
increased activator protein-1 (AP-1) activity and
JunD–DNA binding, and extended the cell cycle [43]
(Fig. 1D). As JNK and c-jun are implicated in cell
cycle regulation [93], it is logical to hypothesize that
the JNK–c-jun–AP-1 pathway mediates the GnRH-
induced antiproliferative effect. This pathway may act
in concert with NF-jB, as it was shown to protect
tumor cells from doxorubicin-induced apoptosis in the
same system. JunD is proposed to act as a modulator
of cell proliferation and to cooperate with the anti-
apoptotic and antiproliferative functions of GnRH.
However, further investigation is necessary to resolve

the observations by others that the GnRH-I agonist
leuprolide and a GnRH-II agonist could not activate
JNK [42,81], and that p38 was implicated in GnRH-II-
induced ovarian cancer cell apoptosis [57] (Fig. 2D).
In addition to the direct antiproliferative signal elic-
ited through PKC-dependent or PKC-independent
pathways, the antiproliferative effect on tumor cells of
GnRH may arise from its ability to activate phospha-
tases and counteract the mitogenic signals induced by
growth factors [94] (Fig. 1A). In OSE and epithelial
ovarian carcinomas, EGF and various growth factors
are secreted and function locally to promote tumor
proliferation and progression [95]. Binding of growth
factors to their cognate receptor tyrosine kinases
induces receptor dimerization and autophosphoryla-
tion. Phosphorylated receptor tyrosine kinases phos-
phorylate adaptor and effector molecules to
subsequently initiate a phosphorylation cascade that is
important for the growth-promoting and tumorigenic
functions of growth factors. Propagation of the phos-
phorylation cascade and its physiological effects can be
terminated by protein phosphatases. It has been shown
that EGFR, when stimulated by EGF, may phosphor-
ylate itself and other cellular substrates, including Src,
in human pancreatic cells; cotreatment with
[D-Trp6]GnRH reversed the effect of EGF and led to
the dephosphorylation of these proteins [96]. In the
plasma membrane of tumor cells, phosphotyrosine
phosphatase (PTP) or serine ⁄ threonine protein phos-
phatase 2A (PP2A) were shown to be activated by

GnRH agonists [50,54,72,97–100], suggesting that
GnRH-I increased the turnover rate of protein phos-
phorylation ⁄ dephosphorylation and that EGFR is a
target of the dephosphorylation activity [72,96]. As a
result, EGFR phosphorylation [72] and the down-
stream signaling and mitogenic effects of EGF were
abrogated, including EGF-induced MAPK activation
[94], immediate early gene c-fos expression [78] and
GnRH and ovarian cancer W K. So et al.
5504 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS
proliferation [92]. Downregulation of receptors for
EGF and ⁄ or insulin-like growth factor-I (IGF-I) by
GnRH antagonist [101] and in ovarian cancer-xeno-
grafted nude mice have been reported [31,102]. Inter-
ference of growth factor signaling by GnRH analogs
has also been demonstrated in prostate cancer cells.
GnRH-I analogs abrogated the mitogenic effects of
EGF and IGF-I and inhibited prostate cancer growth
[103,104] by reducing expression of their receptors
[103–105], as well as by inhibiting EGF- and IGF-I-
induced receptor phosphorylation [103,104] and c-fos
expression. GnRH-II agonist was reported to act in a
similar fashion, i.e. enhancing PTP activity and thus
reducing EGF-induced EGFR phosphorylation,
MAPK activation and c-fos expression [79] (Fig. 2A).
Moreover, GnRH-activated phosphatase activity has
also been implicated in its antiapoptotic function.
Doxorubicin decreased the activity of a crucial
phosphatase in apoptosis control (PP2A), and induced
ovarian cancer cell apoptosis. Cotreatment with the

GnRH-I agonist leuprolide partially restored PP2A
activity and antagonized doxorubicin-induced apopto-
sis [54] (Fig. 1E).
Clinical studies on GnRH agonists and
antagonists in ovarian cancer
In a limited number of studies, GnRH-I agonists have
been evaluated for their potential as second-line ther-
apy in patients with refractory and recurrent ovarian
cancer who had failed at least one chemotherapy regi-
men. In 2001, the European Organization of Research
and Treatment of Cancer Gynecological Cancer Coop-
erative Group completed the largest reported series of
GnRH agonist trials. Seventy-four patients with pro-
gressive ovarian cancer who had previously undergone
platinum-based therapy were treated with the GnRH
agonist triptorelin. No objective responders were
observed. Eleven of 68 evaluable patients (16%) had
stable disease. The median progression-free survival
was 5 months in patients with disease stabilization and
2 months for all evaluable patients. The median sur-
vival for patients with disease stabilization was
17 months, whereas for all patients it was 4 months.
This study showed that treatment with the GnRH
agonist triptorelin has only modest efficacy in patients
pretreated with platinum-containing chemotherapy
[106]. Table 2 summarizes 15 clinical trials, beginning
as early as 1988, that have used three different GnRH
agonists (leuprolide acetate, triptorelin, goserelin) on
relapsed platinum-resistant ovarian cancer patients
(Table 2) [106–120]. The majority of these trials

involved only a limited number of patients.
Three trials have been completed that compared the
use of platinum-based chemotherapy alone or in com-
bination with a GnRH agonist as first-line therapy for
ovarian cancer [121–123]. A prospective randomized
double-blind trial enrolled 135 patients with stage III
or IV epithelial ovarian carcinoma, and showed that
suppression of endogenous gonadotropins by conven-
tional doses of the GnRH agonist triptoreli n produces
no relevant beneficial effects in patients with advanced
ovarian carcinoma who receive standard surgical cyto-
reduction and standard platinum-based chemotherapy
[121]. In the other two studies, patients received carbo-
platin-containing polychemotherapy and cisplatin alone
or chemotherapy plus triptorelin, but no significant dif-
ferences were seen in terms of response, survival and
time to progression [122,123]. The ineffectiveness of the
GnRH agonist in combination with chemotherapy is
postulated to be due to the neutralization of its direct
antiproliferative effects by its antiapoptotic activity, as
demonstrated by the in vitro data [53,54,124].
In vitro data demonstrated that antagonists provided
a greater inhibitory effect on ovarian cancer prolifera-
tion than agonists [29]. Clinically, as GnRH-I antago-
nists do not possess intrinsic gonadotropic activity, the
initial ‘flare-up’ phenomenon, which is common in
agonist treatment, can be avoided. This makes antago-
nists better tolerated and capable of blocking gonado-
tropin secretion within a short time frame [125]. A
clinical trial of the GnRH antagonist cetrorelix was

conducted on 17 patients. All of the patients had
relapsed disease after standard chemotherapy before
entering into the trial. Three patients (18%) experi-
enced a partial remission with cetrorelix treatment that
lasted 2, 6 and 7 months, and six women (35%) had
disease stabilization for 1–12 months. The median
survival was 17 months [126].
Conclusions and future prospects
To date, our understanding of the GnRH system in
tumor cells is still far from complete, especially with
regard to the newly identified GnRH-II isoform and
the ‘putative’ GnRH-II receptor. Although there are
suggestive data supporting the existence of a functional
mammalian GnRH-II receptor and a role of the
GnRH-II receptor in mediating the antiproliferative
effects of GnRH-I antagonists and GnRH-II, direct
evidence for a functional human GnRH-II receptor
and the details of its downstream signaling mechanism
are certainly of great physiological importance and
research interest. The superior antiproliferative effects
of GnRH-II as compared to GnRH-I make GnRH-II
an attractive target for investigation, and the hormonal
W K. So et al. GnRH and ovarian cancer
FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5505
regulation of GnRH-II expression in ovarian cancer
cells is presently under investigation in our laboratory.
The widespread expression of the GnRH receptor in
ovarian carcinomas and the well-documented in vitro
effects, such as antiproliferation and apoptosis,
strongly support the candidacy of GnRH as a promis-

ing therapeutic approach for ovarian cancer. Elucida-
tion of the efficacy and modes of actions of GnRH-I
and GnRH-II, as well as their interactions with growth
factors that are known to be important in ovarian
cancer progression, is undoubtedly warranted.
Acknowledgements
P.C.K.L. is the recipient of a Child & Family Research
Institute Distinguished Scholar Award. W.K.S., J.C.C.
and S.L.P. were recipients of graduate studentship
awards from The Interdisciplinary Women’s Repro-
ductive Health Research Training Program.
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