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Research Article |
Department of OB/GYN,3 Yale University School of Medicine, New Haven, Connecticut 06520
Polish Academy of Sciences Medical Research Center,4 Warsaw 02-106, Poland
Department of GYN/OB,5 Poznan University of Medical Sciences, Poznan 61-701, Poland
ABSTRACT
Endometriosis is characterized by ectopic growth of endometrial tissues. Statins, inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR), have been shown to decrease proliferation of several mesenchymal tissues. Actions of statins may be related to decreased availability of cholesterol as well as intermediate metabolites of the mevalonate pathway downstream of HMGCR. This study was designed to evaluate effects of statins on growth of endometrial stromal cells and to investigate mechanisms of these effects. Human endometrial stromal cells were cultured in the absence and in the presence of serum and with or without mevastatin and simvastatin. DNA synthesis and viable cell numbers were determined. Effects of statins were also evaluated in the presence of mevalonate and squalene. Furthermore, effects on phosphorylation of mitogen-activated protein kinase 3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase [ERK1/2]) were determined. Mevastatin and simvastatin induced a concentration-dependent inhibition of DNA synthesis and viable cell count in chemically defined media and in the presence of serum. Mevalonate, but not squalene, abrogated inhibitory effects of statins on cell proliferation. Statins inhibited MAPK3/1 phosphorylation. This is the first study demonstrating that statins inhibit growth of endometrial stromal cells. This effect is also demonstrable in the presence of a supply of cholesterol and may be related to decreased activation of MAPK3/1. The present observations may be relevant to potential therapeutic use of statins in conditions such as endometriosis.
endometrial stroma, female reproductive tract, kinases, proliferation, signal transduction, statins, uterus
INTRODUCTION
Endometriosis is one of the most common benign gynecologic conditions affecting approximately 6%–10% of women of reproductive age [1–3]. Formation of endometriotic implants requires ectopic attachment and proliferation of endometrial stroma and glands. Prominent features of endometriosis include inflammatory reaction and increased oxidative stress [4, 5]. Women with endometriosis have increased lipid peroxidation and elevated autoantibodies to markers of oxidative stress [6, 7]. Furthermore, it appears that endometriosis is associated with depletion of antioxidant capacity; intraperitoneal levels of vitamin E are decreased, likely because of consumption by oxidation reactions [8]. These observations are also in accord with findings of elevated levels of several enzymes involved in generation and metabolism of reactive oxygen species in endometrial tissues and endometrial implants of women with endometriosis [9–12]. We have recently demonstrated that proliferation of endometrial stroma is stimulated by moderate oxidative stress but inhibited by antioxidants such as vitamin E succinate and ebselen [13].
In view of these considerations and observations, we proposed to study effects of statins on endometrial stroma. Statins are inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase (HMGCR) with intrinsic antioxidant activity [14]. In several biological systems, such as vascular smooth muscle and mesangial cells, statins have been shown to inhibit cell proliferation [15, 16]. Inhibitory actions of statins on proliferation may be due to several mechanisms. First, statin-induced inhibition of HMGCR depletes a broad range of downstream products of the mevalonate pathway, including isoprenyls: geranyl-geranyl pyrophosphate and farnesyl pyrophosphate [16, 17]. Decreased availability of these substances leads to decreased isoprenylation and thus lower activity of small GTPases such as RAS and RHO, resulting in attenuated signaling within important growth-regulating signal transduction pathways [18]. In particular, RAS is an essential component of the mitogen-activated protein kinase (MAPK) system involving mitogen-activated protein kinase 3/1 (MAPK3/1) (also known as extracellular signal-regulated kinase 1/2 [ERK1/2]) whereby activation of MAPK3/1 requires isoprenylated RAS. Statins may also decrease activity of another small GTPase, RAC, which is essential for generation of reactive oxygen species (ROS) by NADPH oxidase [19]. In addition, statins possess direct antioxidant activity [14]. Thus, actions of statins may involve inhibition of growth-promoting signal transduction pathways as well as antioxidant activity, which may also affect growth.
In this report we present new findings demonstrating that statins decrease MAPK3/1 phosphorylation and inhibit growth of endometrial stroma.
MATERIALS AND METHODS
Endometrial samples were obtained from 11 subjects during the follicular phase of the menstrual cycle (age 37.1 ± 1.3 [mean ± SEM]). The study was approved by the Institutional Review Board of Yale University School of Medicine. Stromal cells were purified by enzymatic digestion and subsequently passing them through a sieve [20]. Cells were cultured in F12: DMEM with 1% antibiotic and 10% fetal bovine serum (FBS) and incubated at 37°C with humidified air and 5% CO2 until 70%–80% confluent. Subsequently, the cells were transferred to 96-well plates with serum-free and phenol-free medium at a cell density of 50 000 cells/well. The cells were incubated with mevastatin, simvastatin, mevalonate, and/or squalene (all from Sigma Chemical Co., St. Louis, MO) for 48 h. Proliferation was assessed by determination of DNA synthesis using thymidine incorporation assay [21]. Briefly, [3H] thymidine (1 µCi/well; Amersham Pharmacia Biotech, Arlington Heights, IL) was added to the cells during the last 24 h of culture, the cells were harvested using a multiwell cell harvester (PHD Harvester, Model 290; Cambridge Technology, Inc., Watertown, MA), and radioactivity was determined in a liquid scintillation counter, SL 4000 (Intertechnique, Fairfield, NJ). Each treatment was carried out in at least eight replicates.
Total viable cell number was estimated using a 4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium (MTS) assay (CellTiter 96 AQeuous One, Promega, Madison, WI). This assay involves conversion of MTS to colored formazan by mitochondrial dehydrogenase within living cells; the maximal absorbance of the formazan released to the culture medium is 490 nm [22].
The MAPK3/1 kits ERK1/2 TOTAL and pTpY 185/187 were purchased from Biosource International (Camarillo, CA). Proteins for ELISA tests were obtained as follows. After incubations with treatments, culture dishes with attached cells were transferred to an ice bath and washed twice with ice-cold PBS, then lysis buffer was added (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM PMSF, protease inhibitor cocktail). Lysed cells were transferred to Eppendorf tubes and incubated on ice for 30 min with vigorous vortexing at 5-min intervals. Lysates were centrifuged for 20 min at 20 000 x g. Protein concentration was determined by using standard Bradford colorimetric assay. Supernatants were collected and stored at –80°C or directly subjected to ELISA. ELISA tests were performed according to the manufacturer's instructions. Briefly, protein lysates were added to 96-well plates precoated with capture antibody and incubated for 2 h at room temperature. Subsequently, detecting antibody was added, and incubation continued for 1 h. Next, secondary antibody conjugated with horseradish peroxidase was added, and the mixture was incubated for an additional 30 min. The wells were rinsed four times using washing buffer. The reaction was visualized using stabilized chromogen, which was added for 30 min. Finally, a stop solution was added, and absorbance was read at 405 nm in a spectrophotometer (Biorad model 680).
Statistical Analysis
Baseline data are presented as mean (±SEM). When required, data were logarithmically transformed. Statistical analysis was performed using analysis of variance followed by post hoc pairwise comparisons with the Bonferroni correction.
RESULTS
Dose-Dependent Effects of Statins on DNA Synthesis and Cell Number
Figure 1A presents effects of mevastatin and simvastatin on proliferation, as determined by DNA synthesis assay using radiolabeled thymidine incorporation. Both statins induced a concentration-dependent inhibition of DNA synthesis; mevastatin and simvastatin decreased thymidine incorporation by 53%–83% and 31%–89%, respectively, below control (both significant at up to P < 0.001). Comparable and consistent effects were observed in repeat experiments using separate preparations of endometrial cells; interexperimental coefficients of variation of observed effects were in the range of 6%–20%.
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Since statins inhibit synthesis of cholesterol, which is essential for structural integrity of cells, the observed decline of DNA synthesis may be due to cholesterol depletion. To test this possibility, we also evaluated effects of statins on DNA synthesis in the presence of 5% serum as a rich source of cholesterol (Fig. 1B). In the presence of serum, effects of statins were less pronounced but still highly significant: mevastatin decreased DNA synthesis by up to 65% and simvastatin by up to 49% (both effects significant at up to P < 0.001).
In order to determine the effect of statins on cell number, we have also performed the MTS assay evaluating the number of viable cells. Figure 2A presents effects of statins on estimated cell number in the absence of serum; mevastatin and simvastatin decreased cell number by up to 13% and 44%, respectively. The effect of each statin at the highest concentrations (30 µM) was significant at P < 0.001. Similar observations were made in cultures supplemented with 5% serum, whereby mevastatin decreased cell number by up to 28% and simvastatin by up to 50%; at the highest concentration of statins, effects were significant at P < 0.001.
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Effects of Mevalonate and Squalene
In order to determine whether inhibition of proliferation by statins is mediated by depletion of isoprenoid intermediates, endometrial stromal cells were cultured in the presence of statins and/or mevalonate, an immediate downstream product of HMGCR. As presented in Figure 3A, mevalonate alone increased DNA synthesis by 41%. Mevalonate also reversed the inhibitory effect of mevastatin and partly reversed the inhibitory effect of simvastatin.
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Different results were found in experiments where the cells were cultured in the presence of squalene, an intermediate further downstream in the pathway of cholesterol synthesis. As shown in Figure 3B, squalene alone had no significant effect on DNA synthesis. Squalene also had no effect on mevastatin- and simvastatin-induced inhibition of thymidine incorporation.
Effects on MAPK3/1
One of the possible mechanisms of action of statins on cell proliferation may involve modulation of isoprenylation of small GTPases leading to reduction of activity of signal transduction pathways such as the mitogen-activated-protein-kinase pathway. To test the effects of statins on MAPK, we studied activation of MAPK3/1, as determined by a phosphorylation assay. Both mevastatin and simvastatin significantly inhibited MAPK3/1 phosphorylation by 75% and 86%, respectively (P < 0.005; Fig. 4). Statins had no significant effect on total MAPK3/1. In mevastatin- and simvastatin-treated specimens, total MAPK3/1 was, respectively, at 105 ± 6% and 113 ± 5% of the control value (mean ± SEM).
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DISCUSSION
Regulation of cell proliferation is of critical importance to the function of endometrial tissues under physiological and pathological conditions. Development of new strategies aimed at limiting endometrial growth is particularly relevant to conditions associated with endometrial proliferation, such as endometriosis, adenomyosis, and endometrial hyperplasia.
To our knowledge, this is the first study evaluating the effects of statins on endometrial cells. We have shown that in cultures of human endometrial stroma, 1) statins induce a concentration-dependent inhibition of cell growth, 2) these effects are also demonstrable in the presence of an ample supply of exogenous cholesterol, 3) mevalonate but not squalene abrogates effects of statins, and 4) statins decrease activation of MAPK3/1.
Inhibitory effects of statins on growth were tested by determination of DNA synthesis using a radiolabeled thymidine incorporation assay and by quantification of the number of viable cells using an MTT assay. Both methods provided comparable and complementary but not identical results. The effects on viable cell count were less pronounced than the effects on the rate of DNA synthesis; this is most likely due to the fact that the cell count represents a cumulative and net effect of ongoing proliferation as well as cell death.
Since statins inhibit the cholesterol synthesis (or mevalonate) pathway at the level of HMGCR (Fig. 5), effects of statins may be due not only to depletion of cholesterol but also to decreased levels of intermediates such as isoprenyls, which are essential for posttranslational modification of proteins [16, 17, 23].
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To evaluate the role of cholesterol supply, we tested the effects of mevastatin and simvastatin in the absence and in the presence of serum; under both conditions, statins induced a concentration-dependent decrease of DNA synthesis and cell count. Since the exogenous supply of cholesterol provided by serum was not sufficient to abrogate the effects of statins, it is likely that the actions of statins are not explainable solely by depletion of cholesterol supply. Indeed, this notion is further supported by our observations that supplementation of cultures with squalene did not reverse the effects of statins.
In contrast, supplementation with mevalonate at least partly restored cell proliferation in the presence of statins. Since mevalonate is downstream of HMGCR, it is likely that effects of statins are due to decreased levels of agents in the cholesterol synthesis pathway downstream of mevalonate but upstream of squalene and cholesterol. Potentially important substances that may affect cell proliferation include isoprenyls, which are required for isoprenylation and subsequently activation of small GTPases such as RAS. Activation of RAS is an early step of an important mitogen-activated-protein-kinase pathway: RAS-RAF-MAPK3/1. This pathway may be activated only following isoprenylation of RAS, which facilitates its attachment to the cytoplasmic leaflet of the cellular membrane [24]. Thus, statins may decrease proliferation by inhibiting isoprenylation of Ras, leading to decreased activity of the RAS-RAF-MAPK3/1 pathway. Indeed, consistent with this concept, in the present study we found that both mevastatin and simvastatin inhibit phosphorylation of MAPK3/1.
There is growing evidence that activation of MAPK pathways including MAPK3/1 is important in the pathophysiology of endometriosis [25, 26]. In patients with endometriosis, a microarray analysis of eutopic endometrium identified upregulation of genes involved in RAS-RAF-MAPK pathways [26]. Furthermore, inhibitors of MAPK have been shown to suppress proliferation of endometrial stromal cells [27].
Notably, concentrations of statins, mevalonate, and squalene chosen in this study were based on reports in other in vitro systems assessing effects of statins [16, 28, 29]. It is possible that a different pattern of effects may be observed at other concentrations of these agents; for example, it is conceivable that higher doses of squalene may overcome the inhibitory effects of statins. Such effects are not likely in view of the fact that squalene is a substrate for cholesterol and supply of cholesterol (in serum) did not prevent statin-induced inhibition of endometrial stromal growth.
In conclusion, we propose that statins exert inhibitory effects on growth of endometrial stroma, possibly by decreased activation of MAPK3/1; these effects may be relevant to the potential therapeutic use of statins in conditions such as endometriosis.
FOOTNOTES
1 Supported by NIH grant R01 HD40207 to A.J.D. 
2 Correspondence: Antoni J. Duleba, Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. FAX: 203 7857134; antoni.duleba{at}yale.edu
Received: 15 February 2006.
First decision: 7 March 2006.
Accepted: 27 March 2006.
REFERENCES
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  W. Murk, C. S. Atabekoglu, H. Cakmak, A. Heper, A. Ensari, U. A. Kayisli, and A. Arici Extracellularly Signal-Regulated Kinase Activity in the Human Endometrium: Possible Roles in the Pathogenesis of Endometriosis J. Clin. Endocrinol. Metab., September 1, 2008; 93(9): 3532 - 3540. [Abstract] [Full Text] [PDF]
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denotes means significantly different from mevastatin alone (P < 0.05); 
denotes means significantly different from simvastatin alone (P < 0.05).
