Is 2-Methoxyestradiol an Endogenous Estrogen Metabolite That Inhibits Mammary Carcinogenesis?

[CANCER RESEARCH 58. 2269-2277. June I. 1998] Perspectives in Cancer Research

Bao Ting Zhu1 and Allan H. Conney2

Laboratory for Cancer Research, Department of Chemical Biology, College of Ptiannac\, Rutgers-The Stale University of New Jersey, Piscataway, New Jerse\ 08854-8020

Abstract Estradiol or estrone can be hydroxylated at various positions by NADPH-dependent cytochrome P450 enzymes present in liver and Catechol estrogens (2- or 4-hydroxyestradiol and 2- or 4-hydroxy- extrahepatic target cells (reviewed in Refs. 33 and 34). The chemi estrone) are chemically reactive estrogen metabolites that are O-methyl- cally reactive catechol estrogens (2- or 4-hydroxyestradiol and 2- or ated to less polar monomethyl ethers by catechol-O-methyltransferase, an 4-hydroxyestrone) can be metabolically O-methylated to monomethyl enzyme present in many tissues such as the liver, kidney, brain, placenta, ethers (structures shown in Fig. 1) by COMT,3 an enzyme present in uterus, and mammary gland. In the present report, we review recent studies on the antitumorigenic and antiangiogenic effects of exogenously large amounts in many organs and cells such as liver, kidney, brain, administered 2-methoxyestradiol in vitro and in vivo. We also discuss data placenta, uterus, mammary gland, and RBCs (35-42). Because of the that suggest that endogenous formation of 2-methoxyestradiol (and its ubiquitous distribution of COMT activity in mammalian tissues (35- 2-hydroxyestradiol precursor) may have a protective effect on estrogen- 42) and the very rapid metabolic O-methylation of catechol estrogens induced cancers in target organs. Although the molecular mechanism of as demonstrated both in vitro and in vivo (42-46), large amounts of action of 2-methoxyestradiol is not clear, we suggest that some unique monomethylated estrogen metabolites are produced. A previous study effects of 2-methoxyestradiol may be mediated by a specific intracellular reported that blood concentrations of unconjugated and conjugated effector or receptor that is refractory to the parent hormone, estradiol. 2-methoxyestrone are approximately 4000 and 6000 pg/ml, respec Additional research is needed to identify factors that regulate the meta tively, in pregnant women (47,48). which are over 50-fold higher than bolic formation and disposition of 2-methoxyestradiol in liver and in the concentration of estradiol. Interestingly, 2-methoxyestradiol and target cells and to evaluate the effects of modulating 2-methoxyestradiol formation on estrogen-induced carcinogenesis. 2-methoxyestrone have higher binding affinities for the sex hormone- binding globulin in blood than does estradiol (48), which may be an important factor (in addition to rapid metabolic formation of me- Introduction thoxyestrogens) that contributes to their high circulating levels. Chronic administration of estrogens (such as estradiol and estrone) Most studies showed that methylated catechol estrogens have little results in tumor formation in target organs in several animal models or no binding affinities for the classical estrogen receptor (<0.1% (1-7). Estrogens have also been increasingly implicated as an impor compared with estradiol). and they lack estrogenic activity in the tant etiological factor in the causation of certain types of human uterus (43, 49, 50). An important question is why animals and humans cancer (8-16). Chronic administration of estrogens to postmeno- make large amount of hormonally inactive yet highly lipophilic es pausal women is strongly associated with an increased risk of endo- trogen metabolites that probably have much longer half-lives than metrial cancer (8-12). Although the relationship between estrogen estradiol? Studies on the chemical reactivity and potential genotoxic- and breast cancer is not as strong as that between estrogen and ity of catechol estrogens (23, 25-27, 29-32) have led to the sugges endometrial cancer, there is mounting evidence associating elevated tion that metabolic O-methylation may be a rapid inactivation/detox- breast cancer risk with an increased total life-time exposure to estro ification pathway for these estrogen-derived catechols, as is known for gens (13-16). Epidemiological studies indicate an increased risk of catecholamines. However, there are several studies indicating that breast cancer in women with: (a) high serum levels of estrogens 2-methoxyestradiol may not be just an inactive metabolite of estradiol (comparison of women in the United States with those in Japan and but may have unique biological activities that are not shared with China); (b) prolonged exposure to estrogens (due to early menarche estradiol, 2-hydroxyestradiol, or 4-methoxyestradiol (51-57). and late menopause or long-term use of estrogen supplements); and In this report, we review data that point to the hypothesis that (c) obesity (a cause of increased formation of estrogen by enzymatic endogenously formed 2-methoxyestradiol may have a unique protec aromatization of androgens in adipose cells) (reviewed in Refs. 15 and tive role against estrogen-induced carcinogenesis in estrogen target 16). Although the mechanism of estrogen-induced carcinogenesis is organs. We also propose that some unique actions of 2-methoxyestra still not clear, it is thought that the superpotent mitogenic action of diol may be mediated by a specific but unidentified intracellular estrogenic hormones (5, 14, 17-21), together with the potential geno- receptor or effector that is refractory to the parent hormone, estradiol. toxicity of some reactive estrogen metabolites (e.g.. 4- and 16a- Inhibitory Effect of 2-Methoxyestradiol on the Growth of hydroxylated estrogens; see Refs. 6 and 22-32), may play important Tumor Cells in Vitro and in Vivo roles in the induction of estrogen-associated cancers in target organs of animals and humans. 2-Methoxyestradiol inhibits the proliferation of many human can cer cell lines in vitro (54, 55, 57-61). Human breast cancer cell lines

Received 1/16/98; accepted 3/26/98. were particularly sensitive to the cytotoxic effect of 2-methoxyestra The costs of publication of this article were defrayed in pan by the payment of page diol and several synthetic analogues irrespective of the estrogen charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. receptor status of the cell lines studied (58). An earlier study showed 1To whom requests for reprints should be addressed, at Rutgers University, College of that the inhibitory effect of 2-methoxyestradiol (at a 10 nM concen- Pharmacy. Laboratory for Cancer Research, Department of Chemical Biology, 164 Frelinghuysen Road. Piscataway, NJ 08854-8020. Fax: (732) 445-0687; E-mail: baotingÂ® 3 The abbreviations used are: COMT. calechol-O-methyllransferase; !7ÃŸ-HSD. 17fJ- rci.rutgers.edu. 2 William M. and Myrle W. Garbe Professor of Cancer and Leukemia Research. hydroxysteroid dehydrogenase. 2269

Fig. 1. The structures of monomethylated cat- echo] estradiol metabolites. Estradici is hydroxylated to catechols (2- and 4-hydroxyestradiol| largely by cytochrome P450 enzymes. These cat echols can be rapidly O-melhylated by COMT to form monomethoxy estradiol metabolites. 2-Me- thoxyestradiol and 4-methoxyestradiol are the H,C major monomethylated isomers formed from their respective catechol precursors. Although the structures for estrone and its catechol metab 2-OH-3-MeO-E2 olites are not shown, estrone can also be metab Catecholamines, olized to catechols and then to 0-melhylated quercetin, etc. catechols. Because COMT catalyzes the metab olism of a wide spectrum of endogenous and exogenous catechols. the enzymatic O-methyla- 17ÃŸ-Estradiol(E2) tion of estradiol or estrone catechols can be reg ulated by these non-estrogen catechols (e.g., CH30 hydroxylated flavonoids. catecholamines, and COMT \ 4-MeO-E2 others). P450. cytochrome P450; 2-OH-E-,. 2-hydroxyestradiol; 4-OH-E2. 4-hydroxyestradiol; 2-MeO-E2. 2-melhoxyestradiol; 2-OH-J-MeO- \ E2, 2-hydroxyestradiol ,1-methyl ether; 4-MeO- Â£2,4-methoxyestradiol; 4-OH-3-MeO-E2, 4-hy CH3O droxyestradiol 3-methyl ether. OH 4-OH-3-MeO-E2

tration) on the proliferation of MCF-7 human breast cancer cells was modulate estrogen-induced tumorigenesis in the breast and possibly not shared by estradiol or 2-hydroxyestradiol (55). At considerably other target organs. higher concentrations (>1 piM), 2-methoxyestradiol was highly cyto- Induction of kidney tumors in male Syrian hamsters by chronic toxic to several human cancer cell lines in culture, including HeLa treatment with estradiol or other natural or synthetic estrogens is a cells (a cell line derived from human cervical cancer). MCF-7 breast frequently used animal model for studying the mechanisms of estro cancer cells, Jurkat leukemia cells, and retinoic acid-differentiated gen-induced cancer (2, 6, 63). Recent studies showed that chronic SH-SY5Y neuroblastoma cells (54, 57-60). 2-Methoxyestradiol ap administration of quercetin. a commonly ingested dietary polyphenol pears to target actively dividing cells by disturbing cellular events that is a substrate and also a potent inhibitor of COMT (64, 65), associated with cell proliferation (54, 56). significantly increased the severity of estradiol-induced kidney tumor It is of considerable interest to note that oral administration of formation in male Syrian hamsters (64). Additional studies showed 2-methoxyestradiol (75 mg/kg body weight/day) inhibited the growth that the formation of 2- and 4-methoxyestradiol in vivo is inhibited by of transplanted Meth-A sarcoma and B16 melanoma cells in C3H chronic administration of quercetin (65). This inhibition of the met mice (57), and this treatment also inhibited the growth of a human abolic O-methylation of catechol estrogens is due to a combination of breast carcinoma cell line (MDA-MB-435, estrogen receptor nega three mechanisms (65): (a) a direct competitive inhibition of COMT tive) in immunodeficient mice (61). by quercetin (which is a catechol and an excellent substrate for Inhibitory Effect of 2-Methoxyestradiol on Angiogenesis in Vitro COMT); (b) a noncompetitive inhibition due to increased levels of S-adenosyl-L-homocysteine in the target organ; and (c) a reduced and in Vivo availability in the target organ of S-adenosyl-i.-methionine, the sup 2-Methoxyestradiol has a potent inhibitory effect on the prolifera porting co-substrate for the enzymatic O-methylation of catechol tion of blood-vessel endothelial cells in vitro (ICJO, â€”¿100nM;Ref. estrogens. An inhibition of the COMT-catalyzed O-methylation of 57). Additional studies showed that 2-methoxyestradiol induces ap- catechol estrogens during quercetin administration decreases the pro optosis in cultured arterial endothelial cells in a time- and concentra duction of 2-methoxyestradiol in addition to causing an accumulation tion-dependent manner, and the migration of these vascular endothe of the reactive 2- and 4-hydroxyestradiol intermediates in target lial cells in vitro is also inhibited (62). Administration of this estradiol organs (65). 4-Hydroxyestradiol is a strong renal carcinogen in Syrian metabolite to animals strongly inhibits angiogenesis in vivo (57, 61, hamsters (63, 66), and this compound is believed to contribute to the 62). It is noteworthy that 2-methoxyestradiol is among the most potent carcinogenic action of estradiol (6). It will be of interest to determine endogenous nonprotein inhibitors of in vitro angiogenesis, and its whether other dietary polyphenols that are ingested in large amounts antiangiogenic effect is highly specific and is not shared by several by humans will also enhance estrogen-associated carcinogenesis as closely related structural analogues (57). The strong antiangiogenic well as to determine the relevance of these observations for humans. effect of 2-methoxyestradiol described here as well as its potent It is worth noting that renal carcinogenesis in Syrian hamsters cytostatic effect on the growth of cultured tumor cells (described chronically treated with diethylstilbestrol (a nonsteroidal estrogen) is above) may both contribute to the inhibitory effect of 2-methoxyestra not enhanced by chronic coadministration of quercetin.4 Unlike the diol on tumor growth in animal models (57, 61). facile formation of methylated catechols from estradiol, catechol formation from diethylstilbestrol and the subsequent O-methylation of Decreased 2-Methoxyestradiol Formation Is Associated with an Increased Risk of Estrogen-induced Cancers its catechol intermediates are only very minor metabolic pathways (67, 68). Diethylstilbestrol can directly undergo metabolic redox Because 2-methoxyestradiol inhibits the proliferation of breast can cycling (without conversion to a catechol) to generate free radicals cer cells and angiogenesis in vitro and in vivo (described above), factors that alter the metabolic formation of 2-methoxyestradiol may 4 B. T. Zhu and J. G. Liehr. unpublished data. 2270

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. 2-MhTHOXYKSTRADIOI. AS AN INHIBITOR OF BREAST CANCER and form reactive quinone/semiquinone intermediates (23, 25, 69), with a normal alÃele.This observation is consistent with the proposed which is believed to contribute to diethylstilbestrol-induced carcino- antitumorigenic role of 2-methoxyestradiol and the carcinogenic role genesis. In addition, it was observed that 7.12-dimethylbenz(Â«|anthra- of 4-hydroxyestradiol in human mammary carcinogenesis. It will be cene-induced mammary tumors in rats (70) and azoxymethanol-in- of interest in future studies to determine the COMT enzyme activity duced colonie neoplasms in mice (71) were not increased (but and levels of 2-methoxyestradiol and 4-hydroxyestradiol in the breast significantly decreased) by chronic cotreatment with quercetin. The of the same subjects and to correlate these parameters with the breast inhibitory effects of quercetin on 2-methoxyestradiol formation in cancer risk in women with a normal versus a variant alÃeleof the vitro and in vivo, and its stimulatory effect on estradiol-induced COMT gene. carcinogenesis but not diethylstilbestrol-, 7,l2-dimethylbenz[a)anthracene-, or azoxymethane-induced carcinogenesis (described above) Increased Formation of 2-Hydroxyestradiol and 2- are consistent with the concept that selective stimulation of estradiol- Methoxyestradiol Is Associated with a Decreased Risk of induced carcinogenesis by quercetin is caused by a reduced formation Estrogen-induced Cancers of the antitumorigenic 2-methoxyestradiol. In accord with the inhibitory effect of dietary catechols such as The data discussed above suggest that decreased formation of quercetin and fisetin on COMT-catalyzed O-methylation of catechol 2-methoxyestradiol is associated with increased formation of estradi ol-induced tumors. There are also data (discussed below) suggesting estrogens (64, 65). endogenous catechols may also exert an inhibitory effect on the O-methylation of catechol estrogens. Recent studies have that increased formation of 2-hydroxyestradiol and 2-hydroxyestrone shown that very high concentrations of endogenous catecholamines (products of estrogen 2-hydroxylation metabolism), which subse (substrates and inhibitors of catechol estrogen O-methylation) are quently leads to elevated formation of 2-methoxyestradiol and 2-me- present in male Syrian hamster kidney. CD-I mouse uterus, and thoxyestrone by COMT, is associated with a decreased risk of estro Fisher-344 rat pituitary (72). three target organs that develop estrogen- gen-associated tumors in target organs. induced tumors (1, 2. 4). The concentrations of catecholamines in The formation of spontaneous mammary tumors in female C3H/ these target tissues are up to 50-fold higher than those in nontarget OuJ mice, spontaneous uterine tumors in Oncomice or in Donryu rats, tissues of these animals or in tissues of other strains or species (72). and chemically induced mammary tumors in Sprague Dawley rats is Because catecholamines inhibit the enzymatic O-methylation of cat dependent on the presence of endogenous estrogens (5, 92-94). Stud echol estrogens in vitro at physiologically relevant concentrations ies during the past several years have shown that chronic treatment of (72), we believe that high tissue concentrations of catecholamines animals with indole-3-carbinol stimulates the 2-hydroxylation of es- may inhibit the metabolic formation of 2-methoxyestradiol in addition tradiol (95-97), and this treatment is associated with decreases in to elevating the concentrations of reactive catechol estrogen interme spontaneous mammary tumorigenesis in C3H/OuJ mice (95, 96), diates in target organs. spontaneous uterine tumorigenesis in Oncomice (98) or Donryu rats Earlier studies in female C3H/He mice showed that increased levels (99), and chemically induced mammary tumorigenesis in Sprague of environmental or manipulative stress markedly increased the inci Dawley rats (100). Recent studies in our laboratory showed that dence of spontaneous mammary cancers (73). Interestingly, epidemi chronic administration of 0.05% sodium phÃ©nobarbitalin the drinking olÃ³gica!studies in humans suggest a positive relationship between the water to female C3H/OuJ mice for 16 months very strongly inhibited degree of sustained emotional stress and the risk for developing breast the formation of spontaneous mammary tumors, and this inhibitory cancer (74-76). Because studies in animals and humans both indicate effect of phÃ©nobarbitalwas accompanied by a manyfold increase in that an increased level of stress is accompanied by an increased liver microsomal 2-hydroxylation of estradiol with little or no increase in estradiol 4- or I6a-hydroxylation.^ release of endogenous catecholamines, we suggest a unifying hypoth esis that sustained stress increases the levels of catecholamines. which Earlier studies in humans showed that cigarette smokers have then inhibit the enzymatic O-methylation of catechol estrogens, re enhanced 2-hydroxylation of estradiol in vivo (101-103) and some sulting in decreased formation of 2-methoxyestradiol and increased what lower serum and urinary levels of estradiol and estrone ( 104- accumulation of reactive catechol intermediates in target tissues. This 106). This stimulatory effect of cigarette smoking on estrogen 2-hy hypothesis is in accord with data indicating an association of sustained droxylation was associated with a decreased risk of uterine emotional or environmental stress with an increased breast cancer endometrial cancer (in addition to an increased incidence of osteopo risk. Decreased immune system function with sustained stress has also rosis) in female cigarette smokers (107-109). been postulated to play a role in the increased breast cancer risk Human cytochrome P450 1A1 as well as other P450 isoforms (e.g.. observed (77, 78). 1A2 and 3A4) possess strong catalytic activity for estrogen 2- Several early studies noted marked person-to-person variations in hydroxylation (33, 34). A recent preliminary study suggested that the COMT activity in RBCs (79-81). Additional studies showed that individuals with an Msp\ polymorphism in the CYPIAI gene had a the distribution of COMT activity in an American population has a low basal level of estrogen 2-hydroxylation. and treatment of these polymorphic bimodal pattern (82-84). Genetic analysis revealed that individuals with indole-3-carbinol failed to induce estradiol 2-hy human COMT is encoded by a single gene (a wild-type and a variant droxylation (110). Research on a possibly increased risk of breast alÃele),and approximately one-fourth of the Caucasians studied in cancer in Caucasian women or in African-American women with herited a variant alÃeleencoding a low activity form of the enzyme polymorphisms in the CYPIAI gene has resulted in inconsistent (82, 85-90). We anticipate that individuals who inherit the low conclusions (111, 112). activity form of the COMT gene will be at increased risk for estrogen- It was recently reported that African-American women have a associated breast cancer due to decreased formation of the antitumori lower rate of estrogen 2-hydroxylation than American Caucasian genic 2-methoxyestradiol in addition to retarded inactivation of cat women (113. 114). It is not known whether this difference in estrogen echol estrogen intermediates (particularly 4-hydroxyestradiol, which 2-hydroxylation contributes to the higher breast cancer mortality rate is hormonally active and is carcinogenic). A recent study by Lavigne observed in African-American women than in American Caucasian et al. (91) indicated that postmenopausal women with a variant alÃele women (115). The relative roles of genetic and environmental factors coding for a low activity form of the COMT have an increased risk (odds ratio, 2.18) for developing breast cancer compared with women 5 Unpublished data. 2271

Other hydroxylated estrogen metabolites

2-OH-E2 P45O P450 -*- 4-OH-E2,16a-OH-Ej, etc 2-HydroxylatÃ¬on â€¢¿â€¢c I Estrogen receptor-mediated Genotoxicity mitogenic stimulation

2-MeO-E2 Inhibition

Estrogen-induced carcinogenesis

Fig. 2. A diagram depicling the beneficial effect of estradici 2-hydroxylation and subsequent 2-methoxyestradiol formation on estrogen-induced carcinogenesis. The multiple pathways of oxidative estradici metabolism by NADPH-dependent cytochrome P450 enzymes leads to the formation of: Ui) hormonally active and chemically reactive metabolites (e.g., 4-hydroxyestradiol. 16a-hydroxyestronel: (hi 2-hydroxylated metabolites (e.g.. 2-hydroxyestradiol. 2-hydroxyestrone): and (c) other hydroxylated metabolites. Selectively increasing the 2-hydroxylation pathway of estradici metabolism will not only decrease the availability of the parent hormone, but it will also increase the subsequent formation of 2-methoxyestradiol. a nonpolar estradici metabolite that has potent growth-inhibitory and antiangiogenic actions in mammary tumors. P450, cytochrome P450; Â£,, 170-estradiol: 2-OW-Â£2.2-hydroxyestradiol; 4-OH-E^. 4-hydroxyestradiol; 16a-OH-E,. I6a-hydroxyestrone; 2-MeO-E,. 2-methoxyestradiol.

in explaining these differences in estrogen metabolism in African- estradiol or estrone (129-133) as well as the subsequent O-methyla American and Caucasian-American women have not been explored. tion of the catechol metabolites (39, 40). It is expected that large Consistent with observations associating high levels of estrogen amounts of 2-methoxyestradiol will be produced in these target tis 2-hydroxylation with a reduced cancer risk in animals and humans, sues. Because cultured human breast cancer cells (regardless of their earlier studies with a commonly used hamster kidney tumor model estrogen receptor status) are particularly sensitive to growth inhibition showed that 2-hydroxyestradiol and 2-hydroxyestrone are devoid of by 2-methoxyestradiol (58). increased formation of 2-methoxyestra carcinogenic activity at dose levels where estradici or 4-hydroxyestra diol by the breast is likely to be an important endogenous factor that diol gives rise to a 100% tumor incidence (63, 66). The lack of suppresses the development of estrogen-associated mammary cancers. carcinogenic activity of 2-hydroxylated estrogen metabolites is not There is a need to enhance our understanding of person-to-person completely understood. Although earlier studies suggested that 2- and differences in the formation of 2-methoxyestradiol and of factors that 4-hydroxyestradiol have similar chemical reactivity and genotoxicity influence the formation of 2-methoxyestradiol in normal human breast when tested in vitro (23, 25, 26), 2-hydroxyestradiol appears to have and in breast cancers. Selectively enhancing the endogenous forma a faster rate of metabolism by COMT-catalyzed O-methylation (44. tion of 2-methoxyestradiol in target cells may represent a new ap 72). a more rapid metabolic clearance in vivo (116, 117), and a weak proach for the prevention and treatment of mammary cancers. er hormonal activity in estrogen target organs (118-126) than 4- hydroxyestradiol. Moreover, the COMT-catalyzed O-methylation of Potential Physiological Functions of 2-Methoxyestrone 4-hydroxyestradiol is inhibited by 2-hydroxyestradiol, whereas the O-methylation of 2-hydroxyestradiol is not inhibited by 4-hy Although very high concentrations of 2-methoxyestrone are present droxyestradiol (44). It is likely that 4-hydroxyestradiol may accumu in the human circulation, this estrone metabolite only has about 1% of late in target cells because of inhibition of its O-methylation. Finally, the growth-inhibitory potency of 2-methoxyestradiol in cultured the presence of increased amounts of 2-hydroxylated estrogens will blood-vessel endothelial cells or fibroblasts (57). Because 17ÃŸ-HSDis subsequently lead to increased formation of 2-methoxyestradiol, and a group of intracellular isozymes catalyzing interconversions between the formation of this cytostatic and antiangiogenic metabolite may be estradiol and estrone (134, 135), it is expected that this enzyme an important reason for the lack of carcinogenicity of 2-hydroxylated will also catalyze interconversions between 2-methoxyestradiol and estrogens in animals and for the reduced cancer risk in animals and 2-methoxyestrone (Fig. 3). Accordingly, it is likely that 2-me humans associated with high levels of estrogen 2-hydroxylation. This thoxyestrone is an immediate metabolic precursor of 2-methoxyestra concept is illustrated in Fig. 2. diol. High levels of estradiol are present in estrogen target tissues, and 17ÃŸ-HSDactivity has a cell-specific localization (such as in the cancerous breast tissue contains particularly high levels of estradiol secretory glandular epithelium and proliferative endometrium of the and estrone (127, 128). Many breast cancers also contain high levels uterus: Ref. 136), and this enzyme activity is regulated by certain of enzymes that catalyze the formation of catechol estrogens from hormones during different stages of the normal menstrual cycle (137). 2272

17ÃŸ-HSD(oxidativeactivity) Fig. 3. Metabolic interconversions between estradici and estrone (or between 2-methoxyestradiol and 2-me- 17ÃŸ-HSD(reductiveactivity) thoxyestrone) catalyzed by 17ÃŸ-HSD.The 17j3-oxidative ac HO HO Estradici tivity of 17j3-HSD will convert estradici to estrone, whereas Estrone the 17/3-reductive activity of 17ÃŸ-HSDwill convert estrone to estradici. Although the 17ÃŸ-HSD-cataly/.ed interconversions HjC between estradici and estrone have been studied extensively, the postulated interconversions between 2-methoxyestradiol and 2-methoxyestrone have not been studied. CH3O 17ÃŸ-HSD(oxidativeactivity) 17ÃŸ-HSD(reductiveactivity) HO 2-Methoxyestradiol 2-Methoxyestrone

The uterine 17ÃŸ-HSD(type II) predominantly catalyzes the 17/3- active 16a-hydroxyestrone and loa-hydroxyestradiol) are carcino oxidative conversion of estradiol to estrone (anticipated conversion of genic, whereas the products of the 2-hydroxylation pathway (hormon 2-methoxyestradiol to 2-methoxyestrone; Refs. 134 and 138), whereas ally weak 2-hydroxyestrone and 2-hydroxyestradiol) are not carcino the 17ÃŸ-HSD(type I) present in breast tumors predominantly cata genic. According to this hypothesis, the beneficial effect of increased lyzes the 17ÃŸ-reductiveconversion of estrone to estradiol (anticipated estrogen 2-hydroxylation is because it decreases the pool of endoge conversion of 2-methoxyestrone to 2-methoxyestradiol; Refs. 139 and nous parent estrogen and thereby reduces the formation of 16a- 140). It is noteworthy that the 17/3-reductive activity of 17/3-HSD is hydroxyestrone and 16a-hydroxyestradiol. 16a-Hydroxyestrone, higher in mammary tumors than in normal breast that does not contain which is not only hormonally active but also chemically reactive (28, malignant cells (141-143). This increased 17ÃŸ-HSD(reductive activ 152, 153, 155), may play a role in estrogen-induced mammary cancer. ity) in mammary cancer cells not only provides these cells with a The concept of loa-hydroxylated estrogens as an important etiolog- favorable estrogenic environment for growth due to increased con ical factor in hormonal carcinogenesis, however, is weakened by the version of estrone to estradiol but may also increase the formation of very low carcinogenic activity of 16a-hydroxyestrone and loa-hy the antitumorigenic 2-methoxyestradiol from 2-methoxyestrone. The droxyestradiol (estriol) relative to estradiol or 4-hydroxyestradiol (63, amount of 2-methoxyestradiol formed in situ from 2-methoxyestrone 156). Moreover, some studies suggest that estrogen 16a-hydroxyla- would depend on the availability of 2-methoxyestrone and the level of tion is not markedly different among women with different risks for 17ÃŸ-HSD(reductive activity) in target cells. mammary cancer (114, 155, 157-159). To accommodate the 16a- Although long-term exposure to exogenously administered estra hydroxylation hypothesis with these experimental observations, em diol or estrone has been shown in several animal models to induce phasis has been shifted to the ratios of estrogen 2- to 16o-hydroxy- tumors in their target organs (1-7), surprisingly the very large amount lation as a biomarker for breast cancer risk (98, 152, 153, 155). This of endogenous estrogens present during human full-term pregnancy is shift in emphasis tends to decrease the importance of the absolute associated with a decrease in the risk of human uterine and mammary extent of estrogen 16a-hydroxylation. cancers (144-147). It is possible that endogenous substances that are The recent findings of potent antitumorigenic activity for 2- produced during pregnancy possess protective activity against estro methoxyestradiol suggest a new explanation for the inverse relation gen-dependent tumor formation. Previous studies suggest that proges ship observed previously between the ratios of estrogen 2- to 16a- terone may be one of the inhibitory substances in animals and possibly hydroxylation and breast cancer risk. We believe a major reason that also in humans (148-151). Because human placenta produces large enhanced 2-hydroxylation of estradiol is associated with decreased amounts of estradiol and estrone, and this organ also contains high mammary carcinogenesis is because enhanced 2-hydroxylation in estrogen 2-hydroxylase and COMT activity, it is expected that large creases the formation of 2-methoxyestradiol, and this compound is a amounts of 2-methoxyestrogens will be produced by the placenta. potent antitumorigenic metabolite of estradiol (illustrated in Fig. 2). Consistent with these observations, a previous study (47) reported Possible Mechanisms of Action of 2-Methoxyestradiol that the blood concentration of unconjugated plus conjugated 2-methoxyestrone was â€”¿10,000pg/ml in pregnant women. Although The mechanism for the growth-inhibitory and antiangiogenic ef the level of 2-methoxyestradiol was not reported, it is likely that fects of 2-methoxyestradiol is not known. Observations indicating the substantial amounts are formed from 2-methoxyestrone in target cells ability of 2-methoxyestradiol to induce abnormal spindle formation in by the action of the reductive 17ÃŸ-HSD.The potent growth-inhibitory cultured cells (54), to interact with tubulin at the colchicine binding and antiangiogenic activity of 2-methoxyestradiol may protect against site (56), and to inhibit tubulin polymerization (56, 61 ) have led to the estrogen-induced carcinogenesis in target organs during human preg suggestion that the antitubulin activity of 2-methoxyestradiol may nancy. It will be of considerable interest to test this hypothesis. contribute in an important way to its cytotoxic effect. It should be noted, however, that the concentration of 2-methoxyestradiol or some Importance of 2-Methoxyestradiol for Understanding the Physiological Significance of the Estrogen 2-/16a-Hydroxylation of its synthetic analogues required for inhibition of tubulin polymer ization or colchicine binding to tubulin in vitro is considerably higher Ratio (low P.Mrange) than that required for inhibiting the growth of many Bradlow, Fishman, and their colleagues (98, 152-154) proposed different types of cancer cells in culture ( 10-100 nM; Refs. 56, 58, 59, that the extent of estrogen 16a-hydroxylation or the ratio of estrogen and 61). It is noteworthy that a recent study showed that 2-me 2- to 16a-hydroxylation is an important biomarker for the risk of thoxyestradiol at concentrations sufficient for causing complete met- breast cancer in animals and humans. The core of their hypothesis is aphase arrest in cultured cells does not inhibit the formation of mitotic that the products of the 16a-hydroxylation pathway (hormonally spindles (160). 2273

In cultured H460 or A549 human lung cancer cells (both of which effect; (c) target cells that are particularly sensitive to growth inhibi contain the wild-type p53 gene), treatment with 5 JU.M2-methoxyestra- tion by 2-methoxyestradiol; (d) endogenous and exogenous fact diol increases the wild-type p53 protein levels and induces apoptosis ors that regulate the metabolic formation and disposition of 2- (161). However, 2-methoxyestradiol at the same concentration does methoxyestradiol in liver and in estrogen target cells; and (e) the not induce apoptosis in human lung cancer cells, either without the molecular mechanism(s) underlying the growth-inhibitory and anti- wild-type p53 gene or with a mutated p53 gene (161). These results angiogenic actions of 2-methoxyestradiol. Studies in these and related suggest that 2-methoxyestradiol at relatively high concentrations may areas will not only greatly enhance our understanding of the physio activate the wild-type p53-mediated apoptosis. logical roles of 2-methoxyestradiol, a nonpolar estradiol metabolite Treatment of MCF-7 human breast cancer cells with a low concen formed in large amounts in liver and in estrogen target cells, but these tration (10 nM) of 2-methoxyestradiol caused marked changes (an studies may also offer novel mechanistic insights and strategies for the increase or decrease) in the intracellular cyclic AMP concentration prevention and treatment of estrogen-associated cancers. depending on the phase of the cell cycle, whereas 10 nw estradici or 2-hydroxyestradiol had little or no effect (55). This change of cyclic AMP levels during 2-methoxyestradiol treatment was accompanied References by marked changes in the phosphorylation patterns of various cellular 1. Clifton. K. H., and Meyer. R. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. Is 2-Methoxyestradiol an Endogenous Estrogen Metabolite That Inhibits Mammary Carcinogenesis?

Bao Ting Zhu and Allan H. Conney

Cancer Res 1998;58:2269-2277.

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