2-methoxyestradiol and disorders of female reproductive tissues

2-Methoxyestradiol (2ME) is an endogenous me- tabolite of 17β-estradiol. Once thought of as a mere degrada- tion product, 2ME has gained attention as an important com- ponent of reproductive physiology and as a therapeutic agent in reproductive pathologies such as preeclampsia, endometri- osis, infertility, and cancer. In this review, we discuss the involvement of 2ME in reproductive pathophysiology and sum- marize its known mechanisms of action: microtubule disruption, inhibition of angiogenesis and stimulation of apoptosis. Current- ly, the clinical uses of 2ME as a single agent are limited due to its poor water solubility and thus low bioavailability; however, 2ME analogs and derivatives ha ve been recently developed and tested as cancer treatments. Despite some isolated success stories and ongoing research, 2ME derivatives have not yet provided the expected results. The adjuvant use of 2ME deriv- atives with chemotherapeutic agents is hindered by their intrinsic toxicity confounding the unwanted secondary effects of chemo- therapy. However, due to the well-tested tolerance of the body to high doses of native 2ME, it may the combination of native 2ME with conventional treatments that will offer novel clinical- ly relevant regimens for cancer and other reproductive disorders.

Female reproductive potential starts at menarche and ceases at the menopause. In a woman ’s life, these two stages and the processes in between are determined by the female sex hor- mones. The reproductive cycle is governed by a combination of hormones produced at varying levels throughout the men- strual cycle. Previously, the concept was simple: estrogen and progesterone (the “ovarian” hormones) along with other hor- mones such as oxytocin and prolactin among others played specific roles when required. However, studies in reproductive physiology during the last decade have demonstrated that these hormones are only part of the story, and now their metabolites, previously believed to be intermediates on the road to degradation and excretion, are in fact important players in both reproductive physiology and pathology. A variety of reproductive disorders can disrupt the balance of these hormones, altering the functions of reproductive organs, including the ovaries, Fallopian tubes, uterus, cervix, vagina, vulva, and, at least for the purpose of this review, breast. Amenorrhea, pelvic inflammatory disease, preeclampsia, en- dometriosis, infertility, and cancer are among these disorders. Estrogen Metabolism and the Origin of 2-Methoxyestradiol 2-Methoxyestradiol (2ME) is a naturally occurring metabolite of estradiol. Estrogens can be metabolized into their interme- diaries by members of the cytochrome P450 (Cyp450) family; specifically, CYP1A1 and CYP1B1 are able to convert estro- gens into water-soluble metabolites such as 2- hydroxyestradiol and 4-hydroxyestradiol; subsequently 2ME can be generated by the catechol- O-methyl-transferase (COMT) [ 82]. Under physiological conditions, blood 2ME concentrations in women range from 46 to 70 pg/ml [ 66]. M. P . Pinto: G. I. Owen (*) Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile e-mail: [email protected] R. A. Medina Facultades de Ciencias Biológicas y Medicina, Universidad Andrés Bello, Santiago, Chile G. I. Owen Centro UC Investigación en Oncologia (CITO), Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile G. I. Owen Advanced Center for Chronic Diseases (ACCDiS), Pontificia Universidad Católica de Chile, Alameda 340, Santiago, Chile HORM CANC (2014) 5:274–283 DOI 10.1007/s12672-014-0181-2 A Role for 2ME in Reproductive Physiology Scientific analysis of any malfunction or disorder dictates that the best place to start is to understand the physiology of the process and from this vantage point elucidate the cause of the pathology. The concept that estrogen is metabolized into another active compound (perhaps a future hormone), namely 2ME, was given strength by the observation that endogenous 2ME accelerates oocyte transport in the rat oviduct [ 70]. Although yet to be confirmed in humans, this study also demonstrated that mating halted 2ME effect, with the role of bringing the oocyte down the Fallopian tube (oviduct) taken over by the parental hormone estrogen. 2ME has since been shown to increase overall ovarian weight and has been detect- ed in follicular fluid [ 4, 77]. In follicular fluid, 2ME is spec- ulated to act as a growth inhibitor of ovulatory follicles (by inhibiting angiogenesis) and steroidogenesis [4, 77]. Interest- ingly, low doses of 2ME stimulate granulosa cell proliferation, while higher doses are inhibitory [77]. 2ME is now demonstrated to be a natural component of maternal blood, cord blood, breast milk, and amniotic fluid [3]. In ovine uterine artery endothelial cells derived from pregnant ewes, 2ME increased prostacyclin production in a concentration- and time-dependent manner [46]. In the same model, 2ME treatment was also demonstrated to be anti- angiogenic [78]. 2ME induces invasion of the cytotrophoblast through naturally derived extracellular matrix and thus maybe required to facilitate both appropriate vascular development and oxygenation during pregnancy [56]. Elevated 2ME levels are present during the late stages of pregnancy, during which time breast ductal tissue has differentiated to form alveolar milk-producing structures. In virgin mice, 2ME has been shown to induce mammary ductal dilation and partial mam- mary gland differentiation [42]. These observations reinforce the concept that the 2ME is not merely a degradation product of estradiol but in fact is an independent natural and integra- tive part of female reproductive tract regulation. A Role for 2ME in Reproductive Pathology Reproductive pathologies affect women during their repro- ductive years causing in extreme cases of infertility and preg- nancy loss along with complications in menstruation, concep- tion, labor, and menopausal transition. Endometriosis is char- acterized by the presence of ectopic tissue outside the uterine cavity and affects 6–8 % of women during their reproductive years and is responsible for a high incidence of infertility [10]. A mouse model study using en dometriosis-like lesions showed that these lesions are characterized by hypoxia, resulting in upregulation of the hypoxia-inducible factor 1a (HIF-1α), and the induction of angiogenesis via the vascular endothelial growth factor (VEGF); systemic treatment of these animals with 2ME suppressed the growth of endometriosis- like lesions, reduced HIF-1α and VEGF expression levels and vascular permeability [6]. The authors speculate that 2ME acts through a dual mechanism: an indirect effect by HIF-1 α inhibition, leading to VEGF suppression decreasing angio- genesis, and a direct effect via an inhibition of endothelial cell function [6]. Preeclampsia is a hypertensive disorder of pregnancy char- acterized by placental hypoxia, proteinuria, and fluid reten- tion. Preeclampsia affects approximately 5 % of all pregnan- cies and remains a leading cause of maternal and fetal mor- bidity and mortality [ 80]. In several pregnancy disorders, including preeclampsia, inadequate cytotrophoblast invasion of the uterus occurs. In vitro studies have demonstrated that cytotrophoblast cells treated with 2ME switch to an invasive phenotype when cultured under low oxygen conditions; 2ME treatment also suppressed HIF-1α,t r a n s f o r m i n gg r o w t hf a c t o r beta-3 (TGFβ3), and tissue inhibitor of metalloproteinases-2 (TIMP-2) in these cells. The same study demonstrates in vivo that placentas of COMT −/− mice (2ME deficient) revert their preeclampsia features (higher levels of HIF-1α,T G Fβ3, and TIMP-2) after 2ME administration [ 56]. In patients, a recent study demonstrated that pregnant women that eventually de- veloped preeclampsia displayed significantly lower levels of plasma 2ME at 11–14 weeks [73]. Further reports suggest that the reduction in 2ME synthesis in preeclampsia patients could be explained by alterations in the methionine-homocysteine metabolism or the 17β-estradiol synthesis pathway [72]. Polycystic ovary syndrome (PCOS) is an endocrine disor- der that affects 5 –8 % of women of reproductive age. The features of PCOS are hyperandrogenemia, chronic anovulation, and infertility. In granulosa cells, COMT expres- sion is upregulated by the major contributors to PCOS: insu- lin, dihydrotestosterone, and all-trans retinoic acid, suggesting a role for the dysregulation of 2ME in the development of PCOS and ovulatory dysfunction. Although a genetic analysis of COMT has not been correlated, prolactin levels in women with PCOS varied significantly with COMT haplotypes [ 77, 38]. Future studies are required to determine the role of steroid hormone metabolites in the abovementioned reproductive pathologies, together with pathologies that remain to be ex- amined in the light of 2ME action, such as amenorrhea, pelvic inflammatory disease, ectopic pregnancy, pregnancy loss, and transitional problems at menopause. However, by far, the most reported mention of 2ME in the medical literature comes in relation to cancer and its potential therapeutic use. 2ME and Cancer of the Reproductive Tissues The balance between 2ME and other estrogen metabolites could play a role in carcinogenesis [ 47]; 2-hydroxyestradiol and 4-hydroxyestradiol are endogenous estradiol metabolites HORM CANC (2014) 5:274–283 275 that increase proliferation and the formation of reactive oxy- gen species (ROS) [ 71, 30, 65]; in contrast, 2ME has anti- proliferative effects in reproductive tissues. In human breast cancer biopsies, estrogen 2-hydroxylase and COMT displayed higher levels in tumors and benign neoplasms compared to normal breast tissue, suggesting a role for these metabolites in the bourgeoning cancer cell [ 40]. CYP1A1, CYP1B1, and COMT polymorphisms can bring about toxic levels of 4- hydroxyestradiol and 2ME that may add to an increased risk of ovarian cancer [ 41]. Polymorphisms in COMT (the 2ME- converting enzyme) are linked to numerous disorders, includ- ing altered prefrontal dopaminergic metabolism, fibromyalgia syndrome, Alzheimer’s disease, depression and suicide, ma- ternal stress, and emotional and behavioral problems in chil- dren [ 52, 26, 57, 20, 88]. A specific V al158Met COMT polymorphism has been associated with a decreased risk of uterine leiomyoma [ 25]. In cancers, a COMT V al/V al geno- type is correlated with an increase in endometrial cancer risk [58], while the V al158Met polymorphism is associated to a decreased risk in an Asian population [85]. In a separate study, V al/Met genotype was associated with an increased risk of developing endometrial/ovarian cancer, while a homozygous mutant (Met/Met) demonstrated a decreased risk in Australian and Polish patients [2]. The V al158Met COMT polymorphism has been postulated as a cancer risk factor in the Chinese population; however, some studies have stated that it is not a breast cancer risk factor in Asian population [ 89]. The corre- lation between COMT polymorphisms (and mutations) and cancer risk still needs further study; however, the evidence suggests a high dependency on ethnicity. Despite these isolated reports, most studies have focused on cancer and 2ME. Therefore in the next sections, we will discuss on the effects of 2ME on reproductive tract cancers (including breast) and its potential therapeutic applications. 2ME and Cancer Treatment In 2012, there were an estimated of 14.1 million cancer cases worldwide, of these 6.7 were in women. Cancers of female reproductive tissues combined (ovary, endometrium, cervix, and breast) accounted for 41.5 % of female cancer incidence (World Cancer Research Fund international 2014:www.wcrf. org). In cancer, 2ME is a powerful anti-angiogenic, anti- proliferative, and pro-apoptotic agent [28]; this includes can- cers of reproductive tissues (such as cervical ovarian, endo- metrial, and breast cancer) and others (e.g., lung, colon, kid- ney, prostate and esophagus, stomach, and pancreas among others) [66]. In contrast, normal and non-proliferating tumor cells appear to be more resistant to the anti-proliferative effects of 2ME [51, 50]. Both in vitro and clinical studies consistently show that high circulating levels of 2ME are required (approaching and surpassing micromolar) for its anti-tumorigenic effects [ 75, 44]. During pregnancy, 2ME levels reach their highest with circulating levels that range from 2 to 10 ng/ml [66]. Thus, the concentrations of 2ME (and its more stable derivatives) far exceed these values, and thus, their effects may differ from the physiological effects of 2ME discussed in the previous sec- tions. An exception to this might be the follicular fluid ex- tracted from the ovary. Here, at the site of estradiol production, the 2ME levels reach the micromolar range [ 66, 75]. In addition to its effects as a single agent, the combination of 2ME with other anti-tumorigenic agents has been shown to cause a synergistic inhibition of cancer cell proliferation [ 51 , 76, 43]. At high concentrations, several 2ME anti-proliferative mechanisms have been reported [64], and microarray analysis has reported the transcriptional effects of 2ME in breast cancer cells [84, 83]. The most commonly reported effects of 2ME are microtubule disruption, inhibition of angiogenesis, upreg- ulation of apoptosis, and cell cycle arrest. These and other mechanisms of action are summarized in the sections below. Mechanisms of Action Microtubule Disruption The growth-inhibitory properties of 2ME have been linked to its effects on tubulin polymerization; in fact, recent structural activity analyses suggest that targeting of microtubules is the main mechanism for the anti-proliferative and pro-apoptotic activities of 2ME [ 13]. Destabilization of microtubules also causes cell cycle arrest [28, 14, 59]. As mentioned previously, 2ME suppressed HIF-1α levels. In ovarian cancer cells, HIF-1α suppression by 2ME occurs at both protein levels and its transcriptional activity, and this correlates to a decrease in tubulin polymerization [24]. Mech- anistically, 2ME-induced downregulation of HIF-1α occurs at the post-transcriptional level; HIF-1 α suppression occurs downstream from the interaction with tubulin; this establishes a mechanistic link between the disruption of the microtubule cytoskeleton and the inhibition of angiogenesis [60]. Inhibition of Angiogenesis In vitro studies using EA.hy926 cells demonstrate a decrease in the formation of capillary-like structures upon 2ME treat- ment [1]. A possible mechanism for 2ME inhibition may be through the suppression of HIF-1α, since VEGF is a HIF-1 α target gene suppression of HIF-1a activity will reduce VEGF activity (at mRNA, protein, and secreted levels). Supporting this idea, VEGF secretion is inhibited by 2ME in a dose- dependent manner under both normal and hypoxic conditions. 276 HORM CANC (2014) 5:274–283 Furthermore, 2ME reduce expression, nuclear accumulation, and transcriptional activity of HIF-1 α [60]. Interestingly, the anti-angiogenic activity of 2ME seems to operate indepen- dently from its direct effects on the endothelium; when 2ME is added to breast cancer cells for 48 hr, the resulting conditioned media also inhibits the formation of capillary-like structures in endothelial cells suggesting that 2ME is capable of stimulating breast cancer cells to produce factors that inhibit endothelial cell remodeling [75]. Promotion of Cell Death: Apoptosis and Autophagy In vitro and in vivo studies in human prostate and breast carcinoma cells demonstrate that 2ME induces apoptosis [ 8]. In granulosa cells, 2ME inhibits superoxide dismutase (SOD) enzyme activity [ 5]. In another study, 2ME was shown to induce apoptosis in ovarian cancer but not in normal cells via activation of both the intrinsic and the extrinsic apoptotic pathways. 2ME-mediated apoptosis involved the production of ROS and the activation of both caspase-dependent and caspase-independent pathways. A synergistic apoptotic re- sponse was also reported when 2ME was administered with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) [50]. These data confirm previous results showing that primary human leukemia cells and primary ovarian cancer cells are more sensitive to 2ME than their normal counter- parts, possibly due to the accumulation of ROS [ 37]. Along with apoptosis, 2ME can trigger autophagy. Breast cancer MCF-7 cells treated with a 2ME derivate (2- methoxyestradiol-bis-sulfamate) suffer an increase in lyso- somal staining indicative of autophagy [ 93]; however, this does not occur in the non-tumorigenic MCF-12A cell line [94]. Studies using the 2ME analog ESE-16 demonstrate induction of a crosstalk mechanism that causes apoptosis and autophagic cell death [87]. In human osteosarcoma cells, 2ME induces autophagy (shown by the conversion of the microtubule-associated protein LC3-I to LC3-II), a process not observed in normal (non-cancerous) primary human oste- oblasts [97]. Cell Cycle Arrest As a mechanism of action, 2ME also causes cell cycle arrest at G2/M phase of the cell cycle [74, 50]. This effect appears to be specific to cancer cells, or at least to epithelial cells, as treat- ment of fibroblasts or human umbilical cord vascular endo- thelial cells (HUVEC) with 2ME did not cause G2/M arrest or morphological changes [ 39]. A study in breast cancer cells demonstrated that cell cycle arrest occurs specifically at the prometaphase and is mediated by an upregulation of cyclin B1 and Cdc-2 [ 12]. In esophageal carcinoma cells, 2ME also causes upregulation in cyclin B1 and c-Myc along with G2/ M arrest and subsequent apoptosis [ 19]. In contrast, in endometrial cancer cells, 2ME causes downregulation of cy- clin B1 and phosphorylated Cdc-2 and upregulation of p21 WA F1 /C i p 1that correlates with G2/M arrest and p53 activa- tion [31]. Anti-progestin and Anti-estrogen Hormone signaling pathways are known to act through posi- tive and negative feedbacks. The progesterone receptor (PR) is upregulated by estrogen; however, progesterone-bound PR inhibits estrogen action in the endometrium. Thus, it is not surprising that estrogen metabolites antagonize estrogen ac- tion and known estrogen targets such as the PR. Although

are difficult to interpret (due to its cytotoxicity), 2ME inhibits estrogen-stimulated cell growth in human ovarian cancer OVCAR-3 cells and can block progesterone signaling in the ZR-75-1 breast cancer cell line [ 75]. In the later exam- ple, 2ME was not considered an anti-progestin; however, at micromolar concentrations, it did inhibit progesterone- induced and PR-dependent coagulation and invasion. The mechanism was shown to involve the inhibition of tissue factor (TF) protein, the cellular activator of the coagulation cascade that is also required for cancer cell invasion [ 49]. Signaling Pathway Phosphorylation 2ME can reduce phosphorylation of extracellular signal- regulated kinases 1/2 (ERK1/2) or mitogen-activated protein kinases (MAPKs), yet does not alter the phosphorylation of p38 or Akt. In breast cancer cells, 2ME causes phosphoryla- tion of PR on several serine residues targeted by MAPKs [75]. In lung cancer cells, 2ME-induced radiosensitization is de- pendent on inhibition of Akt and DNA-PKcs pathways but independent of SOD inhibition [ 27]. In ovarian cancer cells [9] and retinoblastoma [ 63], p38 phosphorylation is essential for the pro-apoptotic effect of 2ME. Furthermore, in prostate, breast, liver, and colorectal carcinoma cell lines, 2ME causes activation of c-Jun N-terminal kinase (JNK) and phosphory- lation of Bcl-2, which preceded the induction of apoptosis [9, 16]. The activation of JNK, ERK1/2, and p38 by 2ME has also been reported in MDA-MB-435 breast cancer cells [ 29]. These results demonstrate the cell-specific nature of 2ME action. Is the Estrogen Receptor Required? In ovarian and endometrial cancer cell lines, 2ME induces apoptosis independently of estrogen receptor (ER) presence [51, 50]. 2ME also triggers apoptosis in the ER-negative breast cancer cell line MDA-MB-435 [ 29]. Despite this, mi- croarray studies on breast cancer cells demonstrate that the use of a pure ER antagonist alters 2ME gene regulation [84]. This observation is consistent with previous studies showing that HORM CANC (2014) 5:274–283 277 2ME can bind to the ER (albeit at a significantly lower affinity than 17 β-estradiol) [ 7]. The ability of 2ME to inhibit the growth of ER-negative tumors increases its attractiveness for a therapeutic use in a wider spectrum of cancer patients. Clinical Use Bioavailability for Cancer Use The main limitation for the clinical use of 2ME is its poor water solubility and its low bioavailability [11]. A study using an in situ intestinal recirculation perfusion model in rats showed that 2ME concentrations had no influence on the absorption rate constant [32]. Glucuronidation and subsequent urinary excretion have been reported as a mechanism for 2ME elimination [55]; urine samples from cancer patients showed that <0.01 % of the administered dose of 2ME was excreted unchanged into the urine and approximately 1 % was excreted as glucuronides. Attempts to overcome the limited bioavailability have been made by developing other formulations such as nanosuspensions and poly (organophosphazenes) that act as injectable carriers [17, 11, 21]. In the latter example, a hydro- gel containing a relatively low concentration of 2ME demon- strated improved anti-tumor and anti-angiogenic activity in a mouse orthotopic breast tumor model relative to the traditional delivery method [ 11]. A recent study used 2ME in coated nanoparticles that were administered via inhalation to lungs in rats; results showed that nanoparticles effectively delivered 2ME to lungs enhancing its cytotoxicity without obvious tissue inflammation; demonstrating this method has the po- tential to become an effective and safe treatment of lung cancer [34]. Lipid 2ME nanoparticles have also been used to increase cytotoxicity upon breast cancer, prostate cancer, and glioma cells [ 33]; 2ME liposomes significantly suppressed growth of murine hepatocarcinoma solid tumors [18]. Analogs and Derivatives of 2ME Another approach to overcome the problem of low plasma availability is to engineer modifications into the structure of 2ME to increase its half-life and lower excretion. In adult female rats, where the presence of 2ME is extremely low, the bioavailability of the 2ME derivate 2-methoxyestradiol- 3, 17-bis-sulfamate (2-MeOE2bisMA TE) was reported to reach 85 %. Interestingly, no significant quantities of 2- MeOE2bisMA TE metabolites were detected in plasma after oral or intravenous dosing [44], indicating that this compound is not extensively metabolized. Examining the potency of these metabolites, reports aimed at studying the inhibition of angiogenesis showing that unmodified 2ME at the micromolar range of concentrations causes a mild reduction in tubule formation, while picomolar levels of derivatives 2- MeOE2 bis-sulfamate and 2-EtE2 sulfamate completely abol- ish this process [69]. Recently, a new synthetic 2ME analog (named (8R, 13S, 14S, 17S)-2ethyl-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17- decahydro-6H-cyclopenta(a)phenanthrane-3, 17diyl-bis (sulfamate) or EMBS) effectively suppressed proliferation and induced apoptosis in tumorigenic and non-tumorigenic breast cell lines in vitro [92]. Another analog, named ESE-16, (2-ethyl-3-O-sulfamoyl-estra-1,3,5 (10)16-tetraene), is anti- proliferative on cervical adenocarcinoma cells in culture [87]. Another study shows that three sulfamoylated 2ME analogs trigger apoptosis (via the intrinsic pathway) and tu- bulin depolymerization in HeLa and breast cancer MDA-MB- 231 cells; these compounds reduced cell numbers to 50 % when used at 0.5 μM[ 95]. Collectively, some of these 2ME analogs are currently categorized as second-generation steroid sulfatase (STS) inhibitors (because they are STS and tubulin polymerization inhibitors), and they are characterized by their cytostatic, cytotoxic, and anti-angiogenic properties (for a review see [35]). Clinical Trials S e v e r a lc l i n i c a lt r i a l s( p h a s e sIa n dI I )i n v o l v i n g2 M Ei na variety of tumors and cancers have been completed (NIH, USA www.clinicaltrials.gov ). In a phase I study of 20 patients with solid tumors, the maximum-tolerated dose was not reached even at a dose of 3000 mg bid, treatment had no effect on microvessel density or cell proliferation, and the trial was closed due to extremely low plasma concentrations rela- tive to the administered doses [ 15]. Another phase I study used the 2ME NanoCrystal dispersion (2ME-NCD) formula- tion in 16 patients with refractory solid tumors [ 86]; dose- limiting toxicities included hypophosphatemia (two patients), fatigue (two patients), muscle weakness (one patient), and increased alanine aminotransferase (one patient); in spite of this, treatment was generally well tolerated. In this study, the maximum tolerated dose was determined to be 1,000 mg orally every 6 h. Thirteen patients had stable disease, but there were no confirmed responses. Another trial on 18 patients with platinum-resistant ovarian cancer and primary peritoneal carcinomatosis who received 2ME-NCD 1,000 mg orally four times daily reported that the treatment was well tolerated; there were no objective responses, but seven patients had stable disease as best response. Of those, two had stable disease for more than 12 months [ 62]. A phase I study used the 2ME analog ENMD-1198 in advanced cancer patients and showed that the most common drug-related toxicities were maximum grade 2 fatigue (55 %), nausea/vomiting (37 %), and constipation (34 %). However, 278 HORM CANC (2014) 5:274–283 grade 4 neutropenia (abnormally low number of neutrophils) was observed on two patients, and a maximum tolerated dose was declared. Disease stabilization was observed in five patients, of which a neuroendocrine carcinoma of pancreas, a prostate cancer, and an ovarian cancer patient demonstrated stable dis- ease from eight to 24.5 cycles of ENMD-1198 therapy (from 7 months and 3 weeks to 22 months and 3 weeks). The authors concluded that the analog ENMD-1198 was well tolerated and worthy of additional investigation [98]. In a phase II study with taxane-refractory metastatic castrate-resistant prostate cancer patients, investigators report- ed that 2ME-NCD was well tolerated and showed some evidence of biologic activity but did not appear to have clinically significant activity [ 36]. The study was terminated after only 21 of the projected 50 patients were enrolled, as futility analysis predicted that the primary endpoint of progression-free survival at 6 months was unlikely to be reached. In general, these trials indicate that 2ME is well tolerated; however, there appears to be problems with efficacy, and thus, it may be too soon to evaluate possible long-term side effects. 2ME in Combination Therapy Although clinical trials with 2ME as a single agent report mixed results, the use of 2ME as an adjuvant in cancer has proved more promising. In breast cancer cell lines, combined treatment with 2ME and the anti-estrogen tamoxifen gave favorable results. Interestingly, both 2ME and tamoxifen can act as aromatase gene inhibitors [79, 67]. In vitro, preexposure of endometrial and ovarian cancer cell lines to 2ME has shown to enhance their sensitivity to the apoptotic drug TRAIL [ 51, 50]. Standard chemotherapeutic drugs usually cause damage in both normal and cancer cells; therefore, a further advantage of the 2ME-TRAIL regime is that it en- hances apoptotic behavior in cancer cells maintaining cell viability in normal cells. We speculate that 2ME, alone or in combination with TRAIL, may be an effective treatment for cancers of uterine origin with minimal toxicity to correspond- ing healthy female reproductive tissue. Even more promising are the reports of 2ME enhancing the activity of certain chemotherapies at pharmacological relevant concentrations. Proliferation of MCF-7 breast cancer cells in vitro is significantly reduced by 2ME when added in combination with epirubicine, docetaxel, 5-fluoprouracil, mafosfamide, and carboplatin [ 67, 68]. Combined therapies consisting of 2ME with bort ezomib, arsenic trioxide, or albendazole effectively potentiated cell death in bortezomib- resistant myeloma [ 81], urothelial carcinoma cells [ 53], and colon carcinoma xenografts [23], respectively. The effects of 2ME analogs and its derivatives are not limited to a longer half-life; they also display effects not observed with the endogenous hormone: In fibroblasts, treatment with 2- MeOE2bisMA TE causes a morphological change and induces G2/M arrest but not apoptosis, an effect not observed with 2ME [ 39]. These new effects (possibly related to potency) need to be considered in experimental designs if these deriv- atives are to be incorporated into future clinical trials along with chemotherapeutic agents. The Future for 2ME in the Clinic Our knowledge of the involvement of 2ME in reproductive physiology is still in its infancy. However, the observation that 2ME is involved in the fidelity of follicular development, oocyte transport in the Fallopian tube, and cytotrophobast invasion may open the gates to COMT or 2ME as targets in the future treatment of reproductive abnormalities. Given the observation that pregnant women with preeclampsia have lower levels of 2ME than normal counterparts, there may be an important future role for COMT and 2ME in the treatment of this disorder [48, 91]. Lower 2ME levels have been report- ed months before the clinical manifestation of preeclampsia, suggesting the potential of 2ME for both prediction and pre- vention [ 73, 72]. The observation that 2ME suppressed growth of endometriosis-like lesions also suggests its potential as a treatment against this disorder [ 61, 6]. Although not related directly to reproductive disorders, 2ME through its ability of induce production of nitric oxide (via eNOS) may possess anti-thrombogenic properties that could alleviate ath- erosclerotic symptoms. Furthermore, interventions in hyper- tension, pulmonary hypertension, glomerulosclerosis, and brain injury have been speculated for this metabolite [ 90, 96, 54, 22, 91]. In cancer, the adjuvant use of 2ME may offer a new clinically relevant treatment regime for hormone- dependent and hormone-independent cancers. As phase II clinical trials have demonstrated, 2ME is generally well toler- ated and the combination with chemotherapeutic agents or anti-estrogens (such as tamoxifen) may offer a new alternative against neoplasms of female reproductive tissues [45, 62, 66]. So, the wheel seems to have come full circle for this estrogen metabolite. Starting off its clinical life as a native compound, given as an independent agent, then as an analog, before manifesting as a host of modified derivatives, to finally return to the now likely clinically relevant scenario of the native compound being administrated along with convention- al treatments to offer clinically relevant regimens for cancer and other reproductive disorders. Conflict of Interest and Disclosure The authors have no conflicts of interest or disclosures to declare. Contract Grant Sponsor BMRC CTU06 13CTI-21526, FONDAP ACCDis 15130011, FONDECYT grants 1100870 and 1140970. HORM CANC (2014) 5:274–283 279

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