Current treatment options for women with endometriosis are limited and frequently involve either hormonal manipulation and/or surgery. For many women, the side effects of medical therapy can be as unpleasant as the symptoms of endometriosis and surgical treatment is generally noncurative with a high rate of recurrence [24
]. Therefore, development of better, more effective treatment strategies for women with endometriosis remains a high priority for research and will likely require a more precise understanding of the disease etiology. To this end, we have investigated the impact of simvastatin, a cholesterol-lowering drug with potent anti-inflammatory and antioxidant effects, on endometrial stromal cell adhesion and invasion. The present study has provided insight into not only the mechanisms by which this agent inhibits development of experimental endometriosis, but also provides additional support for pursuing simvastatin as a potential therapy for women with this disease. Specifically, we have demonstrated that: 1) simvastatin decreases adhesiveness of HES cells in vitro; 2) simvastatin decreases invasiveness of HES cells in vitro in a concentration-dependent fashion; 3) the inhibitory effect of simvastatin on invasiveness is abrogated in the presence of GGPP, but not FPP; 4) simvastatin down-regulates MMP2, MMP3, and CD44, and up-regulates TIMP2 gene expression; and 5) addition of GGPP to simvastatin abrogates the inhibitory effect of statin on MMP2, MMP3, and CD44 gene expression.
To our knowledge, this is the first report evaluating the effects of simvastatin on the invasive capacity of HES cells. The present observations provide a novel mechanistic explanation for the findings of our recent in vivo study whereby simvastatin induced a dose-dependent reduction in the number and size of endometriotic implants in a nude mouse model of human endometriosis [13
]. The invasion of the cells across the basement membrane consists of several events: initial attachment-adhesion, degradation of the basement membrane, and finally migration across the membrane. The simvastatin-induced decline in the number of invading HES cells cannot be attributed to a decreased number of cells in culture because during the 24-h culture simvastatin had no effect on the total number of viable cells, as determined by the MTS assay. However, cell motility and hence invasiveness may be affected by the changes in cellular cytoskeleton. Indeed, previously, we have demonstrated that within 24 h, simvastatin alters the morphology of HES cells disrupting the cytoskeleton by disorganizing F-actin fibers, altering cell shape, and inducing cell shrinkage [12
]. Statins, by interfering with isoprenylation (i.e., farnesylation and geranylgeranylation), cause the alteration of the actin cytoskeleton [34
]. Small GTPases—Rho, Rac, and Cdc42—play an important role in the maintenance and rearrangement of the cytoskeleton and cellular polarity [35
]. In particular, Rho activation is involved in signaling pathways stimulating actin stress fiber formation [39
], Rac plays a role in the generation of lamellipodia, and Cdc42 is important in the formation of actin spikes and filopodia [40
]. Moreover, increased expression of RhoA and Rho-associated coiled-coil-forming protein kinase-I (ROCK-I) and ROCK-II was observed in endometriotic stromal cells, suggesting a role of the activation of Rho-ROCK-mediated signaling pathway in the pathogenesis of endometriosis-associated fibrosis [41
]. Statins reduce GGPP and hence decrease geranylgeranylation of Rho, Rac, and Cdc42, leading to an accumulation of these proteins in their inactive form in the cytoplasm and causing detrimental changes in the cell cytoskeleton that leads to a loss of attachment. Furthermore, even though simvastatin had no effect on the total number of viable cells, simvastatin may profoundly alter cellular function by initiation of early apoptotic events such as activation of executioner caspases 3/7; such activation of caspases 3/7 was detected after 24 h exposure to simvastatin [12
Because simvastatin inhibits the first step of the mevalonate pathway, the effects of simvastatin may result from a reduction in the level(s) of any or all the downstream products of this pathway. Among the most important products of mevalonate pathway are the substrates of isoprenylation: FPP and GGPP. The present study demonstrates that the effects of simvastatin on cell invasiveness are most likely due to reduction of GGPP and not FPP. In the presence of GGPP, simvastatin had no effect on cell invasiveness, while in the presence of FPP simvastatin-induced inhibition remained unaffected. These studies clearly indicate the importance of geranylgeranylation, but not farnesylation, in stromal cells invasion in our model and, potentially, in the early events of endometriosis development. These findings are consistent with studies in other biological systems, whereby inhibition of geranylgeranylation resulted in suppression of invasiveness of various neoplastic cells, including breast and thyroid cancer cells [42
Another relevant finding of this study is the observation that simvastatin modulates the relative abundance of mRNAs of MMP2, MMP3, TIMP2, and CD44 by reducing the abundance of mRNAs for MMPs and CD44, which promote adhesiveness and invasiveness, while increasing mRNA for TIMP2, which counteracts invasiveness induced by MMPs. Comparable effects of statins on reduction of several MMPs, including MMP2 and MMP3, have been previously reported in several other in vitro systems in various cell types [44
]. Furthermore, a recent clinical trial in patients with chronic heart failure has demonstrated that statin therapy led to a significant decrease in the circulating levels of MMPs and an increase of TIMP2 [46
]. While the net effect of actions of MMPs and TIMPs is crucial for dynamic tissue remodeling throughout the reproductive cycle [47
], the potential impact of statins on physiologic changes of the endometrium should be address in future in vivo studies. Far less is known regarding the effects of statins on CD44; however, consistent with the present report, a recent study on breast tumor cells has also demonstrated a statin-induced reduction of CD44 expression [48
The present observations of the effects of simvastatin on MMPs, TIMP2, and CD44 provide additional, albeit indirect, evidence in support of anti-invasive effects of simvastatin on HES cells. Notably, changes in the abundance of the above mRNAs may not explain the effects of simvastatin on the invasiveness assay as performed in this study: simvastatin inhibited invasiveness within 24 h, while the effects on mRNAs were detected after 48 h. Consequently, we postulate that simvastatin may affect invasive capacity of HES cells by several independent mechanisms. Because GGPP reversed not only the effects of simvastatin on invasiveness assay but also on the abundance of relevant mRNAs, the common feature of these mechanisms appears to be the importance of statin-induced inhibition of geranylgeranylation. In summary, the findings of this study provide new evidence in support of the concept that simvastatin may exert beneficial and protective effects against the development of endometriosis by reducing the invasive capacity of HES cells.