Endometriosis: Pathogenesis and Treatment 2014 Ed.

16. Potential New Drugs for Endometriosis: Experimental Evidence

Kaei Nasu Yukie Kawano1Masakazu Nishida1Akitoshi Tsuno1Akitoshi Yuge1Wakana Abe1Kentaro Kai1Mamiko Okamoto1 and Hisasshi Narahara1

(1)

Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Idaigaoka 1-1, Hasama-machi, Yufu-shi Oita, 879-5593, Japan

Kaei Nasu

Email: nasu@oita-u.ac.jp

Abstract

Endometriosis, a disease affecting 3–10 % of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Recent basic studies have revealed that the dysregulation of apoptosis, fibrosis, and epigenetic factors plays important roles in the pathogenesis of this enigmatic disease.

Contraceptive steroids, progestogens, agonists of gonadotropin-releasing hormone, androgens, and nonsteroidal anti-inflammatory agents have been used to treat endometriosis. Endometriosis treatments designed to lower circulating estradiol concentrations can be used only for a limited time due to unacceptable side effects. The development of medical treatments based on novel strategies to prevent or treat endometriosis has thus become a research priority.

Regarding the development of novel medical treatments for endometriosis, many researchers have been evaluating new drugs including molecular-targeting agents and herbal medicine as well as the newly developed hormonal agents. This chapter is a review of the findings from recent basic research on the pathogenesis of endometriosis and the evaluations of novel medical treatments for this disease, especially focusing on the inhibitors of nuclear factor-κB, the mevalonate-Rho/ROCK pathway, and histone deacetylase. These agents are now considered promising agents for the treatment and prevention of endometriosis.

Keywords

EndometriosisHistone deacetylase inhibitorMevalonate-Rho/ROCK pathwayNuclear factor-κB

16.1 Introduction

Endometriosis—the benign, estrogen-dependent, tumorlike disease characterized by chronic pelvic pain, dysmenorrhea, dyspareunia, and/or subfertility—is due to the uncontrolled ectopic growth of proliferative endometrial tissue. Women of reproductive age are most commonly affected by endometriosis, which usually occurs in the peritoneum, ovaries, and rectovaginal septum [1]. The symptoms of endometriosis may markedly reduce a woman’s quality of life.

Several surgical and medical strategies have been used to treat endometriosis. Contraceptive steroids, progestogens, and agonists of gonadotropin-releasing hormone, androgens, and nonsteroidal anti-inflammatory agents have been attempted [2]. Endometriosis treatments designed to lower circulating estradiol concentrations can be used only for a limited time due to unacceptable side effects. The current medical treatments inhibit the growth of endometriotic implants by suppressing ovarian steroids and inducing a hypoestrogenic state; they have been demonstrated to be effective in relieving endometriosis-associated pain [2]. However, high recurrence rates, up to 45 %, after the completion of medical treatments remain a significant problem [3]. An international consensus group proposed the development of nonhormonal medical treatments to prevent or treat endometriosis as a research priority [4]. In addition, many research groups have been evaluating new drugs for endometriosis such as molecular-targeting agents and herbal medicine, as well as the newly developed hormonal agents.

In this chapter, we review the recent basic studies on the development of novel medical treatments for endometriosis based on distinct strategies. We focus on the recent publications about the nuclear factor (NF)-κB inhibitors, mevalonate-Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK) inhibitors, and histone deacetylase (HDAC) inhibitors (HDACIs) as potential candidates for the next era in endometriosis treatment and prevention.

16.2 Potential New Drugs

16.2.1 NF-κB Inhibitors

Apoptosis plays a critical role in maintaining tissue homeostasis, and its function is to eliminate excess cells and dysfunctional cells. Apoptosis can be initiated by extracellular or intracellular “death signals,” and it results from a series of related morphologic and biochemical processes. Morphologically, apoptotic cells present with condensed chromatin, multiple membrane-bound organelles (apoptotic bodies), and shrunken appearance. Biochemically, apoptosis is characterized by monomeric or multimeric 180-base pair nucleosomal fragments resulting from the cleavage of double-stranded nuclear DNA [5]. Apoptosis is controlled by the expression of a number of regulatory genes, including c-myc, p53, Fas, NF-κB, and members of the B-cell lymphoma/leukemia (Bcl)-2 family [611].

Histologically, endometriotic tissues and normal proliferative endometrium are similar. However, endometriosis is increasingly being recognized as a condition in which endometriotic cells at the ectopic sites exhibit abnormal proliferative and apoptotic regulation in response to appropriate stimuli [1222]. It has been demonstrated that the degree of apoptosis in endometriotic lesions is less than that in the endometrium of the same patients and that of healthy women [13152325]. The resistance of endometriotic cells to apoptosis is suspected to be either intrinsic or brought about by environmental factors. Aberrantly expressed proliferation-related and apoptosis-related molecules in endometriosis include the decreased expressions of homeobox (HOX) A10 [26] and caspase-1 [27] and the enhanced expressions of c-myc [28], cellular inhibitor of apoptosis protein (cIAP) 1 [29], X chromosome-linked IAP [29], B-cell lymphoma/leukemia (Bcl)-2 [19], Bcl-XL [19], and NF-κB [3031] in endometriotic cells.

The pleiotropic transcription factor NF-κB has been identified as a critical component of several signal transduction pathways [29]. Figure 16.1 shows the proposed functions of NF-κB in the pathogenesis of endometriosis. One important function of NF-κB is its ability to protect cells from apoptosis by activating antiapoptotic genes [910]. Wieser et al. [32] demonstrated the constitutive activation of NF-κB in endometriotic cells. It is suggested that NF-κB has a significant role in the proliferation of endometriotic lesions [3031]. The activation of NF-κB by lipopolysaccharide (LPS) induces the proliferation of endometriotic stromal cells [33].

A313895_1_En_16_Fig1_HTML.gif

Fig. 16.1

Involvement of the NF-κB-mediated pathway in the pathogenesis of endometriosis. ECM extracellular matrix, IκB inhibitor κB, IKK IκB kinase, NIK NF-κB-inducing kinase, MAPKKMAPK kinase, TNF tumor necrosis factor

A number of substances have been presented in the endometriosis literature as NF-κB inhibitors. The reported selective inhibitors of NF-κB include an IκB protease inhibitor, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) [33], thalidomide [34], BAY 11–7085 [35], pyrrolidine dithiocarbamate (PDTC) [3638], costunolide [39], and parthenolide [40]. The urinary preparation human chorionic gonadotropin A (hCG-A) [41], gonadotropin-releasing hormone (GnRH) agonists [30], progesterone [42], and danazol [42] have been demonstrated to inhibit NF-κB activity in endometriosis. NF-κB inhibitors have been shown to significantly block the proliferation of endometriotic stromal cells [333540] and they induce apoptosis and the G0/G1 phase cell-cycle arrest of endometriotic stromal cells [353839]. Additionally, after endometriotic stromal cells were treated with NF-κB inhibitors, the downregulation of the expression of antiapoptotic factors (Bcl-2 and Bcl-XL), inflammatory cytokines (interleukin-6 [IL-6], IL-8, monocyte chemoattractant protein-1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), inflammatory mediators (COX-2 and PGE2), extracellular matrix remodeling mediators (MMP-2, MMP-3, MMP-7, MMP-9), CD44, and vascular endothelial growth factor [VEGF] with simultaneous activation of caspase-3, caspase-8, and caspase-9 was observed [303441].

The suppression of NF-κB activity by proteasome inhibitors also suppresses the proliferation of endometriotic cells in vitro [31]. The NF-κB inhibitors BAY 11–7085 and SN-50 significantly reduced the development of endometriotic lesions in a nude mice model [43]. Takai et al. [40] also demonstrated that parthenolide reduced the growth of endometriotic lesions in a murine model. The antioxidant PDTC reduced the growth and vascularity of experimental peritoneal endometriotic lesions in a rat model [44].

16.2.2 Mevalonate-Rho/ROCK Inhibitors

During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and the alterations of tissue function that are characteristic of this disease [4548]. It has been suggested that type I collagen is a major contributor to endometriosis-associated fibrosis [4649], whereas α-smooth muscle actin (SMA)-positive fibroblastic cells were frequently detected in the fibrotic areas associated with endometriosis of the peritoneum, ovary, rectovaginal septum, and uterosacral ligaments [454850]. An immunohistochemical analysis led Anaf et al. [50] to suggest that endometriotic stromal cells can differentiate to α-SMA-positive myofibroblasts.

To establish a model of fibrosis formation in endometriosis, we used a 3D collagen culture system with human endometriotic stromal cells [5152]. We cultured the cells in floating collagen lattices to reorganize the collagen fibers and make them compact, resulting in contraction of the collagen gels. This culture system provided a model for mechanically relaxed tissue with low tensile strength, comparable to the early stages of an endometriotic lesion. We found that endometriotic stromal cells cultured in floating 3D gels have an enhanced contractile profile compared to normal endometrial stromal cells [51]. This suggested that endometriotic stromal cells may acquire fibrogenetic ability or fail to avoid fibrogenesis during the pathogenesis of endometriosis.

Members of the Rho family of small guanosine triphosphatase (GTPase) are known to regulate cell shape, motility, cell-substratum adhesion, and cell contraction through the reorganization of actin cytoskeletons [5365]. The active form of Rho is GTP-bound [5462], and many polypeptides have been reported as targets of activated Rho, including ROCK-I/p160ROCK and Rho-kinase/ROCK-II [66]. ROCKs phosphorylate the myosin light chain (MLC) [67] and the myosin-binding subunit of myosin phosphatase [68], and they inhibit myosin II regulatory light chain phosphatase activity [68] in cultured fibroblasts. Rho and ROCKs have been implicated in myosin II-dependent force generation [69]. Thus, the activation of ROCKs by Rho can promote the assembly of focal adhesions, actin stress fiber formation, and contraction of non-muscle cells [6268], in which RhoA regulates α-SMA expression [70].

Based on these observations in fibroblasts, we have investigated the signaling pathway underlying endometriotic stromal cell-mediated contractility by using 3D cultures. Our data indicated that human endometriotic stromal cells undergo myofibroblastic differentiation and show increased expression of RhoA, ROCK-I, and ROCK-II proteins, resulting in enhanced contractility [51]. Thus, we evaluated several inhibitors of mevalonate-Rho/ROCK pathways as candidate drugs for the treatment and prevention of endometriosis-associated fibrosis [51527173].

16.2.3 Statins

Statins are potent inhibitors of cholesterol biosynthesis that are widely used to reduce serum cholesterol levels in hyperlipidemic patients [7475]. Statins are divided into three categories: natural statins (i.e., lovastatin and pravastatin), semisynthetic statins (i.e., simvastatin), and synthetic statins (i.e., atorvastatin, fluvastatin, and cerivastatin) [7576]. These three subtypes of statin exhibit markedly different hydrophobicities, with simvastatin as the most hydrophobic and pravastatin as the most hydrophilic.

Statins competitively inhibit 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase to block the conversion of HMG-CoA to L-mevalonate, a rate-limiting step in cholesterol synthesis [77]. By inhibiting the initial step of the cholesterol synthesis pathway, statins reduce the synthesis of several important lipid intermediate compounds including isoprenoids such as farnesyl pyrophosphate (FPP), a precursor of cholesterol, and geranylgeranyl pyrophosphate (GGPP), which is synthesized from FPP [78]. Both FPP and GGPP are important isoprenoid intermediates and serve as lipid attachments for a variety of intercellular proteins to the plasma membrane, including Rho proteins, resulting in their activation [5979]. By inhibiting the activation of Rho proteins, statins also regulate both the Rho/ROCK pathways and the mevalonate pathway [8081].

Simvastatin has been demonstrated to inhibit the proliferation of endometriotic stromal cells as well as the collagen gel contraction mediated by these cells [71]. Attenuation of the endometriotic stromal cell attachment to collagen fibers is involved in this mechanism. Lovastatin also inhibited cell proliferation and angiogenesis in endometriosis [82], whereas simvastatin and mevastatin inhibited the MCP-1 production by endometriotic cells [83]. Atorvastatin also exhibited antiproliferative and anti-inflammatory effects in endometriotic cells [84]. Oktem et al. [85] demonstrated that atorvastatin induced the regression of endometriotic implants in a rat model. Similarly, Bruner-Tran et al. [86] demonstrated that simvastatin induced the regression of endometriotic implants in a nude mouse model.

16.2.4 ROCK Inhibitors

Several selective ROCK inhibitors including Y-27632 and fasudil hydrochloride are now available for basic and clinical studies [58638788]. We demonstrated that Y-27632, a pyridine derivative that acts as a specific inhibitor of ROCK-I and ROCK-II [5863], inhibits endometriotic stromal cell-mediated contractility [51]. Interestingly, Y-27632 was found to exert a greater effect on the contractility of endometriotic stromal cells than on the contractility of normal eutopic endometrial stromal cells, whereas fasudil hydrochloride also inhibited endometriotic stromal cell-mediated contractility, myofibroblastic differentiation, and cell proliferation [72].

16.2.5 Heparin

Heparin is an analog of heparan sulfate, a unique class of macromolecules that is widely expressed on the cell surface and in the extracellular matrix [89]. It is a commonly used anticoagulant drug [90]. Heparin was shown to inhibit the gel contraction mediated by dermal fibroblasts [9192]. We demonstrated that heparin attenuates the contractility of endometriotic stromal cells by suppressing the cells’ attachment to collagen fibers, the inhibition of myofibroblastic differentiation, and the suppression of the Rho/ROCK-mediated pathway [73]. However, the precise target molecule of heparin’s action on endometriotic stromal cells has not yet been elucidated.

16.2.6 HDACIs

Epigenetics refers to the stable inheritance of phenotypes of cells and organisms without changes in DNA sequence or DNA content [93]. The epigenetic phenotypes are conferred via nuclear processes such as DNA methylation and chromatin modifications (e.g., acetylation, biotinylation, isomerization, methylation, phosphorylation, ribosylation, sumoylation, and ubiquitination of histones) and underlie the regulation of all genome functions, including gene expression, DNA replication, and genome stability [9496]. Epigenetic phenotypes are also conferred via microRNA and double-stranded noncoding RNA, which are interconnected and may work together to establish and/or maintain specific gene activity states in normal cells [939697]. Epigenetic processes are known to be involved in development, health, disease, and aging and are responsible for phenomena such as X-chromosome inactivation and genomic imprinting [9899]. Of these epigenetic regulatory mechanisms, histone acetylation is the most studied.

The acetylation levels of histone are controlled by a balance between histone acetyltransferases and histone deacetylases (HDACs). Histone acetyltransferases transfer acetyl groups from acetyl-CoA to lysine residues on the aminoterminal region of histones and activate genetic transcription. Conversely, HDACs restore the positive charge on lysine residues (by removing the acetyl groups) and prevent transcription. HDACs are large multiprotein complexes that target promoter sites through their interaction with sequence-specific transcription. They play an important role in the regulation of gene transcription through the remodeling of chromatin structure and dynamic changes in the nucleosomal packaging of DNA [100].

The hyperacetylation of histones H3 and H4 is often associated with activated transcription, and the hypoacetylation of histones H3 and H4 correlates with transcriptional silencing or repression [101], whereas Choi et al. [102] suggested that the substrates of HDACs are not restricted to histones but include transcriptional regulators, such as p53, E2F-1, Mad-1, BCL-6, and ETO. In this regard, global gene expression analyses have shown that HDACIs affect the expression levels of 2–20 % of genes in the genome, of which about half are upregulated and half downregulated [103].

Accumulating evidence indicates that several epigenetic aberrations are involved in the pathogenesis of endometriosis [104110]. We observed that the levels of acetylated histones H3 and H4 were significantly lower in unstimulated endometriotic stromal cells compared to normal endometrial stromal cells, suggesting that aberrant histone modifications are present in endometriosis [108]. This initial finding encouraged us to evaluate the efficacy of HDACIs for the treatment of endometriosis. Our subsequent experiments demonstrated that HDACIs significantly inhibited the proliferation of endometriotic stromal cells, and they also induced significant levels of cell-cycle arrest at the G0/G1 or G2/M phases and significant apoptosis of these cells [108].

Interestingly, HDACIs showed marginal to weak effects on normal endometrial stromal cells compared to endometriotic stromal cells. Moreover, HDACI treatment significantly inhibited HDAC activity and resulted in the accumulation of acetylated histones H3 and H4 in total cellular chromatin and in the promoter regions of the p16INK4a, p21Waf1/Cip1, p27Kip1, and cell-cycle checkpoint kinase 2 (chk2) genes in these cells. A Western blot analysis revealed the increased protein levels of p16INK4a, p21Waf1/Cip1, p27Kip1, and chk2, the suppression of Bcl-2 and Bcl-XL protein levels, and the activation of caspase-3 and caspase-9 in endometriotic stromal cells after treatment with HDACIs [108]. Recently, we found that the expression of CCAAT/enhancer-binding protein (C/EBP)-α, a tumor suppressor gene, and death receptor 6, a TNF receptor superfamily gene, was epigenetically suppressed by histone deacetylation (our unpublished data).

Histone deacetylation appears to be a potent regulator of gene expression in endometriosis, which raises the prospect of using HDACIs as therapeutic tools in endometriosis [108]. Several classes of HDACIs have been identified, including (a) organic hydroxamic acids [e.g., trichostatin A and suberoylanilide bishydroxamine (SAHA)], (b) short-chain fatty acids [e.g., butyrates and valproic acid (VPA)], (c) benzamides (e.g., MS-275), (d) cyclic tetrapeptides (e.g., trapoxin), and (e) sulfonamide anilides [108]. The molecular events that mediate the biological effects of HDACIs are incompletely understood. Inhibition of HDAC by HDACIs increases histone acetylation and maintains chromatin structure in a more open conformation, resulting in the reactivation of transcriptionally silenced pathways or the suppression of aberrantly expressed genes through the recruitment of repressors [111112]. HDACIs can reactivate genes silenced by promoter hypermethylation as well as the demethylation agents [113]. HDACIs also cause mitotic defects through non-transcriptional mechanisms [114].

We demonstrated that HDACIs, including VPA, SAHA, and apicidin, can inhibit the proliferation, induce cell differentiation and cell-cycle arrest, and stimulate the apoptosis of endometriotic cells [108]. Guo and his colleagues have demonstrated that HDACIs, such as VPA and trichostatin A, can suppress the proliferation, induce cell-cycle arrest, inhibit IL-1β-induced cyclooxygenase-2 expression and NF-κB activation, upregulate peroxisome proliferator-activated receptor γ, p21Waf1/Cip1 and PR-B expression, attenuate invasiveness, and reactivate the silenced E-cadherin gene expression of endometriotic cells [115120]. Romidepsin, an HDACI, was shown to specifically reduce HDAC enzymatic activity, resulting in inhibited cell proliferation, cell-cycle arrest, increased apoptosis, and reduced expression of VEGF mRNA and protein in endometriotic cells [121122]. Treatment with trichostatin A and VPA has been shown to significantly reduce the growth of endometriotic lesions in a murine model [123124]. Histone deacetylation appears to be a potent regulator of gene expression in endometriosis, which raises the prospect of using HDACIs as therapeutic tools in endometriosis [108117].

VPA was used in a pilot study of three patients with endometriosis and adenomyosis who had moderate to severe dysmenorrhea [125]. A VPA dose of 1,000 mg/day was used for 3 months. Complete relief of pain in all cases and an average reduction of one-third in uterine size were reported. The disappearance or reduction of palpable tender nodules in the cul-de-sac was also reported.

16.3 Conclusions

Basic research on endometriosis offers new opportunities to understand the pathogenesis of this enigmatic disease and to provide novel medical treatments other than hormonal therapy. As shown in this chapter, NF-κB inhibitors and mevalonate-Rho/ROCK inhibitors as well as HDACIs seem to be promising therapeutic strategies for the treatment of endometriosis.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 13237327 to K. Nasu, no. 25861500 to Y. Kawano, and no. 23592407 to H. Narahara).

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