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Endocannabinoids modulate apoptosis in endometriosis and adenomyosis

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Adenomyosis that is a form of endometriosis is the growth of ectopic endometrial tissue within the muscular wall of the uterus (myometrium), which may cause dysmenorrhea and infertility. Endocannabinoid mediated apoptotic mechanisms of endometriosis and adenomyosis are not known. We hypothesized that the down regulation of endocannabinoid receptors and/or alteration in their regulatory enzymes may have a direct role in the pathogenesis of endometriosis and adenomyosis through apoptosis. Endocannabinoid receptors CB1 and CB2, their synthesizing and catabolizing enzymes (FAAH, NAPE-PLD, DAGL, MAGL) and the apoptotic indexes were immunohistochemically assessed in endometriotic and adenomyotic tissues. Findings were compared to normal endometrium and myometrium. Endometrial adenocarcinoma (Ishikawa) and ovarian endometriosis cyst wall stromal (CRL-7566) cell lines were furthermore cultured with or without cannabinoid receptor agonists. The IC50 value for CB1 and CB2 receptor agonists was quantified. Cannabinoid agonists on cell death were investigated by Annexin-V/Propidium iodide labeling with flow cytometry. CB1 and CB2 receptor levels decreased in endometriotic and adenomyotic tissues compared to the control group (p=0,001 and p=0,001). FAAH, NAPE-PLD, MAGL and DAGL enzyme levels decreased in endometriotic and adenomyotic tissues compared to control (p=0,001, p=0,001, p=0,001 and p=0,002 respectively). Apoptotic cell indexes both in endometriotic and adenomyotic tissues also decreased significantly, compared to the control group (p=0,001 and p=0,001). CB1 and CB2 receptor agonist mediated dose dependent fast anti-proliferative and pro-apoptotic effects were detected in Ishikawa and ovarian endometriosis cyst wall stromal cell lines (CRL-7566). Endocannabinoids are suggested to increase apoptosis mechanisms in endometriosis and adenomyosis. CB1 and CB2 antagonists can be considered as potential medical therapeutic agents for endometriosis and adenomyosis.
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Acta Histochemica
journal homepage: www.elsevier.com/locate/acthis
Endocannabinoids modulate apoptosis in endometriosis and adenomyosis
Elif Bilgic
a
, Elif Guzel Meydanli
b
, Sevil Kose
c
, Makbule Cisel Aydin
d
, Eda Karaismailoglu
e
,
Irem Akar
c
, Alp Usubutun
d
, Petek Korkusuz
a,
a
Department of Histology and Embryology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
b
Department of Histology and Embryology, Istanbul University Cerrahpasa Medical Faculty, 34093 Istanbul, Turkey
c
Stem Cell Research and Application Center, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
d
Department of Pathology, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
e
Department of Biostatistics, Hacettepe University Faculty of Medicine, 06100 Ankara, Turkey
ARTICLE INFO
Keywords:
Endocannabinoids
Endometriosis
Adenomyosis
Apoptosis
ABSTRACT
Adenomyosis that is a form of endometriosis is the growth of ectopic endometrial tissue within the muscular wall
of the uterus (myometrium), which may cause dysmenorrhea and infertility. Endocannabinoid mediated
apoptotic mechanisms of endometriosis and adenomyosis are not known. We hypothesized that the down
regulation of endocannabinoid receptors and/or alteration in their regulatory enzymes may have a direct role in
the pathogenesis of endometriosis and adenomyosis through apoptosis. Endocannabinoid receptors CB1 and
CB2, their synthesizing and catabolizing enzymes (FAAH, NAPE-PLD, DAGL, MAGL) and the apoptotic indexes
were immunohistochemically assessed in endometriotic and adenomyotic tissues. Findings were compared to
normal endometrium and myometrium. Endometrial adenocarcinoma (Ishikawa) and ovarian endometriosis cyst
wall stromal (CRL-7566) cell lines were furthermore cultured with or without cannabinoid receptor agonists.
The IC50 value for CB1 and CB2 receptor agonists was quantied. Cannabinoid agonists on cell death were
investigated by Annexin-V/Propidium iodide labeling with ow cytometry. CB1 and CB2 receptor levels
decreased in endometriotic and adenomyotic tissues compared to the control group (p = 0,001 and p = 0,001).
FAAH, NAPE-PLD, MAGL and DAGL enzyme levels decreased in endometriotic and adenomyotic tissues
compared to control (p = 0,001, p = 0,001, p = 0,001 and p = 0,002 respectively). Apoptotic cell indexes both
in endometriotic and adenomyotic tissues also decreased signicantly, compared to the control group
(p = 0,001 and p = 0,001). CB1 and CB2 receptor agonist mediated dose dependent fast anti-proliferative
and pro-apoptotic eects were detected in Ishikawa and ovarian endometriosis cyst wall stromal cell lines (CRL-
7566). Endocannabinoids are suggested to increase apoptosis mechanisms in endometriosis and adenomyosis.
CB1 and CB2 antagonists can be considered as potential medical therapeutic agents for endometriosis and
adenomyosis.
1. Introduction
Endometriosis and adenomyosis are dened as the unusual location
of the endometrial tissue at ectopic sites and in the myometrium. They
can cause pelvic pain, dyspareunia, amenorrhea, dysmenorrhea and
infertility (Irving and Clement 2011; Kruse et al., 2012; Lin et al., 2014;
Lo Monte et al., 2013; Sznurkowski and Emerich, 2008; Gao et al.,
2006; Vannuccini et al., 2016; Yang et al., 2013; Yamanaka et al.,
2014). Endometriosis aects a large population and decreases quality of
life. The pathogenesis of the disease remains unclear, although it is
believed to relate with the quite aggressive behavior of endometriotic
cells at migrating ectopic locations and the resistance of these cells to
apoptosis (Agic et al., 2009; Nasu et al., 2011; Sbracia et al., 2016).
Pathogenesis of adenomyosis is similar to endometriosis; adenomyotic
cells have resistance to apoptosis as well (Yamanaka et al., 2014). The
relationship between the endocannabinoids and adenomyosis is not yet
studied.
Endocannabinoids, which are mostly located in the central nervous
system and also in other organ systems, are Cannabis ligands that
specically act through their CB1 and CB2 receptors (Alger and Kim,
http://dx.doi.org/10.1016/j.acthis.2017.05.005
Received 24 October 2016; Received in revised form 9 May 2017; Accepted 9 May 2017
Various data of this study was presented at the XII. National Congress of Histology and Embryology, Ankara, Turkey, May 2730, 2014, and at the XIII. National Congress of
Histology and Embryology, İzmir, Turkey, April 30May 3, 2016.
Corresponding author at: Hacettepe University Faculty of Medicine, Department of Histology and Embryology, Sihhiye 06100, Ankara, Turkey.
E-mail addresses: elifbilgic8@gmail.com (E. Bilgic), elifguzelctf@yahoo.com (E.G. Meydanli), sevil.arslan@hacettepe.edu.tr (S. Kose), ciselaydin@gmail.com (M.C. Aydin),
edaozturk1982@yahoo.com (E. Karaismailoglu), iremakar@yahoo.com (I. Akar), alpusubutun@yahoo.com (A. Usubutun), petek@hacettepe.edu.tr (P. Korkusuz).
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2011; Coskun and Bolkent, 2014; Mercati et al., 2012; Muccioli, 2010;
Scotchie et al., 2015; Yazulla, 2008). The most well-known endocan-
nabinoids are anandamide (AEA) and di-arachidonoylglycerol (2-AG)
(Alger and Kim, 2011; Muccioli, 2010; Scotchie et al., 2015; Yazulla,
2008). AEA is known to act more over the CB1 receptor in the female
genital system, while 2-AG displays its eects usually through the CB2
receptor (Maccarrone, 2009; Taylor et al., 2010). In the female genital
system, endocannabinoids and their receptors are generally located in
the endometrium, the myometrium, the ovarian cortex and the medulla
and the uterine tubes; they have critical roles in menstrual cycle,
ovarian maturation, embryo transplantation and implantation
(Brighton et al., 2011; El-Talatini et al., 2009, 2010; Karasu et al.,
2011; Maccarrone, 2009; Scotchie et al., 2015; Sun et al., 2009; Taylor
et al., 2010).
Recent researches suggested that endocannabinoids are involved in
the pathophysiology of endometriosis in a variety of ways.
Endocannabinoid agonists have anti-proliferative and analgesic eects
on endometriotic cells or patients. Endometriosis-associated pain is
shown to decrease by WIN 55212-2, CB1 and CB2 receptor agonist in
experimental studies or by palmitoylethanolamide in patients with
endometriosis (Cobellis et al., 2011; Dmitrieva et al., 2010; Giugliano
et al., 2013; Indraccolo and Barbieri, 2010; Lo Monte et al., 2013). Cell
proliferation in deep inltrating endometriosis decreased with WIN-
55212-2, both in vitro and in vivo (Leconte et al., 2010). In vitro
stimulatory eect of endocannabinoid agonists on cell migration
moreover was presented (Gentilini et al., 2010; McHugh et al., 2012).
According to this, enhanced endometrial stromal cell migration via CB1
and GPR18 receptor with use of methanandamide, which is another
endocannabinoid agonist, or N-arachidonyl glycine, which is an
endogenous metabolite of anandamide, were shown through the
activation of PI3K/Akt, ERK1/2 or MAPK pathways (Gentilini et al.,
2010; McHugh et al., 2012). Although some activities of endocannabi-
noids are dened, we still do not know the eects of endocannabinoids
on apoptosis in endometriosis. Anandamide leaded to apoptosis
through CB1 receptor and p38 pathway on decidual cells (Almada
et al., 2015; Fonseca et al., 2009).
Given the apoptotic and anti-proliferative eects of endocannabi-
noids, we hypothesized that the down regulation of endocannabinoid
receptors and/or alteration in their regulatory enzymes may have a
direct role in the pathogenesis of endometriosis and adenomyosis
through apoptosis. We aimed to dene the potential apoptosis related
classical receptor mediated eects of endocannabinoids on endome-
triosis and adenomyosis. We investigated the dierences of immune
labelings of CB1 and CB2 receptors, AEA and 2-AG catabolizing and
synthesizing enzymes, as well as apoptotic index between the endome-
triotic and adenomyotic patients and age-matched controls. Depending
on the supposedly pro-apoptotic eects of endocannabinoids (Almada
et al., 2015; Siegmund et al., 2016), endometrial adenocarcinoma cell
line (Ishikawa) and ovarian endometriosis cyst wall stromal cell line
(CRL 7566) were cultured with or without cannabinoid classical
receptor agonists. The xCELLIgence cell impedance based system was
used to calculate the IC50 value for CB1 and CB2 receptor agonists.
Cannabinoid agonistseect on cell death was investigated with ow
cytometry by Annexin-V/propidium iodide labeling. Outcomes of these
experiments may explain endocannabinoid eects of cell survival and
death mechanisms in endometriosis.
2. Materials and methods
2.1. Design
A double blind randomized experimental study was designed. We
received endometrial archive samples belonging to patients having
been diagnosed as endometriotic and adenomyotic from January 2010
to July 2012. Age-matched paran endometrial tissue blocks of 20
endometriosis, 17 adenomyosis patients and 19 normal controls
between 24 and 52 years were obtained from Hacettepe University
Pathology Department (Table 1). Control tissues were obtained from
patients undergoing dilatation and curettage surgery for benign gyne-
cological conditions other than endometrial disease. Control endome-
trial tissues were sub-grouped according to the phase of menstrual cycle
as proliferative (n = 12) and secretory phases (n = 7). The endome-
triotic tissues were also sub-grouped as cystic (n = 9) and non-cystic
(n = 11). The use of endometriotic cells and the paran blocks of
endometrial tissue was approved by the Hacettepe University Non-
invasive Clinical Researches Ethical Committee (TBK 12/05-08), An-
kara, Turkey.
2.1.1. CB1 and CB2 receptors and FAAH, NAPE-PLD, MAGL, DAGL
enzymes immune labeling
56μm Thick paran sections were deparanized. Endogenous
peroxidase activity was blocked by 3% hydrogen peroxide (cat#
216763, Sigma-Aldrich, St. Louis, USA) after antigen retrieval.
Nonspecic binding was blocked with 5% normal mouse serum
(cat#M5905, Sigma-Aldrich, St. Louis, USA) for 30 min. Slides were
incubated with following primary antibodies overnight at 4 °C:
CB1(cat#C2866, rabbit polyclonal;1/100 dilution; Sigma-Aldrich, St.
Louis, USA), CB2 (cat#HPA028718, rabbit polyclonal; 1/100 dilution;
Sigma-Aldrich, St. Louis, USA), FAAH (cat#HPA007425, rabbit poly-
clonal, 1/50 dilution; Sigma-Aldrich, St. Louis, USA), MAGL
(cat#100035, rabbit polyclonal, 1/100 dilution, Cayman, Michigan,
USA), DAGL (cat#ab106979, rabbit polyclonal, 1/100 dilution, Abcam,
Cambridge, USA). Incubation with NAPE-PLD (cat#HPA019832, rabbit
polyclonal, 1/100 dilution, Sigma-Aldrich, St. Louis, USA) was per-
formed overnight at RT. The secondary antibody incubation
(cat#EXTRA3, mouse monoclonal, Sigma-Aldrich, St. Louis, USA) was
performed for 30 min at RT at 1/20 dilution. After washing slides and
incubating with DAB (cat#D3939, Sigma-Aldrich, St. Louis, USA) we
used haematoxylin for counterstaining. Digital images were analyzed
and captured using the Leica DM6000B microscope equipped with a
DFC480 digital camera.
2.1.2. Image analysis
Two pathologists according to pathological criteria for the diseases
selected endometriotic and adenomyotic foci under the microscope (Fu
et al., 2013; Yu et al., 2015).
Table 1
Age-matched control and experimental groups are presented with mean ± standard deviation/median (minmax) of their immune reactivities and apoptotic indexes.
Control n AGE
mean ± SD
CB1
mean ± SD
CB2
mean ± SD
FAAH
mean ± SD
NAPE-PLD
mean ± SD
MAGL
mean ± SD
DAGL
median (min-max)
TUNEL
median (min-max)
Proliferative 12 37,5 ± 1,27 0,80 ± 0,11 0,79 ± 0,10 0,79 ± 0,07 0,73 ± 0,15 0,69 ± 0,09 0,64 (0,490,79) 0,63 (0,00,85)
Secretory 7 41,4 ± 2,5 0,80 ± 0,11 0,76 ± 0,10 0,83 ± 0,11 0,71 ± 0,11 0,76 ± 0,10 0,72 (0500,82) 0,65 (0,40,91)
Endometriosis
Non-cystic 11 40,4 ± 2,07 0,35 ± 0,16 0,35 ± 0,21 0,37 ± 0,26 0,27 ± 0,23 0,22 ± 0,29 0,60 (0,00,66) 0,0 (0,00,70)
Cystic 9 35,6 ± 2,5 0,39 ± 0,20 0,40 ± 0,25 0,34 ± 0,34 0,32 ± 0,29 0,38 ± 0,24 0,52 (0,480,74) 0,11 (0,00,66)
Adenomyosis 17 42,5 ± 0,9 0,37 ± 0,19 0,34 ± 0,32 0,41 ± 0,21 0,43 ± 0,25 0,29 ± 0,26 0,48 (0,00,81) 0,05 (0,00,69)
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[
Ten endometriotic foci or equal amount of glands have been
selected at non-overlapping elds of each endometrial, endometriotic
and adenomyotic sections by the motorized stage module of a Leica
DM6000B microscope (Lin et al., 2014). Photomicrographs of each
focus were generated by the microscope (Leica DM6000B) attached
computerized digital camera (DFC 480, Leica Westlar Germany) and
captured as TIFF at 200×magnication. The bright-eld images were
analyzed quantitatively by image processing program (LAS 3.8 Leica
Inc., Westlar Germany version 3.8). Areas of interest (ROI) consisting of
endometrial, adenomyotic or normal glandular (for control group) foci
have been chosen at the x and y stages at the binary mode; and the total
ROI was calculated for 10 foci. The measurements were done at
minimum 45,876 um
2
- maximum 125,214 um
2
for each endometriotic,
adenomyotic or glandular focus (Hey-Cunningham et al., 2013). Brown
stained particles (immune labeled cells) were counted in the binary
dened area, at counting mode of LAS. Haematoxylin was extracted
from DAB by RGB level of the software. The blue threshold value was
106,49 px for the nuclei, and the brown threshold value was 65,22 px
for peroxidase labeling. The number of total immune reactive cell
percentage was expressed as the ratio of immune positive particles
(both the glandular epithelial and stromal cells) to total ROI.
2.1.3. TUNEL analysis for apoptosis
Slides were rinsed after de-waxing and dehydrating. Endogenous
peroxidase activity was blocked in 3% hydrogen peroxide for 10 min.
Sections were treated with permeabilization solution (0.1% Triton X-
100 in 0.1% sodium citrate) for 8 min at room temperature. We used
the in-situ cell death detection kit (Roche, Indianapolis, IN, USA) for
detecting the DNA fragments. Digital images were analyzed and
captured using the Leica DM6000B microscope equipped with the
DC500 digital camera after DAB incubation and haematoxylin staining.
The apoptotic index was expressed as a percentage of apoptotic
glandular and stromal cells over total glandular and stromal cells at
200×magnication. Average of 4 analyzed non-overlapping elds was
reported (Shen et al., 2010) in every specimen.
2.1.4. Cell culture
Ishikawa cell line (99040201, Sigma, Germany) was used to
simulate normal endometrial glandular cells with their well-known
phenotypic similarity and their response to steroids, resembling the
physiological conditions (Tamm-Rosenstein et al., 2013). Ishikawa cell
line is provided at passage 15 and authenticated by the manufacturer
(European Collection of Authenticated Cell Cultures, London, UK).
Endometriosis cyst wall stromal cell line (CRL-7566, ATCC, USA) was
used at passage 15, to analyze the cannabinoid agonistic eect on
endometriosis model. CRL-7566 is provided at passage 15 and,
authenticated by DNA-based method. Although stated as mycoplasma
free by the providers, the cell lines were tested by EZ-PCR Mycoplasma
test kit (cat#20-700-10, Biological Industries, Kibbutz Beit Haemek,
Israel) before use. Ishikawa cells were incubated in DMEM F-12 with
10% FBS and 2% L-glutamine and 1% pen-strep solution at 37 °C and
5% CO
2.
CRL-7566 cells were incubated in DMEM with % 20 FBS and %
2L-glutamine and % 1 pen-strep solution at 37 °C and 10% CO
2.
Cells
were used for the experiments at passage 18.
2.1.5. Impedance-based real-time cell proliferation analysis
Real-time cell proliferation was assessed with the xCELLIgence
system (Roche Applied Science, Mannheim, Germany; ACEA
Biosciences, San Diego, CA). Disposable 96-well e-plates were coated
with 10 μgr/ml bronectin, cells were seeded and incubated at 37 °C, %
5 CO
2
until cell index was 1 at 22 h (Lowin et al., 2012).
Ishikawa cell line was used to detect half maximal inhibitory
concentration (IC
50
) of selective CB1 and CB2 agonists ACPA (1318,
Tocris Bioscience, Bristol, UK), respectively. ACPA (100 nM, 1 μM,
10 μM, 100 μM) and CB 65 (1 μM, 10 μM, 100 μM) were applied at
dierent concentrations and the IC
50
was calculated accordingly (to
determine the of cannabinoids RTCA software was used) (Fig. 5A and
C). Because our experimental procedure took about 146 h; IC50
concentrations were calculated both at 126th and 46th hours (Fig. 5B
and D). After detecting IC50 concentrations, Ishikawa and CRL-7566
cells were exposed to the determined IC
50
concentration of ACPA
(9.3 ×10
6
M) and CB65 (1.9 ×10
4
M) for 46 h and monitored at
every 15 min for 146 h (Fig. 6).
2.1.6. Flow cytometry analysis
We incubated cells with the determined IC
50
values of ACPA
(9,3 ×10
6
M) and CB65 (1,9 ×10
4
M) for 46 h after expansion.
Cells with and without cannabinoid agonists were analyzed by
Annexin-V/propidium iodide labeling in FACS Aria ow cytometer
(Becton, Dickinson Biosciences, USA) at 46th hour. Cells were classied
as live (Annexin V, PI), necrotic (Annexin V, PI+), early
apoptotic (Annexin V+, PI), and late apoptotic (Annexin V+, PI+)
cells. The acquired data was analyzed by using BD FACSDiva software
v6.1.2 (Becton Dickinson Biosciences, USA) (Fig. 7). The ratio of
apoptosis was reported as early apoptotic percentage plus late apoptotic
percentage in the text.
2.2. Statistics
Distribution of normality of immune labeling was evaluated by the
Shapiro-Wilk test. Age and CB1, CB2, FAAH, NAPE-PLD, MAGL immune
labelings of stromal and glandular cell variables were evaluated by one-
way ANOVA followed by post hoc Tukey testing. DAGL immune
labeling in stromal and glandular cells was not normally distributed.
Therefore, they were evaluated by the Kruskal-Wallis and the Mann-
Whitney U-tests with Bonferroni correction. Correlation analysis was
performed using the Pearsons (for parametric data) or the Spearman
correlation (for nonparametric data) tests. Parametric data were
presented as the mean ± standard error of mean, while others were
presented as minimum, median, and maximum values. Condence
interval was 95% and statistical signicance was dened as p < 0.05.
The SPSS (15.0 version) program and the NCSS-PASS 2007 software
were used for analysis.
3. Results
3.1. CB1 and CB2 receptor immune labeling
CB1 and CB2 receptor immune labeling was cytoplasmic and intense
in both endometrial glandular and stromal cells in the control group
(Fig. 1AF). Immune labeling percentages for CB1 and CB2 receptors in
the experimental groups were signicantly (p = 0,001) lower than that
of the control group (Fig. 1G). The CB1 and CB2 receptor immune
labeling was similar in the endometriosis and the adenomyosis groups.
Endometrial glandular and stromal cells in proliferative and secretory
phases of the control group exhibited similar CB1 receptor immune
labeling. The CB2 receptor labeling of the glandular cells was sig-
nicantly (p = 0,020) higher in the proliferative phase than the
secretory phase however it remained unchanged in the stromal cells
between proliferative and secretory menstrual phases in this group. CB2
immune labeling for glandular epithelial and stromal cells decreased
with age (r = 0,612, p = 0,012; r = 0,53, p = 0,033) in the
adenomyosis group.
3.2. FAAH and NAPE-PLD immune labeling
FAAH and NAPE-PLD enzymes presented a compatible pattern of
immune labeling with CB1 receptor (Fig. 2AF). Immune labeling
analysis indicated that FAAH and NAPE-PLD enzyme immune labeling
were signicantly lower (p = 0,001) in glandular and stromal cells in
the endometriosis and the adenomyosis groups when compared to the
control group (Fig. 2G). Although FAAH enzyme levels in the glandular
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cells was signicantly lower in the proliferative phase when compared
to the secretory phase (p = 0,004), FAAH enzyme immune labeling did
not show any dierence in stromal cells between menstrual phases of
the control group. The control group furthermore showed similar
NAPE-PLD enzyme expression in endometrial glandular and stromal
cells in proliferative and secretory phases.
3.3. MAGL and DAGL enzyme immune labeling
MAGL and DAGL showed a compatible immune labeling pattern
with the CB2 receptor (Fig. 3AF). Immune labeling analysis indicated
that immune labeling of MAGL (p = 0,001 both for glandular and
stromal cells) and DAGL (p = 0,002 for glandular cells) in the experi-
mental groups compared to the control group (Fig. 3GI).
3.4. TUNEL assay for apoptosis
Lower TUNEL positivity was detected in the endometriosis and the
adenomyosis groups compared to the control group in glandular and
stromal cells (p = 0,001) (Fig. 4AE). Apoptotic index revealed no
dierence in the glandular and stromal cells among the phases of the
cycle in the control group and between the endometriosis and the
adenomyosis groups as well as cystic and solid subgroups of endome-
triotic patients.
3.5. Impedance-based real-time cell proliferation analysis
Optimal anti-proliferative eect of ACPA and CB65 at IC50 con-
centrations was at the 46th hour (Fig. 5B and D). The IC
50
concentra-
Fig. 1. AF are endometrial micrographs exhibiting cytoplasmic CB1 (AC) and CB2 (DF) receptor immune labeling on glandular epithelial (*) and stromal cells (**), Haematoxylin
400×. (G) Immune labelings for CB1 and CB2 receptor distribution in control and experimental groups are shown. (*) p = 0,001. Note the signicantly decreased immune labeling in
adenomyosis (C and F) and endometriosis groups (B and E) comparing to control (A and D) in the micrographs and the graphic (n = 19 for control; n = 20 for endometriosis and n = 17
for adenomyosis).
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tions of CB1 and CB2 agonists were detected as 9.3 ×10
6
M for ACPA
and 1.9 ×10
4
M for CB 65 on Ishikawa cells (Fig. 5A and C).
Ishikawa and CRL-7566 endometriotic cells exhibited decreasing
cell indices immediately after application of the IC50 concentration of
ACPA and CB 65. Cell proliferation index for CRL-7566 cells decreased
76% with ACPA and 86% with CB65 (Fig. 6A and B). Cell proliferation
index for Ishikawa cells decreased 95% with ACPA and 81% with CB65
(Fig. 6C and D).
3.6. Flow cytometry analysis
Annexin-V/propidium iodide labeled total (early and late) apoptotic
Ishikawa and CRL-7566 cell numbers increased with ACPA and CB65
exposure compared to the untreated control (Fig. 7). The 71,7% of
Ishikawa cells and 81,7% of CRL-7566 cells were apoptotic (early and
late) with ACPA (Fig. 7A and D). The 80,5% of Ishikawa cells and
78,3% of CRL-7566 cells were apoptotic (early and late) (Fig. 7B and E)
with CB65. In the untreated control group, only 0,8% of Ishikawa cells,
but 76,9% of CRL-7566 cells were apoptotic (early plus late) (Fig. 7C
and F).
4. Discussion
Endometriosis and adenomyosis that co-exist with endometriosis
(Garavaglia et al., 2015), aecting nearly 1015% of the female
population (Jeung et al., 2016) increase the risk of gynecological
malignancies (Krawczyk et al., 2016). Adenomyosis was furthermore
recognized in 1634% of endometrial carcinoma hysterectomy speci-
mens (Gizzo et al., 2016). Endometriotic cells tend to have cancer cell
like aggressive features for migrating to ectopic locations and they are
resistant to apoptosis (Agic et al., 2009; Nasu et al., 2011; Sbracia et al.,
2016). Adenomyotic cells have resistance to apoptosis as well
(Yamanaka et al., 2014).
Cannabinoid receptors (CB1, CB2) and the NAPE-PLD, FAAH,
DAGL, MAGL enzymes exhibited dierent regulation in the glandular
epithelial cells and stromal cells of the normal endometrial, the
Fig. 2. AF are endometrial micrographs showing cytoplasmic AEA catabolizing FAAH (AC) and synthesizing NAPE-PLD (DF) enzyme immune labeling on glandular epithelial (*) and
stromal cells (**), Haematoxylin 400×. (G) Graphic shows the immune labeling for FAAH and NAPE-PLD in control and experimental groups. (*) p = 0,001. Note that both enzymes
signicantly decreased in endometriosis (B and E) and adenomyosis (C and F) groups comparing to control (A and D). (n = 19 for control; n = 20 for endometriosis and n = 17 for
adenomyosis).
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endometriotic and the adenomyotic tissues in this study. Immune
labeling values for CB1 and CB2 receptors in glandular and stromal
cells of endometriosis and adenomyosis groups were lower than that of
the control group. Our ndings for CB1 immune labeling were similar
with the results of Resuehr et al. (2012) who showed that CB1 immune
reactivity decreased in endometriosis compared to the control group,
however they were dierent than that of Leconte et al. (2010) who
suggested no dierence for the CB1 receptor expression level between
endometriosis and controls (Leconte et al., 2010; Resuehr et al., 2012).
CB1 and CB2 receptor distribution in the endometriosis and
adenomyosis groups were not signicantly dierent in our study.
Findings of this study demonstrated lower immune labeling for
cannabinoid receptors in adenomyosis and endometriosis compared
to the control group.
Labeling for NAPE-PLD and FAAH, synthesizing and catabolizing
enzymes of AEA, decreased in glandular epithelial cells and stromal
cells in both endometriosis and adenomyosis compared to the control.
Taylor et al. (2010) showed the existence of the receptors of AEA (CB1)
together with its synthesizing and catabolizing enzymes in the endome-
trium at dierent stages of the menstrual cycle and in the ovary by
immunohistochemistry (Taylor et al., 2010). It is known that AEA is
more common in endometrium than 2-AG at physiological conditions
(Maccarrone, 2009; Taylor et al., 2010). Although Sanchez et al. (2016)
detected increased systemic levels of AEA, 2-AG and OEA in patient
derived serum, lower expressions of CB1 mRNA was detected in the
same casesendometriotic cells compared to controls at secretory phase
of menstruation (Sanchez et al., 2016). Our study is the rst that
searched for the synthesizing and catabolizing enzymes of AEA in
endometriosis and adenomyosis patients. Immune reactivity of synthe-
sizing and catabolizing enzymes were detected to be similar in
endometriosis and adenomyosis. Since the expression of both NAPE-
PLD and FAAH decreased in endometriosis and adenomyosis groups in
line with receptor immune reactivity, we suggest that synthesis and
degrading of AEA get slower together in both epithelial and stromal
cells during the pathogenesis of the disease.
Tissues from the proliferative and secretory phases of the normal
endometrium exhibited the same immune labeling pattern for CB1 in
this study. This nding revealed that CB1 receptor immune reactivity is
not menstrual cycle dependent. This nding correlated well with the
data of Taylor et al. (2010) and colleagues (Taylor et al., 2010).
Fig. 3. AF are endometrial micrographs showing cytoplasmic 2-AG synthesizing MAGL (AC) and 2-AG catabolizing DAGL (DF) enzyme immune labeling on glandular epithelial (*)
and stromal cells (**), Haematoxylin 400×. (G) Graphic shows the immune labeling percentages for MAGL enzyme distribution in control and experimental groups. (*) p = 0,001. (H)
(boxplot graph) shows nonparametric distributed immune labeling scores for DAGL on glandular epithelial cells of all groups respectively. (**) p = 0,002. (n = 19 for control; n = 20 for
endometriosis and n = 17 for adenomyosis).
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[
Resuehr et al. (2012) however reported that secretory phase of the
normal endometrium exhibits increased CB1 immune reactivity
(Resuehr et al., 2012). The strength of our study is the larger number
of patients and also larger panel of labeling comparing to previous
reports.
FAAH immune labeling was higher in the secretory compared to the
proliferative phase in glandular epithelial cells in this study. This
revealed that glandular epithelial cells at secretory phase were inde-
pendent from CB1 receptor activity. We found that immune labeling for
NAPE-PLD was similar at dierent phases of the control group.
Fig. 4. From A to C are endometrial micrographs from control and experimental groups exhibiting apoptotic stromal (**) and epithelial (*) cells undergoing apoptosis with their nuclei
labeled in dark brown. Haematoxylin 400×. D and E show boxplot graphs of apoptotic indices for glandular epithelial and stromal cells respectively (*) p < 0,05. Note that control
group exhibits signicantly higher apoptotic rate comparing to both adenomyosis and endometriosis groups. (n = 19 for control; n = 20 for endometriosis and n = 17 for adenomyosis).
Fig. 5. (A and C) Real time cell proliferation curves of Ishikawa cells following application of dierent doses of ACPA (100 nM, 1 μM, 10 μM, 100 μM) and CB65 (1 μM, 10 μM, 100 μM)
are shown. (B) and (D) are logarithmic graphics representing the calculated value for IC
50
concentration. The anti-proliferative eect of ACPA (9.3 ×10
6
M) and CB65 (1.9 ×10
4
M)
at IC50 concentrations was observed from 46th hour. All plots were generated using the RTCA Software 1.1.
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[
According to Taylor et al. (2010), FAAH enzyme level is higher at late
secretory phase and lower at early proliferative phase of the menstrual
cycle, while NAPE-PLD immune reaction is higher at late secretory and
early proliferative phases than late proliferative and early secretory
phases in glandular epithelial cells of normal endometrium (Taylor
et al., 2010). Our ndings for phase distribution of FAAH are consistent
with the data of Taylor et al. (Taylor et al., 2010). We suggest that the
FAAH enzyme rather than the CB1 receptor or the NAPE-PLD enzyme
regulates endocannabinoid activity. The limitation of our study was
using archive blocks but not fresh tissue samples. Working on archived
paran blocks is a well-established method for evaluating homogenous
patient groups.
Levels of synthesizing and catabolizing enzymes (DAGL and MAGL
respectively) of 2-AG were correlated with CB2 receptor immune
labeling in all groups. DAGL enzyme activities in glandular and stromal
cells increased with age in solid subgroup of endometriosis, suggesting
that 2-AG synthesis decreases with aging at disease. Immune labeling
for both enzymes of 2-AG decreased in glandular and stromal cells in
the experimental groups compared to that of the control group. Our
ndings regarding synthesizing and catabolizing enzymes of 2-AG and
its receptor are original since their immune labeling pattern has not
been studied in the normal endometrium in comparison with endome-
triosis and adenomyosis until now. It is likely that 2-AG might be a
molecule playing an important role in the pathogenesis of endome-
triosis and adenomyosis.
CB2 receptor reactivity was higher in the proliferative phase than
the secretory phase in the control group. This nding was consistent
with the results of Taylor et al. (2010), while the enzyme reactivity
score was similar in both phases (Taylor et al., 2010). These ndings
reveal that the enzymes do not regulate the eect of 2-AG through CB2
receptors in the modulation of the menstrual cycle and these receptors
do not mediate the eects of other endocannabinoids. We suggest 2-AG
activity might be taking an active role in endometrium progressing to
late phases of reproductive ages as we detected increased MAGL
enzyme activity in the proliferative phase on glandular cells with
increasing age. We suggest that the pathophysiological mechanism of
endometriosis could be dierent than adenomyosis. Aging may play
role on dysregulation of endocannabinoids via CB2 receptors, taking
into consideration the decreased CB2 receptor reactivity with increas-
ing age in adenomyosis.
Our data from TUNEL analysis was in line with the literature and
correlated with our immune labeling results. The proliferation capacity
of glandular epithelial and stromal cells of endometrium is higher in
endometriosis than normal endometrium (Agic et al., 2009; Nasu et al.,
2011; Sanchez et al., 2012).
The IC
50
value was conrmed as 9.3 ×10
6
M for ACPA and
1.9 ×10
4
M for CB 65 on Ishikawa cells. ACPA exhibited stronger
anti-proliferative eect on Ishikawa cells and, CB65 caused stronger
anti-proliferation on CRL-7566 respectively. Our study is the rst to
examine the real time direct and dose dependent anti-proliferative
eect of cannabinoid agonists in both control and endometriotic cells.
We report CB65 exhibits stronger pro-apoptotic eect on Ishikawa cells
and, ACPA causes stronger pro-apoptosis on CRL-7566 respectively.
Truthfully, there is more than one cell death mechanism (Galluzzi et al.,
Fig. 6. (AD) Real time cell proliferation curves and bar graphics are shown following application of ACPA (9.3 ×10
6
M) and CB65 (1.9 ×10
4
M) at IC50 concentrations on control
(Ishikawa) and endometriotic (CRL-7566) cells. Note that the cell proliferation indices of CRL-7566 (A and B) and Ishikawa (C and D) cell lines decreased through hours comparing to
untreated controls. All plots were generated using the RTCA Software 1.1.
E. Bilgic et al. $FWD+LVWRFKHPLFD[[[[[[[[[[²[[[
2012). The deviation of our results may be because of the dierent
apoptotic pathways, which play role on endometrial cell death.
Cannabinoid agonists and their receptors have been shown in endo-
metrial cancers (Ayakannu et al., 2013, 2015; Guida et al., 2010). AEA
and 2-AG synthesis and degradation pathways are known in cancer
angiogenesis and overall gene expression levels were reported for
endometrial carcinoma (Ayakannu et al., 2015). Although both syn-
thetic CB1 and CB2 agonists increased the apoptotic cell percentage
compared to control group and decreased the cell proliferation indexes
in our study, the molecular pathways of cannabinoid-dependent cell
mechanisms need to be searched. We suggest that cannabinoid agonists
can potentially inhibit endometriotic cell proliferation. Palmitoyletha-
nolamide was recently used to reduce chronic pelvic pain in endome-
triotic patients (Angioni, 2015). Based upon this nding, we suggest
that cannabinoid agonists can be assessed for their molecular mechan-
isms on endometriotic cell proliferation regression and apoptosis and,
can be a potential therapeutic agent.
In conclusion, endocannabinoids and their receptor distribution on
endometriotic and adenomyotic tissue samples were compared with
healthy endometrial tissue samples. We presented that expression of
endocannabinoid receptors and synthesizing and catabolizing enzymes
and apoptotic cell ratio decrease in endometriosis and adenomyosis,
compared to normal endometrium. Cannabinoid agonist presented anti-
proliferative and apoptotic eect on cell culture.
Conict of interest
The authors listed above have no nancial interest with any
company or organization in the subject matter or materials discussed
in this manuscript.
Acknowledgements
This study was supported by the Scientic and Technological
Research Council of Turkey (TUBITAK, # 112S217).
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
... Other candidates for the potent apoptotic modulators in EMS were described by Bilgic et al. [49]. They showed that downregulation and/or alteration of endocannabinoid receptors could mediate the underlying mechanism of EMS and adenomyosis. ...
... Female/CRL-7566 cell line n = 20 EMS patients, n = 17 adenomyosis and n = 19 controls; n = 12 proliferative, n = 7 secretory; Endometriosis tissues were divided as: n = 9 cystic and n = 11 non-cystic CB1 ↓ CB2 ↓ [49] Female/HEK293T, CRL7566, CRL-11731 cell lines n = 10 OE, n = 10 ovarian cancer, and n = 10 controls miR-191 ↑↓ [50] Female/CRL-7566 cell line n = 20 eutopic and ectopic endometrium ↑ increased. ↓ decreased. ...
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... The results showed non-significant changes in number of samples expressing CNR1, CNR2 and GPR55 during the late proliferative to early secretory period. While CB1 receptor (the protein encoded by the CNR 1 gene) was found to inhibit human decidualization and stimulates apoptosis 38 , studies on CB1 expression in the epithelial glands found no significant regulation across the menstrual cycle 51 , and CB2 expression in stromal cells was similar between the proliferative and the secretory phase 30 . Previous studies have also reported that progesterone exerted minimal effects on CB1 expression in lymphocytes 48,54,58 . ...
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Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni’s method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.
... When endometrial cells were treated with cannabinoid agonist WIN 5512-2, the result was decreased cell proliferation, decreased reactive oxygen species production, and reduction in alpha-smooth muscle actin expression, lending supporting evidence to the anti-inflammatory effects of cannabinoids [73]. Bilgic et al. [74] also found that CB1R and CB2R expressions are decreased in EM tissue compared to control, with concurrently decreased apoptosis indexes. The same study found that exposure of EM tissue to CB1R and CB2 agonists resulted in pro-apoptotic effects. ...
Article
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Endometriosis (EM) is an estrogen-dependent disease characterized by the presence of epithelial, stromal, and smooth muscle cells outside the uterine cavity. It is a chronic and debilitating condition affecting ~10% of women. EM is characterized by infertility and pain, such as dysmenorrhea, chronic pelvic pain, dyspareunia, dysuria, and dyschezia. Although EM was first described in 1860, its aetiology and pathogenesis remain uncertain. Recent evidence demonstrates that the peripheral nervous system plays an important role in the pathophysiology of this disease. Sensory nerves, which surround and innervate endometriotic lesions, not only drive the chronic and debilitating pain associated with EM but also contribute to a growth phenotype by secreting neurotrophic factors and interacting with surrounding immune cells. Here we review the role that peripheral nerves play in driving and maintaining endometriotic lesions. A better understanding of the role of this system, as well as its interactions with immune cells, will unearth novel disease-relevant pathways and targets, providing new therapeutics and better-tailored treatment options.
... Fonseca et al. looked at the effect of different synthetic cannabinoids on endometrial stromal cells noting that WIN 55212-2 induced cell death through the CB1 receptor but that other compounds including JWH-122 had dissimilar results. 24,25 Dmitrieva et al. studied a combination of cannabinoid receptor agonists and antagonists using a rat model which showed CB1 receptor expression in the sensory and sympathetic neurons innervating endometriosis lesions. As part of the study, rats with surgically induced endometriosis treated with WIN 55212-2 were noted to have decreased endometriosis-associated hyperalgesia (demonstrated using electromyographic recording in response to vaginal nociception stimulus). ...
Article
Objective To review the available literature on the effect of cannabis-based products on the female reproductive system and establish if there is any evidence that they benefit or harm patients with endometriosis and therefore if there is sufficient evidence to recommend them. Data Sources An electronic-based search was performed in PubMed, Embase and the Cochrane Database. Reference lists of articles retrieved were reviewed and a grey literature search was also performed. Methods of Study Selection The original database search yielded 264 articles from PubMed, Embase and the Cochrane Database, of which forty-one were included. One hundred and sixty-one studies relating to gynaecological malignancy, conditions unrelated to endometriosis or therapies unrelated to cannabis-based products were excluded. Twelve articles were included from a grey literature search and review of references. Results The majority of available evidence is from laboratory studies aiming to simulate the effects of cannabis-based products on preclinical endometriosis models. Some show evidence of benefit with cannabis-based products. However, results are conflicting and the impact in humans cannot necessarily be extrapolated from this data. Few studies exist looking at the effect of cannabis or its derived products in women with endometriosis – the majority are in the form of surveys and are affected by bias. National guidance was also reviewed: at present this dictates that cannabis-based products can only be prescribed for conditions where there is clear published evidence of benefit and only when all other treatment options have been exhausted. Conclusion Current treatment options for endometriosis often affect fertility and/or have undesirable side effects that impede long-term management. Cannabis-based products have been suggested as a novel therapeutic option that may circumvent these issues. However, there is a paucity of well-designed, robust studies and randomised controlled trials looking at their use in the treatment of endometriosis. In addition, cannabis use has a potential for harm in the long term; with a possible association with ‘cannabis use disorder’, psychosis and mood disturbances. At present, national guidance cannot recommend cannabis-based products to patients in the UK due to lack of clear evidence of benefit. More comprehensive research into the impact of endocannabinoids in the context of endometriosis is required before their use can be recommended or prescribed.
... Ishikawa cells are derived from a well-differentiated adenocarcinoma of human endometrial epithelium that expresses functional steroid receptors for estradiol (E2) and progesterone (P4) (25). The cell line represents an ideal model of normal endometrial epithelium cells owing to its phenotypic similarity and response to steroids, similar to physiological conditions (26). The Ishikawa cells were seeded at a density of 2,000 cells/well for 24 h until they were attached to the 96-well plate for cell proliferation measurement. ...
Article
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Estrogen therapy is widely used as a supplementary treatment after hysteroscopy for female infertility patients owing to its protective function that improves endometrial regeneration and menstruation, inhibits recurrent adhesions, and improves subsequent conception rate. The endometrial protective function of such estrogen administration pre-surgery is still controversial. In the current study, 12 infertility patients were enrolled, who were treated with estrogen before hysteroscopy surgery. Using cutting-edge metabolomic analysis, we observed alterations in the pentose phosphate pathway (PPP) intermediates of the patient’s endometrial tissues. Furthermore, using Ishikawa endometrial cells, we validated our clinical discovery and identified estrogen–ESR–G6PD–PPP axial function, which promotes estrogen-induced cell proliferation.
... Recent studies have suggested that a dysfunction in the endocannabinoid system (ECS) is present in endometriosis patients [17,18], and that aspects of endometriosis-associated pain may be targeted by modulating the ECS [19]. ...
Article
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Background The use of cannabis for symptoms of endometriosis was investigated utilising retrospective archival data from Strainprint Technologies Ltd., a Canadian data technology company with a mobile phone application that tracks a range of data including dose, mode of administration, chemovar and their effects on various self-reported outcomes, including pelvic pain. Methods A retrospective, electronic record-based cohort study of Strainprint TM users with self-reported endometriosis was conducted. Self-rated cannabis efficacy, defined as a function of initial and final symptom ratings, was investigated across the included symptom clusters of cramps, pelvic pain, gastrointestinal pain, nausea, depression, and low libido. Cannabis dosage form, dose and cannabinoid ratio information was also recorded. Results A total number of 252 participants identifying as suffering endometriosis recorded 16193 sessions using cannabis between April 2017 and February 2020. The most common method of ingestion was inhalation (n = 10914, 67.4%), with pain as the most common reported symptom being treated by cannabis (n = 9281, 57.3%). Gastrointestinal symptoms, though a less common reason for cannabis usage (15.2%), had the greatest self-reported improvement after use. Inhaled forms had higher efficacy for pain, while oral forms were superior for mood and gastrointestinal symptoms. Dosage varied across ingestion methods, with a median dose of 9 inhalations (IQR 5 to 11) for inhaled dosage forms and 1 mg/mL (IQR 0.5 to 2) for other ingested dosage forms. The ratio of THC to CBD had a statistically significant, yet clinically small, differential effect on efficacy, depending on method of ingestion. Conclusions Cannabis appears to be effective for pelvic pain, gastrointestinal issues and mood, with effectiveness differing based on method of ingestion. The greater propensity for use of an inhaled dosage delivery may be due to the rapid onset of pain-relieving effects versus the slower onset of oral products. Oral forms appeared to be superior compared to inhaled forms in the less commonly reported mood or gastrointestinal categories. Clinical trials investigating the tolerability and effectiveness of cannabis for endometriosis pain and associated symptoms are urgently required.
... Synthetic agonist of CB1 exhibited a stronger antiproliferative effect on Ishikawa cells and pro-apoptotiс effects on CRL-7566 cells. The CB2 agonist caused stronger antiproliferation on CRL-7566 cells and pro-apoptotiс effects on Ishikawa cells [28]. The properties of endocannabinoid anandamide were investigated using the St-T1b cell line (immortalized stromal cells from endometrium). ...
Article
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Endometriosis is characterized by the formation and development of endometrial tissues outside the uterus, based on an imbalance between proliferation and cell death, leading to the uncontrolled growth of ectopic foci. The potential target for the regulation of these processes is the endocannabinoid system, which was found to be involved in the migration, proliferation, and survival of tumor cells. In this paper, we investigated the effect of endocannabinoid-like compounds from the N-acyl dopamine (NADA) family on the viability of stromal cells from ectopic and eutopic endometrium of patients with ovarian endometriosis. N-arachidonoyldopamine, N-docosahexaenoyldopamine, and N-oleoyldopamine have been shown to have a five-times-more-selective cytotoxic effect on endometrioid stromal cells. To study the mechanisms of the toxic effect, inhibitory analysis, measurements of caspase-3/9 activity, reactive oxygen species, and the mitochondrial membrane potential were performed. It was found that NADA induced apoptosis via an intrinsic pathway through the CB1 receptor and downstream serine palmitoyltransferase, NO synthase activation, increased ROS production, and mitochondrial dysfunction. The higher selectivity of NADA for endometriotic stromal cells and the current lack of effective drug treatment can be considered positive factors for further research of these compounds as possible therapeutic agents against endometriosis.
Book
This book is a printed edition of the Special Issue "Molecular and Cellular Advances in Endometriosis Research" that was published in IJMS
Preprint
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Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to chart the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni’s method. A total of 40 of the 70 genes were present in >90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.
Article
Objective To determine the involvement of endocannabinoid (EC) family member in the pathophysiology of endometriosis (EMS). Design Mass-spectrometry analysis of plasma and tissue samples from EMS patients, controls and mouse model of EMS; mRNA and immunohistochemistry analysis of EMS patient and control samples. Setting Academic teaching hospital and university Patient (s) Endometriosis patients and healthy, fertile control subjects Intervention (s) None Main outcome measure (s) EC analysis in patient plasma, EMS lesions, and healthy endometrial samples. Result (s) While circulating ECs were detected in the plasma samples, no significant changes were observed in EMS patients compared to healthy, fertile controls. However, Palmitoyl Ethanolamide (PEA) levels were significantly higher in the EMS lesions compared to the endometrium of EMS patients. Similarly, genes involved in the EC signaling pathways were differentially expressed in the EMS lesions. Analysis of CB1 and CB2 receptors in the EMS lesions revealed significantly lower CB2 receptor expression, while no significant changes were observed in CB1 receptor expression compared to both endometrium from EMS patients and healthy, fertile controls. PEA levels were significantly elevated in both plasma from EMS mice compared to sham controls and in endometriotic lesions compared to uterine samples. Conclusion (s) Together, we provide the evidence towards dysregulation of members of the ECs in both EMS patients and mouse model of EMS. These findings will advance the knowledge of the role of ECs in EMS and their potential implications as therapeutic targets.
Article
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Endometriosis causes significant chronic pelvic pain, dysmenorrhea, and infertility and affects 10% of all women. In endometriosis, ectopic endometrium surviving after retrograde menstruation exhibits an abnormal immune response characterized by increased levels of activated macrophages and inflammatory cytokines. Particularly, dysfunctional natural killer (NK) cells play an important role in the pathogenesis of the disease by either facilitating or inhibiting the survival, implantation, and proliferation of endometrial cells. NK cells in the peritoneum and peritoneal fluid exhibit reduced levels of cytotoxicity in women with endometriosis. Several cytokines and inhibitory factors in the serum and peritoneal fluid also dysregulate NK cell cytotoxicity. Additionally, increased numbers of immature peripheral NK cells and induction of NK cell apoptosis are evident in the peritoneal fluid of women with endometriosis. The high rate of endometriosis recurrence after pharmaceutical or surgical treatment, which is associated with dysfunctional NK cells, indicates that new immunomodulatory management strategies are required. A good understanding of immune dysfunction would enable improvement of current treatments for endometriosis.
Article
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The aim of the current study was to diagnose the concomitant presence of adenomyosis (AM) in endometrioid endometrial cancer (EEC) in order to evaluate its value as an oncological prognostic marker. A retrospective analysis of 289 patients diagnosed with EEC who underwent total hysterectomy, bilateral salpingo-oophorectomy and pelvic-lymphadenectomy was conducted. The total cohort included 37 patients in Group A (those with concomitant AM and EEC) and 252 patients in Group B (those affected only by EEC). The following factors were evaluated: Presence or absence of AM, tumor grade, depth of myometrial invasion, tumor size, lymphovascular space involvement, lymph node status, peritoneal cytology, concomitant detection of endometrial atypical-hyperplasia or polypoid endometrial features and tumor stage according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Uterine examination of different sections of uterine cervix, corpus, myomas and cervical or endometrial polyps was performed. The diagnosis of AM was confirmed when the distance between the lower border of the endometrium and the foci of the endometrial glands and stroma was >2.5 mm. Parametric and nonparametric statistical tests were performed when possible; continuous variables were analyzed using a Student's t-test, and categorical variables were analyzed by the χ(2) test or Fisher's exact test. The association between FIGO stage and group was determined to be significant: 83.8% of Group A patients were categorized as FIGO stage I, vs. 68.7% of Group B patients. In addition, Group A was associated with lower grades in FIGO stage, myometrial invasion, lymphovascular space involvement, lymph node involvement and tumor size. The findings suggest that the intraoperative evaluation of the presence of AM in patients with EEC may aid surgeons in estimating oncological risk and in selecting the most appropriate surgical treatment.
Article
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Objective. To explore the effects of puerarin to treat endometriosis (EMT) model rats and the possible regulatory mechanisms. Methods. EMT model rats were surgically induced by autotransplantion of endometrial tissues. The appropriate dosage of puerarin to treat EMT model rats was determined by observing the pathologic morphology of ectopic endometrial tissues and by detecting the levels of estradiol (E2) and prostaglandin E2 (PGE2) of both serum and ectopic endometrial tissues. The related genes and proteins of ectopic endometrial tissues were analyzed by Real-time PCR and immunohistochemistry (IHC) to explore the possible mechanisms. Results. Puerarin could reduce the levels of E2 and PGE2 and prevent the growth of ectopic endometrium tissues by inhibiting the expression of aromatase cytochrome P450 (p450arom) and cyclooxygenase-2 (cox-2); puerarin could adjust the anabolism of E2 by upregulating the expression of 17β-hydroxysteroid-2 (17β-hsd-2) and downregulating the expression of 17β-hydroxysteroid-1 (17β-hsd-1) of the ectopic endometrium tissues; puerarin could increase the expression of ERβ and improve the inflammatory microenvironment of EMT model rats. Conclusions. Our data suggest that puerarin has a therapeutic effect on EMT model rats and could be a potential therapeutic agent for the treatment of EMT in clinic.
Article
Endometriosis is a common condition in women of reproductive age. According to several epidemiological studies endometriosis may be associated with increased risk of various malignancies. However, endometriosis-associated malignancy (EAM) is defined by certain histological criteria. About 80 % of EAM have been found in the ovary, whereas 20 % are localized in extragonadal sites like intestine, rectovaginal septum, abdominal wall, pleura and others. Some authors suggest that EAM arise from atypical endometriosis as an intermediate lesion between endometriosis and cancer. Moreover, a number of genetic alterations, like loss of heterozygosity (LOH), PTEN, ARID1 A and p53 mutations have been found in both endometriosis and EAM. Endometriosis-associated ovarian cancer (EAOC) is mostly a well or intermediately differentiated tumor of endometrioid or clear cell histological sub-type. Women affected by EAOC are on average five to ten years younger than non-EAOC patients; in most of the cases EAOC is a low stage disease with favorable clinical outcome. Since EAM is a rare condition systematic data on EAM are still missing. A systematic retrospective study on endometriosis-associated malignancies (EAM study) is currently being conducted by the Endometriosis Research Foundation together with the study groups on ovarian and uterine tumors of the working group for gynecological oncology (AGO) (gyn@mlk-berlin.de).
Article
Cannabinoids and modulators of the endocannabinoid system affect specific mechanisms that are critical to the establishment and development of endometriosis. The aim of this study was to measure the systemic levels of endocannabinoids and related mediators in women with and without endometriosis and to investigate whether such levels correlated with endometriosis-associated pain. Plasma and endometrial biopsies were obtained from women with a laparoscopic diagnosis of endometriosis (n = 27) and no endometrial pathology (n = 29). Plasma levels of endocannabinoids (N-arachidonoylethanolamine [AEA] and 2-arachidonoylglycerol [2-AG]) and related mediators (N-oleoylethanolamine [OEA] and N-palmitoylethanolamine [PEA]), messenger RNA expression of some of their receptors (cannabinoid receptor type 1 [CB1], CB2, transient receptor potential vanilloid type [TRPV1]), and the enzymes involved in the synthesis (N-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D [NAPE-PLD]) and degradation (fatty acid amide hydrolase 1 [FAAH]) of AEA, OEA, and PEA were evaluated in endometrial stromal cells. The systemic levels of AEA, 2-AG, and OEA were elevated in endometriosis in the secretory phase compared to controls. The expression of CB1 was higher in secretory phase endometrial stromal cells of controls versus endometriosis. Similar expression levels of CB2, TRPV1, NAPE-PLD, and FAAH were detected in controls and endometriosis. Patients with moderate-to-severe dysmenorrhea and dyspareunia showed higher AEA and PEA levels than those with low-to-moderate pain symptoms, respectively. The association of increased circulating AEA and 2-AG with decreased local CB1 expression in endometriosis suggests a negative feedback loop regulation, which may impair the capability of these mediators to control pain. These preliminary data suggest that the pharmacological manipulation of the action or levels of these mediators may offer an alternative option for the management of endometriosis-associated pain.
Article
Objective: To investigate the possible correlation between maternal characteristics, in utero and early neonatal life exposures, and the development of endometriosis in adult life. Design: Case-control study. Setting: University hospital. Patient(s): A group of 161 patients with endometriosis and a control group of 230 women undergoing laparoscopy for benign adnexal diseases and free of endometriosis. Intervention(s): All women included in the study were requested to answer a series of questions about their mothers' gestational data and on their own perinatal and early postnatal lives. Main outcome measure(s): Odds ratio, adjusted odds ratios, and 95% confidence intervals for the associations between maternal characteristics during the patient's pregnancy, in utero exposure to obstetrical and perinatal complications, and the type of feeding received during the neonatal period with the development of endometriosis in adult life. Result(s): Mothers of women with endometriosis were significantly more likely to be affected by endometriosis or uterine fibroids, with a higher incidence of smoking during pregnancy. Women with endometriosis were more frequently born prematurely, with a significantly lower birth weight, and their mothers experienced preeclampsia during their pregnancies more often than control subjects. They were also more frequently formula fed than breast fed in early life. However, only prematurity and formula feeding were retained in the multivariate analysis model. Conclusion(s): Among intrauterine and early neonatal exposures, prematurity and formula feeding were risk factors for the development of endometriosis in adult life. Further studies should evaluate the underlying biologic mechanisms.
Article
In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.
Article
Recently, endocannabinoids have emerged as signaling mediators in reproduction. It is widely accepted that anandamide (AEA) levels must be tightly regulated, and that a disturbance in AEA levels may impact decidual stability and regression. We have previously characterized the endocannabinoid machinery in rat decidual tissue and reported the pro-apoptotic action of AEA on rat decidual cells. Cyclooxygenase-2 (COX-2) is an inducible enzyme that plays a crucial role in early pregnancy, and is also a key modulator in the crosstalk between endocannabinoids and prostaglandins. On the other hand, AEA-oxidative metabolism by COX-2 is not merely a mean to inactivate its action, but it yields the formation of a new class of mediators, named prostaglandin-ethanolamides, or prostamides. In this study we found that AEA-induced apoptosis in decidual cells involves COX-2 metabolic pathway. AEA induced COX-2 expression through p38 MAPK, resulting in the formation of prostamide E2 (PME2). Our findings also suggest that AEA-induced effect is associated with NF-kB activation. Finally, we describe the involvement of PME2 in the induction of the intrinsic apoptotic pathway in rat decidual cells. Altogether, our findings highlight the role of COX-2 as a gatekeeper in the uterine environment and clarify the impact of the deregulation of AEA levels on the decidual remodeling process. Copyright © 2015. Published by Elsevier B.V.
Article
The Fas/Fas-Ligand system is an important mediator of apoptosis. We analyzed their expression in tissue specimens obtained from 33 women with severe endometriosis and 18 women without endometriosis. Immunostaining for Fas-Ligand in the eutopic endometrium was stronger in the epithelial cells of secretory phase, while the epithelial cells of endometriotic lesions showed a significantly stronger staining for Fas-Ligand independently from the menstrual phase (P < 0.01). Immunostaining for Fas in the eutopic endometrium showed a reduced staining during the proliferative phase, whereas it was strong in the secretory phase. The epithelial cells of the ectopic endometrium showed a reduced staining for Fas independently from the menstrual phase with respect to the eutopic tissue (P < 0.01). The reduced expression of Fas in the ectopic endometrium with the contemporary higher expression of Fas-Ligand in the corresponding cells suggests a possible immune privilege of this tissue. © The Author(s) 2015.