Melatonin As a Selective Estrogen Enzyme Modulator

Current Cancer Drug Targets, 2008, 8, 691-702 691 Melatonin as a Selective Estrogen Enzyme Modulator

S. Cos*, A. González, C. Martínez-Campa, M.D. Mediavilla, C. Alonso-González and E.J. Sánchez-Barceló

Department of Physiology and Pharmacology, School of Medicine, University of Cantabria, 39011 Santander, Spain Abstract: Melatonin exerts oncostatic effects on different kinds of tumors, especially on hormone-dependent breast cancer. The general conclusion is that melatonin, in vivo, reduces the incidence and growth of chemically-induced mammary tumors in rodents, and, in vitro, inhibits the proliferation and invasiveness of human breast cancer cells. Both studies sup- port the hypothesis that melatonin inhibits the growth of breast cancer by interacting with estrogen-signaling pathways through three different mechanisms: (a) the indirect neuroendocrine mechanism which includes the melatonin down- regulation of the hypothalamic-pituitary-reproductive axis and the consequent reduction of circulating levels of gonadal estrogens, (b) direct melatonin actions at tumor cell level by interacting with the activation of the estrogen receptor, thus behaving as a selective estrogen receptor modulator (SERM), and (c) the regulation of the enzymes involved in the biosynthesis of estrogens in peripheral tissues, thus behaving as a selective estrogen enzyme modulator (SEEM). As melatonin reduces the activity and expression of aromatase, sulfatase and 17-hydroxysteroid dehydrogenase and increases the activity and expression of estrogen sulfotransferase, it may protect mammary tissue from excessive estrogenic effects. Thus, a single molecule has both SERM and SEEM properties, one of the main objectives desired for the breast antitu- moral drugs. Since the inhibition of enzymes involved in the biosynthesis of estrogens is currently one of the first therapeutic strategies used against the growth of breast cancer, melatonin modulation of different enzymes involved in the synthesis of steroid hormones makes, collectively, this indolamine an interesting anticancer drug in the prevention and treatment of estrogen-dependent mammary tumors. Keywords: Melatonin, pineal gland, breast cancer, estradiol, aromatase, sulfatase, sulfotransferase, 17-hydroxysteroid dehydrogenase.

INTRODUCTION host defense and has the potential to be useful as an adjuvant tumor immunotherapeutic agent [11]; e) inhibitory effects of Melatonin, the main hormone secreted by the pineal melatonin on telomerase activity in tumor cells [12]; or gland, has been involved in different physiological actions in f) melatonin’s suppression of fatty acid uptake and metabo- a great variety of biological contexts [1]. The role of mela- lism [13, 14]. tonin in the regulation of cancer cell growth has been widely In this review, we will firstly point out the importance of studied during the last few decades [2-5]. There has been estrogens in the genesis and development of breast cancer considerable study into the oncostatic actions of melatonin on different kinds of tumors and especially on estrogen- and how the intratumoral metabolism and synthesis of estrogens, as a result of the interactions of various enzymes, are dependent breast cancer [5, 6]. It is well established that, in considered to play a very important role in the pathogenesis vivo, experimental manipulations activating the pineal gland, and development of hormone dependent breast carcinoma; or the administration of melatonin, reduce the incidence and secondly, we will underline how the interaction between growth rate of spontaneous or chemically induced mammary estrogens and melatonin has long been described to explain tumors in rodents, while pinealectomy or situations which implicate a reduction of melatonin production usually stimu- the melatonin oncostatic actions and, finally, we will outline the possible mechanisms through which melatonin may in- late mammary carcinogenesis [2, 3, 7], whereas in vitro hibit the growth of breast cancer and how some of these melatonin inhibits breast cancer cell proliferation and mechanisms recently involve interactions with the intratu- invasiveness [2-6]. moral metabolism and biosynthesis of estrogens, with mela- Several mechanisms have been proposed to explain the tonin behaving as a selective estrogen enzyme modulator oncostatic actions of melatonin in breast tumors: a) indirect (SEEM). effects derived from the interaction of melatonin with the neuroendocrine reproductive axis, leading to a down- THE ROLE OF ESTROGENS IN BREAST CANCER regulation of some of the hormones influencing tumor growth, especially gonadal estrogens [1]; b) melatonin anti- Breast cancer continues to be a major cause of death in estrogenic actions directly on the epithelial mammary cells European and American women. The incidence of breast [8, 9]; c) antioxidant effects of melatonin [10]; d) melatonin cancer is rising throughout the world and is the most fre- regulatory effects in hemopoiesis and immunoenhancement, quent cancer in women. There are approximately 1,000,000 which is believed to be closely involved in several aspects of new cases of breast cancer in the world each year [15].

Breast cancer is a classic model of hormone dependent ma- *Address correspondence to this author at the Departamento de Fisiología y lignancy. More than 95% of new cases of breast cancer are Farmacología, Facultad de Medicina, Universidad de Cantabria, Cardenal initially hormone-dependent, whether in pre- or postmeno- Herrera Oria s/n, 39011 Santander, Spain; Tel: 34 942 201988; Fax: 34 942 pausal women, and estradiol plays a main role in their gene- 201903; E-mail: [email protected]

1568-0096/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd. 692 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Cos et al. sis and progression. It is known that breast development at postmenopausal women, except for a small contribution puberty and during sexual maturity is stimulated by 17- from ovarian and/or adrenal testosterone and androstenedi- estradiol, which is the predominant circulating ovarian ster- one, 100% of sex estrogens are synthesized in peripheral oid and the most biologically active hormone in breast tissue. tissues, such as mesenquimal-cells of the adipose tissue and However, there is also considerable evidence that estrogens skin, osteoblasts, condrocites, vascular endothelium, aortic are also mammary carcinogens [16, 17]. The dependence on soft muscle and different areas of the brain such as hypo- ovarian function of breast cancer was first recognized in thalamus (preoptic, anterior and mediobasal areas) and ton- 1896, when Beatson observed regression of advanced and sil, from precursors of adrenal [21]. In this situation, plasma metastatic breast cancer induced by bilateral ovariectomy in levels of estrogens are low, and, however, the concentration premenopausal women. From then on, considerable clinical of estradiol in peripheral tissues such as the mammary gland and experimental studies link cumulative and sustained ex- is high, as a result of the in situ biosynthesis [22, 23]. Yue et posure to estrogens with an increased risk of developing al. reported that in situ synthesis of estrogen predominates breast cancer [18]. The importance of ovarian steroidogene- over uptake from plasma as a means of maintaining breast sis in the genesis of breast cancer is highlighted by the facts tissues estradiol concentrations after menopause [23, 24]. that early menarche and late menopause are associated with Estrogen-dependent breast carcinoma in which in situ con- greater breast cancer risk, whereas late menarche and early version from serum androgen to biologically active estrogens menopause result in a significant reduction of the same. occurs without release into the extracellular space is consid- However, what is not clear are the mechanisms through ered “intracrine” tissue [24]. which estrogens cause cancer. Most hypotheses consider that estrogens binding to ER stimulate cell proliferation, thus INTRACRINOLOGY OF ESTROGEN DEPENDENT increasing the possibility of errors in DNA replication result- HUMAN BREAST CARCINOMA ing in point mutations [19]. Furthermore, direct genotoxic effects of 17-estradiol by itself and its metabolites could There is extensive information that mammary cancer tis- also explain the estrogen-induced carcinogenesis [17, 20]. sue contains all the enzymes responsible for the local biosynthesis of estrogens. The most important precursors (steroids In relation to the role of estrogens in the genesis and C19) for the local synthesis of estrogens are dehydroepyan- growth of mammary tumors, it is important to consider that drosterone (DHEA) and its sulfate (DHEAS), which come two–thirds of breast cancer occur in postmenopausal women, mainly from the adrenal cortex. Fig. (1) is showed a general where ovaries have ceased to be functional and circulating view of the basic enzymatic mechanisms involved in the levels of estrogens are low. In premenopausal women 70% formation and transformation of estrogens in human breast of estrogens come from the ovaries and 30% from the con- cancer. Two principal pathways are involved in the final version of androgens (mainly dehydroepiandrosterone and steps of estrogens formation: the aromatase pathway, which dehydroepiandrosterone sulfate) to estrogens in peripheral transforms androgens into estrogens and the sulfatase path- tissues. After the cessation of the follicular function, in way which converts estrogen sulfates into estrone and estra-

Fig. (1). Enzymatic mechanisms involved in the local production of estrogens in human breast carcinoma tissues. (Abbreviations: DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; 17-HSD type 1, 17-hydroxysteroid dehidrogenase type 1). Melatonin as a Selective Estrogen Enzyme Modulator Current Cancer Drug Targets, 2008, Vol. 8, No. 8 693 diol [25, 26]. The main enzymes involved in the regulation C) In breast cancer tissues the estrogen sulfotransferase of local estrogen production are: (EST) is also present; it is a member of the superfamily of steroid-sulfotransferases which converts es- A) Aromatase is an enzyme complex which consists of trogens into their biologically inactive estrogen sul- two components: aromatase cytochrome P-450 pro- fates [29, 30]. The estrogen sulfotransferase is widely tein and, coupled to it, an ubiquitous flavoprotein, distributed in various normal human tissues, includ- NADPH-cytochrome P-450 reductase [27]. The gene ing the breast, and it is involved in protecting periph- coding for the cytochrome P-450 protein is the largest eral tissues from excessive estrogenic effects. How- of the cytochrome P-450 family. Because its overall ever, in the breast carcinoma tissues the estrogen sul- homology to other members of the P-450 superfamily fotransferase activity and expression tend to be de- is low, aromatase belongs to a separate gene family creased [25, 29, 30]. designated CYP19 [28]. The transcription of the aromatase gene is highly regulated and each tissue can D) 17-hydroxysteroid dehydrogenase (17-HSD) is a regulate the amount of aromatase transcribed in a widely distributed enzyme in mammalian tissues highly specific manner [27]. The catalytic process which catalyzes an interconversion of estrogens or leading to the aromatization of androgens requires the androgens. This enzyme catalyzes the reactions be- sequential transfer of three pairs of electrons and con- tween 17-hydroxysteroids and 17-ketosteroids. At sumes three moles of reduced NADPH in the synthe- least twelve different isoforms of 17-HSD have been sis of one mole of estrogen. The reaction is thought to cloned in humans. Some of these isoforms are reduc- involve three oxidative cycles at the C19 methyl tant and are essential for the gonadal and extrago- group of the steroid substrate which are involved in nadal biosynthesis of estradiol and testosterone. The the conversion of androstenedione to estrone or tes- oxidative isoforms catalize the formation of steroids tosterone to estradiol. In the premenopausal state the of low activity, thus exerting a protective role in dif- major source of aromatase and of its substrates is the ferent tissues, including the mammary gland, against ovary. However, in the postmenopausal state, the the steroid actions. The final step of steroidogenesis is ovary loses its complement of aromatase enzyme, al- the conversion of the weak estrone to the potent bio- though it does continue to secrete androstenedione. logically active estradiol by the action of a reductive 17 -hydroxysteroid dehydrogenase activity (17- The adrenal subsumes the primary role of providing HSD1). The 17-HSD1 converts the relatively inac- substrate for aromatase by directly secreting testos- tive estrone, androstenedione and 5-androstenedione terone and androstenedione. The major source of the into the most potent estradiol, testosterone and aromatase enzyme in postmenopausal women is pe- 5-dihydrotestosterone and viceversa [25, 29, 30]. The ripheral tissues and particularly fat and muscle. In presence of 17-HSD1 has been probed in 50% of the humans, the expression of aromatase has a broader mammary carcinoma in postmenopausal women and tissue distribution and occurs in many organs, includ- also in numerous breast tumor cellular lines, T47D ing ovary, placenta, hypothalamus, liver, muscle, adi- and MCF-7 included [25, 26, 29]. pose tissue and mammary tissue itself. It is well known that the P450 aromatase activity and expres- In normal mammary tissue the local estrogen production sion are much higher in breast cancer tissue than in is displaced towards the production of estrone (low activity) normal mammary tissue, and this is one of the reasons whereas in breast adenocarcinomas the formation of the ac- why the estrogen concentration in this type of tissue tive 17-estradiol predominates. This is because, in normal is very elevated [24, 29]. Two-thirds of breast cancer tissue, the high concentrations of circulating inactive ster- contain aromatase and synthesize biologically signifi- oids, androstenedione and estrone sulfates, are the major cant amounts of estrogens locally in the tumor. The precursor substrates of local estrogen production. Aromatase level of aromatase mRNA expression is highest in the catalyzes androstenedione into estrone and sulfatase hydro- stromal compartment of breast tumors but is also pre- lyzes the estrone sulfates to estrone. Estrone is subsequently sent in epithelial cells as well [28]. converted to potent 17-estradiol by 17-HSD1. In normal tissue, the activity of 17-HSD type 2 (17-HSD2), which B) Sulfatase (STS) is an enzyme which hydrolyzes sev- converts 17-estradiol to estrone, and the EST, which inac- eral sulfated steroids such as estrone sulfate, estradiol tives estrone and 17-estradiol, are high and protect from sulfate, dehydroepiandrosterone sulfate and choles- excessive estrogenic effects. In the breast carcinoma tissue, terol sulfate [29]. The estrogen sulfate can act as an sulfatase, aromatase and 17-HSD1 tend to be overex- estrogen reserve, which could be formed from the sul- pressed, while EST and 17-HSD2 are frequently decreased, fated-estrogens by the estrogen sulfatase, present in which may result in the accumulation of 17-estradiol [30]. the mammary gland in both normal and tumoral tissues. Estrone sulfate has a relatively long half-life in HORMONAL THERAPY IN BREAST CANCER the peripheral blood and the levels of estrone sulfate are 5 to 10 times higher than those of unconjugated Because of the role of estrogens as a mammary carcino- estrogens such as estrone, estradiol and estriol during genic, one of the main objectives in the treatment of breast the menstrual cycle and in postmenopausal women cancer has always been to neutralize the effects of estrogens [25, 29]. In the mammary tumors, the estrone produc- on the breast. The ovariectomy, as mentioned above, was the tion from the sulfated-estrone is ten-time higher than first “antiestrogenic” therapy. Since then, the treatment of that obtained by the aromatase [29]. breast cancer has included localized treatment on the breast 694 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Cos et al. region (surgery and radiotherapy) and general treatment that Some of the major biological actions of melatonin de- centers around chemotherapy and hormonotherapy. The pend on its binding to specific receptors in the target tissues. pharmacological strategies employed to selectively neutral- Two melatonin receptors, termed MT-1 and MT-2, have ize the effects of estrogens on the breast are: a) drugs that act been described in the central nervous system and in numer- through the estrogen receptor interfering with the effects of ous peripheral tissues [34]. They are Gi-protein coupled the endogenous estrogens; this group includes the so-called membrane receptors which decrease intracellular concentra- selective estrogen receptor modulators (SERMs), of which tion of cAMP [34]. Melatonin effects on other second mes- ++ tamoxifen and its derivatives are the most representative sengers such as [Ca ]I, cGMP, diacylglycerol or protein examples that for many years have had a significantly bene- kinase C have been described depending on the cell type ficial effect both on freedom from symptoms of the disease studied [34]. Although controversial, some reports based on and reduction in mortality; b) drugs that interfere with the the lipophilic nature of melatonin have also suggested that synthesis of steroid hormones by inhibiting the enzymes con- this hormone is able to bind and activate nuclear receptor trolling the interconversion from androgenic precursors: the members of the family of retinoid orphan nuclear receptors SEEMs (selective estrogen enzyme modulators). At present, (RZR/ROR) [35]. It has not, though, been possible to sub- antiaromatases which include steroidal (formestane, exeme- sequently confirm this action mechanism. Alternative stane, etc) as well as non-steroidal (anastrozole, letrozole, mechanisms of melatonin actions, not mediated by receptors, etc.) compounds are extensively used in breast cancer treat- have been also proposed. Melatonin may act at intracellular ment with positive benefits [31, 32]. Recent data, in trials sites, binding to cytosolic calmodulin and thus affecting the with breast cancer patients, have shown that the utilization of calcium signaling and modulating cytoskeletical structural selective agents to regulate other enzymes involved in the proteins [36]. Finally, recent in vivo and in vitro studies have biosynthesis and transformation of estrogens (estrogen sul- shown that melatonin is a potent direct scavenger of hy- fotransferases, sulfatases and 17-hydroxysteroid deshidro- droxyl radicals and other oxygen centered free radicals, pro- genases type 1), in combination, with antiaromatase drugs, tecting cells against oxidative damage [10]. may also have possible important therapeutic potential as an Different physiological actions, in a great variety of bio- endocrine therapy for the total blockade of local estrogens in logical contexts, have been attributed to melatonin. In mam- breast cancer and may provide new possibilities in the treat- mals, this indolamine is involved in the synchronization of ment of this disease [24, 25]. circadian rhythms as well as in seasonal reproduction [1, 37]. A considerable number of reports have appeared describing MELATONIN, THE MAIN PINEAL HORMONE the effects of melatonin on the immune system, which suggests that melatonin has immunomodulatory actions [11]. Fifty years ago, melatonin, the main biologically active Melatonin has also been described as an efficient free radical substance secreted by the pineal gland, was isolated by scavenger with antioxidant properties [10]. Lerner and collaborators [33]. Melatonin synthesis from tryptophan is made in successive steps involving different MELATONIN AND ESTROGENS IN BREAST CAN- enzymes. The rate-limiting step in this biosynthetic pathway CER is the activity of the enzyme arylalkylamine Nacetyltransferase (NAT), which catalyzes the transforma- The pineal gland is a neuroendocrine organ which, tion of serotonin to N-acetylserotonin, the direct precursor of through the synthesis and secretion of melatonin, plays a melatonin, and is controlled by the light-dark cycle. The main role in the regulation of reproductive physiology. regulation of the NAT activity depends on the suprachias- Melatonin was formerly considered as a hormone controlling matic nucleus of hypothalamus which projects axons to the seasonal reproduction in wild animals through its effects on paraventricular nucleus of hypothalamus and from there to the hypothalamic-pituitary reproductive axis [1, 37]. In sea- the thoracic spinal cord, where the axons synapses with sonally breeding mammals, melatonin controls the reproduc- preganglionic sympathetic neurons. These neurons project to tive function through the activation of receptor sites within the superior cervical ganglion, which sends postganglionic the hypothalamic-pituitary axis thus driving the levels of terminals to the pineal gland. The release of norepinephrine gonadal activity [38, 39]. Melatonin down-regulation of the from these sympathetic terminals induces the activity of the ovarian estrogen secretion has been observed in a variety of NAT and drives the synthesis of melatonin. The neurons of mammals (the so-called long breeders) [1] whereas in others, the suprachiasmatic nucleus act as an endogenous oscillator such as sheep (short breeders), control of reproduction de- with period lengths close to 24 h, which is synchronized by pends on the melatonin-induced up-regulation of the synthe- the light/dark signals perceived by the eyes and transmitted sis of estrogens during the winter [40]. In humans the role of melatonin on the reproductive system is not completely clear by the retinohypothalamic tract. The light-dark cycle thus and even controversial. An inverse relationship between se- entrains the rhythm of melatonin synthesis. Circulating lev- rum melatonin and ovarian activity [41] and a certain role of els of melatonin are low during the day and show a large melatonin in the modulation of the neuroendocrine- increase at night. The nocturnal rise of melatonin is abol- reproductive axis has been proposed [42]. Furthermore, di- ished, or substantially decreased, in animals exposed to con- rect stimulatory effects of melatonin on ovarian steroi- stant light whereas, in animals kept in constant darkness, dogenesis have been demonstrated in human granulosa-luteal melatonin is secreted following the endogenous rhythm cells [43]. However, some other reports indicate no effect of driven by the suprachiasmatic nucleus of the hypothalamus melatonin on estrogen production by granulosa cells [44]. [1, 34]. Together, these data suggest that melatonin modulates ovar- Melatonin as a Selective Estrogen Enzyme Modulator Current Cancer Drug Targets, 2008, Vol. 8, No. 8 695 ian function although, in humans, this control on gonadal with enhanced pineal function or those treated with mela- activity has not been solidly demonstrated. Based on the role tonin, in contrast to pinealectomized animals or to those of the pineal in inhibiting gonadal maduration and sex hor- animals with decreased melatonin levels, have: (a) an in- mone secretion in mammals, Cohen and collaborators, in crease in tumor latency or time elapsing between the admini- 1978 [45], proposed a possible relationship between the pin- stration of the carcinogen and the appearance of palpable eal function and the etiology of breast cancer and introduced, mammary tumors, (b) a lower tumor incidence, (c) a reduc- for the first time, the hypothesis that diminished function of tion in the number and size of the tumors, (d) a lower rate of the pineal gland may promote the development of breast tumor growth, (e) a more frequent incidence of tumor regres- cancer in human beings. They supported their hypothesis by sion in previously induced tumors, (f) a decrease in serum indirect data such as a strong correlation between pineal cal- estradiol and pituitary FSH and LH, and (g) a decrease in the cification (interpreted as diminished pineal function) and the estrogen receptor concentration at tumor level [2, 3]. incidence of breast cancer and a low incidence of breast can- The growth of chemically-induced estrogen receptor- cer in psychiatric patients taking chlorpromazine, which positive mammary tumors in ovariectomized rats treated raises serum melatonin. The authors suggested that a de- daily with exogenous estradiol was significantly reduced crease in pineal function, whatever its cause, decreases mela- when these animals were subjected to some of the experi- tonin levels and induces a relative “hyperestrogenism”, mental manipulations known as enhancers of pineal- which underlies the development of breast cancer in human dependent effects (anosmia, underfeeding or exposure to beings [45]. Since then, numerous studies have suggested an cold, associated with light deprivation) [2, 3]. These animals association between melatonin levels and cancer progression subjected to increased pineal function, which had lower tu- [4, 14, 46]. Tamarkin and collaborators [47] stated that the mor growth than control and pinealectomized animals, also amplitude of the nightime peak of plasma melatonin is di- showed a reduction in the concentration of the estrogen re- minished in women with estrogen receptor-positive breast ceptor at the tumor level. In this case, these antitumor effects cancer versus those with estrogen receptor-negative disease could not be explained by a pineal-dependent decline in cir- or healthy, matched controls. Whether the decline in the noc- culating estrogens, since serum estradiol concentrations were turnal peak of plasma melatonin represents a long-term pin- kept stable because of the exogenous administration of ster- eal failure or a change that occurs at the time of breast cancer oids and the lack of changes in the rate of metabolism of development is not clear. An inverse correlation between steroids. The results of these experiments and other similar nuclear grade and tumor melatonin concentration and a posi- ones suggested that melatonin may directly counteract the tive association between tumor melatonin and estrogen re- effects of estrogens at tumor level. The direct antiestrogenic ceptor status suggested that high tumor melatonin concentra- effects of melatonin have been established from in vitro tion may reflect the differentiation state and constitute a studies mainly with the estrogen-sensitive MCF-7 human good prognostic marker [48]. Pineal melatonin secretion may breast cancer cell line. At mammary tumor cell level, mela- be modified in quantity as well as in rhythmicity in breast tonin interferes with the estrogen receptor and counteracts cancer patients [49]. The relationship between pineal func- the effects of estrogens, thus behaving as a selective estrogen tion and risk of breast cancer has also been proposed because receptor modulator (SERM). In these estrogen-responsive of the low incidence of breast cancer among blind women MCF-7 cells, melatonin exerts a direct antiproliferative ef- [50] as well as the inverse association between breast cancer fect, including a decrease in cell number, DNA content and incidence and degree of visual impairment [51]. thymidine incorporation [54-56]. This effect is dependent on Melatonin represents an important nocturnal output com- the presence of estrogen receptors in the cells. The fact that ponent of the circadian system which exerts an important only human breast cancer cell lines that express estrogen influence over many physiologic activities. Indirect evidence receptors have been found to be responsive to the antimito- is emerging that disruption of circadian clock activity is as- genic effects of melatonin, strengthen the hypothesis that sociated with cancer in humans [52]. It has recently been melatonin’s oncostatic actions are mediated through its ef- described that the exposure to light at night and the endo- fects at different levels of the estrogen signalling pathway crine disruption induced by light pollution increases the [55]. The link between the antiproliferative effect of mela- growth of DMBA-induced mammary adenocarcinomas in tonin on MCF-7 cell growth and the estrogen-response path- rats [53]. In healthy premenopausal female human subjects way is further supported: (a) by the ability of melatonin to the exposure to bright white light at night has been also pos- block, under different culture systems (monolayer and clono- tulated to result from the enhancement of human breast on- genic soft agar) the mitogenic effect of estradiol [2, 9, 54]; cogenesis via disruption of the circadian oncostatic mela- (b) by the melatonin blockade of the estrogen-rescue of tonin signal [46]. This kind of experiment encourages the tamoxifen-inhibited cells in clonogenic agar and monolayer study of melatonin-based treatments to reduce the risk of culture [9]; (c) by the ability of tamoxifen, a non-steroidal carcinogenesis. antiestrogen, to reduce the antiproliferative action of melatonin, which suggests that both tamoxifen and melatonin Besides clinical and epidemiological studies, there is evi- may be working through the same or related mechanisms dence from in vivo studies on animal models that supports [55]; (d) by melatonin’s down-regulation of both ER protein the hypothesis that melatonin oncostatic actions on hormone- and ER mRNA expression in a time- and dose-dependent dependent mammary tumors are based on antiestrogenic actions. Most in vivo studies have used, as an animal model, manner in MCF-7 cells [8, 57]; (e) by the melatonin modulation of estrogen-regulated proteins, growth factors and pro- the chemically induced (7,12-dimethylbenz(a)anthracene or tooncogenes (TGF , c-myc, pS2, progesterone receptor, c- N-nitrosomethylurea) mammary cancer in rats [see revisions fox, TGF ) in human breast cancer cells [58, 59]; (f) by the 2 and 3] and the general conclusions are that the animals 696 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Cos et al. enhanced growth suppression effects of melatonin in MT1- Most of the in vitro studies which have demonstrated the transfected ER-positive cells in comparison to parental and direct effects of melatonin on the control of the enzymes vector-transfected MCF-7 cells but, however, MT1- involved in the biosynthesis and transformation of estrogens overexpression did not induce a melatonin-sensitive pheno- in breast cancer cells were carried out on a MCF-7 human type in ER-negative breast cancer cells [60]; (g) by the en- breast cancer cell line, which was derived from a pleural hanced inhibition of ER mRNA expression induced by effusion of a woman with metastatic carcinoma of the breast melatonin in MT1-transfected MCF-7 cells [60]; and (h) by [62]. This cell line is a good model because, in addition to the ability of melatonin to counteract the estradiol-induced melatonin receptors [60, 63], it contains Arom, STS, invasiveness of mammary tumor cells [61]. 17HSD1 and EST enzymes [25, 26, 29], the enzymes involved in the intratumoral biosynthesis and transformation of The oncostatic properties of melatonin based on its ability to interact with the estrogen receptors has been reviewed estrogens. elsewhere [6, 7]. Therefore, in this review what is exclu- As indicated above, one of the major pathways involved sively summarized is the recent information on the on- in the synthesis of estrogens in mammary tumor cells is the costatic properties of melatonin based on its ability to regu- aromatization of androstenedione to estrone by aromatase. late the activity and expression of some enzymes involved in MCF-7 human breast cancer cells respond to the presence of the biosynthesis of estrogens in peripheral tissues. androgens, like testosterone, in the culture medium increasing their proliferation. Physiological concentrations of mela- MELATONIN AS A SELECTIVE ESTROGEN EN- tonin are able to counteract the testosterone-induced cell ZYME MODULATOR proliferation dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells In all the experimental models (in vivo and in vitro stud- [64]. In particular, melatonin reduces the aromatase activity ies) relating melatonin with tumor growth, estradiol reaches of MCF-7 cells both at basal conditions and when aromatase the tumor tissue from sources different from the mammary activity is stimulated by cAMP or cortisol [64]. The greatest gland tissue (ovaries in in vivo models and estradiol added to inhibition of the aromatase activity has been obtained with the culture medium in in vitro models) [2, 3]. However, as 1 nM melatonin, the same concentration that gives the high- we have previously commented, in the breast cancer of est antiproliferative and anti-invasive effects of MCF-7 cells postmenopausal women, estrogens are synthesized in the [2, 54, 61]. In addition by RT-PCR, it has been shown that mammary tissue by transformation from androgens precur- melatonin also down-regulates aromatase mRNA steady sors of adrenal origin or from biologically inactive estrogens. state levels in these cells (Fig. (2)) [64]. Transfection of the MT1 melatonin receptor in MCF-7 cells significantly de-

Fig. (2). Effects of physiological concentration of melatonin (1 nM) on aromatase (Arom), sulfatase (STS), 17-hydroxysteroid dehydrogenase type 1 (17-HSD1) and estrogen sulfotransferase (EST) activity and mRNA expression in MCF-7 human breast cancer cells. Data are expressed as the percentage of the untreated-control group (mean ± S.E.M.). a, p<0.001 vs unpretreated-control cells; b, p<0.01 vs unpretreated-control cells. Modified from Cos et al. 2005 [64] and González et al. 2008 [78]. Melatonin as a Selective Estrogen Enzyme Modulator Current Cancer Drug Targets, 2008, Vol. 8, No. 8 697 creases aromatase activity of the cells and MT1-transfected trogens from androgens, due to the aromatase action, is sup- cells show a level of aromatase activity that is 50 % of vec- pressed by the administration of melatonin (Fig. (3)) [66]. tor-transfected MCF-7 cells [65]. The proliferation of estro- The growth-stimulatory effects of testosterone on the tumors gen-sensitive MCF-7 cells in an estradiol-free medium but in depend exclusively on locally formed estrogens, since no presence of testosterone (an indirect measure of aromatase changes in serum estradiol have been described in testoster- activity) is strongly inhibited by melatonin in those cells one-treated rats. In tumors from animals treated with mela- overexpressing the MT1 receptor. This inhibitory effect of tonin the lowest microsomal aromatase activity have been melatonin on cell growth is higher in MT1 transfected cells described (Fig. (3)). This finding supports that melatonin than in vector transfected ones. In MT1-transfected cells, could exert at least part of its antitumor effects on hormone- aromatase activity is inhibited by melatonin. The same con- dependent mammary tumors by inhibiting the aromatase centrations of melatonin do not significantly influence at the activity of the tumor tissue and then reducing the intratu- same level the aromatase activity of vector-transfected cells. moral conversion of androgens to estrogens [66]. MT melatonin receptor transfection also induces a signifi- 1 Aminoglutethimide, the first non-steroidal aromatase in- cant 55 % inhibition of aromatase steady-state mRNA ex- hibitor used clinically in mammary cancer, is a reversible pression in comparison to vector-transfected MCF-7 cells. In competitive inhibitor which binds competitively to the heme addition, in MT -transfected cells melatonin treatment is able 1 moiety of aromatase. When melatonin has been used in to inhibit aromatase mRNA expression and 1 nM melatonin combination with an aromatase inhibitor such as aminoglu- induces a higher and significant downregulation of aro- tethimide, it has been reported an increase in the efficacy of matase mRNA expression than in vector-transfected cells the competitive inhibitor used in treating breast cancer [71]. [65]. These findings point to the importance of MT mela- 1 A sequential regimen of melatonin (1 nM) followed 24 hrs tonin receptor in mediating melatonin’s oncostatic action in later by aminoglutethimide (100 M) induces a significantly MCF-7 human breast cancer cells and confirm MT mela- 1 higher decrease in MCF-7 cell aromatase activity to below tonin receptor as a major mediator in the melatonin signaling the values obtained in unpretreated cells and a significant pathway in breast cancer. inhibition in the aromatase mRNA expression as compared This modulator effect of melatonin on the enzyme which to cells exposed to the same doses of aminoglutethimide, but controls the conversion from androgenic precursors to estro- without melatonin pretreatment. The aminoglutethimide in- gens has been also described in vivo, in rats bearing DMBA- hibitory effect is more potent when MCF-7 cells are pre- induced mammary tumors [66]. The growth of these mam- exposed to melatonin [71]. Melatonin pretreatment increases mary tumors is estrogen-dependent and the ovariectomy sig- the reduction of the aromatase activity of cells exposed to nificantly reduces the size and number of the tumors, while aminoglutethimide as a result of the decrease in the aro- the administration of testosterone to ovariectomized animals matase mRNA expression. Aminoglutethimide modulation is able to maintain the tumor growth at the control animals’ on aromatase expression is suppressed by adenylate cyclase level. The stimulatory effect of tumor development induced inhibitors, thus indicating that a cAMP-dependent mecha- by testosterone, which depends on the local synthesis of es- nism might be involved [72]. Again cAMP has been sug-

Fig. (3). Changes in DMBA-induced tumor size and microsomal aromatase activity in rats 9 weeks after ovariectomy (O) and treatment with testosterone (T) and melatonin (M). Data are expressed as the percentage of the control (C) group (mean ± S.E.M.). a, p<0.05 vs C; b, p<0.001 vs C; c, p<0.001 vs OT. Modified from Cos et al. 2006 [66]. 698 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Cos et al. gested as one of the possible links between melatonin, ami- Since estrone sulfate is quantitatively the most important noglutethimide and aromatase activity and expression in precursor of estradiol [25] and the estrogen sulfatase activity human breast cancer cells. is 40-500 times higher than aromatase activity in breast can- The antiaromatase effect of melatonin has been shown cer tissues [26, 77], the interest of these enzymes as targets not only in breast cancer cells. In C6 glioma cells, which for anticancer drugs seems clear. In recent years, it has been express estrogen receptors and also have the ability to syn- demonstrated that some progestins (promegestone, thesize estrogens from androgens, melatonin also counteracts nomegestrol acetate, medrogestone, dyhidrogesterone, norel- the growth stimulatory effects of testosterone, which is de- gestromin), various synthetic steroids (tibolone and its me- pendent on the local synthesis of estrogens from testoster- tabolites) as well as other steroidal (sulfamates) and non- one, reduces the aromatase activity of C6 cells and down- steroidal compounds are potent inhibitors of sulfatase [25, regulates aromatase mRNA steady state levels [73]. By anal- 26]. Melatonin, above all, at physiological (1 nM) concentra- ogy to the implications of aromatase in other forms of estro- tions also reduces the synthesis of biologically active estrogen-sensitive tumors, it is conceivable that the modulation of gens in MCF-7 cells, through the inhibition of the conversion the aromatase by melatonin may play a role in the growth of from biologically inactive forms of estrogens to bioactive glioblastomas. unconjugated estrogens via sulfatase [78]. It has been demonstrated that the mRNA expression of the enzyme responsi- It is now known that expression of the aromatase gene ble for converting estrone-sulfate and estradiol-sulfate into (CYP19) is regulated in a tissue-specific manner by the use estrone and estradiol respectively, is also significantly re- of alternative promoters. The aromatase gene (CYP19) is in duced by melatonin (Fig. (2)) [78]. Synthetic antiestrogens, normal mammary glands under the control of promoter I.4 like tamoxifen and its derivates, seem to exert some of their and is regulated by the combined action of a glucocorticoid effects on breast tumors by also blocking estrogen biosyn- and a member of the class I cytokine family (e.g., interleu- thesis through inhibition of sulfatase activity and/or expres- kin-6, interleukin-11, oncostatin-M, leukemia inhibitory fac- sion [25, 79]. tor) [74]. The 5’-upstream region of promoter I.4 contains a glucocorticoid response element which can bind transcrip- The last step of steroidogenesis in peripheral tissues is the conversion of the weak estrone to the potent biologically tion factors of the ‘signal transducer and activator of transcription’ family. Cytokines, in the presence of glucocorti- active estradiol by the activity of 17-HSD1 [25, 30] and it coids, regulate aromatase gene expression via the promoter has been suggested that this enzyme might be a marker for I.4. However, in mammary cancer cells aromatase expres- hormone-dependent breast cancer [80]. It has been reported sion is primarily regulated by increased activity of the that the production of 17-estradiol from estrone in MCF-7 proximally located promoters II and I.3, which are regulated cells decreases (2- to 3-fold) in the presence of 1 nM mela- by cAMP and factors that regulate cAMP levels [67]. Pros- tonin [78]. Furthermore, physiological or pharmacological doses of melatonin also decreased 17-HSD1 expression, taglandin E2 is an important regulator of aromatase gene expression via promoters II and I.3. [74]. It is known that mela- especially with physiological doses of the indoleamine (a 6- tonin, through a membrane-bound Gi protein-coupled recep- fold reduction of 17-HSD1 expression with 1 nM mela- tor (MT-1), down-regulates cAMP in different cell types [68, tonin) (Fig. (2)) [78]. Effects of melatonin on the steroi- 69]. Since, in MCF-7 cells, it has been demonstrated that dogenic activity of the testes, mediated by an inhibition of melatonin at nanomolar concentration reduces the forskolin- 17-HSD activity and/or expression, have also been previ- induced increase of cAMP [69] and, in murine mammary ously described in rat and hamster [81, 82]. A significant tissue, melatonin decreases cAMP and increases cGMP in decrease in the activity of 17-HSD was described in the both a dose- and time- dependent way [70], it has been sug- testes of melatonin-treated rats maintained under normal gested that melatonin could modulate aromatase through its light-dark schedules [81]. In isolated Leydig cells from ac- modulatory activity on cAMP [64]. In addition, a protective tive testes of adult hamsters kept in a long-day (14 h light, 10 effect of melatonin on inflammatory tissue, which is corre- h dark) photoperiod and from regressed testes of adult ani- lated with suppression of cyclooxygenase-2, the enzyme that mals exposed to a short-day photoperiod during 16 weeks (6 h light, 18 h dark), melatonin significantly decreased the catalyzes the synthesis of prostaglandin-E2 [75] has also been reported, thus melatonin might also regulate aromatase expression of P450 side chain cleavage, 3-hydroxysteroid dehydrogenase and 17-hydroxysteroid dehydrogenase [82]. through a modulatory activity of prostaglandin-E2 synthesis in breast tumor cells. Antiestrogens such as tamoxifen, 4-hydroxytamoxifen or ICI 164384 can also inhibit by competition the 17-HSD1 ef- Over the last decade many studies have been carried out fects in both rat mammary and human breast tumor tissues to identify potential aromatase stimulator factors. Interleu- [25, 26]. kin-6 has been one of the most potent factors detected. Cul- tured fibroblasts derived from normal or malignant breast Since the sulfoestrogens are the biologically inactive tissue were also found to be a rich source of interleukin-6. forms of the estrogens, another possibility to control the tis- Other cytokines and growth factors that can stimulate aro- sular concentration of active estradiol is to look for ways to matase activity include tumor necrosis factor-, oncostatin stimulate the enzymes involved in the sulfate formation. In M, leukaemia inhibitory factor and insulin-like growth factor this concern, it is remarkable that melatonin, at physiological type I [76]. Since melatonin is able to regulate the cytokine doses, can increase almost 2-times the estrogen sulfotrans- production in immunocompetent cells [11], the actions of ferase activity and up to 3-times the estrogen sulfotransferase melatonin on cytokine production in breast tumors may also expression in MCF-7 (Fig. (2)) [78]. be another link between melatonin and tumor estrogen syn- Most melatonin actions on enzymes involved in the intra- thesis. tumoral metabolism and biosynthesis of estrogens in breast Melatonin as a Selective Estrogen Enzyme Modulator Current Cancer Drug Targets, 2008, Vol. 8, No. 8 699

Fig. (4). Expression of enzymes related to the local production of estrogens in human normal tissue and breast carcinoma tissue. In the breast carcinoma tissue STS, Arom and 17-HSD1 tend to be overexpressed, while EST is decreased. Melatonin decreases the expression of aromatase, sulfatase and 17-HSD1 and increases the estrogen sulfotransferase expression. Thus, melatonin tends to modify the expression of the enzymes involved in the local synthesis of estrogens, causing it to be similar to the expression of enzymes in the mammary normal tissue. tumors are at physiological doses in the nanomolar range, the normal tissue. Melatonin is able not only to interact with the same concentration with which melatonin obtains the highest estrogen-signaling pathway, but also to modulate the biosyn- antiproliferative and anti-invasive effects [2, 61]. However, thesis of estrogens directly at mammary tumor cell level. other mechanisms by which melatonin exert its oncostatic Little is known about the factors regulating estrogen sul- actions, such as by its antioxidant or immunoenhancement fatase, 17-HSD1 and sulfotransferase expression in hu- effects, have been described using very high pharmacologi- mans. However, it is now evident that cytokines, growth cal concentrations of melatonin [10, 11]. factors and PGE2 have crucial roles in regulating estrogen As pointed out above, in the normal breast tissue the lo- synthesis in breast tumors. Interleukin-6 and tumor necrosis cal estrogen production is displaced towards the production factor- can stimulate the activities of all the enzymes in- of estrone, whereas in the breast carcinoma tissues what pre- volved in estrogen synthesis [76]. The activities of the aro- dominates is the formation of the active 17-estradiol, be- matase, 17-HSD1 and estrone sulfatase are all increased in cause in normal tissue, the high concentrations of circulating a co-ordinated manner by the actions of cytokines such as inactive steroids, androstenedione and estrone sulfates, are interleukin-6 and tumor necrosis factor-, and PGE2. Macro- the major precursor substrates of local estrogen production phages and lymphocytes, which invade many breast tumors, (Fig. (4)). In the breast carcinoma tissue, aromatase, which are thought to be an important source of factors that can catalyzes androstenedione into estrone, sulfatase, which hy- stimulate estrogen synthesis in malignant breast tissue. As drolyzes the estrone sulfates to estrone, and 17-HSD1, pointed out above, since melatonin is able to modulate the which converts the estrone to the potent 17-estradiol, tend cytokine production in immunocompetent cells [11], the ac- to be overexpressed, while the expression of estrogen sul- tions of melatonin on cytokines and PGE2 production in fotransferase, the enzyme that inactivates estrone and breast tumors may also be other mechanisms which might 17-estradiol, is frequently decreased, which may result in modulate the activity and expression of the enzyme tumor the accumulation of 17-estradiol in breast cancer tissues estrogen synthesis. (Fig. (4)) [30]. However, melatonin since decreases the activity and expression of aromatase, sulfatase and 17-HSD1 CONCLUSIONS and increases the activity and expression of estrogen sulfotransferase (Fig. (5)), it may protect mammary tissue from Estrogens, besides being involved in the growth and dif- excessive estrogenic effects and, as shown in Fig. (4), it ferentiation of the normal mammary gland, contribute tends to modify the activity and expression of the enzymes greatly to the genesis, growth and development of breast involved in the local synthesis of estrogens causing them to cancer and some of the mammary tumors require estrogens be similar to the expression of enzymes in the mammary for continued growth. Melatonin, the main secretory product 700 Current Cancer Drug Targets, 2008, Vol. 8, No. 8 Cos et al.

Fig. (5). Sites of action of melatonin as selective estrogen enzyme modulator in human breast cancer cells. (Abbreviations: DHEA, dehydroepiandrosterone; DHEA-S, dehydroepiandrosterone sulfate; 17-HSD type 1, 17-hydroxysteroid dehydrogenase type 1). of the pineal gland, acts as a regulator of neoplastic cell from excessive estrogenic effects. Melatonin is a naturally growth, particularly on those tumors corresponding to the occurring antiestrogen which shares properties with most of mammary gland, especially on estrogen-dependent mam- the synthetic drugs used to either interact with estrogens at mary tumors. Melatonin reduces the estrogen-mediated de- the estrogen receptor level (SERMs), or to decrease the bio- velopment of breast cancer on the basis of two different synthesis of estrogens at tumor level (SEEMs). Both proper- mechanisms: indirect neuroendocrine mechanisms, such as ties of melatonin are focused to selectively neutralize the the melatonin down-regulation of the neuroendocrine repro- effects of estrogens on the breast, one of the main objectives ductive axis and the consequent reduction of estrogenic hor- of the antitumor pharmacological therapeutics. All these ac- mones responsible for the normal and pathological growth of tions at different levels of the estrogen-signaling pathways, the mammary gland, and, on the other hand, direct melatonin collectively, make melatonin an interesting anticancer drug actions at the tumor cell level. Evidence from in vivo studies in the prevention and treatment of estrogen-dependent on animal models and in vitro studies on human breast can- mammary tumors and opens up new and interesting possi- cer cell lines supports the hypothesis that melatonin on- bilities in clinical applications of melatonin in breast cancer. costatic actions on hormone-dependent mammary tumors are mainly based on antiestrogenic actions and they are due to ACKNOWLEDGEMENTS melatonin interactions on the tumor cells’estrogen-signaling pathway. At the level of the mammary tumor cell, melatonin This work was supported by grants from the Spanish interacts with the estrogen-response pathway and counteracts MCYT (SAF2007-60659 and 62762). the effects of estrogens, thus behaving as a selective estrogen receptor modulator. Recently, melatonin has been described REFERENCES as also being able to regulate the activity of some enzymes (aromatase, sulfatase, 17-hydroxysteroid dehydrogenase, [1] Reiter, R. J. The pineal and its hormones in the control of reproduc- estrogen sulfotransferase) responsible for the local synthesis tion in mammals. Endocr. Rev. 1980, 1, 109-131. of estrogens, thus behaving as a selective estrogen enzyme [2] Cos, S.; Sánchez-Barceló, E. J. Melatonin and mammary patho- modulator. Melatonin decreases the activity and expression logical growth. Front. Neuroendocrinol. 2000, 21, 133-170. of some enzymes like aromatase, sulfatase and 17 - [3] Cos, S.; Sánchez-Barceló, E. J. Melatonin, experimental basis for a possible application in breast cancer prevention and treatment. His- hydroxysteroid dehydrogenase, whose function is increase tol. Histopathol. 2000, 15, 637-647. local estrogens, and increases the activity and expression of [4] Blask, D. E.; Sauer, L. A.; Dauchy, R. T. Melatonin as a chronobi- the estrogen sulfotransferase, an enzyme whose role in the otic/ anticancer agent: cellular, biochemical and molecular mecha- mammary tissue is to sulfonate estrogens to inactive estrogen nisms of action and their implications for circadian-based cancer sulfates (Fig. (5)). All these melatonin actions on sex steroid- therapy. Curr. Top. Med. Chem. 2002, 2, 113-132. producing enzymes may serve to protect mammary tissue Melatonin as a Selective Estrogen Enzyme Modulator Current Cancer Drug Targets, 2008, Vol. 8, No. 8 701

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Received: January 25, 2008 Revised: May 22, 2008 Accepted: September 12, 2008