{"paper_id":"382c6780-b00e-4e87-8ba0-afbd4559fcbb","body_text":"REVIEW\n2-Methoxyestradiol and Disorders of Female\nReproductive Tissues\nMauricio P. Pinto & Rodolfo A. Medina & Gareth I. Owen\nReceived: 9 April 2014 / Accepted: 16 April 2014 / Published online: 25 April 2014\n# Springer Science+Business Media New Y ork 2014\nAbstract 2-Methoxyestradiol (2ME) is an endogenous me-\ntabolite of 17β-estradiol. Once thought of as a mere degrada-\ntion product, 2ME has gained attention as an important com-\nponent of reproductive physiology and as a therapeutic agent\nin reproductive pathologies such as preeclampsia, endometri-\nosis, infertility, and cancer. In this review, we discuss the\ninvolvement of 2ME in reproductive pathophysiology and sum-\nmarize its known mechanisms of action: microtubule disruption,\ninhibition of angiogenesis and stimulation of apoptosis. Current-\nly, the clinical uses of 2ME as a single agent are limited due to its\npoor water solubility and thus low bioavailability; however,\n2ME analogs and derivatives ha ve been recently developed\nand tested as cancer treatments. Despite some isolated success\nstories and ongoing research, 2ME derivatives have not yet\nprovided the expected results. The adjuvant use of 2ME deriv-\natives with chemotherapeutic agents is hindered by their intrinsic\ntoxicity confounding the unwanted secondary effects of chemo-\ntherapy. However, due to the well-tested tolerance of the body to\nhigh doses of native 2ME, it may the combination of native\n2ME with conventional treatments that will offer novel clinical-\nly relevant regimens for cancer and other reproductive disorders.\nIntroduction\nFemale reproductive potential starts at menarche and ceases at\nthe menopause. In a woman ’s life, these two stages and the\nprocesses in between are determined by the female sex hor-\nmones. The reproductive cycle is governed by a combination\nof hormones produced at varying levels throughout the men-\nstrual cycle. Previously, the concept was simple: estrogen and\nprogesterone (the “ovarian” hormones) along with other hor-\nmones such as oxytocin and prolactin among others played\nspecific roles when required. However, studies in reproductive\nphysiology during the last decade have demonstrated that\nthese hormones are only part of the story, and now their\nmetabolites, previously believed to be intermediates on the\nroad to degradation and excretion, are in fact important\nplayers in both reproductive physiology and pathology. A\nvariety of reproductive disorders can disrupt the balance of\nthese hormones, altering the functions of reproductive organs,\nincluding the ovaries, Fallopian tubes, uterus, cervix, vagina,\nvulva, and, at least for the purpose of this review, breast.\nAmenorrhea, pelvic inflammatory disease, preeclampsia, en-\ndometriosis, infertility, and cancer are among these disorders.\nEstrogen Metabolism and the Origin\nof 2-Methoxyestradiol\n2-Methoxyestradiol (2ME) is a naturally occurring metabolite\nof estradiol. Estrogens can be metabolized into their interme-\ndiaries by members of the cytochrome P450 (Cyp450) family;\nspecifically, CYP1A1 and CYP1B1 are able to convert estro-\ngens into water-soluble metabolites such as 2-\nhydroxyestradiol and 4-hydroxyestradiol; subsequently 2ME\ncan be generated by the catechol- O-methyl-transferase\n(COMT) [ 82]. Under physiological conditions, blood 2ME\nconcentrations in women range from 46 to 70 pg/ml [ 66].\nM. P . Pinto: G. I. Owen (*)\nDepartamento de Fisiología, Facultad de Ciencias Biológicas,\nPontificia Universidad Católica de Chile, Santiago, Chile\ne-mail: gowen@bio.puc.cl\nR. A. Medina\nFacultades de Ciencias Biológicas y Medicina, Universidad Andrés\nBello, Santiago, Chile\nG. I. Owen\nCentro UC Investigación en Oncologia (CITO), Pontificia\nUniversidad Católica de Chile, Alameda 340, Santiago, Chile\nG. I. Owen\nAdvanced Center for Chronic Diseases (ACCDiS), Pontificia\nUniversidad Católica de Chile, Alameda 340, Santiago, Chile\nHORM CANC (2014) 5:274–283\nDOI 10.1007/s12672-014-0181-2\n\nA Role for 2ME in Reproductive Physiology\nScientific analysis of any malfunction or disorder dictates that\nthe best place to start is to understand the physiology of the\nprocess and from this vantage point elucidate the cause of the\npathology. The concept that estrogen is metabolized into\nanother active compound (perhaps a future hormone), namely\n2ME, was given strength by the observation that endogenous\n2ME accelerates oocyte transport in the rat oviduct [ 70].\nAlthough yet to be confirmed in humans, this study also\ndemonstrated that mating halted 2ME effect, with the role of\nbringing the oocyte down the Fallopian tube (oviduct) taken\nover by the parental hormone estrogen. 2ME has since been\nshown to increase overall ovarian weight and has been detect-\ned in follicular fluid [ 4, 77]. In follicular fluid, 2ME is spec-\nulated to act as a growth inhibitor of ovulatory follicles (by\ninhibiting angiogenesis) and steroidogenesis [4, 77]. Interest-\ningly, low doses of 2ME stimulate granulosa cell proliferation,\nwhile higher doses are inhibitory [77].\n2ME is now demonstrated to be a natural component of\nmaternal blood, cord blood, breast milk, and amniotic fluid\n[3]. In ovine uterine artery endothelial cells derived from\npregnant ewes, 2ME increased prostacyclin production in a\nconcentration- and time-dependent manner [46]. In the same\nmodel, 2ME treatment was also demonstrated to be anti-\nangiogenic [78]. 2ME induces invasion of the cytotrophoblast\nthrough naturally derived extracellular matrix and thus maybe\nrequired to facilitate both appropriate vascular development\nand oxygenation during pregnancy [56]. Elevated 2ME levels\nare present during the late stages of pregnancy, during which\ntime breast ductal tissue has differentiated to form alveolar\nmilk-producing structures. In virgin mice, 2ME has been\nshown to induce mammary ductal dilation and partial mam-\nmary gland differentiation [42]. These observations reinforce\nthe concept that the 2ME is not merely a degradation product\nof estradiol but in fact is an independent natural and integra-\ntive part of female reproductive tract regulation.\nA Role for 2ME in Reproductive Pathology\nReproductive pathologies affect women during their repro-\nductive years causing in extreme cases of infertility and preg-\nnancy loss along with complications in menstruation, concep-\ntion, labor, and menopausal transition. Endometriosis is char-\nacterized by the presence of ectopic tissue outside the uterine\ncavity and affects 6–8 % of women during their reproductive\nyears and is responsible for a high incidence of infertility [10].\nA mouse model study using en dometriosis-like lesions\nshowed that these lesions are characterized by hypoxia,\nresulting in upregulation of the hypoxia-inducible factor 1a\n(HIF-1α), and the induction of angiogenesis via the vascular\nendothelial growth factor (VEGF); systemic treatment of these\nanimals with 2ME suppressed the growth of endometriosis-\nlike lesions, reduced HIF-1α and VEGF expression levels and\nvascular permeability [6]. The authors speculate that 2ME acts\nthrough a dual mechanism: an indirect effect by HIF-1 α\ninhibition, leading to VEGF suppression decreasing angio-\ngenesis, and a direct effect via an inhibition of endothelial cell\nfunction [6].\nPreeclampsia is a hypertensive disorder of pregnancy char-\nacterized by placental hypoxia, proteinuria, and fluid reten-\ntion. Preeclampsia affects approximately 5 % of all pregnan-\ncies and remains a leading cause of maternal and fetal mor-\nbidity and mortality [ 80]. In several pregnancy disorders,\nincluding preeclampsia, inadequate cytotrophoblast invasion\nof the uterus occurs. In vitro studies have demonstrated that\ncytotrophoblast cells treated with 2ME switch to an invasive\nphenotype when cultured under low oxygen conditions; 2ME\ntreatment 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\nbeta-3 (TGFβ3), and tissue inhibitor of metalloproteinases-2\n(TIMP-2) in these cells. The same study demonstrates in vivo\nthat placentas of COMT\n−/− mice (2ME deficient) revert their\npreeclampsia features (higher levels of HIF-1α,T G Fβ3, and\nTIMP-2) after 2ME administration [ 56]. In patients, a recent\nstudy demonstrated that pregnant women that eventually de-\nveloped preeclampsia displayed significantly lower levels of\nplasma 2ME at 11–14 weeks [73]. Further reports suggest that\nthe reduction in 2ME synthesis in preeclampsia patients could\nbe explained by alterations in the methionine-homocysteine\nmetabolism or the 17β-estradiol synthesis pathway [72].\nPolycystic ovary syndrome (PCOS) is an endocrine disor-\nder that affects 5 –8 % of women of reproductive age. The\nfeatures of PCOS are hyperandrogenemia, chronic\nanovulation, and infertility. In granulosa cells, COMT expres-\nsion is upregulated by the major contributors to PCOS: insu-\nlin, dihydrotestosterone, and all-trans retinoic acid, suggesting\na role for the dysregulation of 2ME in the development of\nPCOS and ovulatory dysfunction. Although a genetic analysis\nof COMT has not been correlated, prolactin levels in women\nwith PCOS varied significantly with COMT haplotypes [ 77,\n38]. Future studies are required to determine the role of steroid\nhormone metabolites in the abovementioned reproductive\npathologies, together with pathologies that remain to be ex-\namined in the light of 2ME action, such as amenorrhea, pelvic\ninflammatory disease, ectopic pregnancy, pregnancy loss, and\ntransitional problems at menopause. However, by far, the most\nreported mention of 2ME in the medical literature comes in\nrelation to cancer and its potential therapeutic use.\n2ME and Cancer of the Reproductive Tissues\nThe balance between 2ME and other estrogen metabolites\ncould play a role in carcinogenesis [ 47]; 2-hydroxyestradiol\nand 4-hydroxyestradiol are endogenous estradiol metabolites\nHORM CANC (2014) 5:274–283 275\n\nthat increase proliferation and the formation of reactive oxy-\ngen species (ROS) [ 71, 30, 65]; in contrast, 2ME has anti-\nproliferative effects in reproductive tissues. In human breast\ncancer biopsies, estrogen 2-hydroxylase and COMT displayed\nhigher levels in tumors and benign neoplasms compared to\nnormal breast tissue, suggesting a role for these metabolites in\nthe bourgeoning cancer cell [ 40]. CYP1A1, CYP1B1, and\nCOMT polymorphisms can bring about toxic levels of 4-\nhydroxyestradiol and 2ME that may add to an increased risk\nof ovarian cancer [ 41]. Polymorphisms in COMT (the 2ME-\nconverting enzyme) are linked to numerous disorders, includ-\ning altered prefrontal dopaminergic metabolism, fibromyalgia\nsyndrome, Alzheimer’s disease, depression and suicide, ma-\nternal stress, and emotional and behavioral problems in chil-\ndren [ 52, 26, 57, 20, 88]. A specific V al158Met COMT\npolymorphism has been associated with a decreased risk of\nuterine leiomyoma [ 25]. In cancers, a COMT V al/V al geno-\ntype is correlated with an increase in endometrial cancer risk\n[58], while the V al158Met polymorphism is associated to a\ndecreased risk in an Asian population [85]. In a separate study,\nV al/Met genotype was associated with an increased risk of\ndeveloping endometrial/ovarian cancer, while a homozygous\nmutant (Met/Met) demonstrated a decreased risk in Australian\nand Polish patients [2]. The V al158Met COMT polymorphism\nhas been postulated as a cancer risk factor in the Chinese\npopulation; however, some studies have stated that it is not a\nbreast cancer risk factor in Asian population [ 89]. The corre-\nlation between COMT polymorphisms (and mutations) and\ncancer risk still needs further study; however, the evidence\nsuggests a high dependency on ethnicity.\nDespite these isolated reports, most studies have focused\non cancer and 2ME. Therefore in the next sections, we will\ndiscuss on the effects of 2ME on reproductive tract cancers\n(including breast) and its potential therapeutic applications.\n2ME and Cancer Treatment\nIn 2012, there were an estimated of 14.1 million cancer cases\nworldwide, of these 6.7 were in women. Cancers of female\nreproductive tissues combined (ovary, endometrium, cervix,\nand breast) accounted for 41.5 % of female cancer incidence\n(World Cancer Research Fund international 2014:www.wcrf.\norg).\nIn cancer, 2ME is a powerful anti-angiogenic, anti-\nproliferative, and pro-apoptotic agent [28]; this includes can-\ncers of reproductive tissues (such as cervical ovarian, endo-\nmetrial, and breast cancer) and others (e.g., lung, colon, kid-\nney, prostate and esophagus, stomach, and pancreas among\nothers) [66]. In contrast, normal and non-proliferating tumor\ncells appear to be more resistant to the anti-proliferative effects\nof 2ME [51, 50].\nBoth in vitro and clinical studies consistently show that\nhigh circulating levels of 2ME are required (approaching and\nsurpassing micromolar) for its anti-tumorigenic effects [ 75,\n44]. During pregnancy, 2ME levels reach their highest with\ncirculating levels that range from 2 to 10 ng/ml [66]. Thus, the\nconcentrations of 2ME (and its more stable derivatives) far\nexceed these values, and thus, their effects may differ from the\nphysiological effects of 2ME discussed in the previous sec-\ntions. An exception to this might be the follicular fluid ex-\ntracted from the ovary. Here, at the site of estradiol production,\nthe 2ME levels reach the micromolar range [ 66, 75]. In\naddition to its effects as a single agent, the combination of\n2ME with other anti-tumorigenic agents has been shown to\ncause a synergistic inhibition of cancer cell proliferation [ 51\n,\n76, 43]. At high concentrations, several 2ME anti-proliferative\nmechanisms have been reported [64], and microarray analysis\nhas reported the transcriptional effects of 2ME in breast cancer\ncells [84, 83]. The most commonly reported effects of 2ME\nare microtubule disruption, inhibition of angiogenesis, upreg-\nulation of apoptosis, and cell cycle arrest. These and other\nmechanisms of action are summarized in the sections below.\nMechanisms of Action\nMicrotubule Disruption\nThe growth-inhibitory properties of 2ME have been linked to\nits effects on tubulin polymerization; in fact, recent structural\nactivity analyses suggest that targeting of microtubules is the\nmain mechanism for the anti-proliferative and pro-apoptotic\nactivities of 2ME [ 13]. Destabilization of microtubules also\ncauses cell cycle arrest [28, 14, 59].\nAs mentioned previously, 2ME suppressed HIF-1α levels.\nIn ovarian cancer cells, HIF-1α suppression by 2ME occurs at\nboth protein levels and its transcriptional activity, and this\ncorrelates to a decrease in tubulin polymerization [24]. Mech-\nanistically, 2ME-induced downregulation of HIF-1α occurs at\nthe post-transcriptional level; HIF-1 α suppression occurs\ndownstream from the interaction with tubulin; this establishes\na mechanistic link between the disruption of the microtubule\ncytoskeleton and the inhibition of angiogenesis [60].\nInhibition of Angiogenesis\nIn vitro studies using EA.hy926 cells demonstrate a decrease\nin the formation of capillary-like structures upon 2ME treat-\nment [1]. A possible mechanism for 2ME inhibition may be\nthrough the suppression of HIF-1α, since VEGF is a HIF-1 α\ntarget gene suppression of HIF-1a activity will reduce VEGF\nactivity (at mRNA, protein, and secreted levels). Supporting\nthis idea, VEGF secretion is inhibited by 2ME in a dose-\ndependent manner under both normal and hypoxic conditions.\n276 HORM CANC (2014) 5:274–283\n\nFurthermore, 2ME reduce expression, nuclear accumulation,\nand transcriptional activity of HIF-1 α [60]. Interestingly, the\nanti-angiogenic activity of 2ME seems to operate indepen-\ndently from its direct effects on the endothelium; when 2ME is\nadded to breast cancer cells for 48 hr, the resulting conditioned\nmedia also inhibits the formation of capillary-like structures in\nendothelial cells suggesting that 2ME is capable of stimulating\nbreast cancer cells to produce factors that inhibit endothelial\ncell remodeling [75].\nPromotion of Cell Death: Apoptosis and Autophagy\nIn vitro and in vivo studies in human prostate and breast\ncarcinoma cells demonstrate that 2ME induces apoptosis [ 8].\nIn granulosa cells, 2ME inhibits superoxide dismutase (SOD)\nenzyme activity [ 5]. In another study, 2ME was shown to\ninduce apoptosis in ovarian cancer but not in normal cells\nvia activation of both the intrinsic and the extrinsic apoptotic\npathways. 2ME-mediated apoptosis involved the production\nof ROS and the activation of both caspase-dependent and\ncaspase-independent pathways. A synergistic apoptotic re-\nsponse was also reported when 2ME was administered with\ntumor necrosis factor-related apoptosis-inducing ligand\n(TRAIL) [50]. These data confirm previous results showing\nthat primary human leukemia cells and primary ovarian cancer\ncells are more sensitive to 2ME than their normal counter-\nparts, possibly due to the accumulation of ROS [ 37]. Along\nwith apoptosis, 2ME can trigger autophagy. Breast cancer\nMCF-7 cells treated with a 2ME derivate (2-\nmethoxyestradiol-bis-sulfamate) suffer an increase in lyso-\nsomal staining indicative of autophagy [ 93]; however, this\ndoes not occur in the non-tumorigenic MCF-12A cell line\n[94]. Studies using the 2ME analog ESE-16 demonstrate\ninduction of a crosstalk mechanism that causes apoptosis\nand autophagic cell death [87]. In human osteosarcoma cells,\n2ME induces autophagy (shown by the conversion of the\nmicrotubule-associated protein LC3-I to LC3-II), a process\nnot observed in normal (non-cancerous) primary human oste-\noblasts [97].\nCell Cycle Arrest\nAs a mechanism of action, 2ME also causes cell cycle arrest at\nG2/M phase of the cell cycle [74, 50]. This effect appears to be\nspecific to cancer cells, or at least to epithelial cells, as treat-\nment of fibroblasts or human umbilical cord vascular endo-\nthelial cells (HUVEC) with 2ME did not cause G2/M arrest or\nmorphological changes [ 39]. A study in breast cancer cells\ndemonstrated that cell cycle arrest occurs specifically at the\nprometaphase and is mediated by an upregulation of cyclin B1\nand Cdc-2 [ 12]. In esophageal carcinoma cells, 2ME also\ncauses upregulation in cyclin B1 and c-Myc along with G2/\nM arrest and subsequent apoptosis [ 19]. In contrast, in\nendometrial cancer cells, 2ME causes downregulation of cy-\nclin B1 and phosphorylated Cdc-2 and upregulation of\np21\nWA F1 /C i p 1that correlates with G2/M arrest and p53 activa-\ntion [31].\nAnti-progestin and Anti-estrogen\nHormone signaling pathways are known to act through posi-\ntive and negative feedbacks. The progesterone receptor (PR)\nis upregulated by estrogen; however, progesterone-bound PR\ninhibits estrogen action in the endometrium. Thus, it is not\nsurprising that estrogen metabolites antagonize estrogen ac-\ntion and known estrogen targets such as the PR. Although\nresults are difficult to interpret (due to its cytotoxicity), 2ME\ninhibits estrogen-stimulated cell growth in human ovarian\ncancer OVCAR-3 cells and can block progesterone signaling\nin the ZR-75-1 breast cancer cell line [ 75]. In the later exam-\nple, 2ME was not considered an anti-progestin; however, at\nmicromolar concentrations, it did inhibit progesterone-\ninduced and PR-dependent coagulation and invasion. The\nmechanism was shown to involve the inhibition of tissue\nfactor (TF) protein, the cellular activator of the coagulation\ncascade that is also required for cancer cell invasion [ 49].\nSignaling Pathway Phosphorylation\n2ME can reduce phosphorylation of extracellular signal-\nregulated kinases 1/2 (ERK1/2) or mitogen-activated protein\nkinases (MAPKs), yet does not alter the phosphorylation of\np38 or Akt. In breast cancer cells, 2ME causes phosphoryla-\ntion of PR on several serine residues targeted by MAPKs [75].\nIn lung cancer cells, 2ME-induced radiosensitization is de-\npendent on inhibition of Akt and DNA-PKcs pathways but\nindependent of SOD inhibition [ 27]. In ovarian cancer cells\n[9] and retinoblastoma [ 63], p38 phosphorylation is essential\nfor the pro-apoptotic effect of 2ME. Furthermore, in prostate,\nbreast, liver, and colorectal carcinoma cell lines, 2ME causes\nactivation of c-Jun N-terminal kinase (JNK) and phosphory-\nlation of Bcl-2, which preceded the induction of apoptosis [9,\n16]. The activation of JNK, ERK1/2, and p38 by 2ME has\nalso been reported in MDA-MB-435 breast cancer cells [ 29].\nThese results demonstrate the cell-specific nature of 2ME\naction.\nIs the Estrogen Receptor Required?\nIn ovarian and endometrial cancer cell lines, 2ME induces\napoptosis independently of estrogen receptor (ER) presence\n[51, 50]. 2ME also triggers apoptosis in the ER-negative\nbreast cancer cell line MDA-MB-435 [ 29]. Despite this, mi-\ncroarray studies on breast cancer cells demonstrate that the use\nof a pure ER antagonist alters 2ME gene regulation [84]. This\nobservation is consistent with previous studies showing that\nHORM CANC (2014) 5:274–283 277\n\n2ME can bind to the ER (albeit at a significantly lower affinity\nthan 17 β-estradiol) [ 7]. The ability of 2ME to inhibit the\ngrowth of ER-negative tumors increases its attractiveness for\na therapeutic use in a wider spectrum of cancer patients.\nClinical Use\nBioavailability for Cancer Use\nThe main limitation for the clinical use of 2ME is its poor\nwater solubility and its low bioavailability [11]. A study using\nan in situ intestinal recirculation perfusion model in rats\nshowed that 2ME concentrations had no influence on the\nabsorption rate constant [32]. Glucuronidation and subsequent\nurinary excretion have been reported as a mechanism for 2ME\nelimination [55]; urine samples from cancer patients showed\nthat <0.01 % of the administered dose of 2ME was excreted\nunchanged into the urine and approximately 1 % was excreted\nas glucuronides.\nAttempts to overcome the limited bioavailability have been\nmade by developing other formulations such as\nnanosuspensions and poly (organophosphazenes) that act as\ninjectable carriers [17, 11, 21]. In the latter example, a hydro-\ngel containing a relatively low concentration of 2ME demon-\nstrated improved anti-tumor and anti-angiogenic activity in a\nmouse orthotopic breast tumor model relative to the traditional\ndelivery method [ 11]. A recent study used 2ME in coated\nnanoparticles that were administered via inhalation to lungs in\nrats; results showed that nanoparticles effectively delivered\n2ME to lungs enhancing its cytotoxicity without obvious\ntissue inflammation; demonstrating this method has the po-\ntential to become an effective and safe treatment of lung\ncancer [34]. Lipid 2ME nanoparticles have also been used to\nincrease cytotoxicity upon breast cancer, prostate cancer, and\nglioma cells [ 33]; 2ME liposomes significantly suppressed\ngrowth of murine hepatocarcinoma solid tumors [18].\nAnalogs and Derivatives of 2ME\nAnother approach to overcome the problem of low plasma\navailability is to engineer modifications into the structure of\n2ME to increase its half-life and lower excretion. In adult\nfemale rats, where the presence of 2ME is extremely low,\nthe bioavailability of the 2ME derivate 2-methoxyestradiol-\n3, 17-bis-sulfamate (2-MeOE2bisMA TE) was reported to\nreach 85 %. Interestingly, no significant quantities of 2-\nMeOE2bisMA TE metabolites were detected in plasma after\noral or intravenous dosing [44], indicating that this compound\nis not extensively metabolized. Examining the potency of\nthese metabolites, reports aimed at studying the inhibition of\nangiogenesis showing that unmodified 2ME at the\nmicromolar range of concentrations causes a mild reduction\nin tubule formation, while picomolar levels of derivatives 2-\nMeOE2 bis-sulfamate and 2-EtE2 sulfamate completely abol-\nish this process [69].\nRecently, a new synthetic 2ME analog (named (8R, 13S,\n14S, 17S)-2ethyl-13-methyl-7, 8, 9, 11, 12, 13, 14, 15, 16, 17-\ndecahydro-6H-cyclopenta(a)phenanthrane-3, 17diyl-bis\n(sulfamate) or EMBS) effectively suppressed proliferation\nand induced apoptosis in tumorigenic and non-tumorigenic\nbreast cell lines in vitro [92]. Another analog, named ESE-16,\n(2-ethyl-3-O-sulfamoyl-estra-1,3,5 (10)16-tetraene), is anti-\nproliferative on cervical adenocarcinoma cells in culture\n[87]. Another study shows that three sulfamoylated 2ME\nanalogs trigger apoptosis (via the intrinsic pathway) and tu-\nbulin depolymerization in HeLa and breast cancer MDA-MB-\n231 cells; these compounds reduced cell numbers to 50 %\nwhen used at 0.5 μM[ 95]. Collectively, some of these 2ME\nanalogs are currently categorized as second-generation steroid\nsulfatase (STS) inhibitors (because they are STS and tubulin\npolymerization inhibitors), and they are characterized by their\ncytostatic, cytotoxic, and anti-angiogenic properties (for a\nreview see [35]).\nClinical Trials\nS 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\nvariety of tumors and cancers have been completed (NIH,\nUSA www.clinicaltrials.gov ). In a phase I study of 20\npatients with solid tumors, the maximum-tolerated dose was\nnot reached even at a dose of 3000 mg bid, treatment had no\neffect on microvessel density or cell proliferation, and the trial\nwas closed due to extremely low plasma concentrations rela-\ntive to the administered doses [ 15]. Another phase I study\nused the 2ME NanoCrystal dispersion (2ME-NCD) formula-\ntion in 16 patients with refractory solid tumors [ 86]; dose-\nlimiting toxicities included hypophosphatemia (two patients),\nfatigue (two patients), muscle weakness (one patient), and\nincreased alanine aminotransferase (one patient); in spite of\nthis, treatment was generally well tolerated. In this study, the\nmaximum tolerated dose was determined to be 1,000 mg\norally every 6 h. Thirteen patients had stable disease, but there\nwere no confirmed responses. Another trial on 18 patients\nwith platinum-resistant ovarian cancer and primary peritoneal\ncarcinomatosis who received 2ME-NCD 1,000 mg orally four\ntimes daily reported that the treatment was well tolerated;\nthere were no objective responses, but seven patients had\nstable disease as best response. Of those, two had stable\ndisease for more than 12 months [ 62].\nA phase I study used the 2ME analog ENMD-1198 in\nadvanced cancer patients and showed that the most common\ndrug-related toxicities were maximum grade 2 fatigue (55 %),\nnausea/vomiting (37 %), and constipation (34 %). However,\n278 HORM CANC (2014) 5:274–283\n\ngrade 4 neutropenia (abnormally low number of neutrophils)\nwas observed on two patients, and a maximum tolerated dose\nwas declared. Disease stabilization was observed in five patients,\nof which a neuroendocrine carcinoma of pancreas, a prostate\ncancer, and an ovarian cancer patient demonstrated stable dis-\nease from eight to 24.5 cycles of ENMD-1198 therapy (from\n7 months and 3 weeks to 22 months and 3 weeks). The authors\nconcluded that the analog ENMD-1198 was well tolerated and\nworthy of additional investigation [98].\nIn a phase II study with taxane-refractory metastatic\ncastrate-resistant prostate cancer patients, investigators report-\ned that 2ME-NCD was well tolerated and showed some\nevidence of biologic activity but did not appear to have\nclinically significant activity [ 36]. The study was terminated\nafter only 21 of the projected 50 patients were enrolled, as\nfutility analysis predicted that the primary endpoint of\nprogression-free survival at 6 months was unlikely to be\nreached. In general, these trials indicate that 2ME is well\ntolerated; however, there appears to be problems with efficacy,\nand thus, it may be too soon to evaluate possible long-term\nside effects.\n2ME in Combination Therapy\nAlthough clinical trials with 2ME as a single agent report\nmixed results, the use of 2ME as an adjuvant in cancer has\nproved more promising. In breast cancer cell lines, combined\ntreatment with 2ME and the anti-estrogen tamoxifen gave\nfavorable results. Interestingly, both 2ME and tamoxifen can\nact as aromatase gene inhibitors [79, 67]. In vitro, preexposure\nof endometrial and ovarian cancer cell lines to 2ME has\nshown to enhance their sensitivity to the apoptotic drug\nTRAIL [ 51, 50]. Standard chemotherapeutic drugs usually\ncause damage in both normal and cancer cells; therefore, a\nfurther advantage of the 2ME-TRAIL regime is that it en-\nhances apoptotic behavior in cancer cells maintaining cell\nviability in normal cells. We speculate that 2ME, alone or in\ncombination with TRAIL, may be an effective treatment for\ncancers of uterine origin with minimal toxicity to correspond-\ning healthy female reproductive tissue.\nEven more promising are the reports of 2ME enhancing the\nactivity of certain chemotherapies at pharmacological relevant\nconcentrations. Proliferation of MCF-7 breast cancer cells\nin vitro is significantly reduced by 2ME when added in\ncombination with epirubicine, docetaxel, 5-fluoprouracil,\nmafosfamide, and carboplatin [ 67, 68]. Combined therapies\nconsisting of 2ME with bort ezomib, arsenic trioxide, or\nalbendazole effectively potentiated cell death in bortezomib-\nresistant myeloma [ 81], urothelial carcinoma cells [ 53], and\ncolon carcinoma xenografts [23], respectively. The effects of\n2ME analogs and its derivatives are not limited to a longer\nhalf-life; they also display effects not observed with the\nendogenous hormone: In fibroblasts, treatment with 2-\nMeOE2bisMA TE causes a morphological change and induces\nG2/M arrest but not apoptosis, an effect not observed with\n2ME [ 39]. These new effects (possibly related to potency)\nneed to be considered in experimental designs if these deriv-\natives are to be incorporated into future clinical trials along\nwith chemotherapeutic agents.\nThe Future for 2ME in the Clinic\nOur knowledge of the involvement of 2ME in reproductive\nphysiology is still in its infancy. However, the observation that\n2ME is involved in the fidelity of follicular development,\noocyte transport in the Fallopian tube, and cytotrophobast\ninvasion may open the gates to COMT or 2ME as targets in\nthe future treatment of reproductive abnormalities. Given the\nobservation that pregnant women with preeclampsia have\nlower levels of 2ME than normal counterparts, there may be\nan important future role for COMT and 2ME in the treatment\nof this disorder [48, 91]. Lower 2ME levels have been report-\ned months before the clinical manifestation of preeclampsia,\nsuggesting the potential of 2ME for both prediction and pre-\nvention [ 73, 72]. The observation that 2ME suppressed\ngrowth of endometriosis-like lesions also suggests its potential\nas a treatment against this disorder [ 61, 6]. Although not\nrelated directly to reproductive disorders, 2ME through its\nability of induce production of nitric oxide (via eNOS) may\npossess anti-thrombogenic properties that could alleviate ath-\nerosclerotic symptoms. Furthermore, interventions in hyper-\ntension, pulmonary hypertension, glomerulosclerosis, and\nbrain injury have been speculated for this metabolite [ 90, 96,\n54, 22, 91]. In cancer, the adjuvant use of 2ME may offer a\nnew clinically relevant treatment regime for hormone-\ndependent and hormone-independent cancers. As phase II\nclinical trials have demonstrated, 2ME is generally well toler-\nated and the combination with chemotherapeutic agents or\nanti-estrogens (such as tamoxifen) may offer a new alternative\nagainst neoplasms of female reproductive tissues [45, 62, 66].\nSo, the wheel seems to have come full circle for this\nestrogen metabolite. Starting off its clinical life as a native\ncompound, given as an independent agent, then as an analog,\nbefore manifesting as a host of modified derivatives, to finally\nreturn to the now likely clinically relevant scenario of the\nnative compound being administrated along with convention-\nal treatments to offer clinically relevant regimens for cancer\nand other reproductive disorders.\nConflict of Interest and Disclosure The authors have no conflicts of\ninterest or disclosures to declare.\nContract Grant Sponsor BMRC CTU06 13CTI-21526, FONDAP\nACCDis 15130011, FONDECYT grants 1100870 and 1140970.\nHORM CANC (2014) 5:274–283 279\n\nReferences\n1. Aranda E, Owen GI (2009) A semi-quantitative assay to screen for\nangiogenic compounds and compounds with angiogenic potential\nusing the EA.hy926 endothelial cell line. Biol Res 42(3):377–389\n2. 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