Resveratrol Inhibits Development of Experimental Endometriosis In Vivo and Reduces Endometrial Stromal Cell Invasiveness In Vitro

Abstract

Endometriosis is a common gynecologic disorder characterized by ectopic attachment and growth of endometrial tissues. Resveratrol is a natural polyphenol with antiproliferative and anti-inflammatory properties. Our objective was to study the effects of resveratrol on human endometriotic implants in a nude mouse model and to examine its impact on human endometrial stromal (HES) cell invasiveness in vitro. Human endometrial tissues were obtained from healthy donors. Endometriosis was established in oophorectomized nude mice by intraperitoneal injection of endometrial tissues. Mice were treated with 17β-estradiol (8 mg, silastic capsule implants) alone (n = 16) or with resveratrol (6 mg/mouse; n = 20) for 10–12 and 18–20 days beginning 1 day after tissue injection. Mice were killed and endometrial implants were evaluated. A Matrigel invasion assay was used to examine the effects of resveratrol on HES cells. We assessed number and size of endometriotic implants in vivo and Matrigel invasion in vitro. Resveratrol decreased the number of endometrial implants per mouse by 60% (P < 0.001) and the total volume of lesions per mouse by 80% (P < 0.001). Resveratrol (10–30 μM) also induced a concentration-dependent reduction of invasiveness of HES by up to 78% (P < 0.0001). Resveratrol inhibits development of endometriosis in the nude mouse and reduces invasiveness of HES cells. These observations may aid in the development of novel treatments of endometriosis.

Resveratrol administration decreases development of endometriotic implants in a nude mouse endometriosis model as well as invasiveness of human endometrial stromal cells in culture.

INTRODUCTION

Endometriosis, defined as the presence of endometrial glands and stroma outside the uterus, is one of the most common benign disorders, affecting approximately 6%–10% of women of reproductive age [1]. Most frequent presentations of endometriosis involve pelvic pain and infertility. Although the etiology of this condition is still debated, the most widely accepted concepts invoke retrograde menstruation followed by ectopic attachment of endometrial tissues. Development of endometriotic lesions is thought to involve degradation of the extracellular matrix, invasion of the peritoneum, and subsequent growth of endometrial stroma and glands. This process is associated with increased oxidative stress and inflammation [2, 3]. Currently available therapies include surgical excision and various medical treatments such as the use of gonadotropin-releasing hormone analogs, aromatase inhibitors, and progestins. Unfortunately, these approaches often have only modest success and are associated with significant risk of complications and side effects; consequently, the search continues for new, safe, and effective long-term treatments.

One of the potential novel candidates for treatment of endometriosis is resveratrol (trans-3,5,4′-trihydoxystilbene). Resveratrol is a natural phytoalexin: a polyphenol synthesized by plants following ultraviolet radiation and fungal infections. Significant concentrations of resveratrol have been detected in grapes, berries, nuts, and red wine. Growing evidence indicates that this compound possesses antineoplastic, anti-inflammatory, and antioxidant properties [4]. Resveratrol has also been shown to exhibit profound in vitro and in vivo growth-inhibiting and apoptosis-inducing activities in several biological systems, including cancer cell lines and animal models of carcinogenesis [4–10]. These properties have been linked to inhibition of proliferation in association with cell cycle arrest and, in some systems, with increased apoptotic cell death [6–10]. Mechanisms of action of resveratrol further include multiple cellular targets affecting various signal transduction pathways, including AKT, RPS6KB2 (p70S6K), mitrogen-activated protein kinase 1/3 (MAPK1/3; ERK1/2), STAT3, MAPK14 (p38), protein kinase C, and peroxisome proliferator-activated receptors (PPAR) gamma [11–13]. Importantly, several of these pathways are relevant to the pathophysiology of endometriosis, especially in relation to their impact on inflammatory processes [14–19].

Further supporting a concept that resveratrol may be a therapeutic agent for treatment of endometriosis, it has been demonstrated that administration of this compound in vivo leads to reduction of mRNA expression of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A (HMG_CoA) reductase (HMGCR) [20]. Hence, resveratrol may share with statins the ability to inhibit the mevalonate pathway. This observation is relevant to the present study, because we and other investigators have reported that statins (competitive inhibitors of HMG-CoA reductase activity) protect against the development of experimental endometriosis and inhibit proliferation of human endometrial stromal (HES) cells [21–24].

In view of the above considerations, we proposed a hypothesis that resveratrol may have an inhibitory effect on the development of endometriosis. This study was designed to evaluate the effects of resveratrol on the development of human endometriotic implants in a nude mouse model and to test whether it affects invasiveness of HES cells in vitro.

MATERIALS AND METHODS

Human Endometrial Tissues

Human endometrial tissues (n = 9) were obtained from biopsies using a Pipelle suction curette (Unimar, Inc.); all biopsies were collected during the proliferative phase (Days 9–12) of the menstrual cycle from women age 18–45 exhibiting normal menstrual cycles and no history of endometriosis. Individuals using hormone therapy such as oral contraceptive pills or progestins for the preceding 3 mo were excluded. Biopsies were washed in prewarmed, phenol red-free Dulbecco modified Eagle medium/Ham F-12 medium (DME/F-12; Sigma) to remove residual blood and mucus prior to culturing. Informed consent was obtained prior to biopsy. This study was approved by the Institutional Review Board and Committee for the Protection of Human Subjects from Vanderbilt University, Yale University, and the University of California-Davis.

Human Tissue Preparation for Endometriosis Model

Tissues from endometrial biopsies were dissected into small cubes (∼1 mm³) and 8–10 pieces of tissue per treatment group were suspended in tissue culture inserts (Millipore). Tissue fragments were maintained under serum-free conditions in DME/F-12 supplemented with 1% insulin-transferrin-selenium (ITS+; Collaborative Biomedical), 0.1% Excyte (Miles Scientific), and 1× antibiotic/antimycotic solution and incubated at 37°C in a humidified chamber with 5% CO₂. All tissues were incubated in the presence of 1 nM 17β-estradiol (Sigma) and maintained in culture for 24 h prior to injection into mice. All mouse experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee in accordance with the Animal Welfare Act.

Establishment of Experimental Endometriosis

Five-week-old female nude mice (NCr strain) were purchased following oophorectomy from Harlan Sprague Dawley. Upon completion of a 1-wk acclimation period, mice were implanted with a slow-release silastic capsule containing 8 mg 17β-estradiol (in cholesterol) 24 h prior to injection of human tissues. Endometrial tissues, obtained and cultured as described above, were washed in PBS and injected into mice intraperitoneally along the ventral midline just below the umbilicus. Each experiment was conducted using samples from separate donors. At the end of each experiment, mice were killed by cervical dislocation under anesthesia.

Preparation of Estradiol Pellets for Animal Treatment

Estradiol-filled silastic capsules were made using estradiol 17-β (Sigma) and silastic tubing (Dow Corning Silastic Laboratory Tubing; 1.6 mm i.d.; 0.8 mm o.d.; wall thickness 3.2 mm); both ends were sealed with Type A medical silicone (Factor II, Inc.). Prior to use in the experimental animal model, steroid-filled capsules were washed in sterile PBS containing 1% antibiotic/antimycotic solution followed by three washes in sterile PBS without additives.

Evaluation of the Effect of Resveratrol on Development of Experimental Endometriosis

In six experiments mice received 17β-estradiol 24 h prior to injection of human endometrial tissues as described above. Twenty-four hours after tissue injection, mice were randomly assigned to one of the following treatment regimens: vehicle only or resveratrol (6 mg/mouse; Sigma). Resveratrol was dissolved in 100% ethanol and then diluted with sterile water to achieve the correct dose in 200 μl of 25% ethanol (i.e., 131 mM resveratrol). Resveratrol or 25% ethanol vehicle was provided to mice by gavage daily for 10–12 days or for 18–20 days.

Twenty-four hours after the last treatment, the mice were killed by cervical dislocation under isoflurane anesthesia and examined for the presence, number, and size of lesions. Lesions were measured in two dimensions, the larger denoted a and the smaller denoted b, and total volume, calculated by standard methodology using the formula V = a × b² × 0.5. Lesions were photographed, then removed and formalin fixed for standard microscopy.

Immunohistochemical Evaluations of Endometriotic Lesions

Excised lesions were immediately placed in 10% buffered formalin overnight and subsequently embedded in paraffin. Five-micron sections were deparaffinized and immunohistochemical staining was performed.

To evaluate proliferative activity and DNA repair in endometriotic lesions, staining for MKI67 (Ki-67) and proliferating cell nuclear antigen (PCNA) was performed. MKI67 protein is considered to be a specific marker of proliferation and is present during all active phases of the cell cycle [25]. PCNA is present throughout the G1, S, and G2 phases of the cell cycle and is considered a sensitive index of proliferation as well as DNA repair [26–28]. Detection of MKI67 and PCNA was carried out using, respectively, MKI67 H-300 and PCNA sc-56 primary antibodies from Santa Cruz Biotechnology.

Apoptosis was evaluated by detection of DNA fragmentation using TUNEL assay [29]. Briefly, a commercially available kit (DeadEnd Colorimetric TUNEL System; Promega) was used according to manufacturer's instructions. The assay involves identification of nicks in the DNA, which are identified by terminal deoxynucleotidyl transferase. One hundred cells in 10 nonadjacent high-power fields were evaluated for each of four specimens per group.

Analysis of immunohistochemical staining was performed by the HSCORE, a semiquantitative method allowing the incorporation of both the number of stained cells and the intracellular staining intensity [30, 31]. To determine the intensity of the stain, the following scale was used: no stain = 0, weak staining = 1, moderate staining = 2, and intense staining = 3. The HSCORE was calculated by H = Σ Pi(I + 1), where Pi is the percentage of stained cells in each intensity category and I the intensity of staining (0, 1, 2, or 3). The endometrial glandular epithelium and endometrial stroma were individually analyzed for the percentage of cells in each staining category. Statistical analyses were performed using the Mann-Whitney test.

Isolation of Human Endometrial Stromal Cells

Human endometrial stromal (HES) cells used for invasion assay were isolated following enzymatic digestion of endometrial fragments and subsequently passing of the cells through a 70-μm sieve (BD Falcon) [32]. Cells were then cultured at 37°C in humidified air and 5% carbon dioxide in Dulbecco modified Eagle medium (DMEM) supplemented with 1% antibiotic and charcoal/dextran-treated fetal bovine serum (FBS) and 1 nM estradiol.

Effect of Resveratrol on Invasiveness of Human Endometrial Stroma

Twenty-four-well plates with transwell inserts (6.5-mm diameter) with 8.0-μm pore size polycarbonate membrane (Transwell Permeable Supports) were used for the assay. The membranes of precooled inserts were coated using 40 μl of Matrigel (ECM gel, growth factor reduced, without phenol red, from Engelberth-Holm-Swarm mouse sarcoma; Sigma-Aldrich) diluted to a final protein concentration of 1.2 mg/ml with cold phenol red-free culture media without FBS [33]. The Matrigel layers were dried for 2 h and then rehydrated by adding warm phenol red-free, serum-free DMEM and then incubated at 37°C for 30 min [34, 35].

The cells were trypsinized, washed, and suspended in phenol red-free, serum-free DMEM with 1 nM 17β-estradiol and transferred to the transwell inserts (50 000 cells/transwell insert). The lower chambers of the wells were filled with 600 μl of phenol red-free DMEM with 1 nM estradiol and 10% charcoal/dextran-treated FBS used as a source of chemoattractants [33–35]. Subsequently, the cells were cultured for 24 h without (control) or with resveratrol (10–30 μM) added to the transwell insert.

After 24 h, noninvading cells were removed from the top of the transwell inserts by scraping, and the cells invading the Matrigel were fixed in 4% paraformaldehyde for 30 min, washed in PBS, stained in crystal violet for 60 min, and washed several times in PBS. The number of invading cells was assessed under a light microscope (magnification 10×). The mean number of invading cells was calculated from four replicates and analyzed as a percentage of control. The experiment was repeated three times using cells from different donors.

To exclude the possibility of toxicity of resveratrol on the endometrial cells, the effect of resveratrol on the number of viable cells was evaluated as follows: cells were seeded at a density of 15 000 cells/well in 96-well plates and cultured in phenol red-free, serum-free DMEM with 1 nM estradiol without additives (control) or with resveratrol (10–30 μM). Determination of the number of viable cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was performed after 24 h. Two hours before the termination of cultures, CellTiter 96 AQueous One Solution reagent (Promega), which consists of a tetrazolium compound (MTS) and an electron coupling reagent (phenazine ethosulfate) was added to the endometrial stromal cells, and then absorbance was measured using a microplate reader (Fluostar Omega; BMG). MTS was bioreduced to formazan, presumably by NADPH or NADH produced by dehydrogenase enzymes present in metabolically active cells. The quantity of formazan product as measured by absorbance at 490 nm is proportional to the number of living cells [36].

Statistical Analysis

Comparisons between groups were performed using the Student t-test. Normality of the distribution was assessed by Shapiro-Wilk W test. In the absence of normal distribution, values were transformed logarithmically and subsequently the Student t-test was performed. If logarithmic transformation did not normalize the distribution, comparisons were carried out by the Mann-Whitney U-test. When performing multiple comparisons, Bonferroni correction was used.

Comparisons of categorical variables (i.e., presence or absence of endometriotic implants) were performed using the Fisher exact test. Statistical analysis was performed using the JMP program, version 8 (SAS Institute). Results are presented as means ± SEM.

RESULTS

Effects of Resveratrol on Experimental Endometriosis

Resveratrol exerted a significant inhibitory impact on development of endometrial implants in this nude mouse model of endometriosis, reducing the proportion of animals developing endometriosis as well as decreasing the number of lesions per mouse and the total volume of lesions per mouse. Overall, in six separate experiments 100% (16/16) of control mice developed endometriotic lesions, a significantly greater proportion than the 65% (13/20) of mice receiving resveratrol (Fisher exact test: P = 0.01).

When pooling results from all experiments, resveratrol treatment reduced the average number of endometriotic lesions per mouse by 60% (P < 0.0001) and the total volume of lesions per mouse by 80% (P < 0.0001). As presented in Figure 1A, a significant reduction of the number of lesions per mouse was observed following 10–12 days of treatment (decrease by 67% in four separate experiments) and a comparable trend was observed following 18–20 days of treatment (decrease by 45%, two separate experiments). In a similar fashion, resveratrol significantly reduced the total volume of lesions per mouse by 75% after 10–12 days and by 88% after 18–20 days (Fig. 1B).

FIG. 1.

Effect of resveratrol on the number (A) and volume (B) of lesions per mouse; each bar represents mean ± SEM. The figure summarizes results of four experiments carried out for 10–12 days and two experiments carried out for 18–20 days.

Figure 2 presents representative gross appearance of endometriotic lesions in control mice in comparison to animals receiving resveratrol for 10 and 20 days. On gross examination, lesions obtained from control mice were larger and typically highly vascularized whereas lesions from resveratrol-treated mice exhibited a decrease in size and were typically poorly vascularized.

FIG. 2.

Gross morphology of experimental endometriotic lesions established by proliferative phase human endometrium in nude mice. Treatment was initiated 24 h following human tissue injection, and mice were killed after 10 days of treatment with vehicle (A), after 10 days of resveratrol treatment (B), and after 20 days of resveratrol treatment (C). Original magnification ×15.

Immunohistochemical Evaluation of Endometriotic Lesions: Markers of Proliferation and Apoptosis

To evaluate the proliferative activity of endometriotic lesions, a specific marker of proliferation, MKI67, was evaluated (Fig. 3). Lesions exposed to resveratrol, in comparison to control specimens, had significantly lower HSCOREs in stroma (0.56 ± 0.05 vs. 1.67 ± 0.07; P < 0.01) and in glands (1.48 ± 0.05 vs. 1.98 ± 0.12; P < 0.01). In addition, lesions were evaluated for PCNA staining. PCNA is present in dividing cells as well as in cells undergoing DNA repair [26–28]. The average HSCORE of the endometrial stroma was 3.8-fold greater in resveratrol vs. control specimens (1.32 ± 0.05 vs. 0.35 ± 0.09; P < 0.01). A trend towards a greater HSCORE was also noted in the endometrial glands of lesions from resveratrol-treated animals in comparison to controls (1.39 ± 0.20 vs. 0.86 ± 0.25; P = 0.15).

FIG. 3.

Immunohistochemical evaluation of endometriotic implants from control (CTL, panels on the left) and resveratrol-treated animals (RES, panels on the right). Staining was performed for MKI67, PCNA, and TUNEL. Original magnification ×200.

Immunohistochemical evaluation for apoptosis by TUNEL staining revealed that lesions from mice exposed to resveratrol had a significantly greater HSCORE than control mice. In endometrial stroma, the average HSCORE from the resveratrol-treated group was 2-fold greater than that from the control group: 0.88 ± 0.04 vs. 0.43 ± 0.05 (P < 0.05). In endometrial glands, HSCORE for TUNEL staining was also significantly greater in the resveratrol-treated group than in the control group: 1.67 ± 0.12 vs. 0.40 ± 0.15 (P = 0.01). Figure 3 illustrates typical TUNEL staining in endometriotic implants from resveratrol-treated and control animals.

Effect of Resveratrol on Invasiveness of Human Endometrial Stroma

In three separate experiments using tissues from three different subjects, 24-h exposure to resveratrol induced a concentration-dependent decline in the invasiveness of HES cells. As illustrated in Figure 4, resveratrol at doses of 10 and 30 μM induced 40% and 78% decreases of invasiveness, respectively (both at P < 0.001). The effect of resveratrol at 30 μM was significantly greater than the effect of resveratrol at 10 μM (P < 0.01).

FIG. 4.

Effects of resveratrol on invasiveness of HES cells cultured for 24 h in the absence (C, control) or in the presence of resveratrol (10 and 30 μM). Each bar represents mean ± SEM. Means with no superscripts in common are significantly different (P < 0.05).

Because the invasiveness assay relies on counting cells invading Matrigel, reduced numbers of cells seen following resveratrol treatment may have been due, at least in part, not to reduced invasiveness but to resveratrol-induced reduction of the number of cells (i.e., reduced proliferation and/or increased apoptosis). To verify that the observed effects were indeed related to reduced invasiveness, in parallel experiments, cells were cultured in the absence (control) or in the presence of identical concentrations of resveratrol and the cell number was evaluated by MTS assay. In all experiments, 24-h exposure to resveratrol had no significant effect on the number of viable cells.

DISCUSSION

The present study has shown that: 1) administration of resveratrol in vivo results in a significant decrease in the number and size of endometriotic lesions, 2) lesions from animals exposed to resveratrol exhibit evidence of increased apoptotic activity, and 3) resveratrol in cultures of HES induces a concentration-dependent reduction of invasiveness.

To our knowledge, this is the first report evaluating effects of resveratrol on an animal model of endometriosis and on endometrial tissues. Resveratrol reduced development of experimental endometriosis, as evidenced by a decreased proportion of animals with endometriotic lesions, a lower number of lesions, and a smaller volume of lesions. These protective effects of resveratrol may be related to various mechanisms such as reduction of proliferation of endometrial cells, increased cell death (e.g., apoptosis), and/or reduced ability to attach and to implant. Indeed, in various other biological systems resveratrol has been shown to inhibit cell proliferation, especially in neoplastic cell cultures [6–10]. Resveratrol also induced apoptosis in several cell types and cancer cell lines, including, among others, 3T3-L1 adipocytes, epidermal cells, neuroblastoma, prostate cancer, colon cancer, leukemia, and medulloblastoma [6, 11, 37]. The concept that resveratrol may prevent the development of endometriosis by induction of apoptosis is supported by our histochemical studies demonstrating an increase in TUNEL staining of endometrial implants. Effects of resveratrol on proliferation of endometriotic tissues are less clear. Immunohistochemical evaluation of implants from resveratrol-treated animals revealed a decrease of MKI67 staining but an increase in PCNA staining. Notably, MKI67 is a more specific marker of proliferation than PCNA; furthermore, PCNA may increase not only in proliferating tissues but also in tissues attempting to repair DNA [28]. Interestingly, resveratrol, along with other hydroxystilbenes, has been shown to induce DNA nicking [38].

We observed comparable effects of resveratrol on the number and volume of lesions (Fig. 1) following either shorter (10–12 days) or longer (18–20 days) duration of treatment; hence, it is likely that the primary mode of action of resveratrol may be related to prevention from, or at least reduction of, implantation of endometrial tissues. Another argument in favor of a role of resveratrol as an agent protecting from development of endometriosis at the level of attachment and implantation of tissues stems from in vitro studies on HES cells. As presented in Figure 4, resveratrol induced a concentration-dependent reduction of invasiveness into the Matrigel; hence, it is tempting to speculate that comparable actions of resveratrol may have been involved in the reduction of peritoneal attachment of endometriotic implants in vivo. However, invasiveness and peritoneal attachment should not be equated, and these processes may be mediated by separate mechanisms. It is also possible that resveratrol may induce regression of established lesions; this concept is supported by the observation that endometriotic lesions appeared smaller in mice receiving resveratrol for 18–20 days in comparison to 10–12 days.

At present, mechanisms of resveratrol action on endometrial cells remain unknown. Resveratrol is involved in regulation of a broad range of intracellular targets, including activation of sirtuin 1 (SIRT1) [39]. SIRT1 is a NAD+-dependent class II protein deacetylase regulating lipid metabolism by deacetylation of modified lysine residues on histones and various transcriptional regulators [40]. Downstream targets of SIRT1 may include, among others, AKT, TRP53, MAPK1/3, and PPAR gamma [11–13]. In addition, resveratrol may also act independently of SIRT1, for example by inhibition of proliferation of some cell types via mechanisms involving inhibition of AKT and MAPK1/3 [41]. These signal transduction pathways have been shown to play a role in the pathophysiology of endometriosis [14, 42]. Importantly, resveratrol significantly affects these targets at micromolar concentrations, which are potentially attainable pharmacologically [43]. In a recent phase 1 study of pharmacokinetics in healthy volunteers, the levels of resveratrol exceeded 2 μM and the levels of conjugated metabolites of resveratrol were up to 8-fold higher [44].

It should be noted that the present study was carried out using endometrial tissues from women without endometriosis. Because endometrium from women with endometriosis and endometriotic implants differ from normal endometrium, further studies should also evaluate effects of resveratrol on eutopic and ectopic endometrial tissues from women with endometriosis.

A clinically important issue is whether the dose of resveratrol used in this study corresponds to a reasonable dose for human use. At present we can offer only a speculative answer. A recent study suggested that dose translation from animal to human studies should use body surface area as a factor [45]. Using this approach, a dose of 6 mg per mouse translates to approximately an equivalent of 1000 mg for a 60-kg human. To date there are few published studies on the use of resveratrol in clinical trials, but ongoing trials registered with ClinicalTrials.gov indicate that typical doses of resveratrol range from 75 to 5000 mg per day [46, 47]. With regard to the concentrations of resveratrol used in the invasiveness assay, they correspond to the concentrations used in other in vitro studies [6, 7, 9, 10, 48]. The bioavailability of resveratrol in human and in rodent models is in the micromolar range and the concentrations of metabolites of resveratrol are nearly an order of magnitude greater [6, 44, 49].

In summary, our study demonstrates that resveratrol holds promise as a potential novel treatment of endometriosis. Notably, the present findings represent only a first step towards potential use of resveratrol in the treatment of endometriosis in patients. To date, although resveratrol is widely used as a nutritional supplement, it has not been approved by the FDA for any clinical application and its long-term safety has yet to be proven.