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Journal of Ethnopharmacology 140 (2012) 391– 397
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology
journa l h o me page: www.elsev ier .com/ locate / je thpharm
eer (Cervus elaphus) antler extract suppresses adhesion and migration ofndometriotic cells and regulates MMP-2 and MMP-9 expression
i-Hyun Kima,b, Yeong-In Yanga,b, Ji-Hye Ahna,b, Jong-Gyu Leea,b, Kyung-Tae Leea, Jung-Hye Choia,b,c,∗
Department of Life and Nanopharamceutical Science, Kyung Hee University, Seoul 130-701, Republic of KoreaDepartment of Oriental Pharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of KoreaResearch Center for New Nano Bio Fusion Technology, Kyung Hee University, Seoul 130-701, Republic of Korea
r t i c l e i n f o
rticle history:eceived 2 September 2011eceived in revised form 20 January 2012ccepted 22 January 2012vailable online 31 January 2012
eywords:ndometriosiseproductive pharmacology
a b s t r a c t
Ethnopharmacological relevance: Deer antler has been used for centuries as medicine for a wide range ofhealth problems, including various women’s diseases. However, there is a safety concern related to itsuse in hormone-sensitive conditions, such as breast cancer and endometriosis. In the present study, weinvestigated the effect of deer (Cervus elaphus) antler extract (DAE) on adhesion and migration of humanendometriotic cells.Materials and methods: Adhesion, wound-healing, and transwell migration assays were performed inendometriotic cells 11Z and 12Z. Expression of matrix metalloproteinase (MMP)-2, MMP-9, TNF-�, andIL-6 were measured by real-time RT-PCR and Western blot analysis.
raditional medicine Asia & Oceaniaeer antlerdhesionound healingMP
Results: DAE (50 and 100 �g/ml) decreased the adhesion of 11Z and 12Z cells on peritoneal mesothelialMet5-A cells. Wound-healing and transwell migration assays revealed that DAE (50 and 100 �g/ml)inhibited migration in 11Z and 12Z cells. It was further demonstrated that treatment with DAE (50 and100 �g/ml) significantly decreased the levels of MMP-2, MMP-9, TNF-�, and IL-6.Conclusions: These results indicate that DAE is a potential anti-endometriotic agent to inhibit the adhesionand migration of endometrial cells through the suppression of various related molecules.
© 2012 Elsevier Ireland Ltd. All rights reserved.
. Introduction
Endometriosis is a common gynaecological disorder in whichunctioning endometrial glands and stroma are aberrantly presentn sites outside the uterine cavity. It affects 5–15% of women inhe reproductive phase, and the main clinical features are infer-ility and chronic pelvic pain, including dysmenorrhoea. In fact,ndometriosis accounts for over 20% of all cases of infertility inomen (Bulun, 2009). The establishment of endometriotic lesions
n the peritoneal cavity requires adhesion, migration, invasion,nd proliferation of the ectopic endometrial tissue (Nisolle andonnez, 1997; Van Langendonckt et al., 2002; Giudice and Kao,004). The molecular mechanism of endometriosis has been a focusf active scientific investigations over the past decade. For exam-
le, the expression levels of matrix metalloproteinase-2 (MMP-2)nd matrix metalloproteinase-9 (MMP-9) were higher in womenith endometriosis than their healthy counterparts (Osteen et al.,∗ Corresponding author at: Department of Life and Nanopharmaceutical Science,yung Hee University, Dongdaemun-Gu, Hoegi-Dong 130-701, Seoul, Republic oforea. Tel.: +82 2 9612246; fax: +82 2 9612172.
E-mail addresses: [email protected], [email protected] (J.-H. Choi).
378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2012.01.032
2003). In fact, the enzymes play important roles in the ectopicadhesion, invasion and implantation, and neovascularisation of theendometrium (Chen et al., 2004; Li et al., 2006).
Various therapies have been used for treating endometrio-sis, including surgical and medical strategies. Current medi-cal therapy consists mainly of hormonal suppressive therapyin which the medication causes a downregulation of thehypothalamus–pituitary–ovarian pathway. Those medical treat-ments are effective for relieving pain during treatment but have ahigh recurrence rate and significant side effects, such as hot flashesand genital atrophy (Vignali et al., 2005). Thus, novel therapeuticstrategies are urgently needed to improve the clinical managementof patients with endometriosis.
Deer antler (termed “Nokyong” in Korean and “Lu rong” inChina) has been used in the East for centuries as a key compo-nent for prevention and/or treatment of a wide range of healthproblems including cardiovascular, immunological, and gynaeco-logical disease (Huang, 1997). These traditional uses are supportedby several studies showing that deer antler has anti-inflammatory
effects (Takikawa et al., 1972; Zhang et al., 2000; Kim et al., 2003),anti-whiplash activities (Chen et al., 1992; Sunwoo et al., 1998;Blob and Snelgrove, 2006), and anti-aging activities (Dai et al.,2011). In Asia and Russia, deer antler has been used for a variety of
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ynaecological conditions, such as menstrual disorder, menopause,nd infertility. However, it is generally believed that deer velvethould be avoided for women with hormone-sensitive diseases,uch as endometriosis and breast cancer. To the best of our knowl-dge, there is no scientific evidence of the bioactivity and safetyf deer antler for gynaecological diseases. In the present study, wenvestigate the effect of deer antler extract (DAE) on endometriosisnd the associated molecular mechanism.
. Materials and methods
.1. Materials
Powdered antler from Cervus elaphus Linnaeus was obtainedrom Kyung Hee Oriental Medical Hospital (Kyung Hee University,eoul, South Korea) as i.p. injection grade for humans, originatingrom deer born and bred in New Zealand. Because deer antler hasraditionally been extracted by water in oriental medicine, we used
similar extraction method using boiling water for 48 h, filtration,nd lyophilisation (yield: 36.1%). The powdered extract (DAE; pH.8 ± 0.056) was dissolved in saline and then filtered through a 0.22-m syringe filter. Based on the Korean Herbal Pharmacopoeia, lossf dry weight and ash content were analysed, which were 11.16%nd 31.65%, respectively. RNase was purchased from Sigma Chem-cal Co. (St. Louis, MO, USA). Antibodies for MMP-9, �-actin, andhe peroxidase-conjugated secondary antibodies were purchasedrom Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). MMP-2ntibody was purchased from Cell Signaling Technology, Inc. (Bev-rly, MA, USA). Cell Tracker CMFDA was obtained from InvitrogenPaisley, UK). 24-Well transwell plate was purchased from SPL Life-cience (Pocheon-si, South Korea)
.2. Cell culture
Immortalized human endometriotic epithelial cells (11Z and2Z) (Zeitvogel et al., 2001) were maintained in Dulbecco’sodified Eagle’s medium (DMEM)/F12 medium supplementedith 10% fetal bovine serum and 100 U/ml penicillin G and
00 g/ml streptomycin (Life Technologies, Grand Island, NY) in aumidified atmosphere of 5% CO2–95% air at 37 ◦C. The endometri-tic cells were generously provided by Dr. Starzinski-PowitzJohann-Wolfgang-Goethe-Universitaet, Germany). Human peri-oneal methothelial cells (PMCs) Met-5A were maintained in
edium 199 medium supplemented with 10% fetal bovine serum,00 U/ml penicillin G and 100 g/ml streptomycin (Life Technolo-ies, Grand Island, NY, USA), and 400 nM hydrocortisone (Sigmahemical Co, St. Louis, MO, USA) in a humidified atmosphere of% CO2–95% air at 37 ◦C. Met-5A cells were purchased from ATCCAmerican Type Culture Collection).
.3. Attachment assay
The Met-5A PMCs were grown to confluence on 96-well plates.1Z and 12Z cells were labelled with cell tracker green CMFDA.1Z and 12Z cells (20,000 cells per well) in DMEM/F12, with andithout DAE, were added to each PMC well, and all assays were
un in triplicate. Cells were incubated for 1 h at 37 ◦C in 5% CO2,nd the plates were inverted and washed in phosphate-bufferedolution with calcium and magnesium. Total fluorescence was readt a wavelength of 490 nm. The percentage of attached cells wasalculated by multiplying the control group fluorescence by 100.
.4. Wound healing assay
To determine cell motility, 11Z and 12Z cells (5 × 105 cells perell) were seeded in 12-well tissue culture plates and grown to
macology 140 (2012) 391– 397
80–90% confluence. After aspirating the medium, the centres ofthe cell monolayers were scraped with a sterile micropipette tip tocreate a denuded zone (gap) of constant width. Subsequently, cel-lular debris was washed with PBS, and the 11Z and 12Z cells wereexposed to DAE (50 and 100 �g/ml). The wound closure was moni-tored and photographed at 0, 2, 8, and 24 h. To quantify the migratedcells, pictures of the initial wounded monolayers were comparedwith corresponding pictures of cells at the end of incubation. Artifi-cial lines fitting the cut edges were drawn on pictures of the originalwounds and overlaid on the pictures of cultures after incubation.Cells that migrated across the lines were counted in six randomfields from each triplicate treatment, and data are presented as themean ± SD.
2.5. Transwell-migration assay
An in vitro transwell-migration assay was performed using a 24-well transwell unit (8 �m pore size) with polyvinylpyrrolidone-freepolycarbonate (PVPF) filters. The filters were washed thoroughlyin PBS and dried immediately before use. Cells were placed in theupper part of the transwell plate and incubated with DAE for 24 hat 37 ◦C. The cells that migrated to the lower surface of the mem-brane were fixed with methanol and stained with 0.5% crystal violetfor 10 min. Finally, we determined migrative phenotypes by count-ing the cells that migrated to the lower side of the filter usingmicroscopy at 200×.
2.6. Western blot analysis
Cells were washed with ice-cold PBS and extracted in proteinlysis buffer (Intron, Seoul, South Korea). Protein concentrationswere determined by the Bradford assay. Protein samples of celllysates were mixed with equal volumes of 5× SDS sample buffer,boiled for 4 min, and then separated by 10–12% SDS-PAGE gels.After electrophoresis, proteins were transferred to polyvinylidenedifluoride membranes. The membranes were blocked in 2.5%non-fat dry milk for 30 m, washed, and incubated overnightat 4 ◦C with specific antibodies against MMP-2, MMP-9, and�-actin in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). The membranes were washed three times to remove theprimary antibodies, and incubated for 1 h with a horseradishperoxidase-conjugated secondary antibody (1:1000–2000).After washing three times with TBS-T, immuno-positive bandswere visualised by enhanced chemiluminescence and analysedusing ImageQuant Las-4000 (GE Healthcare Life Science, Tokyo,Japan).
2.7. RNA isolation and real time RT-PCR analysis
Total RNA was prepared using the TRIzol reagent (InvitrogenCanada, Burlington, ON, Canada), according to the manufac-turer’s instructions. Total RNA was reverse transcribed intofirst-strand cDNA (Amersham Pharmacia Biotech, Oakville, ON,Canada) following the manufacturer’s procedure. The synthesizedcDNA was used as a template for polymerase chain reaction(PCR) amplification. Real-time PCR was performed using Ther-mal Cycler Dice Real Time PCR System (Takara, Japan). Theprimers used for SYBR Green real-time RT-PCR were as follows:for MMP-2, sense primer, 5′-ACCGCGACAAGAAGTATGGC-3′,and antisense primer, 5′-CCACTTGCGGTCATCATCGT-3′; forMMP-9, sense primer, 5′-CGATGACGAGTTGTGGTCCC-3′ andantisense primer, 5′-TCGTAGTTGGCCGTGGTACT-3′; for IL-6,
sense primer, 5′-CCCAGGAGAAGATTCCAAA-3′, and antisenseprimer, 5′-TTGTTTTCTGCCAGTGCCTC-3′; for TNF-�, sense primer,5′-AGCACTGAAA GCATGATCCG-3′ and antisense primer, 5′-GGCCAGAGGGCTGATTAGAG-3′; for GAPDH, sense primer,
opharmacology 140 (2012) 391– 397 393
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Fig. 1. The effect of DAE on the attachment ability of endometriotic cells. Endometri-otic 11Z and 12Z cells were labelled with Cell Tracker Green CMFDA. A confluentmonolayer of Met-5A cells was established on a 96-well plate. Attachment of 11Z(A) and 12Z (B) cells to Met-5A cells is seen at 1 h. Cell attachment is defined as thepercentage of attached cells relative to control group cells. The columns represent
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J.-H. Kim et al. / Journal of Ethn
′-GAGTCAACGGATTTGGTCGT-3′ and antisense primer, 5′-TGATTTTGGAGGGATCTCG-3′. A dissociation curve analysis ofMP-2, MMP-9, IL-6, TNF-�, and GAPDH showed a single peak.
CRs were carried out for 45 cycles using the following conditions:enaturation at 95 ◦C for 5 s, annealing at 57 ◦C for 10 s, andlongation at 72 ◦C for 20 s. Mean Ct of the gene of interest wasalculated from triplicate measurements and normalized with theean Ct of a control gene, GAPDH.
.8. Statistical analysis
Data shown are the mean ± SD of three individual experimentserformed in triplicate. Statistical analysis was performed by thetudent’s t-test and P < 0.05 was considered statistically significant.
. Results
.1. The effect of DAE on endometriotic cell adhesion
Once endometrial tissue is introduced into the peritoneal cav-ty, it must attach, invade deep into the peritoneum covered by
mesothelial layer to establish a vascular supply, and prolifer-te. To investigate the effect of DAE on the cellular adhesionf endometriotic cells to the human mesothelial layer, adhesionssays using endometriotic cells (11Z and 12Z) and peritonealesothelial cells were performed in the presence or absence ofAE. As shown in Fig. 1, pre-incubation of endometriotic cells withAE (50 and 100 �g/ml) inhibited the adhesion of both endometri-tic cells to human peritoneal mesothelial cells Met-5A. The MTT
ssay revealed that this extract, up to 200 �g/ml, does not affecthe viability of 11Z, 12Z, and Met-5A cells during a period of 48 hdata not shown). Thus, the anti-adhesive effect observed was notttributable to cytotoxic effects.ig. 2. The effect of DAE on the wound-healing ability of endometriotic cells. Scratches
llowed to grow for another 24 h in the presence or absence of different concentrations oixels. The columns represent the mean of three individual experiments performed in tri
the mean of three individual experiments performed in triplicate; bars, SD. *P < 0.05,statistical significance compared with controls.
3.2. The effect of DAE on endometriotic cell migration
To examine the effect of DAE on cell migration, we performedwound healing assays and transwell-migration assays. To comparethe differences in migratory behaviour, images were printed at thesame size, and wound closure was determined after the indicatedtimes (8 and 24 h) compared to control. At 24 h, the wound washealed approximately 60% in both 11Z and 12Z cells. As shown inFig. 2, DAE significantly reduced the cell motility in both 11Z and
12Z cells, compared with control. Treatment of endometriotic cellswith DAE at 100 �g/ml for 24 h showed that only approximately40% of cells had migrated. The migration assay using transwells alsowere made in confluent cultures, and 11Z (A) and 12Z (B) endometrial cells weref DAE. The distances covered by the cells (wound width) were plotted in terms of
plicate; bars, SD. *P < 0.05, statistical significance compared with controls.
394 J.-H. Kim et al. / Journal of Ethnopharmacology 140 (2012) 391– 397
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analogues, oestrogen/progestin combined oral contraceptives, andprogestins, are effective for relieving pain during treatment butdo not minimise the high recurrence rate and have signifi-cant side effects (Garai et al., 2006). Thus, novel therapeutic
ig. 3. The effect of DAE on the migration ability of endometriotic cells. Migration oell migration is depicted as the percentage of invading cells relative to control groriplicate; bars, SD. *P < 0.05, statistical significance compared with controls.
emonstrated that DAE (50 and 100 �g/ml) effectively inhibitedell migration in endometriotic 11Z and 12Z cells (Fig. 3).
.3. The effect of DAE on the expression of MMP-2 and MMP-9 inndometriotic cells
Matrix metalloproteinases (MMPs) are essential for extracellu-ar matrix remodelling and may contribute to the development ofndometriosis (Osteen et al., 2003). It is known that MMP-2 andMP-9 play important roles in the ectopic adhesion, invasion, and
mplantation and neovascularisation of the endometrium (Li et al.,006; Chen et al., 2009). First, protein expression of MMP-2 andMP-9 was investigated by Western blot after treatment with DAE
50 and 100 �g/ml) for 24 h. Treatment of 11Z and 12Z cells withAE significantly decreased MMP-2 and MMP-9 expression (Fig. 4).
n addition, real-time RT-PCR was performed to investigate theffect of DAE on mRNA expression of MMP-2 and MMP-9. As shownn Fig. 5, DAE significantly suppressed mRNA expression of MMP-2nd MMP-9 in 11Z and 12Z cells. These results suggest that DAE reg-lates the expression of MMP-2 and MMP-9 at the transcriptional
evel.
.4. The effect of DAE on the expression of TNF- ̨ and IL-6 inndometriotic cells
Several studies have demonstrated that the levels of inflamma-ory cytokines such as interleukin 6 (IL-6) and tumour necrosisactor-alpha (TNF-�) are up-regulated in endometriosis (Xaviert al., 2006; Cho et al., 2007; Martinez et al., 2007; Othman Eel et al.,008). To investigate the effect of DAE on TNF-� and IL-6 mRNA lev-ls, real-time RT-PCR was performed on endometriotic cells afterreatment with DAE (50 and 100 �g/ml) for 24 h. As shown in Fig. 6,
AE markedly suppressed the mRNA expression of TNF-� and IL-6n 11Z and 12Z cells. These data suggest that DAE suppresses thenflammatory condition by regulating the levels of TNF-� and IL-6n endometriotic cells.
(A) and 12Z (B) cells after treatment with different concentrations of DAE for 24 h.lls. The columns represent the mean of three individual experiments performed in
4. Discussion
Current medical therapies for endometriosis, including non-steroidal anti-inflammatory agents, androgenic agents, GnRH
Fig. 4. The effect of DAE on MMP-2 and MMP-9 protein expression in endometrioticcells. Cells were treated with various concentrations of DAE for 24 h. MMP-2 andMMP-9 protein levels from whole-cell lysates were analysed by Western blot in (A)11Z and (B) 12Z cells. �-Actin was used as a loading control.
J.-H. Kim et al. / Journal of Ethnopharmacology 140 (2012) 391– 397 395
Fig. 5. The effect of DAE on MMP-2 and MMP-9 mRNA levels in endometriotic cells.Cells were treated with various concentrations of DAE for 24 h. Total RNA was puri-fied and subjected to real-time RT-PCR for MMP-2 and MMP-9 with GADPH as anits
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Fig. 6. The effect of DAE on the expression of IL-6 and TNF-� in endometriotic cells.Cells were treated with 50 �g/ml DAE for 24 h. Total RNA was purified and subjectedto real-time RT-PCR for TNF-� and IL-6 with GADPH as an internal control in 11Z (A)
nternal control in (A) 11Z and (B) 12Z cells. The columns represent the mean ofhree individual experiments performed in triplicate; bars, SD. *P < 0.05, statisticalignificance compared with controls.
trategies are essential to improve clinical management of patientsith endometriosis.
Deer antler, traditionally used as a tonic and a valuable drugn Asia, has anti-hypertensive, erythropoietic, anti-stress, anti-nflammatory, and immunopotentiation effects (Takikawa et al.,972; Zhang et al., 2000; Kim et al., 2003). For example, it has beentudied as a treatment for several immune-related diseases, such asheumatoid arthritis (Kim et al., 2003, 2004, 2005). Treatment witheer antler extract resulted in an inhibition of arthritis develop-ent and immune responses to type II collagen (Kang et al., 2006).eer antler extract reduced protease activity and damages due toxidants and free radicals in the synovial fluid of rats with rheuma-oid arthritis (Kim et al., 2008a). DAE diminished the developmentf polyarthritis and eliminated the joint swelling and distortionbserved during the acute phase and the chronic phase of the dis-ase in rats (Kim et al., 2008b). In a phase II trial, DAE showed greatmprovement for rheumatoid arthritis compared to placebo (Allent al., 2002). The production of proinflammatory cytokines, such asNF-� and IL-6, by lipoteichoic acid was significantly reduced afterreatment with DAE in Raw 264.7 cells and peritoneal macrophageells (Dai et al., 2011). In addition, the effect of DAE on bone healthas been studied. Chloroform extract of deer antler inhibited osteo-last differentiation and bone resorption by suppression of ERK,kt, and IKB via receptor activator of NF-kappaB ligand (RANKL)
n mouse bone marrow cultures (Kim et al., 2008a). Deer antler
mproved gait, performance in daily life activities, and vitality inogs with osteoarthrosis (Moreau et al., 2004).Deer antler has been widely used for gynaecological condi-ions, especially for treating infertility and menstrual problems and
and 12Z (B) cells. The columns represent the mean of three individual experimentsperformed in triplicate; bars, SD. *P < 0.05, statistical significance compared withcontrols.
alleviating the symptoms of menopause. Indeed, oriental prescrip-tions that include deer antler are very common for women. Onthe other hand, it is generally believed that deer velvet shouldbe avoided by women with hormone-sensitive diseases such asendometriosis and breast cancer. Despite its common use and thesafety issue issues associated with deer antler, little is known aboutthe precise mechanism of this traditional medication in gynae-cological diseases, such as endometriosis. In the present study,we have investigated the effect of deer antler water extract onendometriosis using an in vitro endometriosis model. 11Z and 12Zcell lines used in this study were established from active endometri-otic lesions from women with endometriosis. These lines havebeen demonstrated to retain the phenotypic characteristics andseveral in vivo properties of the active phase of endometriosis(Zeitvogel et al., 2001; Banu et al., 2008). The typical characteris-tics of endometriotic cells are adhesion, migration, invasion, andproliferation (Banu et al., 2008). The results showed that DAEmarkedly decreased the adhesion and migration of endometrioticcells without any change in cell viability. Although some studieshave suggested that endometriotic cells are invasive in clinical fea-tures and in in vitro experiments, the present study showed that 11Zand 12Z cells were only slightly invasive, and no significant changeswere observed following DAE treatment (data not shown).
MMPs are a family of zinc-dependent endopeptidases that
can degrade the collagen components of extracellular matrix.They regulate migration, invasion, proliferation, and apoptosis invarious types of cells (Sternlicht and Werb, 2001). MMP-2 andMMP-9 possess the strongest degrading effect on collagen IV, the
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rincipal component of basement membranes. MMP-2 and MMP-, by degrading extracellular cellular matrix (ECM) and promotinghe release of key factors, play a critical role in the pathogenesisf endometriosis (Liotta and Stetler-Stevenson, 1991; Kato et al.,002; Gilabert-Estelles et al., 2007). In addition, MMP-2 and MMP-
are elevated in the urine of patients with endometriosis comparedo control (Becker et al., 2010). An immunohistochemical studyevealed MMP-9 expression is higher in endometriosis than prolif-rative endometrium (Shaco-Levy et al., 2008). In an in vivo study,nduction of endometriosis increased the levels of MMP-2 and
MP-9 in peritoneal cells and fluid in BALB/c mice (Chen et al.,010). In the present study, we have demonstrated that DAE sig-ificantly inhibits MMP-2 and MMP-9 mRNA and protein in both1Z and 12Z cells, suggesting that DAE regulates the expression ofMP-2 and MMP-9 at the transcriptional level. Because MMPs have
een implicated in the migration of endometriotic cells, down-egulation of MMP-2 and MMP-9 may mediate the inhibition ofigration by DAE in endometriotic cells.It has been suggested that the endometrium or the peritoneal
nvironment of women with endometriosis is abnormal and pro-otes the establishment and maintenance of disease (Seli and Arici,
003). Several studies have demonstrated an increase in a numberf inflammatory cytokines, including IL-6, IL-8, and MCP-1 in theeritoneal fluid of women with the disease compared to disease-ree women. The increased levels of cytokines may induce someathogenic conditions for endometriosis (Gazvani and Templeton,002). The pro-inflammatory cytokine TNF-� was also elevated notnly in the peritoneal fluid, but also in the serum of women withndometriosis (Wu and Ho, 2003; Agic et al., 2006). Indeed, theres a positive correlation between peritoneal levels of TNF-� and theize and number of active lesions (Harada et al., 2010). In additiono its pro-inflammatory functions, TNF-� has been shown to stim-late the expression of MMPs in endometrial tissue (Braundmeiernd Nowak, 2006). Elevated levels of TNF-� and IL-6 in women withndometriosis are widely known to be possible biomarkers in theiagnosis of endometriosis (Wu and Ho, 2003; Agic et al., 2006).
n the present study, we have demonstrated that DAE reduces thexpression of TNF-� and IL-6 in endometriotic cells. This resultmplies that DAE may mediate the anti-endometriotic effects byegulating the expression of the pro-inflammatory cytokines TNF-
and IL-6. However, further study is required to investigate thenderlying molecular mechanisms.
In summary, these results suggest that DAE inhibits endometri-tic cell migration and their ability to bind to mesothelial cells. Theechanism of DAE action is thought to be mediated by its ability
o regulate MMP-2 and MMP-9 expression. In addition, DAE alsoownregulates the levels of TNF-� and IL-6, regarded as biomarkersf endometriosis, as well as pro-inflammatory cytokines, suggest-ng that DAE is effective for treating endometriosis. Taken together,AE is a potential anti-adhesive and anti-metastatic agent, whoseroperties may expand future clinical research on endometriosis.
cknowledgements
This work was supported by the Korea Science and Engi-eering Foundation (KOSEF) grant (to JHC) funded by the Koreaovernment (MEST) (No. 2009-0068979). We sincerely thankr. Starzinski-Powitz (Johann-Wolfgang-Goethe-Universitaet,ermany) for providing endometriotic epithelial cells as generousifts.
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