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The Journal of Clinical Endocrinology and Metabolism logoLink to The Journal of Clinical Endocrinology and Metabolism
. 2010 Apr 28;95(7):3453–3459. doi: 10.1210/jc.2010-0072

Simvastatin Induces Apoptosis and Alters Cytoskeleton in Endometrial Stromal Cells

Anna Sokalska 1, Donna H Wong 1, Amanda Cress 1, Piotr C Piotrowski 1, Izabela Rzepczynska 1, Jesus Villanueva 1, Antoni J Duleba 1
PMCID: PMC2928904  PMID: 20427495

Abstract

Context: Statins are competitive inhibitors of 3-hydroxy-3methylglutaryl-coenzyme A reductase, with antimitotic, antioxidant, antiinflammatory, and immunomodulatory properties. Recent studies have shown that statins reduce the growth of human endometrial stromal (HES) cells and protect from the development of endometriosis in animal models.

Objectives: The present study was conducted to evaluate the effects of simvastatin on apoptosis and cytoskeleton of HES cells.

Design and Setting: In vitro experiments were performed in the university research laboratory.

Patients: HES cells were obtained from endometrial biopsies collected from nine subjects in the proliferative phase of their menstrual cycle.

Main Outcome Measures: The effect of simvastatin (10 and 30 μm) and/or geranylgeranyl pyrophosphate (GGPP, 30 μm) on caspase 3 and 7 activity, DNA fragmentation, and HES cell morphology was evaluated.

Results: Simvastatin induced significant time- and concentration-dependent apoptotic effects on HES cells as determined by increased activity of executioner caspases and DNA fragmentation. Simvastatin also caused profound alterations in HES cell morphology and F-actin cytoskeleton. This effect was abrogated by geranylgeranyl pyrophosphate, an important product of the mevalonate pathway.

Conclusions: Simvastatin induces apoptosis and disruption of the cytoskeleton of HES cells by reducing isoprenylation in cultures of human endometrial stroma. The present findings may lead to the development of novel treatments for endometriosis involving statins.

Simvastatin induces apoptosis and disrupts the cytoskeleton of human endometrial cells by reducing isoprenylation.

Endometriosis is one of the most common gynecological disorders, affecting up to 10% of women in reproductive age (1,2,3). By definition, the development of endometriosis involves growth of endometrial tissues outside the uterus. It is often associated with chronic pelvic pain and infertility; in addition to detrimental effects on the quality of life, the economic cost of endometriosis in the United States alone has been estimated to be over $20 billion annually (4). Currently available treatments of this condition are only modestly successful and are associated with significant side effects.

Recently, we proposed that statins may provide a novel and effective treatment of endometriosis. In our first in vivo animal study, we evaluated the effects of one of the statins, simvastatin, on the development of endometriosis-like implants in a nude mouse model (5). In that model, implantation of human endometrial xenografts resembles human endometriosis. We have demonstrated that a 10-d course of simvastatin resulted in a profound dose-dependent decrease in the number and volume of endometriotic implants. These effects of simvastatin may be related, at least in part, to the disruption of the growth of endometrial stroma. Indeed, the in vitro studies have demonstrated that simvastatin induced a concentration-dependent inhibition of human endometrial stromal (HES) cell proliferation, as evidenced by reduced DNA synthesis and a decreased number of viable cells (6). Furthermore, we found that this effect of simvastatin was due to the inhibition of the mevalonate pathway but was not dependent on the availability of cholesterol (6). Simvastatin was also shown to inhibit the proliferation of stromal cells derived from human endometriotic implants in ovaries (endometriomas) (7).

Simvastatin, like other statins, is a competitive inhibitor of 3-hydroxy-3methylglutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway (8). The mevalonate pathway consists of a series of reactions starting with acetyl-coenzyme A and involving the formation of farnesyl pyrophosphate (FPP): the substrate for several biologically important agents including cholesterol, isoprenylated proteins, coenzyme Q (ubiquinone), and dolichol (8). Among the most crucial to cellular function appear to be components of the pathway leading to isoprenylation of proteins: FPP and geranylgeranyl-pyrophosphate (GGPP). Isoprenylation consists of attachment of FPP (farnesylation) or GGPP (geranylgeranylation) to the carboxyl terminus of proteins (9). This posttranslational modification facilitates membrane attachment and the function of several families of proteins including Ras and Ras-related GTP binding proteins (small GTPases) (9). The functions of these proteins depend on association with the cytoplasmic leaflet of the cellular membrane: farnesylation of Ras and geranylgeranylation of Rho, Rac, and Cdc42. Statins can impair both geranylgeranylation and farnesylation by depletion of GGPP and FPP and hence may affect several signal transduction steps relevant to regulation of proliferation, apoptosis, and cell morphology.

To further investigate the effects of simvastatin on endometrial tissues, this study was designed to test the hypothesis that the inhibitory effect of simvastatin on the growth of HES is related to the disruption of isoprenylation, which in turn leads to increased apoptosis and disruption of the cytoskeleton.

Subjects and Methods

Acquisition of human tissues

Endometrial tissues were obtained from nine subjects (ages, 18–45 yr) during the proliferative phase of the menstrual cycle. All subjects were undergoing procedures such as tubal ligation or surgeries for benign gynecological conditions such as chronic pelvic pain. None of the subjects was using oral contraceptive pills or any other hormonal treatment that could affect endometrial function. The use of human tissues was approved by the University of California Davis Institutional Review Board. Written consent was obtained from all subjects. HES cells were obtained after enzymatic digestion of endometrial fragments and subsequently passing the cells through a 70-μm sieve (BD Falcon, Bedford, MA) (10). Isolation, characterization, and separation of HES from epithelial/glandular cells has been described previously by others (11,12,13). Cells were then cultured at 37 C in humidified air and 5% carbon dioxide in DMEM with 1% antibiotic and 10% fetal bovine serum. The cells were then transferred to 96-well plates (15,000 cells/well for Caspase-3/7 Activity Assay; 25,000 cells/well for TUNEL Assay) or to eight-well culture slides (15,000 cells/well) and cultured until 60–70% confluency. Subsequently, the culture media was changed to phenol-red free and serum-free DMEM, and the cells were incubated for an additional 24 h before adding the treatments. Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

Caspase-3/7 activity assay

Apoptosis in HES cells was evaluated by measuring activity of executioner caspases 3 and 7 using the Apo-ONE Homogeneous Caspase-3/7 Assay Kit (Promega, Madison, WI), following the manufacturer’s instructions. The cells were replaced with fresh media (phenol red-free and serum-free DMEM) without or with estradiol (1 nm), and were then cultured without (control) or with simvastatin (10–30 μm) for 6–48 h and/or GGPP (30 μm). Caspase-3/7 activity was measured in a microplate reader (Fluostar Omega; BMG Lab Technologies, Durham, NC) at excitation wavelength 485 nm and emission wavelength 520 nm. Caspase-3/7 activity was expressed per number of total viable cells.

Total viable cell number was estimated using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium (MTS) assay CellTiter 96 AQueous One Solution (Promega). This assay involves conversion of MTS to colored formazan by mitochondrial dehydrogenase within metabolically active cells. The quantity of formazan product as measured by absorbance at 490 nm is proportional to the number of living cells (14). Protein quantification was performed using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) based on the Bradford method. Experiments were repeated 16 times using the endometrial samples obtained from nine patients—13 times in the absence of estradiol and three times in the presence of 1 nm estradiol.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay

TUNEL assay evaluated DNA fragmentation in HES cell cultures using the HT TiterTACS Assay Kit (Trevigen, Gaithersburg, MD), according to the manufacturer’s instructions. The cells were replaced with fresh media with estradiol (1 nm), and then treated with simvastatin (10–30 μm) and/or GGPP (30 μm) for 48 h. Subsequently, the cells were fixed with 3.7% buffered formaldehyde solution for 7 min, washed with PBS, permeabilized with 100% methanol for 20 min, washed twice with PBS, digested with proteinase K for 15 min, quenched with 3% hydrogen peroxide, washed with distilled water, labeled with terminal deoxynucleotidyl transferase, and incubated at 37 C for 1 h in a humidified chamber, and then treated with stop buffer. The cells were incubated with TACS-Sapphire substrate, and the colorimetric reaction was stopped with 0.2 n HCl after 30 min. Negative controls were labeled without the terminal deoxynucleotidyl transferase, and positive controls were generated using TACS-Nuclease to create DNA breaks. The colorimetric reaction was measured in a microplate reader (Fluostar Omega; BMG) at absorbance 450 nm. The experiment was repeated two times using endometrial samples obtained from two patients.

Cell immunofluorescence

The HES cells were cultured without (control) or with simvastatin (10 μm) and/or GGPP (30 μm) for 24 and 48 h and then subjected to 4′,6-diamidino-2-phenylindole and F-actin staining to observe nuclear and cellular morphological changes. Approximately 15,000 HES cells per well were seeded in duplicate in eight-well culture slides (BD Biosciences, Bedford, MA). Briefly, the cells were fixed with 4% paraformaldehyde in PBS for 30 min, washed three times with PBS, blocked with 1% BSA in PBS for 30 min, washed twice with PBS, and stained with Texas red-phalloidin and DAPI (Molecular Probes, Carlsbad, CA). Slides were then examined under an Olympus BX61 fluorescent microscope at 40× magnification (Olympus America, Melville, NY). Apoptotic cells were morphologically identified based on disrupted or disorganized F-actin filaments. The experiment was repeated three times using endometrial samples obtained from three patients.

Statistical analysis

Comparisons between the groups were performed using ANOVA followed by post hoc comparisons of means, when appropriate values were logarithmically transformed before analysis. Normality of distribution was assessed by Shapiro-Wilk W test. In the absence of normality, nonparametric testing (Kruskal-Wallis) was used. Statistical analysis was performed using JMP program version 8 (SAS Institute, Cary, NC). Results are presented as means ± sem.

Results

Effects of simvastatin on caspase activation

To determine whether simvastatin affects activity of caspases 3 and 7, HES cells were cultured for 6, 12, 24, and 48 h in the absence or presence of simvastatin (10 and 30 μm) (Fig. 1). Exposure to simvastatin at either concentration had no significant effect at 6 and 12 h. However, at 24 h, simvastatin at 10 and 30 μm increased activity of caspases 3 and 7, respectively, by 43% (P < 0.002) and 72% (P < 0.001) compared with control levels. In a similar fashion, simvastatin at 10 and 30 μm after a 48-h treatment increased caspase 3 and 7 activity, respectively, by 43 and 93% (P < 0.001).

Figure 1.

Figure 1

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Time-course of the effect of simvastatin (10 and 30 μm) on activity of executioner caspases 3 and 7. HES cells were cultured in chemically defined media for 6, 12, 24, and 48 h in the absence (control) or in the presence of simvastatin. Caspase 3/7 activity was determined using Apo-ONE Homogeneous Caspase-3/7 Assay. Caspase activity was calculated per number of viable cells (determined by MTS assay) and expressed as percentage of control at each tested time-point. Each mean is determined from at least eight replicates. Each bar represents mean ± sem. At each time-point, means were compared using ANOVA with post hoc pairwise comparisons using Bonferroni correction. Means with no superscripts in common are significantly different (P < 0.05).

To determine whether simvastatin-induced activation of caspases 3/7 was related to reduced availability of GGPP, HES cells were cultured for 48 h without or with simvastatin (10 μm) and/or GGPP (30 μm) (Fig. 2). GGPP abrogated the stimulatory effect of simvastatin and reduced the activity of caspases 3/7 to a level comparable to that observed in control cultures. Comparable effects were observed in the absence and in the presence of 1 nm estradiol (Fig. 2, A–C). Furthermore, as presented in Fig. 2, B and C, the effects were similar when the activities of caspases 3/7 were expressed per number of viable cells and per milligram of protein.

Figure 2.

Figure 2

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A, Effect of simvastatin (10 μm) and GGPP (30 μm) on activity of executioner caspases 3 and 7. HES cells were cultured for 48 h in chemically defined media in the absence (control) or in the presence of simvastatin and/or GGPP in the absence of estradiol. Activity of caspases 3/7 was determined as described in Fig. 1. Each mean is determined from at least four replicates. Each bar represents mean ± sem; means with no superscripts in common are significantly different (P < 0.05). B, Effects of simvastatin (10 μm) and GGPP (30 μm) in the presence of estradiol (1 nm). Activity of caspases 3/7 was calculated per number of viable cells (determined by MTS assay). Each bar represents mean ± sem. Means with no superscripts in common are significantly different (P < 0.05). C, Effects of simvastatin (10 μm) and GGPP (30 μm) in the presence of estradiol (1 nm). Activity of caspases 3/7 was calculated per milligram of protein. Each bar represents mean ± sem. Means with no superscripts in common are significantly different (P < 0.05).

Effects of simvastatin on DNA fragmentation

Another approach to assess apoptosis involved the TUNEL assay quantifying DNA fragmentation in HES cells cultured for 48 h. As presented in Fig. 3, simvastatin alone (10 and 30 μm) induced an approximately 2.6-fold increase of TUNEL signal above control levels (P < 0.01). Exposure of HES cells to GGPP (30 μm) had no significant effect on DNA fragmentation. However, GGPP partly reversed the effects of simvastatin, reducing the TUNEL signal by 25–44% below the level observed in the presence of simvastatin alone; this effect was statistically significant for simvastatin 10 μm (P = 0.02).

Figure 3.

Figure 3

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Effect of simvastatin (10 and 30 μm) and GGPP (30 μm) on apoptosis (DNA fragmentation) of HES cells. Apoptosis was determined by TUNEL assay (HT TiterTACS Assay Kit; Trevigen, Gaithersburg, MD). Cells were exposed to simvastatin and/or GGPP for 48 h in the presence of 1 nm estradiol in chemically defined media, fixed, and labeled according to the HT TiterTACS protocol before colorimetric analysis. Each mean is determined from at least four replicates. Each bar represents mean ± sem; means with no superscripts in common are significantly different (P < 0.05).

Impact of simvastatin on the cytoskeleton

The effects of simvastatin on the F-actin cytoskeleton are presented in Fig. 4. In the absence of simvastatin, F-actin fibers are organized in parallel bundles. After a 24-h exposure to simvastatin alone, the F-actin fibers are disorganized, and their net loosened and disassembled. The shape of the cells appears indistinct. After a 48-h exposure to simvastatin, the cells are shrunken, with irregular outlines and the absence of identifiable fibers. GGPP alone has no significant effect on the distribution of the F-actin fibers after both 24- and 48-h exposures. At both time-points, GGPP markedly reversed the effects of simvastatin and restored F-actin cytoskeleton organization.

Figure 4.

Figure 4

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A and B, Effect of simvastatin (10 μm) and GGPP (30 μm) on morphology of HES cells in the presence of 1 nm estradiol. Cells were cultured for 24 h (A) and 48 h (B), then fixed, stained and visualized under a fluorescent microscope (40× magnification) as described in Subjects and Methods. Nuclear staining with 4′,6-diamidino-2-phenylindole and F-actin was used to observe morphological changes. a, Control cultures; b, cultures in the presence of GGPP alone; c, cultures in the presence of simvastatin alone; d, cultures in the presence of simvastatin and GGPP.

Discussion

The present study demonstrates that in cultures of human endometrial stroma: 1) simvastatin induces a wave of apoptosis and profound morphological changes including cell shrinkage and derangement of the cytoskeleton; and 2) these effects of simvastatin are abrogated in the presence of GGPP. These effects were observed both in the absence and in the presence of 1 nm estradiol.

The regulation of apoptotic death of endometrial stromal cells is likely to be an important mechanism controlling the growth of endometrial tissues. Under pathological conditions, excessive growth of endometrial tissues may significantly contribute to the development of endometriosis. Although the cause(s) of endometriosis remain unknown, growing evidence points to inflammation as an important factor involved in the pathophysiology of this condition. Peritoneal fluid in women with endometriosis contains increased levels of various proinflammatory cytokines including IL-1, IL-6, IL-17A, and TNFα (15,16,17). Furthermore, there is also evidence that endometriosis is associated with increased markers of oxidative stress in parallel with reduction of the levels of antioxidants such as vitamin E (18,19,20).

The above data provide a basis for the development of new treatments for endometriosis using antiinflammatory and antioxidant agents. In this context, statins are particularly promising because they have been shown to possess both antiinflammatory and antioxidant activities in various biological models (21,22,23). Furthermore, growing evidence also points at statins as drugs with antiproliferative and antineoplastic properties (24).

It is likely that the ability of simvastatin to induce HES cell apoptosis may represent an important mechanism contributing to the statin-induced inhibition of HES cell growth in vitro and protection from growth of endometrial implants in a nude mouse model of endometriosis (5,6). The present findings show that simvastatin induces a wave of apoptotic cell death, as evidenced by the observation of several hallmarks of this process including stimulation of the activity of executioner caspases 3/7, DNA fragmentation, and ultimately morphological changes in cellular appearance, including the disruption of the cytoskeleton, the alterations in cell shape, and cell shrinkage.

Remarkably, GGPP exerted a clear-cut protective effect from all the above actions of simvastatin, indicating that these actions are mediated via the reduction of geranylgeranylation. Geranylgeranylation is a process of posttranslational modification of proteins consisting of attachment of lipophilic GGPP molecule at the C terminus of a variety of proteins including small GTPases: Rho, Rac, and Cdc42. Consequently, these proteins may anchor to the cellular membranes and be activated. Rho, Rac, and Cdc42 play an important role in many key cellular functions including protection from apoptosis, maintenance and rearrangement of the cytoskeleton, and cellular polarity (25,26,27,28). In particular, Rho activation is involved in signaling pathways stimulating actin stress fiber formation (29), whereas Rac plays a role in the generation of lamellipodia and Cdc42 in the formation of actin spikes and filopodia (30). Statins reduce GGPP and hence decrease geranylgeranylation of Rho, Rac, and Cdc42 leading to an accumulation of these proteins in an inactive form in the cytoplasm (Fig. 5) and causing detrimental changes in the cell cytoskeleton leading to loss of attachment (31).

Figure 5.

Figure 5

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Proposed mechanisms of action of simvastatin on apoptosis and morphology of HES cells. Inhibition of the mevalonate pathway results in decreased availability of GGPP and hence reduced activity of Rac, Rho, and Cdc42. HMG-CoA, 3-Hydroxy-3methylglutaryl-coenzyme A.

The simvastatin-induced reduction of geranylgeranylation may also reduce the ability of cells to generate reactive oxygen species (ROS). Specifically, the reduced isoprenylation of Rac prevents the activation of a major intracellular source of superoxide and other ROS: reduced nicotinamide adenine dinucleotide phosphate oxidase (32). At moderate concentrations, ROS serve as second messengers involved in intracellular signaling including the stimulation of cellular proliferation and the expression of growth-inducing genes (33,34). Rac-dependent ROS have been shown to be of key importance in protecting cells from apoptosis (35). Consequently, simvastatin may induce apoptosis, at least in part, via the reduction of Rac activity and the consequent decrease in ROS.

The present observed mechanisms of actions on endometrial cells are likely shared by other statins. Indeed, the inhibitory effects on endometrial/endometriotic cells were also observed in experiments involving lovastatin and atorvastatin (36,37). Thus, in three-dimensional cultures of endometrial cells, Esfandiari et al. (38) observed that lovastatin reduced the proliferation of stromal cells and abolished angiogenesis. Atorvastatin induced the regression of endometriotic implants in surgically induced experimental endometriosis in a rat autotransplantation model (39). Most recently, atorvastatin was shown to inhibit the expression of proliferative, inflammatory, and angiogenic genes in endometrial and endometriotic cells (37).

Although this report demonstrates consistent and significant effects of simvastatin on HES, it is important to note that eutopic and ectopic endometrial tissues are functionally different, and further studies should also assess the effects of statins on ectopic/endometriotic tissues.

In summary, the present study provides new insight into the actions of statins on endometrial cells, demonstrating that statin-induced apoptosis and alterations of cellular skeleton are mediated, at least in part, by the reduction of geranylgeranylation.

Footnotes

This work was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/National Institutes of Health through Cooperative Agreement U54 HD052668, as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research.

Disclosure Summary: A.J.D. received grant support from the NICHD (04/01/07 to the present). The other authors have nothing to declare.

First Published Online April 28, 2010

Abbreviations: FPP, Farnesyl pyrophosphate; GGPP, geranylgeranyl-pyrophosphate; HES, human endometrial stromal; ROS, reactive oxygen species; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.

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