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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2006-2486
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 8 3213-3218
Copyright © 2007 by The Endocrine Society

Metformin Suppresses Interleukin (IL)-1ß-Induced IL-8 Production, Aromatase Activation, and Proliferation of Endometriotic Stromal Cells

Yuri Takemura, Yutaka Osuga, Osamu Yoshino, Akiko Hasegawa, Tetsuya Hirata, Yasushi Hirota, Emi Nose, Chieko Morimoto, Miyuki Harada, Kaori Koga, Toshiki Tajima, Tetsu Yano and Yuji Taketani

Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan

Address all correspondence and requests for reprints to: Yutaka Osuga, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Context: Metformin, a widely used treatment for diabetes that improves insulin sensitivity, also has both antiinflammatory properties and a modulatory effect on ovarian steroid production, two actions that have been suggested to be efficacious in therapy for endometriosis.

Objective: To determine whether metformin may be effective for the treatment of endometriosis, we evaluated the effects of this agent on inflammatory response, estradiol production, and proliferation of endometriotic stromal cells (ESCs).

Design: ESCs derived from ovarian endometriomas were cultured with various concentrations of metformin.

Main Outcome Measures: IL-8 production, mRNA expression and aromatase activity, and 5-bromo-2'-deoxyuridine incorporation in ESCs were measured.

Results: Metformin dose-dependently suppressed IL-1ß-induced IL-8 production, cAMP-induced mRNA expression and aromatase activity, and 5-bromo-2'-deoxyuridine incorporation in ESCs.

Conclusion: These results suggest that further investigation into the unique therapeutic potential of metformin as an antiendometriotic drug is warranted.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
METFORMIN IS A widely used antidiabetic agent that improves insulin sensitivity (1). In reproductive medicine, the drug has been successfully used for the treatment of polycystic ovary syndrome, an etiology of which is suggested to be insulin resistance (2). Metformin may also reduce obesity-associated inflammatory status and other inflammatory responses (3, 4, 5), and has reduced serum C-reactive protein levels in women with polycystic ovary syndrome (6). In addition, it has direct effects on steroidogenesis in ovarian granulosa cells and thecal cells (7, 8).

Endometriosis is an estrogen-dependent enigmatic disease that deteriorates the health of women of reproductive age (9, 10). A large body of evidence suggests that the peritoneal inflammatory environment stimulates progress of the disease (10, 11, 12, 13, 14), and we and others have shown that antiinflammatory drugs have therapeutic potential for the disease (15, 16, 17, 18). Metformin, in addition to antiinflammatory properties, has a possible modulatory effect on local steroid production, suggesting it may be active against endometriosis.

Endometriosis is characterized by inflammation, estrogen dependency, and proliferation of endometriotic cells. Here, we have evaluated the effects of metformin on markers of these pathophysiological processes in endometriotic cells as a first step toward evaluating its therapeutic application in this condition.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Reagents and materials

Type I collagenase and antibiotics (penicillin, streptomycin, and amphotericin B) were purchased from Sigma-Aldrich (St. Louis, MO). DMEM/Ham’s F12 (F-12) medium, 0.25% trypsin-EDTA, and 0.4% trypan blue stain were from Life Technologies, Inc. (Grand Island, NY). 1, 1-Dimethylbiguanide hydrochloride (metformin), 8-bromo-cAMP, and 4-androstene-3, 17-dione (androstenedione) were obtained from Sigma-Aldrich. Recombinant IL-1ß was purchased from Genzyme/Techne (Minneapolis, MN). Charcoal-stripped fetal bovine serum (FBS) was from HyClone (Logan, UT). Deoxyribonuclease I was from Invitrogen (Carlsbad, CA).

Collection of samples

Endometriotic tissues were obtained from women undergoing laparoscopy or laparotomy for ovarian endometriomas. In total, 38 women aged 24–45 yr were recruited to the present study. All women had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. Symptoms of the women were pain (n = 26), infertility (n = 2), both pain and infertility (n = 6), and neither (n = 4). Menstrual phases at operation were proliferative in 18 patients and secretory in 20. Stages of endometriosis were III (n = 17) and IV (n = 21). The endometriotic tissue samples were collected from the cyst walls of ovarian endometriomas under sterile conditions for primary cell cultures.

The experimental procedures were approved by the institutional review board of the University of Tokyo, and signed informed consent for use of the sample was obtained from each woman.

Isolation and culture of human endometriotic stromal cells (ESCs)

Human ESCs were isolated and cultured as described previously (12, 19, 20). Fresh endometriotic specimens collected in sterile medium were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/F-12, containing 25 mg/ml type I collagenase and 15 U/ml deoxyribonuclease I, for 2–3 h at 37 C, and separated using serial filtration. Debris was removed with a 100-µm nylon cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ), and dispersed epithelial glands were eliminated by filtration through a 70-µm nylon cell strainer (Becton Dickinson and Co.). ESCs in the filtrate were collected by centrifugation and resuspended in phenol red-free DMEM/F-12 containing 10% charcoal-stripped FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B. The ESCs were seeded in a 100-mm culture plate and kept at 37 C in a humidified 5% CO2/95% air atmosphere. At the first passage, the cells were plated into 6-, 24-, 48-, or 96-well culture plates (Becton Dickinson and Co.) at a density of 2 x 105 cells/ml in medium supplemented with 10% FBS.

The purity of the stromal cell preparations was more than 95%, as judged by positive cellular staining for vimentin and negative cellular staining for cytokeratin, CD45, CD68, and von Willebrand factor.

Measurement of IL-8

When the ESCs were approaching confluence, media were removed and replaced with fresh media and antibiotics, and the cells were cultured in serum-free media for an additional 12 h. Subsequently, the cells were incubated with or without metformin (10, 100, and 1000 µM) in serum-free media for 24 h and then stimulated with 5 ng/ml IL-1ß in serum-free media for 24 h, according to our previous study (14, 20, 21). Metformin was dissolved in distilled water and then diluted 1:1000 in the medium. The doses of metformin used in the present study are similar to those used in other studies examining in vitro effects of metformin (4, 22). In addition, the peak plasma concentration of the drug at a standard dosage is approximately 10–20 µM (23, 24). Because local application of an antiendometriotic drug is desirable (25), we also tested metformin at relatively high concentrations. Concentrations of IL-8 in conditioned culture media were measured using a specific ELISA kit (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Data were standardized by total protein of cell lysates.

RNA extraction, reverse transcription, and real-time quantitative PCR of aromatase

When the ESCs were approaching confluence, media were replaced with fresh media and antibiotics, and the cells were cultured in serum-free media for an additional 12 h. Subsequently, the cells were incubated with or without metformin (10, 100, and 1000 µM) for 24 h, and then stimulated with 1 mM cAMP for 24 h. Total RNA was extracted from the ESCs, using an RNeasy minikit (QIAGEN, Hilden, Germany). The quality of the total RNA thus obtained was confirmed by determining appropriate sharp 28S and 18S rRNA bands by agarose gel electrophoresis. One microgram of total RNA was reverse transcribed in a 20-µl volume using ReverTra Ace -{alpha}- (TOYOBO Co., Ltd., Osaka, Japan).

Aromatase mRNA expression was assessed by real-time quantitative PCR using a LightCycler according to the manufacturer’s instructions (Roche Diagnostic GmbH, Mannheim, Germany). Aromatase primers (sense, 5'-CAGAGGCCAAGAGTTTGAGG-3'; antisense, 5'-ACACTAGCAGGTGGGTTTGG-3') were chosen to amplify a 243-bp fragment. Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (TOYOBO Co., Ltd.) were used to measure GAPDH mRNA levels so that expression of aromatase mRNA could be normalized to RNA loading for each sample. PCR conditions were as follows: for aromatase, 30 cycles at 95 C for 15 sec, 64 C for 8 sec, and 72 C for 10 sec; and for GAPDH, 25 cycles at 95 C for 15 sec, 64 C for 10 sec, and 72 C for 18 sec. All these PCR conditions were followed by melting curve analysis.

Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and their identities were confirmed using an ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).

Aromatase assay

The effect of metformin on aromatase activity was evaluated by measuring estrone levels in conditioned media of ESCs cultured with androstenedione. ESCs were seeded into 24-well plates at a density of 1 x 105 cells per well in 500 µl of the culture medium. After the ESCs were approaching confluence, the medium was replaced with fresh medium containing 2.5% FBS. The cells were incubated with or without metformin (10, 100, and 1000 µM) for 24 h and then stimulated with 1 mM cAMP for 24 h. After the treatments, 5 nM androstenedione was added to each well, and ESCs were incubated for another 24 h. The conditioned media were collected, centrifuged, and stored at –80 C for measurement of estrone levels.

Measurement of estrone

Concentrations of estrone in conditioned culture media were measured using a specific Estrone EIA kit (Yanaihara Institute Inc., Shizuoka, Japan) according to the manufacturer’s protocol (26). Data were standardized by total protein of cell lysates.

5-Bromo-2'-deoxyuridine (BrdU) proliferation assay

The effect of metformin on the proliferation of ESCs was examined by measuring incorporation of BrdU into DNA. The assay was performed using a Biotrak cell proliferation ELISA system (Amersham Biosciences, Little Chalfont, UK), as previously described (12, 19, 20). Briefly, ESCs were seeded into 96-well plates at a density of 5 x 103 cells per well in 100 µl culture medium. After 24 h, the medium was replaced with fresh medium containing 2.5% FBS. After 24 or 48 h of treatment with or without metformin (10, 100, and 1000 µM), 100-µl BrdU solutions were added and incubated at 37 C for an additional 2 h. The culture medium was then removed, and the cells were fixed, and the DNA was denatured by the addition of fixative at 200 µl/well. The peroxidase-labeled anti-BrdU bound to the BrdU incorporated in newly synthesized, cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant color was read at 450 nm in a DigiScan microplate reader (ASYS Hithec GmbH, Eugendorf, Austria).

Measurement of lactate dehydrogenase (LDH) release and trypan blue exclusion test

LDH release measurement and trypan blue exclusion test were conducted to examine the effect of metformin treatment on cell viability. After ESCs were treated with or without metformin (10, 100, and 1000 µM) for 24 h, conditioned media were collected, and the cells were dissociated with 0.25% trypsin-EDTA and collected by centrifugation at 200 g for 5 min. The release of LDH into conditioned culture media was measured using a specific Cytotoxicity Detection kit (Roche Diagnostic GmbH) according to the manufacturer’s protocol. Assay medium and 2% triton X-100 solution were used for low control and high control, respectively. The ESCs were resuspended in 0.2% trypan blue solution. The number of total or trypan blue stained ESCs was counted using a microscope. Trypan blue-stained ESCs were considered dead.

Statistical analysis

Data were evaluated using ANOVA with post hoc analysis for multiple comparisons. P values < 0.05 were accepted as significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects of metformin on IL-1ß-induced IL-8 production in ESCs

IL-8 is a proinflammatory cytokine that has been implicated in the pathogenesis of the disease (11, 12, 14, 19, 20, 21, 27). We performed dose-response experiments to determine the effect of metformin on the production of IL-8 in ESCs. The concentrations of IL-8 in all samples were above the lower limit of the assay. Preincubation with metformin significantly decreased IL-1ß-induced IL-8 production in ESCs in a dose-dependent manner compared with controls. The maximal effect was observed at 1000 µM, but significant decreases were seen at 10 µM (Fig. 1Go and Table 1Go).


Figure 1
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  		FIG. 1. Effects of metformin on IL-1ß-induced IL-8 production in ESCs. ESCs were incubated with or without metformin (10, 100, and 1000 µM) for 24 h, and then stimulated with (closed bars) or without (open bars) IL-1ß (5 ng/ml) for 24 h. At the end of the incubation period, conditioned media were collected and assayed for concentrations of IL-8 by ELISA. Values are the mean ± SEM of quadruplicate cultures. The results are representative of four separate experiments using samples from different women. *, P < 0.05 vs. IL-1ß without metformin.

 

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  		TABLE 1. Combined data of responses of ESCs obtained from different patients

 
Effects of metformin on cAMP-induced mRNA expression and activity of aromatase in ESCs

Because endometriosis is estrogen dependent, we evaluated the effect of metformin on the expression of aromatase, a critical enzyme for the local production of estrogens that drive the development of the disease (28). We conducted dose-response experiments to determine the effect of metformin on cAMP-induced aromatase mRNA expression in ESCs (Fig. 2AGo). Metformin decreased cAMP-induced aromatase mRNA levels in ESCs in a dose-dependent manner. The maximum decrease was observed at 1000 µM, but significant decreases were seen at 100 µM.


Figure 2
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  		FIG. 2. Effects of metformin on mRNA expression (A) and activity (B) of aromatase induced by cAMP in ESCs. ESCs were incubated with or without metformin (10, 100, and 1000 µM) for 24 h, and then stimulated with (closed bars) or without (open bars) cAMP (1 mM) for 24 h. A, At the end of the incubation period, the total RNA isolated from the ESCs was reverse transcribed and amplified by real-time PCR using primers for aromatase. The data were calculated by subtracting the signal threshold cycles of the internal standard (GAPDH) from aromatase. Values are the mean ± SEM of six independent experiments. *, P < 0.05 vs. cAMP without metformin. B, At the end of the incubation period, androstenedione was added, and cells were incubated for a further 24 h, and conditioned media were collected and assayed for aromatase activity by Estrone EIA kit. Values represent the mean ± SEM of quadruplicate cultures. The results are representative of four separate experiments using samples from different women. *, P < 0.03 vs. cAMP without metformin.

 
To measure aromatase activity, ESCs were preincubated with metformin for 24 h and then stimulated with cAMP for 24 h. After incubation with 5 nM androstenedione for another 24 h, concentrations of estrone were measured. The concentrations of estrone in all conditioned culture media were above the lower limits of the assay. As depicted in Fig. 2BGo and Table 1Go, metformin decreased cAMP-induced aromatase activity in ESCs in a dose-dependent manner, with the maximal effect being observed at 1000 µM, and significant decreases seen at 10 µM.

Effects of metformin on BrdU incorporation in ESCs

Dose-response experiments were conducted to determine the effect of metformin on DNA synthesis, as a marker of cell proliferation, in ESCs. Metformin at concentrations between 10 and 1000 µM dose dependently inhibited BrdU incorporation into DNA in ESCs at 24 and 48 h of the treatment, the maximal effect being observed at 1000 µM (Fig. 3Go and Table 1Go).


Figure 3
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  		FIG. 3. Effects of metformin on BrdU incorporation in ESCs. The effect of metformin on the proliferation of ESCs was examined by measuring BrdU into DNA by using a cell proliferation ELISA system. ESCs were treated with or without metformin (10, 100, and 1000 µM) for 24 (A) or 48 h (B). Values are the mean ± SEM of sextuplicate cultures. The results are representative of four separate experiments using samples from different women. *, P < 0.01 vs. control.

 
Effects of metformin on cell viability of ESCs

Metformin did not increase LDH release from ESCs, nor did it increase the number of trypan blue-stained ESCs (Table 2Go).


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  		TABLE 2. Effects of metformin on LDH release from ESCs and trypan blue staining of ESCs

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we have demonstrated that metformin suppressed the production of IL-1ß-induced IL-8, the activation of aromatase, and the proliferation of ESCs. These effects of metformin would all be expected to limit the development of endometriosis.

IL-1ß-induced secretion of IL-8 from endometriotic cells has been proposed to be a driver of endometriosis progression (29, 30). IL-8 levels are increased in the peritoneal fluid of women with endometriosis (31, 32). Interestingly, metformin has suppressed IL-8 release from human adipose tissue in vitro (33), and a recent report demonstrated that metformin inhibited IL-1ß-induced release of IL-6 and IL-8 in human vascular wall cells (4). Although we show here that metformin can inhibit IL-1ß-induced secretion of IL-8 from ESCs, at the same doses, metformin did not inhibit secretion of IL-8 from eutopic endometrial stromal cells (data not shown). Thus, metformin seems to exert its antiinflammatory role by reducing proinflammatory cytokine secretion in specific cell types.

Because endometriosis is an estrogen-dependent disease, local production of estrogen in endometriotic tissues is suggested to be important for the growth of the lesion. Numerous reports demonstrate abundant aromatase expression and elevated local estrogen production in endometriotic tissues (28), suggesting that aromatase is responsible for the local production of estrogen. Cases of endometriosis have also been successfully treated with aromatase inhibitors (34, 35, 36, 37, 38). A fascinating proposed mechanism is that increased prostaglandin estradiol stimulates aromatase activity via cAMP and increases estrogen production in endometriotic lesions (28). In the present study, cAMP-stimulated aromatase activity was suppressed with metformin in ESCs. Thus, metformin could be expected to suppress estrogen levels in endometriotic tissues. Interestingly, metformin has inhibited FSH and insulin-stimulated progesterone and estradiol production in granulosa cells (8). Together, metformin may inhibit endometriosis through suppression of both ovarian and local production of estrogens.

We also show that metformin inhibited BrdU uptake of ESCs. An antiproliferative effect of metformin has also been demonstrated in leptin-stimulated vascular smooth muscle cells (39). Combined with the antiinflammatory and antiestrogenic effect of metformin, the direct antiproliferative effect on ESCs supports its therapeutic potential for endometriosis. Consistent with this, one case report described regression of atypical endometrial hyperplasia that was resistant to progestin therapy after metformin treatment (40).

What is an intracellular mechanism that underlies these diverse effects of metformin in ESCs? AMP-activated protein kinase (AMPK) is a known target of metformin action in various cells (22, 41). It is increasingly being shown to have pleiotropic actions in the regulation of the endocrine system. However, to date, the role of AMPK in endometriosis is unknown. Adiponectin is also an activator of AMPK. We have recently shown that adiponectin stimulated AMPK and inhibited inflammatory cytokine production in endometrial cells (21). In addition, we reported that serum and peritoneal fluid adiponectin levels were decreased in women with endometriosis (42, 43). Together, these suggest that AMPK may be involved in the antiinflammatory effects of metformin demonstrated in ESCs in the present study.

Recently, peroxisome proliferator-activated receptor (PPAR)-{gamma} agonists, ciglitazone (44), and rosiglitazone (45) have regressed endometriotic lesion in a rat endometriosis model. Interestingly, like metformin, these PPAR-{gamma} agonists are also widely used antidiabetic drugs. Similar to metformin, PPAR-{gamma} agonists exert antiinflammatory (46) and antiproliferative effects (47), which are likely to mediate their antiendometriotic properties.

It has been argued that ovarian endometriosis is a different entity from peritoneal endometriosis (48), as exemplified by a recent study that showed different patterns of gene expression between ovarian and nonovarian endometriosis (49). Due to technical limitations, we only used endometriotic cells from ovarian endometrioma, and it remains possible that nonovarian endometriotic cells may show different responses to metformin from the present results.

In summary, we have shown that metformin can inhibit IL-1ß-induced IL-8 production, aromatase activation, and proliferation of ESCs. These findings suggest a unique therapeutic potential for metformin as an antiendometriotic drug.


   Footnotes
 
This work was partially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Welfare.

Disclosure Information: The authors have nothing to declare.

First Published Online May 15, 2007

Abbreviations: AMPK, AMP-activated protein kinase; BrdU, 5-bromo-2'-deoxyuridine; ESC, endometriotic stromal cell; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LDH, lactate dehydrogenase; PPAR, peroxisome proliferator-activated receptor.

Received November 13, 2006.

Accepted May 7, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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