Simvastatin reduces steroidogenesis by inhibiting Cyp17a1 gene expression in rat ovarian theca-interstitial cells.

Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, theca-interstitial hyperplasia, and increased androgen production by theca cells. Previously, our group has demonstrated that statins (competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway) reduce proliferation of theca-interstitial cells in vitro and decrease serum androgen levels in women with PCOS. The present study evaluated the effect of simvastatin on rat ovarian theca-interstitial cell steroidogenesis. Because actions of statins may be due to reduced cholesterol availability and/or isoprenylation of proteins, the present study also investigated whether steroidogenesis was affected by cell- and mitochondrion-permeable 22-hydroxycholesterol, isoprenylation substrates (farnesyl-pyrophosphate [FPP] and geranylgeranyl-pyrophosphate [GGPP]), as well as selective inhibitors of farnesyltransferase (FTI) and geranylgeranyltransferase (GGTI). Theca-interstitial cells were cultured for 12, 24, and 48 h with or without simvastatin, GGPP, FPP, FTI, GGTI, and/or 22-hydroxycholesterol. Simvastatin decreased androgen levels in a time- and concentration-dependent fashion. This inhibitory effect correlated with a decrease in mRNA levels of Cyp17a1, the gene encoding the key enzyme regulating androgen biosynthesis. After 48 h, GGPP alone and FPP alone had no effect on Cyp17a1 mRNA expression; however, the inhibitory action of simvastatin was partly abrogated by both GGPP and FPP. The present findings indicate that statin-induced reduction of androgen levels is likely due, at least in part, to the inhibition of isoprenylation, resulting in decreased expression of CYP17A1.

Simvastatin decreases invasiveness of human endometrial stromal cells.

Recently we reported that statins, the competitive inhibitors of the key enzyme regulating the mevalonate pathway, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), decrease proliferation of human endometrial stromal (HES) cells. Furthermore, we found that simvastatin treatment reduces the number and the size of endometrial implants in a nude mouse model of endometriosis. The present study was undertaken to investigate the effect of simvastatin on HES cell invasiveness and on expression of selected genes relevant to invasiveness: matrix metalloproteinase 2 (MMP2), MMP3, tissue inhibitor of matrix metalloproteinase 2 (TIMP2), and CD44. Because statin-induced inhibition of HMGCR reduces the production of substrates for isoprenylation-geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP)-the effects of GGPP and FPP were also evaluated. Simvastatin induced a concentration-dependent reduction of invasiveness of HES cells. This effect of simvastatin was abrogated by GGPP but not by FPP. Simvastatin also reduced the mRNA levels of MMP2, MMP3, and CD44, but increased TIMP2 mRNA; all these effects of simvastatin were partly or entirely reversed in the presence of GGPP. The present findings provide a novel mechanism of action of simvastatin on endometrial stroma that may explain reduction of endometriosis in animal models of this disease. Furthermore, the presently described effects of simvastatin are likely mediated, at least in part, by inhibition of geranylgeranylation.

Antioxidants induce apoptosis of rat ovarian theca-interstitial cells.

Regulation of growth of ovarian theca-interstitial tissues is essential for normal ovarian development and function. Reactive oxygen species are involved in modulation of signal transduction pathways, including regulation of tissue growth and apoptosis. Previously, we have demonstrated that antioxidants inhibit proliferation of theca-interstitial cells. This report evaluates the effects of antioxidants on apoptosis of rat theca-interstitial cells. The cells were cultured in chemically defined media without or with vitamin E succinate and ebselen. Apoptosis was evaluated by cytochemical assessment of nuclear morphology, activity of executioner caspases 3 and 7, and determination of staining with annexin V in combination with propidium iodide. Both tested antioxidants induced significant morphological changes consistent with apoptosis, including chromatin condensation, nuclear shrinkage, and pyknosis. Antioxidants also induced other hallmarks of apoptosis including increased activity of caspases 3/7 as well as increased staining with annexin V. The present findings demonstrate that antioxidants with distinctly different mechanisms of action induce a series of events consistent with the process of apoptosis in ovarian mesenchyme. These observations may be of translational-clinical relevance, providing mechanistic support for the use of antioxidants in the treatment of PCOS, a condition associated with excessive growth and activity of theca-interstitial cells.

Effects of resveratrol on proliferation and apoptosis in rat ovarian theca-interstitial cells.

Polycystic ovary syndrome (PCOS) is characterized by ovarian dysfunction and associated with ovarian theca-interstitial (T-I) cell hyperplasia, hyperinsulinemia, systemic inflammation and oxidative stress. This in vitro study tested whether rat T-I cell growth with or without insulin can be altered by resveratrol, a natural polyphenol with anti-carcinogenic, anti-inflammatory, anti-proliferative and antioxidant properties. Rat T-I cells were cultured with and without resveratrol and/or insulin, and the effects on DNA synthesis, number of viable cells and markers of apoptosis were evaluated. Resveratrol alone induced a potent concentration-dependent inhibition of cell growth by inhibiting DNA synthesis, decreasing the number of viable cells and increasing the activity of executioner caspases 3 and 7; these effects of resveratrol counteracted the pro-proliferative and anti-apoptotic effects of insulin. Immunofluorescence analysis of cells incubated with resveratrol showed concentration- and time-dependent morphological changes consistent with apoptosis. The present findings indicate that resveratrol promotes apoptosis to reduce rat T-I cell growth in vitro as well as inhibiting insulin-induced rat T-I cell growth. This suggests a possibility that resveratrol and/or mechanisms mediating its effect may be relevant to the development of novel treatments for PCOS, which is characterized by both excessive ovarian mesenchyma growth and hyperinsulinemia.

Role of isoprenylation in simvastatin-induced inhibition of ovarian theca-interstitial growth in the rat.

Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting step of the mevalonate pathway. The pleiotropic effects of statins may be due to inhibition of cholesterol synthesis, as well as decreased availability of several biologically important intermediate components of the mevalonate pathway, including two substrates for isoprenylation (farnesyl pyrophosphate [FPP] and geranylgeranyl pyrophosphate [GGPP]). Recently, we demonstrated statin-induced inhibition of ovarian theca-interstitial cell proliferation in vitro, as well as reduction of testosterone levels in women with polycystic ovary syndrome (PCOS). This study evaluates the relative contribution of inhibition of isoprenylation and/or cholesterol availability to the modulation of theca-interstitial proliferation. Rat theca-interstitial cells were cultured in chemically defined media with or without simvastatin, FPP, GGPP, squalene, and/or two membrane-permeable forms of cholesterol (25-hydroxycholesterol and 22-hydroxycholesterol). Simvastatin inhibited DNA synthesis and the count of viable cells. The effects of simvastatin were partly abrogated by FPP and GGPP but not by squalene or cholesterol. Inhibition of farnesyl transferase and geranylgeranyl transferase reduced cell proliferation. The present findings indicate that simvastatin inhibits proliferation of theca-interstitial cells, at least in part, by reduction of isoprenylation. These observations provide likely mechanisms explaining clinically observed improvement of ovarian hyperandrogenism in women with PCOS.

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis. METHODS: Rat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 µM) and/or resveratrol (3-10 µM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test. RESULTS: Resveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin. CONCLUSIONS: The present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

Resveratrol potentiates effect of simvastatin on inhibition of mevalonate pathway in human endometrial stromal cells.

CONTEXT: Growth of endometriotic lesions in rodent model of endometriosis is inhibited by resveratrol, a natural polyphenol with antiproliferative and antiinflammatory properties, and simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) activity. OBJECTIVE: The objective of the investigation was to study the mechanism of action of resveratrol and its interactions with simvastatin, focusing on cholesterol biosynthesis and HMGCR gene expression and protein activity in primary cultures of human endometrial stromal (HES) cells. METHODS: HES cells were obtained from healthy volunteers. Biosynthesis of cholesterol was assessed by measuring the conversion of [(14)C]acetate to [(14)C]cholesterol. HMGCR mRNA transcripts were quantified by real-time PCR, protein expression by Western blot analysis, and enzyme activity by measuring the conversion of [3-(14)C]3-hydroxy-3-methyl-glutaryl-coenzyme A to [(14)C]mevalonic acid lactone in HES cell microsomes. RESULTS: Resveratrol inhibited cholesterol biosynthesis, HMGCR mRNA, and enzyme activity. Simvastatin inhibited cholesterol biosynthesis and enzyme activity but increased HMGCR mRNA and protein expression. Resveratrol potentiated the inhibitory effects of simvastatin on cholesterol biosynthesis and HMGCR enzyme activity and abrogated the stimulatory effects of simvastatin on HMGCR mRNA transcripts and protein expression. CONCLUSIONS: Resveratrol inhibits key steps of the mevalonate pathway by mechanisms that are partly complementary to and partly comparable with simvastatin via reducing both expression and activity of HMGCR. A combination of resveratrol and simvastatin may be of potential clinical relevance to development new treatments of human endometriosis.

Letrozole increases ovarian growth and Cyp17a1 gene expression in the rat ovary.

OBJECTIVE: To evaluate the effects of letrozole on ovarian size and steroidogenesis in vivo, as well as on proliferation and steroidogenesis of theca-interstitial cells alone and in coculture with granulosa cells using an in vitro model. DESIGN: In vivo and in vitro studies. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): In vivo effects of letrozole were studied in intact rats receiving either letrozole (90-day continuous-release SC pellets, 400 µg/d) or placebo pellets (control group). In in vitro experiments, theca cells were cultured alone or in coculture with granulosa cells in the absence or presence of letrozole. MAIN OUTCOME MEASURE(S): Deoxyribonucleic acid synthesis was determined by thymidine incorporation assay; steroidogenesis by mass spectrometry; and steroidogenic enzyme messenger RNA (mRNA) expression by polymerase chain reaction. RESULT(S): In vivo, letrozole induced an increase in ovarian size compared with the control group and also induced a profound increase of androgen, LH levels, and Cyp17a1 mRNA expression. Conversely, a decrease in Star, Cyp11a1, and Hsd3b1 transcripts was observed in letrozole-exposed rats. In vitro, letrozole did not alter either theca cell proliferation or Cyp17a1 mRNA expression. Similarly, letrozole did not affect Cyp17a1 transcripts in granulosa-theca cocultures. CONCLUSION(S): These findings suggest that letrozole exerts potent, but indirect, effect on growth of rat ovary and dramatically increases androgen levels and Cyp17a1 mRNA expression, the key enzyme regulating the androgen biosynthesis pathway. The present findings reveal novel mechanisms of action of letrozole in the rat ovary.

Effects of resveratrol on growth and function of rat ovarian granulosa cells.

OBJECTIVE: To evaluate the effects of resveratrol on growth and function of granulosa cells. Previously, we demonstrated that resveratrol exerts profound proapoptotic effects on theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Granulosa cells were cultured in the absence or presence of resveratrol. MAIN OUTCOME MEASURE(S): DNA synthesis was determined by thymidine incorporation assay, apoptosis by activity of caspases 3/7, cell morphology by immunocytochemistry, steroidogenesis by mass spectrometry, antimüllerian hormone (AMH), and vascular endothelial growth factor (VEGF) expression by polymerase chain reaction and Western blot. RESULT(S): Resveratrol induced a biphasic effect on DNA synthesis, whereby a lower concentration stimulated thymidine incorporation and higher concentrations inhibited it. Additionally, resveratrol slightly increased the cell number and modestly decreased the activity of caspases 3/7 with no effect on cell morphology or progesterone production. However, resveratrol decreased aromatization and VEGF expression, whereas AMH expression remained unaltered. CONCLUSION(S): Resveratrol, by exerting cytostatic but not cytotoxic effects, together with antiangiogenic actions mediated by decreased VEGF in granulosa cells, may alter the ratio of theca-to-granulosa cells and decrease vascular permeability, and therefore may be of potential therapeutic use in conditions associated with highly vascularized theca-interstitial hyperplasia and abnormal angiogenesis, such as those seen in women with polycystic ovary syndrome.

Resveratrol reduces steroidogenesis in rat ovarian theca-interstitial cells: the role of inhibition of Akt/PKB signaling pathway.

Polycystic ovary syndrome is characterized by theca-interstitial hyperplasia and increased expression of steroidogenic genes, leading to excessive androgen production. Resveratrol, a natural polyphenol, promotes apoptosis and reduces rat theca-interstitial cell growth, in part by inhibiting the mevalonate pathway and decreasing the availability of substrates of isoprenylation [farnesyl-pyrophosphate (FPP) and geranylgeranyl-pyrophosphate (GGPP)]. This study evaluated the effect of resveratrol on rat theca-interstitial cell steroidogenesis. Because resveratrol may activate sirtuins, this study also investigated whether steroidogenesis was affected by sirtuin inhibitors (nicotinamide, sirtinol). Theca-interstitial cells were cultured with or without resveratrol (1-10 µm), GGPP (30 µm), FPP (30 µm), nicotinamide (1 mm), and/or sirtinol (10 µm). Resveratrol did not affect progesterone levels but reduced androgen production in a concentration-dependent fashion (androstenedione by up to 78% and androsterone by up to 76%). This inhibitory effect correlated with a decrease in mRNA expression of genes regulating androgen production, especially Cyp17a1 (by up to 73%). GGPP and FPP had no effect on androgen levels and Cyp17a1 mRNA levels and did not alter the effects induced by resveratrol. Similarly, sirtuin inhibitors did not reverse resveratrol-induced inhibition of steroidogenesis. However, resveratrol decreased activity of serine-threonine kinase/protein kinase B pathway, a cell-signaling pathway involved in ovarian steroidogenesis. The present findings indicate that resveratrol reduces androgen production primarily by inhibiting Cyp17a1 mRNA expression, and this inhibition may be mediated, in part, by blocking the activity of the serine-threonine kinase/protein kinase B pathway. These findings may be of clinical relevance to conditions associated with excessive production of androgens by theca cells, such as polycystic ovary syndrome.

Resveratrol inhibits the mevalonate pathway and potentiates the antiproliferative effects of simvastatin in rat theca-interstitial cells.

OBJECTIVE: To examine the mechanisms of action of resveratrol and its interaction with simvastatin on growth and the mevalonate pathway in rat theca-interstitial cells. DESIGN: In vitro study. SETTING: Research laboratory. ANIMAL(S): Immature Sprague-Dawley female rats. INTERVENTION(S): Theca-interstitial cells were cultured in the absence or presence of resveratrol, simvastatin, mevalonic acid, farnesyl pyrophosphate, and/or geranylgeranyl pyrophosphate. MAIN OUTCOME MEASURE(S): DNA synthesis was assessed by thymidine incorporation assay; 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) expression and activity were evaluated with the use of quantitative real-time polymerase chain reaction, Western blot analysis, and HMGCR activity assay. Cholesterol synthesis was determined by the conversion of [(14)C]-acetate to [(14)C]-cholesterol. RESULT(S): Resveratrol potentiated the simvastatin-induced inhibition on cell proliferation in a concentration-dependent manner. Inhibitory effects of resveratrol were partly abrogated by the addition of mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Resveratrol reduced HMGCR expression and activity, and decreased cholesterol synthesis. In contrast, simvastatin inhibited HMGCR activity with a compensatory increase in HMGCR expression. Resveratrol counteracted this effect of simvastatin on HMGCR expression but augmented the simvastatin-induced inhibition on HMGCR activity and cholesterol synthesis. CONCLUSION(S): Resveratrol inhibits the mevalonate pathway via distinctly different mechanisms than statins. These observations demonstrate a novel mechanism of action of resveratrol and underscore the potential translational/clinical relevance of resveratrol interactions with simvastatin.

Statins inhibit growth of human theca-interstitial cells in PCOS and non-PCOS tissues independently of cholesterol availability.

CONTEXT: Polycystic ovary syndrome (PCOS) is associated with ovarian enlargement, prominent theca-interstitial hyperplasia, and excessive androgen production. Recent clinical trials have demonstrated that statins, 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors, decrease androgen levels in women with PCOS. OBJECTIVE: The present study evaluated the effect of statins on proliferation of human ovarian theca-interstitial cells. DESIGN AND SETTINGS: In vitro experiments were performed in the university research laboratory. PATIENTS: Human theca-interstitial cells were isolated from ovaries of PCOS (n=4) and non-PCOS (n=4) patients. MAIN OUTCOME MEASURES: The cells were incubated for 48 h without additives (control) or with simvastatin (3-30 µm), mevastatin (3-30 µm), and/or the cell- and mitochondrion-permeable form of cholesterol (22-hydroxycholesterol; 10 µm). To determine whether the effects of statins could be affected by leukocytes, the experiment was carried out on cells not purified of leukocytes and cells purified using anti-CD-45 immunomagnetic beads. The effect of statins on proliferation was evaluated by determination of DNA synthesis using radiolabeled thymidine-incorporation assay and by quantification of viable cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. RESULTS: Statins induced an inhibition of DNA synthesis in both the absence and the presence of 22-hydroxycholesterol; furthermore, 22-hydroxycholesterol alone also inhibited DNA synthesis. These effects of statins and 22-hydroxycholesterol were confirmed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium assay. Comparable inhibition of proliferation was observed in cells obtained from women with and without PCOS and in cell preparations treated and not treated with anti-CD-45 immunomagnetic beads. CONCLUSIONS: Statins inhibit proliferation of human theca-interstitial cells irrespective of the availability of cholesterol and independently of leukocytes both in normal and PCOS ovaries.

Simvastatin induces apoptosis and alters cytoskeleton in endometrial stromal cells.

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 mum) and/or geranylgeranyl pyrophosphate (GGPP, 30 mum) 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.