PENGARUH GNRH AGONIS TERHADAP

APOPTOSIS DALAM KASUS

ENDOMETRIOSIS

Noor Pramono Noerpramana

HIFERI, Yogyakarta, 18 November 2017

INTRODUCTION
 The etiology of endometriosis (EDT) is
associated with retrograde migration of eutopic
endometrial tissue outside the uterine cavity.
Mechanisms of endometriotic lesions
development include:
- hormonal factors (e.g. estrogens),
- immunological factors,
- apoptosis and cell growth, and
- angiogenesis.
Fig. Courtesy ref22-Amodel of the most relevant molecular pathways in epithelial and stromal ectropic endometrial cells involved in
the pathophysiology of endometriosis. *Major proinflammatory cytokines are produced in both peritoneal macrophages and
endometriotic cells **Altered peritoneal cell mediated immunity seen in endometriosis is related to inflammatory cytokine expression
profile.
Advances in Reproductive Sciences, 2016, 4, 53-73
 Synthetic gonadotropin-releasing hormone (GnRH) analogues (both
agonists and antagonists) are frequently used in the treatment of
human reproductive pathologies, including EDT.

 GnRH and GnRH receptors have been isolated from eutopic and ectopic
endometrium

 In vitro, endometrial epithelial cultures(EEC) from patients with

endometriosis

 GnRH analogues (GnRH agonist, GnRH a, leuprolide acetate and GnRH

antagonist, GnRH ant, Antide) enhanced apoptosis in EEC, this was
accompanied by:
- Increase in expression of the pro-apoptotic protein Bax and FasL, and
- Decrease in expression of the anti-apoptotic protein Bcl-2.
 The administration of long-lasting GnRH agonists have:
- Cental effect: reduces the endogenous secretion of
gonadotropins by a mechanism of pituitary
downregulation  regression of endometriotic implants
due to the induction of hypoestrogenism.
- Direct effect on extra-pituitary tissues, such as
endometrium and ovary. Reports: it directly inhibit the
proliferation of various types of steroid-dependent
cancers, including: the breast, ovary and endometrium
(GnRH and GnRH receptors have been isolated from
both eutopic and ectopic endometrium).
 GnRHa appear to have a direct effect in
endometrial cells cultures, by enhancing the
percentage of apoptotic cells and decreasing the
release of pro-mitogenic cytokines such as IL-1B
and VEGF (angiogenesis)

 The percentage of apoptotic cells can be assessed

by the acridine orange-ethidium bromide
technique in endometrial cultures, under basal
conditions and after exposure to GnRHa, GnRHant,
or both
APOPTOSIS
 There are two different mechanisms of cell death: necrosis
and apoptosis.

 Necrosis: the viable cell becomes leaky, forms blebs and the
resulting cellular and nuclear lysis causes a release of the
cell’s contents leading to inflammation.

 Apoptosis, or programmed cell death;

- Kills unwanted cells but without inducing an immune
response or an inflammatory reaction.
-The viable cell shrinks, the chromatin condenses, budding
occurs, and the apoptotic bodies are phagocytozed by white
blood cells, so there is no resulting inflammation.
FIG. COMPARISON OF THE CELLULAR CHANGES THAT OCCUR DURING APOPTOSIS AND
NECROSIS
GYNECOL OBSTET INVEST 2008;66(SUPPL 1):10–18
Two major pathways towards apoptosis:

 Mitochondrial pathway (intrisic pathway):

The Bcl-2 family of proteins constitutes a critical intracellular
checkpoint.
The ratio of anti- to pro- apoptotic molecules, such as Bcl-2/Bax,
consitutes a rheostat that sets the threshold of susceptibility to
apoptosis, which utilizes organelles such as mitochondria to amplify
death signal. This interaction, Bax antagonizes Bcl-2 (Bcl-2 to prolong
cell survival)
 Death receptor pathway (extrinsic pathway):
The Fas-Fas ligand (FasL) system is the most studied mechanism in the
death receptor pathway because it is the primary mechanism for
induction of apoptosis in cell and tissues.
Fas (=CD95=Apo-1=TNFRSF6) is a prototypical member of TNF receptor
family, and induces apoptosis via cross-linking with FasL in various type
of cell.
When FasL binds to Fas, the target cell undergoes apoptotic cell death
with characteristic nuclear condensation and DNA fragmentation.
Whichever pathway is triggered, the cell
changes which result are mediated by a family
of cysteine aspartic acid specific proteases
(caspases), which occur as inactive precursors
(procaspases) that are proteolytically converted
to an active state following an apoptotic
stimulus.
 Intrinsic pathway:
-requires disruption of the mitochondrial membrane and the release
of mitochondrial proteins, such as cytochrome C.
-Cytochrome C is released into the cytoplasm, along with the other
two cytosolic protein factors, apoptotic protease activating factor-1
(Apaf-1) and procaspase-9, promoting the assembly of a caspase-
activating complex termed the apoptosome, which in turn initiates
the apoptotic caspase cascade.
-Regulators of cytochrome C release are the Bcl-2 family of proteins,
which can be either pro-apoptotic or anti-apoptotic.
-Cross-communication occurs between the extrinsic and intrinsic
pathways, and factors produced in one pathway can stimulate the
other.
 Extrinsic pathway:
-is initiated through binding of the transmembrane
death receptors, such as the Fas, TNF, and TRAIL
receptors, located on the cell membrane with their
respective ligands.
-This binding triggers aggregation of the receptors and
recruitment of an adaptor protein known as Fas-
associated death domain protein (FADD) within the cell
cytoplasm.
-FADD, in turn, recruits caspase-8 to form the death-
inducing signal complex (DISC) which initiates the
caspase activation cascade.
FIG. THE INTRINSIC AND EXTRINSIC APOPTOSIS PATHWAYS. REPRODUCED WITH
PERMISSION FROM MACFARLANE AND WILLIAMS
GYNECOL OBSTET INVEST 2008;66(SUPPL 1):10–18
 In the human endometrium, Bcl-2, Bax, Fas
and FasL are expressed throughout the
menstrual cycle

 The involvement of Bax and Bcl-2 in regulating

the decreasing eutopic endometrial apoptosis
in patients with endometriosis, and Fas and
FasL are diminished in eutopic endometrium
from endometriosis patients
ANGIOGENESIS
 Highly regulated process of angiogenesis that
occurs within the female reproductive tract is
critical for normal reproduction, including follicular
maturation, selection and normal function of the
corpus luteum, and endometrial growth and
remodeling.
 The pathogenesis of EDT involves the implantation
of exfoliated endometrium which is dependent for
its survival on the development of an extensive
blood supply – by the process of angiogenesis –
both within and surrounding the ectopic tissue.
ANGIOGENESIS CONT’
 Vascular development: vasculogenesis and
angiogenesis.
 Vasculogenesis: The differentiation of endothelial
cells from embryologic mesoderm and the
assembly of these differentiated cells into discrete
blood vessels
 Angiogenesis: The proliferation and migration of
endothelial cells from pre-existing vessels and
leads to the sprouting or remodeling of new
vessels in a given area.
ANGIOGENESIS CONT’
 There are two main regulators of angiogenesis:
--vascular endothelial growth factors (VEGFs) and
--angiopoietins.

 VEGF family of angiogenic molecules involved in both

physiological angiogenesis and a number of pathological
conditions that are characterized by excessive angiogenesis.

 VEGF is:
A highly specific mitogen for vascular endothelial cells, and
Not only induces angiogenesis, but also
Works as a survival factor for tumour and endothelial cells,
protecting them from apoptosis
ANGIOGENESIS CONT’

 VEGF levels are elevated in peritoneal fluid from patients

with EDT and in the endometriotic tissue itself.
 The expression of VEGF in endometrial cells has been found
to be potentiated by a variety of cytokines, including IL-1.
 VEGF-A:
--is a potent mitogen and migratory stimulus for endothelial
cells and has been found to be involved in both physiological
and pathological conditions.
--that it is produced by endometrial cells and is a critical
factor involved in the pathogenesis of the disease.
 Visualization of ectopic endometrial implants by laparoscopy
shows them to be highly vascularized.
 An immunological basis, IL-1B is a pleiotrophic cytokine involved in
the inflammatory immune response:
- to act as a growth factor (mitogenic effect)
- induces the protection of different cells from apoptotic death

 IL-1B and VEGF are produced by endometrial cells

 Infammatory cytokines, such as IL-1B and IL-6, elevated in the
peritoneal fluid of women with endometriosis.
 In endometriotic cells as in other tissues, expression of VEGF to be
potentiated by a variety of cytokines, especially IL-1B

 In view of the above evidence it has therefore been postulated that

GnRH analogues interfere with endometrial cell growth by regulation
of apoptotic and angiogenic mechanisms.
HUMAN REPRODUCTION, VOL.25, NO.3 PP. 642–653, 2010
ADVANCED ACCESS PUBLICATION ON DECEMBER 15, 2009 DOI:10.1093/HUMREP/DEP437

CHANGES IN TISSUE INFLAMMATION, ANGIOGENESIS AND APOPTOSIS IN ENDOMETRIOSIS,

ADENOMYOSIS AND UTERINE MYOMA AFTER GNRH AGONIST THERAPY
KHALEQUE NEWAZ KHAN1,5, MICHIO KITAJIMA1, KOICHI HIRAKI1, AKIRA FUJISHITA2, ICHIRO SEKINE3, TADAYUKI
ISHIMARU4, AND HIDEAKI MASUZAKI1

 GnRHa was able to reduce the inflammatory reaction and

angiogenesis; and to induced apoptosis in tissues from women
with endometriosis, adenomyosis, and uterine myoma. These
multiple biological effects at the tissue level may be involved in
the regression of these reproductive diseases.

 The reduction in inflammatory reaction and angiogenic response

was determined by a decrease in the amount of Mo infiltration
and micro-vessel density in the biopsy specimens derived from
GnRHa-treated women  could be due to the direct effect of
GnRHa at the tissue level or indirect effect of decrease estrogen
level. Estradiol may play a role in the recruitment of immune cells
such as Mo.
 The decrease in inflammatory response among GnRHa users
might be the dual effect of systemic and local suppression of
estrogen. GnRHa has been reported to decrease the
expression of aromatase cytochrome P450 in the eutopic
endometrium from women with endometriosis, adenomyosis
or leiomyoma

 Apoptosis to be detected using Nuclear morphological

features of an apoptotic cell were considered as TUNEL-
positive stained nucleus, i.e. shrinkage of the nucleus with
condensed chromatin and/or densely aggregated marginal
chromatin or dot-like or drop-like condensed nuclear
fragments.
INVET. 2012, 14(1): 101-110

APOPTOSIS INDUCED BY A GNRH ANALOGUE IN A GRANULOSA CELL LINE FROM BOVINE OVARY (BGC-1)
APOPTOSIS INDUCIDA POR UN ANÁLOGO DE GNRH EN UNA LÍNEA CELULAR DE GRANULOSA DE
OVARIO BOVINO (BGC-1)
CRUZANS, P.R.1; CAROU, M.C.1; LORENZO, M.S.1; LOMBARDO, D.M.1 *

 GnRHR have been identified in ovarian granulosa cells (GC) and luteal cells (LC).
The activation of GnRHR in GC regulates gonadal function.

 Studies using GnRHa in rat ovaries, in vivo and in vitro, showed an increase in
follicular atresia.

 To study the regulation of apoptosis in an established line of bovine granulosa

cells (BGC-1) using leuprolide acetate (LA) as GnRHa and ANTIDE as competitive
antagonist.

 Results: increase in the proportion of apoptotic cells (induced for 24 and 28 hs

with 100 nM LA) by morphological technique (DAPI staining and Hematoxilin),
membrane markers expression (Annexin V-FITC and PI detected by flow
cytometry) and biochemical techniques such as determining caspase 3 activity.
Apoptosis was partially inhibited by ANTIDE, showing per se activity when
caspase 3 activity was evaluated.
 The intracellular pathways regulating the apoptotic
process revealed the existence of a mechanism
that involves the activation of the intrinsic
pathway, through the analysis of protein
expression of Bcl-2 family (Bax and Bcl-2) and the
inhibition of extrinsic pathway by inhibiting the
activity of the enzym phospholipase D (PLD). It is
proposed that ANTIDE works as antagonist of the
intrinsic pathway but as extrinsic pathway agonist
as inhibiting the activity of PLD.
 Apoptosis is programmed cell death which:
 involves an orderly process genetically regulated by the
synthesis of proteins such as Bcl-2 family,
 includes pro and anti-apoptotic genes, caspases, and
 initiator and effectors proteins that culminate with the
orderly DNA fragmentation.
 In BGC-1, apoptosis induced by LA dose-dependent
manner via the intrinsic pathway by stimulating the
expression of Bax, and through the extrinsic pathway by
inhibiting PLD activity. ANTIDE antagonist character is
questionable considering the results presented.
WENG ET AL. JOURNAL OF TRANSLATIONAL MEDICINE 2014, 12:306

GNRH AGONISTS INDUCE ENDOMETRIAL EPITHELIAL CELL APOPTOSIS VIA GRP78

DOWN-REGULATION
HUINAN WENG1,2†, FENGHUA LIU2†, SHUIWANG HU1, LI LI2 AND YIFENG WANG3*

 GnRHa induce endometrial epithelial cell apoptosis via

GRP 78 down-regulation. Provide an important
molecular framework for further evaluation of GnRHa
therapy.

 Apoptosis, or programmed cell death:

-is a physiological process that plays a critical role in
maintaining tissue homeostasis, killing unwanted cells
without inducing immune responses or inflammatory
reactions.
-Has been implicated in both normal development and
disease.
 Both GnRH and GnRH receptors have been isolated from the eutopic
and ectopic endometrium, supporting a direct function for GnRHa in
endometrial growth. GnRHa induces apoptosis in both eutopic and
ectopic endometrial cells from patients with endometriosis. GnRHa
induces apoptosis and reduces cell proliferation in ovarian,
endometrial and breast cancer cell line.

 In this study compared the proteomic profiles of endometriosis in

patients before and after GnRHa therapy to identify proteins involved
in the mechanisms of GnRHa action.

 LA promoted the apoptosis of eutopic endometrial epithelial cells and

inhibited the expression of anti-apoptotic factor GRP78. LA induces
endometrial epithelial cell apoptosis throught the downregulation of
GRP78.
CONCLUSIONS
 Gonadotropin-releasing hormone (GnRH) analogues may act
directly on endometrial cells and inhibit their growth and
proliferation by regulation of apoptotic and angiogenic
mechanisms.

 Eutopic endometrial cells from patients with endometriosis show

an increased proliferation rate and are less susceptible to cell
death by apoptosis than those from subjects without the disease.

 GnRH analogue, leuprorelin, inhibits cell proliferation and

increases the apoptotic rate in eutopic endometrial cell cultures,
an effect that appears to be mediated by an increase in the
expression of the pro-apoptotic proteins Bax and FasL and a
decrease in the expression of the anti-apoptotic protein Bcl-2.
CONCLUSIONS CONT’
 Angiogenesis is an important process in the development of endometrial
tissue, and it is regulated by vascular endothelial growth factors (VEGFs)
and angiopoietins.

 VEGF levels are elevated in peritoneal fluid and endometriotic tissue from
patients with endometriosis.The expression of VEGF is potentiated by a
variety of cytokines, including IL-1 _ .

 Leuprorelin reduces the production of VEGF-A and IL-1B in eutopic

endometrial cell cultures, it could inhibit the development of endometriosis.

 GnRH analogues appear to be effective in reducing the growth of

endometrial cells:
- not only due to their classical pituitary endocrine effects,
- but also via a direct effect on the endometrial cells themselves.
TERIMA KASIH