Embed Size (px)
Citation preview
1
1
Decorin induced by progesterone plays a crucial role in suppressing endometriosis 2
3
Yoshihiro Ono1, Yoshito Terai
1, Akiko Tanabe
1, Atsushi Hayashi
1, Masami Hayashi
1, 4
Yoshiki Yamashita1, Satoru Kyo
2, and Masahide Ohmichi
1 5
6
1; Department of Obstetrics and Gynecology, Osaka Medical College, Osaka, Japan 7
2; Department of Obstetrics and Gynecology, Graduate School of Medical Science, 8
Kanazawa University, Kanazawa, Japan 9
10
Address, correspondence and reprint requests to: 11
Yoshito Terai, M.D., 12
Department of Obstetrics and Gynecology 13
Osaka Medical College 14
2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan 15
16
Running Title: Progestin suppresses endometriosis via decorin 17
Tel No. +81(Japan)-72-683-1221 18
Fax No. +81(Japan)-72-681-3723 19
21
22
Page 1 of 58 Accepted Preprint first posted on 22 September 2014 as Manuscript JOE-14-0393
Copyright © 2014 by the Society for Endocrinology.
2
Abstract 23
Dienogest, a synthetic progestin, was shown to be effective against 24
endometriosis, although it is still unclear how it affects the ectopic endometrial cells. 25
Decorin was shown to be a powerful endogenous tumor repressor acting in a paracrine 26
fashion to limit tumor growth. 27
Our objectives were to examine the direct effects of progesterone and dienogest 28
on the in vitro proliferation of the human ectopic endometrial epithelial and stromal cell 29
lines, and evaluated how decorin contributes to this effect. We also examined the 30
decorin mRNA expression in 50 endometriosis patients. 31
The growth effects of both cell lines were inhibited in a dose-dependent 32
manner by both decorin and dienogest. Using a CHIP assay, it was noted that 33
progesterone and dienogest directly induced the binding of the decorin promoter in the 34
EMOsis cc/TERT cells (immortalized human ovarian epithelial cells) and CRL-4003 35
cells (immortalized human endometrial stromal cells). Progesterone and dienogest also 36
led to significant induced cell cycle arrest via decorin by promoting p21 production in 37
both cell lines in a dose-dependent manner. Decorin also suppressed the expression of 38
MET in both cell lines. We confirmed the decorin mRNA expression in the patients 39
treated with dienogest was higher than that in the control group. 40
Page 2 of 58
3
In conclusion, the decorin induced by dienogest appears to play a crucial role 41
in suppressing endometriosis by exerting antiproliferative effects and inducing cell 42
cycle arrest via the p21 production of human ectopic endometrial cells and eutopic 43
endometrial stromal cells. 44
45
Key words; Decorin; progesterone; dienogest; endometriosis; cell cycle arrest; p21 46
47
48
Page 3 of 58
4
Introduction 49
Endometriosis is a chronic, benign disease which is characterized by the 50
presence of endometrium-like tissue outside the uterine cavity, primarily on the ovaries. 51
It is a major cause of symptoms like pelvic pain, dysmenorrhea, dyspareunia and 52
infertility. This condition affects from 6-10% of females of reproductive age and often 53
relapses after surgical therapy (Giudice and Kao, 2004; Guo, 2009; Eskenazi and 54
Warner, 1997). Gonadotropin-releasing hormone (GnRH) agonists are an established 55
therapy for endometriosis. Although GnRH agonists provide effective pain relief and 56
reduce the progression of endometriotic implants (Brown et al., 2010), they are 57
associated with symptoms of estrogen deprivation, like hot flushes, vaginal dryness, 58
headache and decreased libido. In addition, the involvement of GnRH agonists in bone 59
demineralization restricts their use to six months in the absence of add-back therapy 60
(Brown et al., 2010; Jee et al., 2009). Progestins are a class of compounds that mimic 61
the activity of progesterone and are a recommended treatment for the pain associated 62
with endometriosis, but a number of agents in this class are associated with androgenic 63
effects and weight gain at the doses that are required for efficacy (Vercellini et al., 64
2009). 65
Dienogest (17a-cyanomethyl-17b-hydroxy-estra-4,9-dien-3-one) is a 66
Page 4 of 58
5
fourth-generation progestin with potent oral progestational activity without any 67
systemic androgenic activity (Sitruk, 2004a,b; Sasagawa et al.,2008). Clinical trials 68
have shown that dienogest provides effective pain relief equivalent to GnRH agonists, a 69
reduction of endometriotic lesions and a favorable safety and tolerability profile in 70
patients with endometriosis (Cosson et al., 2002; Köhler et al., 2010; Strowitzki et al., 71
2010a,b; Momoeda et al., 2009; Felice et al., 2012). The effects of dienogest on 72
endometriosis are associated not only with its anti-ovulatory activity (Irahara et al., 73
2007), but also with a direct effect on the proliferation or cytokine production in stromal 74
cells from eutopic and ectopic endometrial tissues (Okada et al., 2001; Horie et al., 75
2005; Fu et al., 2008). In a recent report, dienogest was shown to have a direct effect on 76
endometrial epithelial cells (Shimizu et al., 2009). However, it is still unclear how 77
dienogest affects endometriotic cells to contribute to its therapeutic effect on 78
endometriosis. 79
Decorin, a prototype member of the small leucine-rich proteoglycan family 80
(Iozzo, 1998; Kresse et al., 1993; Neame and Kay, 2000), is a stromal 81
proteoglycan synthesized chiefly by fibroblasts, stressed vascular endothelial cells and 82
smooth muscle cells (Danielson et al., 1997; Keene et al., 2000, Zhang et al., 2006; 83
Zhang et al., 2009). It has multiple functions, including regulation of collagen 84
Page 5 of 58
6
fibrillogenesis and maintenance of tissue integrity (Weber et al., 1996; Iozzo, 1997; 85
Fairlie et al., 1998). Decorin is also known to have high expression in various normal 86
tissues, including the uterus (Su et al., 2004). It can regulate multiple cellular functions 87
because of its ability to bind to a variety of molecules in both the extracellular matrix, as 88
well as on the cell surface (Seidler and Dreier, 2008; Goldoni and Iozzo, 2008). Decorin 89
was recently reported to sequester multiple growth factors, such as transforming growth 90
factor (TGF)–β1, and directly antagonizes several members of the receptor tyrosine 91
kinase (RTK) family (Iozzo, 1997; Fairlie et al., 1998; Seidler and Dreier, 2008), 92
including the epidermal growth factor receptor (EGFR), the insulin-like growth factor 93
receptor I (IGF-IR) (Schaefer et al., 2007) and the hepatocyte growth factor receptor 94
(MET) (Iozzo and Sanderson, 2011; Goldoni et al., 2009). Moreover, reduced decorin 95
within tumor stromal cells has been established as a poor prognostic factor for invasive 96
breast cancer and in murine models of spontaneous breast cancer with mammary gland 97
carcinogenesis (Goldoni and Iozzo, 2008). Decorin is a powerful endogenous tumor 98
repressor acting in a paracrine fashion to limit tumor growth and angiogenesis (Thomas 99
and Liliana, 2012). However, it is still unclear how decorin affects endometrial 100
epithelial cells or endometrial stromal cells contributing to its therapeutic effect on 101
endometriosis. 102
Page 6 of 58
7
In the present study, we examined the direct effects of dienogest on the in vitro 103
proliferation of the human endometrial epithelial and stromal cell lines, and evaluated 104
how decorin contributes to this effect. 105
106
Page 7 of 58
8
Materials and Methods 107
Materials 108
Human recombinant decorin was purchased from R&D Systems (Minneapolis, 109
MN, USA). The rabbit monoclonal anti-human decorin antibody (product code 110
ab151988) used for immunoblotting and immunohistochemistry and the mouse 111
monoclonal anti-human beta-actin antibody used for immunoblotting was purchased 112
from Abcam (Cambridge, MA, USA). The mouse monoclonal anti-human p21 antibody 113
(product code 554228) used for immunoblotting was purchased from BD Biosciences 114
(San Jose, CA, USA). The rabbit monoclonal anti-human c-Met antibody (product code 115
LS-C49950) used for immunoblotting and immunohistochemistry was purchased from 116
LifeSpan Biosciences, Inc. (Seattle, WA, USA). A goat anti-human decorin antibody 117
used as a neutralizing antibody for decorin was purchased from R&D Systems 118
(Minneapolis, MN, USA). The BD Falcon™ 96-well microplates used for the cell 119
proliferation assays were from purchased from BD (Franklin Lakes, NJ, USA). A rabbit 120
anti-PR antibody (H-190) used in the ChIP assay was purchased from Santa Cruz 121
Biotechnology (Santa Cruz, CA, USA) 122
123
Patients and tissue collection 124
Page 8 of 58
9
Fifty patients who received surgical treatment between January 2002 and July 125
2012 were included in this study. All patients were under treatment in the Department of 126
Obstetrics and Gynecology of Osaka Medical College. This was a retrospective 127
cross-sectional case-controlled study of human tissue samples approved by the 128
Institutional Review Board of Osaka Medical College. Written informed consent was 129
obtained from all patients participating in the study. The inclusion criteria were: age 130
older than 20 years and no more than 50 years at the time of the surgical procedure, the 131
presence of regular menstrual cycles (24–35 day interval) with the exception of those 132
treated with dienogest, a fourth-generation progestin with a potent oral progestational 133
activity without systemic androgenic effects, the absence of any evidence of past or 134
recent pelvic inflammatory disease and no history of any hormonal treatment for at least 135
12 months at baseline. Trans-vaginal ultrasonography was performed for all patients, 136
and showed mainly hypoechoic cystic masses in the ovaries, and the presence of ovarian 137
endometriomas was confirmed before surgery by magnetic resonance imaging (MRI), 138
which showed high-intensity areas on both T1- and T2-weighted images. The main 139
indication for pre-surgical treatment was the patient’s personal preference after carefully 140
explaining the treatment options. Tissue specimens were obtained from patients treated 141
with dienogest at a dose of 2 mg (Dienogest group; n=25) for three to five months 142
Page 9 of 58
10
before surgery. Simultaneous sampling of ovarian endometrioma capsules was 143
performed during laparoscopic surgery for adnexal masses consistent with ovarian 144
endometrioma. The scoring of endometriosis in each case was documented according to 145
the American Society for Reproductive Medicine (ASRM) (American Society for 146
Reproductive Medicine, 1997). The diagnosis of endometriosis was confirmed 147
histologically. The samples obtained from the endometrioma were immediately frozen 148
in liquid nitrogen for further RT-PCR analyses, fixed in 10% formaldehyde and then 149
routinely processed for paraffin embedding for a histological analysis. 150
151
Cell lines 152
We used a human immortalized epithelial cell line derived from an ovarian 153
endometrioma, EMOsis-CC/TERT, and primary cultured stromal cells derived from 154
ovarian endometrioma, HMOsis scl2 and HMOsis scl3, which were established by Dr. 155
Satoru Kyo (Kanazawa University, Japan). We also used the CRL-7566 endometriosis 156
cell line and CRL-4003 immortalized human endometrial stromal cells, which were 157
purchased from the American Type Culture Collection (Manassas, VA, USA). 158
EMOsis-CC/TERT cells were grown in DMEM supplemented with 10% fetal bovine 159
serum in an atmosphere of 5% CO2 at 37 °C (Bono et al., 2012). The CRL-4003 cells 160
Page 10 of 58
11
were cultured in growth media (DMEM/ F12 10% FBS, 1% BD Insulin, Transferrin, 161
Selenous (ITS) +Premix Universal Culture Supplement (Catalog#354352, BD, Franklin 162
Lakes, NJ)), in an atmosphere of 5% CO2 at 37°C (Lockwood and Nemerson, 1993; 163
Krikun and Mor, 2004). Human stromal cells derived from ovarian endometrioma, 164
another kind gift from Dr. Satoru Kyo, were used to confirm that the CRL-4003 cells 165
possessed similar characteristics to primary cultured endometrioid stromal cells. 166
167
RNA extraction and semi-quantitative reverse transcription-polymerase chain 168
reaction (RT-PCR) 169
Total RNA from homogeneized tissue or from cultured EMOsis-CC/TERT cells 170
or CRL-4003 cells was obtained using the RNeasy Mini kit (Qiagen, Germantown, MD, 171
USA), and 2 µg were reverse-transcribed with Superscript II RNase H-reverse 172
transcriptase (Invitrogen, Carlsbad, CA, USA) using random primers according to the 173
manufacturer’s instructions. The cDNA (1 µl) was amplified using 0.1 µM of each 174
primer, 1 U of Taq DNA polymerase (Roche Diagnostics, Mannheim, Germany), PCR 175
buffer with 1.5 mM MgCl2, and 0.25 mM dNTPs in a 20 µl reaction volume in a 176
PTC200 Thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA). The amplification 177
conditions were as follows: initial denaturation at 94°C for 3 min, followed by 33 cycles 178
Page 11 of 58
12
comprising denaturation at 94°C for 30 sec, annealing at the optimized temperature for 179
each set of primers for 30 sec and extension at 72°C for 30 sec. The final extension was 180
carried out for 5 min at 72°C. The products were analyzed on 2.0% (w/v) agarose gels 181
stained with 0.5 mg/ml ethidium bromide (Sigma-Aldrich) and visualized under an 182
ultraviolet transilluminator. The product size was approximated using a 100-bp DNA 183
ladder (Bangalore Genei, Bangalore, India). The negative control did not contain 184
reverse transcriptase (RT) enzyme in the reaction mixture. The primers used were as 185
follows: decorin (DCN)-F: 5’-AAATATTGTGCAAGGCCCGG-3’, and reverse, 186
DCN-R: 5’-TTTTGCTGCCTGAGTCATCG-3’ , ERα-F: 5’- 187
AGAGATGCTCCATGCCTTTG-3’, and reverse, ERα-R: 5’- 188
GCAGACAGGGAGCTGGTTCA-3’ , PR AB-F: 5’- 189
AACACGTCAGTGGGCAGATG-3’, and reverse, PR AB-R: 5’- 190
GCAGCAATAACTTCAGACATC-3’, PR B-F: 5’- TACCTCACCTGCAGCCTTCT-3’, 191
and reverse, PR B-R: 5’- GCAACAGCCAGCACAAGATA-3’ GAPDH 192
-F:5’-AGCCACATCGCTCAGACA-3’, and reverse, GAPDH -R: 193
5’-GCCCAATACGACCAAATCC-3’ 194
The experiments were repeated at least three times. 195
196
Page 12 of 58
13
One-step real-time polymerase chain reaction (real-time PCR) 197
Commercially available TaqMan Gene Expression Assay kits (Applied 198
Biosystems) were used to assess the human glyceraldehyde-3-phosphate dehydrogenase 199
(GAPDH; Hs02758991_g1), human beta actin (ACTB; Hs01060665_g1 ), human 200
RNA18S5 (Hs01060665_g1) and human decorin (Hs00754870_s1) gene expression 201
levels. All primers were designed using the Primer Express software program (version 202
1.0; Perkin-Elmer Applied Biosystems, Carlsbad, CA) from the GeneBank database 203
according to the manufacturer’s protocol. The cDNA template was amplified in a 20 µL 204
reaction containing 1 x TaqMan Universal PCR Master Mix (Perkin-Elmer Applied 205
Biosystems) and 200 nM forward and reverse primers and 100 nM TaqMan probe. 206
Thermal cycling conditions were as follow: 95°C for 15 seconds followed by 60°C for 207
one minute for 45 cycles in each case during One-step real-time PCR (Perkin-Elmer 208
Applied Biosystems). Quantification cycle (Cq) values were used as an endpoint, and 209
were defined as the PCR cycle number during which the fluorescence generated by the 210
amplification crossed the threshold. The reproducibility of the assay was tested for 211
beta-actin, RNA18S5 and GAPDH transcripts by calculating the coefficient of variation 212
(CV) of repeated amplifications of the same samples, both in the same PCR and in 213
different reactions. The average CV was 10.9%, 19.2% and 4.1%, respectively, for these 214
Page 13 of 58
14
transcripts. Therefore, GAPDH was selected as the endogenous reference gene in this 215
study. Expression levels of decorin mRNA were normalised to GAPDH mRNA, 216
calculated from the triplicate of CT values using the ∆∆CT method and expressed 217
relative to one of the specimens that was assigned the value 1. 218
219
Proliferation assay 220
The EMOsis CC/TERT cells in 10% fetal bovine serum and CRL-4003 cells in 221
growth media were seeded in 96-well plates at a density of 2×104 cells per well. The 222
cells were then incubated for 48 h in the absence or presence of 0.5, 1.5, 3 or 6 µg/ml of 223
decorin or 50, 100, 200 or 500 nM of dienogest. The concentrations of dienogest that 224
exhibited antiproliferative effects on cells were within the range of the serum dienogest 225
concentrations achieved with the dose (2 mg/day) used to treat endometriosis (10 and 226
140 nmol/l), as indicated in the previous study by Foster and Wilde (Foster and Wilde, 227
1998). CellTiter 96 AQueous (MTS) One Solution reagent (Promega, Tokyo, Japan) 228
was added to each well, and the absorbance was recorded at 490 nm using a Corona 229
SH-1000 lab absorbance microplate reader (Corona Electric Co.Inc, Ibaraki, Japan). The 230
cell numbers were then calculated by using a standard curve correlating the absorbance 231
to the cell number counted under a microscope. All experiments were carried out in 232
Page 14 of 58
15
quadruplicate, and the cell viability was expressed as the ratio of the number of viable 233
cells with decorin or dienogest stimulation to that of cells without stimulation. 234
235
Western blot analysis 236
EMOsis-CC/TERT cells and CRL-4003 cells treated with or without 237
progesterone or dienogest were washed twice with ice-cold phosphate-buffered saline 238
and lysed using Pierce RIPA Buffer (Thermo Fisher Scientific, MA, USA). Equal 239
amounts of whole cell protein lysates were separated by SDS polyacrylamide gel 240
electrophoresis and electrotransferred to nitrocellulose membranes. Nonspecific antigen 241
sites were blocked with 10% bovine serum albumin in 1x Tris-buffered saline for 1h. 242
The Western blot analyses were performed with primary antibodies against decorin 243
(1:250 dilution), c-Met (1:1000 dilution), p21 (1:500 dilution) or beta-actin (1:1000 244
dilution) overnight at 4°C.. The immunoreactive bands in the immunoblots were 245
visualized with horseradish peroxidase-coupled goat anti-rabbit immunoglobulin using 246
an enhanced chemiluminescence Western blotting system (ECL Plus, GE healthcare 247
Life Sciences, Pittsburgh, PA, USA). Additional membranes were analyzed by 248
chemiluminescence prior to incubation with the primary antibody to confirm that the 249
reactive band observed in the immunoblotting corresponded to a protein recognized 250
Page 15 of 58
16
specifically by the primary antibody. 251
The experiment was performed three times, and the ratio of cell cycle 252
distribution was expressed as the mean ± SD. 253
254
Flow cytometry 255
To analyze the cell cycle distribution, CRL-4003 cells and EMOsis CC/TERT 256
cells were plated in six-well plates at a density of 2 × 105 per well, then the EMOsis 257
cc/TERT cells were cultured in 10% fetal bovine serum and the CRL-4003 cells were 258
cultured in growth media until they reached 70% to 80% confluence. The cells were 259
harvested after being incubated for 24 h in the absence or presence of 3 or 6 µg/ml of 260
decorin or 100 or 500 µM of dienogest, then the cell proliferation was evaluated by 261
measuring the distribution of the cells in the different phases of the cell cycle by flow 262
cytometry using the Cycle TEST PLUS DNA Reagent Kit (BD Pharmingen), which is 263
based on the measurement of the DNA content of nuclei labeled with propidium iodide, 264
according to the manufacturer's instructions. Briefly, cells were trypsinized (250 µL of 265
trypsin buffer) for 10 min at room temperature, and then a trypsin inhibitor (200 µL) 266
and RNase buffer were added and allowed to react for 10 min at room temperature. 267
Finally, propidium iodide staining solution (200 µL) was added and incubated for 10 268
Page 16 of 58
17
min in the dark on ice. Samples were immediately analyzed in the EC800 flow 269
cytometer (Sony Biotechnology Inc). The FlowJo version 9 software program (Tree Star, 270
Inc. Ashland, OR, USA) was used for the cell cycle analysis. The experiment was 271
performed three times, and the ratio of cell cycle distribution was expressed as the mean 272
± SD. 273
274
Enzyme-linked immunosorbent assay (ELISA) 275
EMOsis cc/TERT and CRL-4003 cells were seeded at 1×106 cells per well in 276
6-well plates and cultured in growth media until they reached 70% to 80% confluence. 277
The cells were then starved for 16 hours. The culture supernatants were collected after 278
being incubated for 48 hours with vehicle (PBS) or 10, 100 or 500 nM of dienogest, and 279
the level of decorin was assayed with 100 µl of cell-free culture supernatant using the 280
Decorin Human ELISA Kit (Abcam, Cambridge, MA) according to the 281
manufacturer’s instructions. Absorbance was read at 450 nm with the Corona SH-1000 282
lab absorbance microplate reader (Corona Electric Co. Inc., Ibaraki, Japan). The sample 283
concentrations were determined via interpolation based on the standard curve. The assay 284
was performed three times, and the colony number was expressed as the mean ± SD. 285
286
Page 17 of 58
18
Chromatin immunoprecipitation (ChIP) assays 287
The ChIP assay was conducted using the ChIP-IT® Express Chromatin 288
Immunoprecipitation Kit (ACTIVE MOTIF, CA, USA) according to the manufacturer’s 289
protocol. Briefly, 1 × 107 EMOsis cc/TERT and CRL-4003 cells were incubated in 290
growth medium. After incubation overnight, formaldehyde (37%) was directly added to 291
the culture at a final concentration of 1%, and the cells were incubated for 15 min at 292
37°C to cross-link the protein to DNA. The cells were pelleted and resuspended in 600 293
µl of lysis buffer supplemented with 3 µl of protease inhibitor cocktail and 50 mM 294
phenylmethylsulfonyl fluoride, and were incubated on ice for 10 min. The nuclear 295
fraction was resuspended in shearing buffer (supplemented with 1.05 µl protease 296
inhibitor cocktail), and was sonicated with a sonicator. The sheared chromatin solution 297
was used for each ChIP assay with 3 µg anti-PR antibody (H-190, Santa Cruz) or rabbit 298
IgG as a negative control. After that, the 1157 ~ 666-bp region of the decorin promoter 299
encompassing the progesterone response element (PRE) that was predicted by the 300
Matinspector software program (Genomatix Software GmbH, München, Germany) was 301
amplified by conventional PCR using the following primers: F: 302
5’-AAATATTGTGCAAGGCCCGG-3’ and R: 5’-TTTTGCTGCCTGAGTCATCG-3’. 303
All of the ChIP assays were repeated at least three times with similar results, and 304
Page 18 of 58
19
representative results for conventional PCR are shown. 305
306
Small interfering RNA (siRNA) transfection (decorin siRNA) 307
siRNA specific for decorin and the scrambled control was purchased from Invitrogen. 308
The cells were transfected using the Lipofectamine reagent (Invitrogen) according to the 309
manufacturer’s instructions. Briefly, oligomer-Lipofectamine plus complexes were 310
prepared as follows. A total of 20 pmol of siRNA oligomer was diluted in 50 µl of 311
Opti-MEM (Invitrogen), and the Lipofectamine plus solution was mixed gently before 312
use, after which a 1-µl aliquot was diluted in 50 µl of Opti-MEM, mixed gently and 313
incubated for five minutes at room temperature. The diluted oligomer was then 314
combined with the diluted Lipofectamine plus, mixed gently and incubated for another 315
20 minutes at room temperature, and the oligomer-Lipofectamine plus complexes were 316
added to each well containing cells and medium and mixed gently by rocking the plate 317
back and forth. The cells were subsequently incubated at 37°C in a CO2 incubator for 24 318
hours and prepared for each assay. 319
320
Immunohistochemistry (IHC) and scoring 321
The endometrioma samples were fixed in formalin and embedded in paraffin. 322
Page 19 of 58
20
Deparaffinized and rehydrated sections (4 µm) were then autoclaved in 0.01 mol/l 323
citrate buffer (pH 6.0) for 15 minutes at 121℃ for antigen retrieval. The endogenous 324
peroxidase activity was blocked with a 0.3% solution of hydrogen peroxide in methanol 325
for 30 minutes, and the sections were subsequently incubated at 4℃ for 12 hours with 326
anti-decorin rabbit antibodies (1:100 dilution; Abcam) or anti-Met rabbit antibodies 327
(1:100 dilution; LifeSpan BioSciences). The sections were then washed with 1X 328
phosphate-buffered saline (PBS) and incubated with Histofine simple stain MAX PO 329
(multi; Nichirei) for 30 minutes at room temperature. Finally, the sections were washed 330
with 1X PBS, and the signals were visualized after incubation with 331
H2O2/diaminobenzidine substrate solution for five minutes. Tissue 332
immunohistochemical (IHC) staining was evaluated under light microscopy. The tissue 333
IHC staining was analyzed blindly and independently by two examiners using a four 334
point semi-quantitative scale for intensity: 3+ (very strong), 2+ (strong), 1+ 335
(moderate/weak) and 0 (no staining) (Cawthorn et al., 2012). In cases where there was a 336
disagreement between the examiners, the cores were reviewed together and a consensus 337
was reached. 338
339
340
Page 20 of 58
21
Statistical analyses 341
All experiments were performed in triplicate, except for the cell proliferation 342
assay. The statistical calculations were performed using the StatView statistical software 343
package (SAS Institute, Cary, NC), and the statistical significance of each difference 344
was determined using the Kruskal-Wallis and Mann - Whitney U test, or a paired t-test, 345
as appropriate. A value of p < 0.05 was considered to be statistically significant. 346
347
Results 348
Progesterone treatment promoted the expression and secretion of decorin in the 349
EMOsis cc/TERT and CRL-4003 cells 350
EMOsis cc/TERT cells, which are immortalized human epithelial cells derived 351
from an ovarian endometrioma, and CRL-4003 cells, which are immortalized human 352
endometrial stromal cells, were treated with vehicle (PBS), 1.5 µg/ml progesterone or 353
100 nmol/L dienogest for 12 hours. The coefficient of variation (CV) of the mRNA 354
expression (quantification cycle) in all cell samples tested for GAPDH, ACTB and 355
RNA18S5 was 4.1%, 10.9% and 19.2%, respectively. Therefore, normalization against 356
GAPDH alone met the standards described in the MIQE guidelines (Bustin et al., 2009). 357
The decorin mRNA expression was significantly increased in both the EMOsis cc/TERT 358
Page 21 of 58
22
cells and CRL-4003 cells treated with progesterone or dienogest compared with the 359
control cells by performing real-time PCR (p < 0.05) (Fig. 1A). 360
Next, we ascertained whether progesterone and dienogest promoted decorin 361
synthesis in the EMOsis cc/TERT and CRL-4003 cells by performing a Western blot 362
analysis. The expression levels of the decorin protein in EMOsis cc/TERT cells and 363
CRL-4003 cells treated with progesterone or dienogest were significantly increased 364
compared with the untreated cells (p < 0.05) (Fig. 1B). 365
We measured the concentrations of decorin produced by the EMOsis cc/TERT 366
and CRL-4003 cells using the Decorin Human ELISA Kit. The decorin concentration 367
significantly increased in a dose-dependent manner following dienogest treatment (p < 368
0.05) (Fig. 1C), suggesting that progestin stimulation promoted decorin secretion in 369
both cell lines. Interestingly, the concentration of decorin in the cultured medium of the 370
untreated CRL-4003 cells was higher than that observed in the cultured medium 371
EMOsis cc/TERT cells (p < 0.05) (Fig. 1C). 372
373
Dienogest inhibits the proliferation of EMOsis cc/TERT and CRL-4003 cells via the 374
effects on decorin 375
We examined whether dienogest and decorin inihibited the proliferation of 376
Page 22 of 58
23
EMOsis cc/TERT cells and the CRL-4003 cells. The growth of the EMOsis cc/TERT 377
cells and CRL-4003 cells was inhibited in a dose-dependent manner by dienogest (Fig. 378
2A). However, these findings were cancelled by transfection with the decorin siRNA 379
and the addition of a decorin-neutralizing antibody (Fig. 2A). Moreover, the growth of 380
the EMOsis cc/TERT cells and CRL-4003 cells was also inhibited by decorin in a 381
dose-dependent manner (Fig. 2A), and these findings were also cancelled by the 382
addition of a decorin-neutralizing antibody (Fig. 2B). These data suggest that dienogest 383
and decorin possess cytostatic effects, and that dienogest apparently inhibits cell growth 384
via its effects on decorin. 385
386
Progesterone and dienogest play crucial roles in stimulating the decorin gene 387
expression in EMOsis cc/TERT and CRL-4003 cells 388
To directly evaluate whether progesterone and dienogest play a role in 389
stimulating decorin expression, we evaluated whether the progesterone receptor (PR) 390
was recruited to the promoter of the decorin gene by performing the chromatin 391
immunoprecipitation (ChIP) assay, as seen in Figure 3A. The decorin promoter region 392
between -1010 bp and -679 bp contains several half-progesterone response elements 393
(hPRE), two TATA-boxes and one CAAT-box (Santra et al., 1994). Therefore, ChIP 394
Page 23 of 58
24
primers for the decorin promoter region were designed to cover the region from -1157 395
bp and -666 bp. The EMOsis cc/TERT and CRL-4003 cells were exposed to vehicle 396
(PBS), 1.5 µg/ml progesterone or 100 nmol/L dienogest overnight, and then processed 397
for the ChIP assay. Progesterone and dienogest induced the binding of the PR to the 398
hPRE binding site of the decorin promoter in both EMOsis cc/TERT and CRL-4003 399
cells (Fig. 3B). These results suggest that progesterone and dienogest play a crucial role 400
in stimulating the decorin gene expression in EMOsis cc/TERT cells and CRL-4003 401
cells. 402
403
Progesterone and dienogest induce cell cycle arrest via the actions of decorin by 404
promoting p21 production and suppressing the expression of Met in EMOsis 405
cc/TERT and CRL-4003 cells 406
To determine whether the anti-proliferative effects of decorin synthesis 407
promoted by dienogest were accompanied by an effect on the cell cycle profile, EMOsis 408
cc/TERT cells and CRL-4003 cells were treated with vehicle (PBS), dienogest or 409
decorin for 16 hours, and then processed for PI flow cytometry to determine the 410
distribution of cells in the various stages of the cell cycle. As shown in Figure 4A , cells 411
treated with either dienogest or decorin exhibited a significant increase in G0/G1 phase 412
Page 24 of 58
25
cells and a reduction in S and G2-M phase cells compared with both untreated cells and 413
the effects of dienogest and decorin were dose-dependent manner (p<0.05). Therefore, 414
the dienogest and decorin treatment induced cell cycle arrest in EMOsis cc/TERT cells 415
and CRL-4003 cells. 416
Next, we examined the expression of p21in EMOsis cc/TERT and CRL-4003 417
cells treated with progesterone or dienogest. As shown in Figure 4B, treatment with 418
progesterone and/or dienogest led to a significant decrease in the expression of Met and 419
significant increase in the expression of p21 in both the EMOsis cc/TERT and 420
CRL-4003 cells compared with that observed in the untreated cells (p < 0.05 for both). 421
However, these findings were cancelled by the addition of a decorin-neutralizing 422
antibody as well as transfection with the decorin siRNA (Fig. 4C). These observations 423
indicate that decorin directly increased the expression of p21 in both the EMOsis 424
cc/TERT and CRL-4003 cells. 425
426
Dienogest treatment promoted the expression of the decorin in ovarian endometrioma 427
Based on the results that progesterone and dienogest have anti-proliferative 428
effects via the upregulation on decorin, we sought to examine the clinical relevance of 429
decorin in patinents with endometrioma. A total of 50 females who underwent surgical 430
Page 25 of 58
26
treatment for endometrioma were evaluated in this study. They were divided in two 431
groups, 25 females who did not receive any medical treatment before surgery (control 432
group) and 25 females who received dienogest (a fourth-generation progestin) (Table 1). 433
There were no relevant group differences in terms of the age, VAS score, cyst size or 434
ASRM score at baseline. The use of concomitant medications recorded in 435
patient-maintained diaries, including analgesic medications for endometriosis, did not 436
differ significantly between the groups at baseline. The mean (± SD) VAS score at 437
baseline was 35.6 ± 20.4 mm in the control group and 48.3 ± 26.1 mm in the dienogest 438
group. The mean (± SD) ASRM score at baseline was 39.5 ±17.9 in the control group 439
and 38.8 ± 20.3 in the dienogest group. The mean (± SD) cyst size at baseline was 440
60.7±14.9 mm in the control group and 57.5±15.5 mm in the dienogest group. There 441
were no significant differences between the two groups. However, the mean (± SD) 442
post-treatment cyst size was 44.7±12.8 mm in the dienogest group, which was 443
significantly different than the pre-treatment cyst size in this group (p=0.0002). 444
We investigated whether pre-surgical treatment with dienogest attenuated the 445
expression of decorin in the ectopic endometrium. The CV of GAPDH, ACTB and 446
RNA18S5 mRNA expression (quantification cycle) in all patient samples tested was 447
4.1%, 10.9% and 19.2%. Therefore, normalization against GAPDH alone met the 448
Page 26 of 58
27
standards described in the MIQE guidelines (Bustin et al., 2009). The endometrioma 449
samples obtained from the patients who received dienogest treatment showed 450
significantly higher expression of decorin mRNA compared with that observed in the 451
control tissues using real-time PCR (Fig. 5A). Moreover, we confirmed that the 452
expression of decorin in the ectopic endometrium of dienogest treatment group was 453
significantly higher than that observed in the control tissue using an 454
immunohistochemical analysis (staining intensity of epithelial cells: control, 0.17±455
0.38; dienogest, 2.39±0.61; staining intensity of stromal cells: control, 1.17±0.38; 456
dienogest, 2.94±0.24; p<0.05). We also confirmed that the expression of Met was 457
significantly lower in the ectopic endometrium of the dienogest treatment group 458
(staining intensity of epithelial cells: control, 2.33±0.69; dienogest, 0.94±0.64; 459
staining intensity of stromal cells: control, 2.67±0.59; dienogest, 0.78±0.65; p < 0.05) 460
(Fig. 5B). 461
462
Page 27 of 58
28
Discussion 463
In the present study, we showed that the endometriotic cells in the patients 464
treated with dienogest had a higher decorin mRNA expression levels than those in the 465
endometriotic cells in the untreated patients. A previous report demonstrated that 466
dienogest inhibited the proliferation of immortalized human endometrial epithelial cells 467
by suppressing cyclin D1 gene expression (Shimizu et al., 2009), however, this did not 468
occur in endometrial stromal cells. Elsewhere, it was reported that dienogest showed 469
antiproliferative effects on both human eutopic and ectopic endometrial stromal cells 470
(Fu et al., 2008; Okada et al., 2001). However, the previous studies have not 471
demonstrated how progesterone directly affects the proliferation of the human 472
endometriosis cells. We herein demonstrated, for the first time, that progesterone and 473
dienogest have anti-proliferative effects via their impact on decorin. Moreover, we 474
demonstrated that this was the case not only for human endometriotic epithelial cells, 475
but also endometrial stromal cells. In the current study, we used CRL-4003 cells, an 476
immortalized human endometrial stromal cell line, not ectopic endometrioid stromal 477
cells. However, we confirmed that the CRL-4003 cells identified both the hormonal 478
status (ER and PR) and expression of decorin mRNA treated with dienogest in the 479
primary cultured endometriotic stromal cells (HMOsis scl2, HMOsis scl3) and 480
Page 28 of 58
29
CRL-7566 endometriosis cell line (Supplemental figure). Our findings indicate that both 481
CRL-4003 cells and ectopic endometrioid stromal cells have identical characteristics for 482
testing the effects of dienogest and decorin on stromal cells, with respect to the 483
convenience of experimentation. 484
We demonstrated that progesterone and dienogest directly induced the binding 485
of the PR to the PRE binding site of the decorin promoter in endometriotic epithelial 486
cells and endometrial stromal cells by the CHIP assay, and that decorin was synthesized 487
by the endometriotic epithelial cells and endometrial stromal cells. Decorin induced cell 488
cycle arrest in endometrial epithelial cells and endometrial stromal cells. It has 489
previously been shown that decorin induces cell cycle arrest by inducing the 490
cyclin-dependent protein kinase inhibitor, p21 Waf1/Cip1
, decreasing the activity and 491
abundance of multiple cyclins and activating pro-apoptotic pathways (Iozzo and 492
Sanderson, 2011). We clarified the signaling pathway involving decorin and p21 493
synthesis, as the dienogest-induced increase in the expression of p21 in the 494
endometriotic epithelial cells and stromal cells was significantly reduced by both the 495
addition of a decorin-neutralizing antibody and transfection with the decorin siRNA. 496
Consequently, our study showed that the direct antiproliferative effects of progesterone 497
and dienogest occur via the induction of G0/G1 cell-cycle arrest via decorin, and that 498
Page 29 of 58
30
progesterone and dienogest lead the upregulation of p21 synthesis via the upregulation 499
of decorin in human endometriotic epithelial cells and endometrial stromal cells. 500
The prototypic members of the small leucine-rich proteoglycan family include 501
not only decorin, but also biglycan, lumican and fibromodulin (Iozzo, 1998; Kresse et 502
al., 1993; Neame and Kay, 2000). The patterns of expression of these prototype 503
members of the small leucine-rich proteoglycan family in the uterus were found to be 504
estrogen- or progesterone-dependent in mouse experiments (Salgado et al., 2011). 505
Although we could not evaluate whether the other small leucine-rich proteoglycans, 506
including biglycan, lumican and fibromodulin, affect the endometriotic epithelial cells 507
or endometrial stromal cells (contributing to its therapeutic effect on endometriosis), 508
decorin is known to have the most potent tumor-suppressive effects compared with 509
other molecules. 510
The anti-tumor effects of decorin were recently reported in various malignant 511
tumors, including uterine cervical carcinoma cells (Grant et al., 2002), ovarian cancer 512
(Nash et al., 1999), colon carcinoma cells (Santra et al., 1995), breast cancer cells (Reed 513
et al., 2005) and pancreatic cancer cells (Koninger et al., 2004). The previous reports 514
indicated that decorin is a powerful inhibitor of tumor angiogenesis in several malignant 515
cell lines (Grant et al., 2002). Endometriosis has been shown to be highly dependent on 516
Page 30 of 58
31
angiogenesis for the maintenance and growth of endometriosis (Nap et al., 2004). 517
Decorin leads to the downregulation of Met, which was known as hepatocyte growth 518
factor receptor, an established mediator of malignant transformation, invasion, 519
metastasis (Zhang et al., 2003). In this study, decorin suppressed the expression of Met 520
in endometriotic epithelial cells and endometrial stromal cells, which contributed to its 521
therapeutic effects on endometriosis. 522
These findings indicated that the estrogen-independent anti-proliferative effects 523
of decorin on endometriotic epithelial cells and endometrial stromal cells may 524
contribute to the effectiveness against endometriosis, without causing hypoestrogenic 525
side effects, such as bone loss and hot flashes. Moreover, decorin has minimal toxicity, 526
because it is a naturally ubiquitous proteoglycan (Nap et al., 2004). Further 527
examinations will be necessary to determine the optimum strategy for obtaining 528
therapeutic benefits of decorin treatment in the patients with endometriosis. 529
In summary, we demonstrated that progesterone and dienogest inhibit the 530
proliferation of immortalized human endometriosis epithelial cells and stromal cells via 531
the upregulation of decorin. Moreover, we demonstrated that decorin has significant 532
antiproliferative effects on human endometriotic epithelial cells and endometrial stromal 533
cells due to its actions in inducing cell cycle arrest via p21 synthesis in endometriotic 534
Page 31 of 58
32
tissues. 535
536
537
Author’s roles 538
YO conducted clinical evaluation, and contributed to sample collection and manuscript 539
writing and performed most experiments. YT, AT and MH designed this study, 540
analyzed data and wrote the manuscript. TT, AH and SK were involved in clinical 541
material collection and analysis. YY was involved in study design and data 542
interpretation. YT and MO were involved in study conception and design, data analysis, 543
drafting the article and its final edition. All authors gave their final approval of the 544
submitted version. 545
546
Acknowledgments 547
This work was supported by JSPS KAKENHI Grant Numbers 25462621 (to Y. Terai) 548
and Takeda Science Foundation (to A. Tanabe). 549
550
Funding 551
This work was supported by JSPS KAKENHI Grant Numbers 25462621 (to Y. Terai) 552
and Takeda Science Foundation (to A. Tanabe). 553
554
Conflict of interest statement 555
Page 32 of 58
33
The authors declare no conflicts of interest in association with this study. 556
557
Page 33 of 58
34
References 558
American Society for Reproductive Medicine (2006) Revised American Society for 559
Reproductive Medicine classification of endometriosis. Fertil Steril. 67, 817-821. 560
561
Bono Y, Kyo S, Takakura M, Maida Y, Mizumoto1 Y, Nakamura M, Nomura K, 562
Kiyono T, Inoue M (2012) Creation of immortalised epithelial cells from ovarian 563
endometrioma. Br J Cancer. 106, 1205-1213. 564
565
Brown J, Pan A, Hart RJ (2010) Gonadotrophin-releasing hormone analogues for pain 566
associated with endometriosis. Cochrane Database Syst Rev. 12, CD008475. 567
568
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, KubistaM, Mueller R, Nolan 569
T, Pfaffl MW, Shipley GL et al. (2009) The MIQE guidelines: minimum information 570
for publication of quantitative real-time PCR experiments. Clinical Chemistry. 55, 571
611–622. 572
573
Cawthorn TR, Moreno JC, Dharsee M, Tran-Thanh D, Ackloo S, Zhu PH, Sardana G, 574
Chen J, Kupchak P, Jacks LM et al (2012) Proteomic Analyses Reveal High Expression 575
Page 34 of 58
35
of Decorin and Endoplasmin (HSP90B1) Are Associated with Breast Cancer Metastasis 576
and Decreased Survival. PLoS ONE. 7, e30992. 577
578
Cosson M, Querleu D, Donnez J, Madelenat P, Konincks P, Audebert A, Manhes H 579
(2002) Dienogest is as effective as triptorelin in the treatment of endometriosis after 580
laparoscopic surgery: results of a prospective, multicenter, randomized study. Fertil 581
Steril. 77, 684-692. 582
583
Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV (1997) 584
Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin 585
fragility. J Cell Biol. 136, 729-743. 586
587
Eskenazi B, Warner ML (1997) Epidemiology of endometriosis. Obstet Gynecol Clin 588
North Am. 24, 235-258. 589
590
Fairlie DP, West ML, Wong AK (1998) Towards protein surface mimetics. Curr Med 591
Chem. 5, 29-62. 592
593
Page 35 of 58
36
Felice P, Daniela H, Christian S, Thomas F, Christoph G, Stefano L, Lucia L, Thomas S 594
(2012) Reduced pelvic pain in women with endometriosis: efficacy of long-term 595
dienogest treatment. Arch Gynecol Obstet. 285, 167-173. 596
597
Foster RH, Wilde MI. (1998) Dienogest. Drugs. 56, 825–833. 598
599
Fu L, Osuga Y, Morimoto C, Hirata T, Hirota Y, Yano T, Taketani Y (2008) Dienogest 600
inhibits BrdU uptake with G0/G1 arrest in cultured endometriotic stromal cells. Fertil 601
Steril. 89, 1344-1347. 602
603
Giudice LC, Kao LC (2004) Endometriosis. Lancet. 64, 1789-1799. 604
605
Goldoni S, Humphries A, Nystro¨m A, Sattar S, Owens RT, Mc- Quillan DJ, Ireton K, 606
Iozzo RV (2009) Decorin is a novel antagonistic ligand of the Met receptor. J Cell Biol. 607
185, 743-754. 608
609
Goldoni S, Iozzo RV (2008) Tumor microenvironment: Modulation by decorin and 610
related molecules harboring leucine-rich tandem motifs. Int J Cancer. 123, 2473-2479. 611
Page 36 of 58
37
612
Grant DS, Yenisey C, Rose RW, Tootell M, Santra M, Iozzo RV (2002) Decorin 613
suppresses tumor cell-mediated angiogenesis. Oncogene. 21, 4765-4777. 614
615
Guo SW (2009) Recurrence of endometriosis and its control. Hum Reprod Update. 15, 616
441-461. 617
618
Horie S, Harada T, Mitsunari M, Taniguchi F, Iwabe T, Terakawa N (2005) 619
Progesterone and progestational compounds attenuate tumor necrosis factor 620
alpha-induced interleukin-8 production via nuclear factor kappa B inactivation in 621
endometriotic stromal cells. Fertil Steril. 83, 1530-1535. 622
623
Iozzo RV, Sanderson RD (2011) Proteoglycans in cancer biology, tumor 624
microenvironment and angiogenesis. J Cell Mol Med. 15, 1013-1031. 625
626
Iozzo RV (1997) The family of the small leucine-rich proteoglycans: key regulators of 627
matrix assembly and cellular growth. Crit Rev Biochem Mol Biol. 32, 141-174. 628
629
Page 37 of 58
38
Iozzo, RV (1998) Matrix proteoglycans: From molecular design to cellular function. 630
Annu Rev Biochem. 67, 609-652. 631
632
Irahara M, Harada T, Momoeda M, Taketani Y (2007) Hormonal and histological study 633
on irregular genital bleeding in patients with endometriosis during treatment with 634
dienogest, a novel progestational therapeutic agent. Reprod Med Biol. 6, 223-228. 635
636
Jee BC, Lee JY, Suh CS, Kim SH, Choi YM, Moon SY (2009) Impact of GnRH agonist 637
treatment on recurrence of ovarian endometriomas after conservative laparoscopic 638
surgery. Fertil Steril. 91, 40-45. 639
640
Keene DR, San Antonio JD, Mayne R, McQuillan DJ, Sarris G, Santoro SA, Iozzo 641
RV(2000) Decorin binds near the C terminus of type I collagen. J Biol Chem. 275, 642
21801-21804. 643
644
Köhler G, Faustmann TA, Gerlinger C, Seitz C, Mueck AO (2010) A dose-ranging 645
study to determine the efficacy and safety of 1, 2, and 4 mg of dienogest daily for 646
endometriosis. Int J Gynaecol Obstet. 108, 21-25. 647
Page 38 of 58
39
648
Koninger J, Giese NA, Francesco di Mola F, Berberat P, Giese T, Esposito I, Bachem 649
MG, Friess H (2004) Overexpressed Decorin in Pancreatic Cancer: Potential Tumor 650
Growth Inhibition and Attenuation of Chemotherapeutic Action. Clin Cancer Res. 10, 651
4776-4783. 652
653
Kresse, H, Hausser, H, Scho¨nherr, E (1993) Experientia (Basel). 49, 403-416 654
655
Krikun G, Mor G (2004) A novel immortalized human endometrial stromal cell line 656
with normal progestational response. Endocrinology. 145, 2291-2296. 657
658
Lockwood CJ, Nemerson Y (1993) Progestational regulation of human endometrial 659
stromal cell tissue factor expression during decidualization. J Clin Endocrinol Metab. 76, 660
231-236. 661
662
Momoeda M, Harada T, Terakawa N, Aso T, Fukunaga M, Hagino H, Taketani Y 663
(2009) Long-term use of dienogest for the treatment of endometriosis. J Obstet 664
Gynaecol Res. 35,1069-1076. 665
Page 39 of 58
40
666
Nap AW, Griffioen AW, Dunselman GA, Bouma-ter Steege JC, Thijssen VL, Evers JL, 667
Groothuis PG (2004) Antiangiogenesis Therapy for Endometriosis. J Clin Endocrinol 668
Metab. 89, 1089-1095. 669
670
Nash MA, Loercher AE, Freedman RS (1999) In Vitro Growth Inhibition of Ovarian 671
Cancer Cells by Decorin : Synergism of Action between Decorin and Carboplatin. 672
Cancer Res. 59, 6192-6196. 673
674
Neame PJ, Kay CJ (2000) Small leucine-rich proteoglycans. In: Iozzo RV, eds. 675
Proteoglycans, Structure, Biology and Molecular Interactions. Marcel Dekker, Inc, New 676
York. 201-35 677
678
Okada H, Nakajima T, Yoshimura T, Yasuda K, Kanzaki H (2001) The inhibitory effect 679
of dienogest, a synthetic steroid, on the growth of human endometrial stromal cells in 680
vitro. Mol Hum Reprod. 7, 341-347. 681
682
Page 40 of 58
41
Reed CC, Waterhouse A, Kirby S, Kay P, Owens RT, McQuillan DJ, Izzo RV (2005) 683
Decorin prevents metastatic spreading of breast cancer. Oncogene. 24, 1104-1110. 684
685
Salgado RM, Favaro RR, Zorn TM (2011) Modulation of small leucine-rich 686
proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone. 687
Reprod Biol Endocrin. 9, 22. 688
689
Santra M, Danielson KG, Iozzo RV (1994) Structural and Functional Characterization 690
of the Human Decorin Gene Promoter. J Biol Chem. 269, 579-587. 691
692
Santra M, Skorski T, Calabretta B, Lattime EC, Iozzo RV (1995) De novo decorin gene 693
expression suppresses the malignant phenotype in human colon cancer cells. Proc Natl 694
Acad Sci USA. 92, 7016-7020. 695
696
Sasagawa S, Shimizu Y, Kami H, Takeuchi T, Mita S, Imada K, Kato S, Mizuguchi K 697
(2008) Dienogest is a selective progesterone receptor agonist in transactivation analysis 698
with potent oral endometrial activity due to its efficient pharmacokinetic profile. 699
Steroids. 73, 222-231. 700
Page 41 of 58
42
701
Schaefer L, Tsalastra W, Babelova A, Baliova M, Minnerup J, Sorokin L, Gro¨ ne HJ, 702
Reinhardt DP, Pfeilschifter J, Iozzo RV et al. (2007) Decorin-mediated regulation of 703
fibrillin-1 in the kidney involves the insulin-like growth factor-I receptor and 704
Mammalian target of rapamycin. Am J Pathol. 170, 301-315. 705
706
Seidler DG, Dreier R (2008) Decorin and its galactosaminoglycan chain: extracellular 707
regulator of cellular function? IUBMB Life. 60, 729-733. 708
709
Shimizu Y, Takeuchi T, Mita S, Mizuguchi K, Kiyono T, Inoue M, Kyo S (2009) 710
Dienogest, a synthetic progestin, inhibits the proliferation of immortalized human 711
endometrial epithelial cells with suppression of cyclin D1 gene expression. Mol Hum 712
Reprod. 15, 693-701. 713
714
Sitruk WR (2004) New progestogens: a review of their effects in perimenopausal and 715
postmenopausal women. Drugs Aging. 21, 865-883. 716
717
Sitruk WR (2004) Pharmacological profile of progestins. Maturitas. 47, 277-283. 718
Page 42 of 58
43
719
Strowitzki T, Faustmann A, Christoph G, Seitz C (2010) Dienogest in the treatment of 720
endometriosis-associated pelvic pain: a 12-week, randomized, double-blind, placebo 721
–controlled study. Eur J Obstet Gynecol Reprod Biol. 151, 193-198. 722
723
Strowitzki T, Marr J, Gerlinger C, Faustmann T, Seitz C (2010) Dienogest is as 724
effective as leuprolide acetate in treating the painful symptoms of endometriosis: a 725
24-week, randomized, multicentre, open-label trial. Hum Reprod. 25, 633-641. 726
727
Su AI, Wiltshire T, Batalov S, Lapp H, Ching KA, Block D, Zhang J, Soden R, 728
Hayakawa M, Kreiman G et al (2004) A gene atlas of the mouse and human 729
protein-encoding transcriptomes. Proc Natl Acd Sci USA. 101, 6062-6067. 730
731
Thomas N, Liliana S (2012) A Guardian from the Matrix. Am J Pathol. 181, 380–387. 732
733
734
Page 43 of 58
44
Vercellini P, Somigliana E, Viganò P, Abbiati A, Barbara G, Crosignani PG (2009) 735
Endometriosis: current therapies and new pharmacological developments. Drugs. 69, 736
649-675. 737
738
Weber IT, Harrison RW, Iozzo RV (1996) Model structure of decorin and implications 739
for collagen fibrillogenesis. J Biol Chem. 271, 31767-31770. 740
741
742
Zhang G, Chen S, Goldoni S, Calder BW, Simpson HC, Owens RT, McQuillan DJ, 743
Young MF, Iozzo RV, Birk DE (2009) Genetic evidence for the coordinated regulation 744
of collagen fibrillogenesis in the cornea by decorin and biglycan. J Biol Chem. 284, 745
8888-8897. 746
747
Zhang G, Ezura Y, Chervoneva I, Robinson PS, Beason DP, Carine ET, Soslowsky LJ, 748
Iozzo RV, Birk DE (2006) Decorin regulates assembly of collagen fibrils and 749
acquisition of biomechanical properties during tendon development. J Cell Biochem. 98, 750
1436-1449. 751
752
Page 44 of 58
45
Zhang YW, Su Y, Volpert OV, Vande Woude GF (2003) Hepatocyte growth 753
factor/scatter factor mediates angiogenesis through positive VEGF and negative 754
thrombospondin 1 regulation. Proc Natl Acad Sci USA. 100, 12718-12723. 755
756
Page 45 of 58
46
Figure legends 757
Figure 1. Progesterone treatment promoted the expression and secretion of the 758
decorin in EMOsis cc/TERT cells and CRL-4003 cells. 759
(A) Progesterone treatment promoted the decorin mRNA expression in EMOsis 760
cc/TERT cells and CRL-4003 cells. The mRNA expression of decorin was determined 761
by real time PCR from the total RNA obtained from CRL-4003 cells and EMOsis 762
cc/TERT cells cultured with or without decorin or dienogest for 12 hours. The mRNA 763
levels of decorin were normalized to those of GAPDH mRNA. The data were processed 764
by the comparative Ct method and expressed as a fold-increase relative to the basal 765
transcription level in the control. Bars indicate the standard error. Significant differences 766
are indicated by an asterisk. *p<0.05, **p<0.01. (B) Progesterone treatment 767
promoted the expression of the decorin in EMOsis cc/TERT cells and CRL-4003 cells. 768
CRL-4003 cells and EMOsis cc/TERT cells were harvested and used to prepare cell 769
lysates after treatment with or without dienogest or progesterone for 24 h. The lysates 770
were subjected to SDS-PAGE and blotted with anti-decorin (upper panel) or anti-β-actin 771
(lower panel) antibodies. The lower panel shows the densitometric quantification of the 772
Western blot analysis normalized to the β-actin expression and expressed as a 773
fold-increase relative to the basal transcription level in the control. The mean ± SD of 774
Page 46 of 58
47
three determinations is shown. *p<0.05. (C) Progesterone treatment promoted the 775
secretion of the decorin in EMOsis cc/TERT cells and CRL-4003 cells. CRL-4003 776
cells and EMOsis cc/TERT cells were treated with dienogest at various concentrations 777
for 48 hours, and the concentrations of decorin produced by the EMOsis cc/TERT and 778
CRL-4003 cells were measured using the Decorin (DCN) Human ELISA Kit. The data 779
are shown as the mean ± SD (N = 5). * indicates a significant (p< 0.05) difference 780
compared with the untreated control group, while ※indicates a significant (p< 0.05) 781
difference compared with the untreated control EMOsis cc/TERT cells. 782
783
Figure 2. Progesterone treatment inhibited the proliferation of EMOsis cc/TERT 784
cells and CRL-4003 cells in a decorin-dependent manner 785
(A) Progesterone treatment inhibits the proliferation of EMOsis cc/TERT cells and 786
CRL-4003 cells in a decorin-dependent manner. CRL-4003 cells, EMOsis cc/TERT 787
cells and EMO sis cc/TERT cells transfected the siRNA specific for decorin (si-DCN) 788
were treated with dienogest at various concentrations with or without the decorin 789
neutralizing antibody (5 µg/mL) for 48 hours, and the proliferation was measured by the 790
MTS assay. (E) Decorin treatment inhibits the proliferation of EMOsis cc/TERT cells 791
and CRL-4003 cells in a decorin-dependent manner. CRL-4003 cells and EMOsis 792
Page 47 of 58
48
cc/TERT cells were treated with decorin at various concentrations with or without a 793
decorin neutralizing antibody (5 µg/mL) for 48 hours, and the proliferation was 794
measured by the MTS assay. The data are shown as the means ± SD (N = 5), and * 795
indicates a significant (p< 0.05) difference compared with the untreated control. 796
797
Figure 3. Dienogest and progesterone play a crucial role in stimulating the decorin 798
gene expression in EMOsis cc/TERT and CRL-4003 cells. 799
(A) The half-PRE island is located in the Decorin promoter. The nucleotide sequence 800
and putative regulatory elements of the 5’-flanking region of the human decorin gene. 801
The coding sequence of the human decorin gene is indicated by bold and capital letters. 802
The blue underlined letters and red double underlined letters indicate the CAAT-box and 803
TATA-box, respectively. The large A residue at position +1 with an arrowhead on top 804
indicates the transcriptional initiation site. Boxed letters indicate the half-PRE sites. The 805
arrows indicate the primer used for PCR amplification in the ChIP assay. 806
(B) Progesterone and dienogest directory stimulated the decorin gene expression. The 807
ChIP assay, performed as described in the Materials and Methods section, showed the 808
expression of the decorin promoter in control medium or progesterone or 809
dienogest-stimulated EMOsis cc/TERT and CRL-4003 cells. An anti-PR antibody 810
Page 48 of 58
49
(Santa Cruz) was used for immunoprecipitation. The input DNA represents PCR 811
products from chromatin pellets before immunoprecipitation. IgG was used as the 812
negative control, and normal rabbit Anti-IgG antibodies were used. The PCR primer 813
was designed as shown in the Materials and Methods section. 814
815
Figure 4. Dienogest induces cell cycle arrest in EMOsis cc/TERT cells and 816
CRL-4003 cells by promoting p21 production, and affects the Met expression. 817
(A) Inhibition of cell cycle progression by decorin and dienogest. EMOsis cc/TERT 818
cells and CRL-4003 cells were exposed to 100 nmol/L or 500 nmol/L dienogest or 3 819
µg/ml or 6 µg/ml decorin overnight, or were left untreated, and then processed for PI 820
flow cytometry. The cell cycle characteristics (G0-G1: S: G2-M%) of EMOsis cc/TERT 821
cells: control (52.64 ± 0.67: 29.45 ± 0.92: 17.91 ± 1.08), 100 nM dienogest (77.21 ± 822
0.18＊: 19.73 ± 0.36＊: 14.15 ± 1.81), 500 nM dienogest (80.38 ± 0.57＊: 12.14 ± 0.32＊: 823
7.4 ± 0.37＊), 3 µg/ml decorin (67.59 ± 0.95＊: 19.82 ± 0.04＊: 12.61 ± 0.83), 6 µg/ml 824
decorin (66.39 ± 0.26＊: 14.51 ± 0.64＊: 8.28 ± 0.56＊) and CRL-4003 cells: control 825
(57.23 ± 0.10: 20.46 ± 0.63: 22.31 ± 0.71), 100 nM dienogest (66.57 ± 0.38＊: 15.23 ± 826
0.09: 18.2 ± 0.26), 500 nM dienogest (78.07 ± 0.69＊: 8.32 ± 0.17＊: 8.32 ± 0.91＊), 3 827
µg/ml decorin (65.05 ± 0.50＊: 15.22 ± 0.75: 19.73 ± 0.27), 6 µg/ml decorin (75.31 ± 828
Page 49 of 58
50
0.47＊: 9.41 ± 0.29＊: 15.28 ± 1.08＊). The data are shown as the means ± SD (N = 5), 829
and * indicates a significant (p< 0.05) difference compared with the untreated control. 830
The panel shows the proportion of cells in the G0/G1 phase (black), S phase (gray) and 831
G2/M phase (white). (B) The p21 expression was increased and the Met expression 832
was decreased by dienogest and progesterone in a decorin-dependent manner. 833
Proteins were extracted from CRL-4003 cells and EMOsis cc/TERT cells after 834
treatment with 100 nmol/L dienogest, 100 nmol/L progesterone or 100 nmol/L 835
dienogest with or without 5 µg/mL of a decorin-neutralizing antibody for 24 h. The 836
Western blot analysis was performed with anti-Met antibodies, anti-p21 antibodies and 837
anti-β-actin antibodies. The lower panel shows the densitometric quantification of the 838
Western blot analysis normalized to the β-actin expression and expressed as a 839
fold-increase relative to the basal transcription level in the control. The mean ± SD of 840
three determinations is shown. *p<0.05 (C) Decorin and p21 expression is increased 841
and Met expression is decreased by dienogest and progesterone in a 842
decorin-dependent manner. Proteins were extracted from CRL-4003 cells, EMOsis 843
cc/TERT cells and EMO sis cc/TERT cells transfected with decorin siRNA cultured 844
with or without progesterone or dienogest for 24 hours. The Western blot analysis was 845
performed with anti-decorin antibodies, anti-p21 antibodies, anti-Met antibodies and 846
Page 50 of 58
51
anti-β-actin antibodies. The lower panel shows the densitometric quantification of the 847
Western blot analysis normalized to the β-actin expression and expressed as a 848
fold-increase relative to the basal transcription level in the control. The mean ± SD of 849
three determinations is shown. *p<0.05 850
851
Figure 5. Progesterone treatment promoted the expression of the decorin in 852
ovarian endometrioma. 853
(A) The decorin mRNA expression in the patients with ovarian 854
endometrioma. A total of 50 cases were selected for the study, and RNA was extracted 855
from surgically obtained endometrioma tissue samples from the control group (n=25), 856
dienogest group (n=25). The mRNA levels of decorin were measured by real-time PCR 857
and normalized to those of GAPDH mRNA. The data were processed by the 858
comparative Ct method and expressed as a fold-increase relative to the basal 859
transcription level in the control. Bars indicate the standard error. Significant differences 860
are indicated by an asterisk. *p<0.05 861
(B) The decorin and Met expression in ovarian endometrioma. 862
Representative examples of immunohistochemical sequential sections stained with 863
decorin and Met among the ovarian endometrioma samples obtained from 864
Page 51 of 58
52
endometriosis patients who underwent oophorectomy. A representative section from the 865
no preoperative treatment control group is on the left, and a representative section from 866
the dienogest preoperative treatment group is on the right. The black filled arrows 867
indicate the epithelial cells and the white filled arrows indicate the stromal cells. 868
869
Supplemental figure legends 870
supplemental figure 1.: (A) We evaluated the expression of ER mRNA and PR mRNA, 871
in the primary cultured cells (HMOsis scl2 and HMOsis scl3) which were stromal cells 872
derived from ovarian endometrioma and CRL-7566 endometriosis cell line using 873
RT-PCR. HMOsis scl2, HMOsis scl3 and CRL-7566 cells were identified as ER and PR 874
positive as with EMOsis cc/TERT cells and CRL-4003 cells. (B) We examined that the 875
mRNA expression of decorin of HMOsis scl2, HMOsis scl3 and CRL-7566 cells with 876
or without dienogest treatment. The HMOsis scl2, HMOsis scl3 and CRL-7566 cells 877
treated with dienogest showed significantly higher expression of decorin mRNA. 878
Page 52 of 58
254x190mm (96 x 96 DPI)
Page 53 of 58
254x190mm (96 x 96 DPI)
Page 54 of 58
254x190mm (96 x 96 DPI)
Page 55 of 58
190x254mm (96 x 96 DPI)
Page 56 of 58
254x190mm (96 x 96 DPI)
Page 57 of 58
Table 1. Patient characteristics according to the treatment group
Characteristic Control Dienogest p value
Patient number 25 25 NS
Age (years, mean) (± S.D.) 35.5±6.23 33.1±6.86 NS
Duration of drug administration (weeks, mean) - 21.6±11.5 -
ASRM score (mean) 39.5±17.9 38.7±20.3 NS
Pre-treatment cyst size (maximum diameter mm, mean) 60.7±14.9 57.5±15.5 NS
Post-treatment cyst size (maximum diameter mm, mean) - 44.7±12.8＊ -
Pre-treatment pelvic pain VAS (mm, mean) 35.6±20.3 48.3±20.3 NS
Post-treatment pelvic pain VAS (mm, mean) - 20.6±15.1 -
ASRM score: American Society for Reproductive Medicine score, VAS score: Visual Analog Scale score, S.D.:
Standard deviation. ＊:paired t test p value between the pre and post treatment cyst size : 0.0002
Page 58 of 58
