Molecular Mechanisms of Melatonin Anticancer Effects

Part 2. Light, the Pineal, Melatonin, Circadian Disruption and Cancer

Integrative Cancer Therapies8(4) 337 –346© The Author(s) 2009Reprints and permission: http://www. sagepub.com/journalsPermissions.navDOI: 10.1177/1534735409353332http://ict.sagepub.com

Molecular Mechanisms of Melatonin Anticancer Effects

Steven M. Hill, PhD,1 Tripp Frasch, BA,1 Shulin Xiang, PhD,1 Lin Yuan, MD,1 Tamika Duplessis, PhD,1 and Lulu Mao PhD1

Abstract

The authors have shown that, via activation of its MT1 receptor, melatonin modulates the transcriptional activity of various nuclear receptors and the proliferation of both ERa+ and ERa- human breast cancer cells. Employing dominant-negative (DN) and dominant-positive (DP) G proteins, it was demonstrated that Gai2 proteins mediate the suppression of estrogen-induced ERa transcriptional activity by melatonin, whereas the Gaq proteins mediate the enhancement of retinoid-induced RARa transcriptional activity by melatonin. In primary human breast tumors, the authors’ studies demonstrate an inverse correlation between ERa and MT1 receptor expression, and confocal microscopic studies demonstrate that the MT1 receptor is localized to the caveoli and that its expression can be repressed by estrogen and melatonin. Melatonin, via activation of its MT1 receptor, suppresses the development and growth of breast cancer by regulation of growth factors, regulation of gene expression, regulation of clock genes, inhibition of tumor cell invasion and metastasis, and even regulation of mammary gland development. The authors have previously reported that the clock gene, Period 2 (Per2), is not expressed in human breast cancer cells but that its reexpression in breast cancer cells results in increased expression of p53 and induction of apoptosis. The authors demonstrate that melatonin, via repression of RORa transcriptional activity, blocks the expression of the clock gene BMAL1. Melatonin’s blockade of BMAL1 expression is associated with the decreased expression of SIRT1, a member of the Silencing Information Regulator family and a histone and protein deacetylase that inhibits the expression of DNA repair enzymes (p53, BRCA1 & 2, and Ku70) and the expression of apoptosis-associated genes. Finally, the authors developed an MMTV-MT1-flag mammary knock-in transgenic mouse that displays reduced ductal branching, ductal epithelium proliferation, and reduced terminal end bud formation during puberty and pregnancy. Lactating female MT1 transgenic mice show a dramatic reduction in the expression of b-casein and whey acidic milk proteins. Further analyses showed significantly reduced ERa expression in mammary glands of MT1 transgenic mice. These results demonstrate that the MT1 receptor is a major transducer of melatonin’s actions in the breast, suppressing mammary gland development and mediating the anticancer actions of melatonin through multiple pathways.

Keywords

melatonin, cancer, circadian, mammary, clock

Introduction

The pineal hormone melatonin is synthesized and secreted at night in response to darkness and is repressed by light.1 Thus, melatonin production in mammals and humans shows a diurnal variation rising and falling throughout the night and day, respectively. The circadian synthesis and secretion of melatonin is modulated by a “master biological clock” located in the suprachiasmatic nucleus of the hypothala-mus.2 The literature demonstrates that the biological functions of this indolamine are expansive; its actions as a chronobiotic hormone entraining circadian rhythms to the light–dark cycle are probably the best described.3 Melato-nin has been used for several decades to treat sleep disorders by world travelers and to help reset the body’s internal

clock. During the past several decades, there has been an explosion of work to identify the different actions of mela-tonin, especially in relation to cancer. This work on the anticancer effects of melatonin span in vivo and in vitro studies demonstrating both the direct and indirect actions of melatonin on cancer cells and its chemo preventive and therapeutic actions.

Melatonin is an indolamine that is produced in a number of tissues (eg, retina, gut); however, the pineal gland is its

1Tulane University, New Orleans, LA, USA

Corresponding Author:Tripp Frasch, Tulane University, 1430 Tulane Avenue, SL-49, New Orleans, LA 70112, USAEmail: [email protected]

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338 Integrative Cancer Therapies 8(4)

principal site of synthesis. Melatonin is synthesized from the amino acid tryptophan in the pineal gland in response to the onset of darkness. Once produced in the pineal gland, melatonin, being highly lipophilic, diffuses into the blood stream to be delivered to distant organs and also into the cerebrospinal fluid to bathe the hypothalamus and central nervous system. Melatonin’s ability to effect biological activity appears to be related to 2 major mechanisms: anti-oxidant activity and receptor-mediated activity. Our studies have focused on both in vivo and in vitro antitumor actions of melatonin. Principally, we have worked first to under-stand the cellular and molecular actions of melatonin on

breast cancer cells and more recently on normal breast epi-thelial cells.

Melatonin Inhibits the Proliferation of Human Breast Cancer CellsThe anticancer action of melatonin appears to be heavily weighted toward suppression of cell proliferation. We have demonstrated that physiologic concentrations of melatonin, corresponding to peak daytime (10-11 M) and peak night-time (10-9 M) serum values in humans, can significantly suppress the proliferation of both estrogen receptor alpha (ERa)-positive (MCF-7, T47D, ZR-75-1) and ERa- negative (MDA-MB-468) human breast cancer cells in vitro. The growth-inhibitory actions of melatonin show a linear dose response with nanomolar concentrations inhibit-ing proliferation, and micromolar concentrations being without effect on the proliferation of MCF-7 breast cancer cells.4 Melatonin, however, does not inhibit the proliferation of MDA-MB-231, MDA-MB-330, or BT-20 ERa-negative human breast tumor cells lines. Interestingly, work by Dauchy et al5 has demonstrated that melatonin can inhibit the proliferation and growth of ERa-negative and proges-terone receptor–negative human breast tumor xenographs in nude rats. The antiproliferative actions of melatonin on human breast cancer cells in vitro have been confirmed by numerous laboratories6,7 and have also been seen in other tumor types, including prostate cancer, ovarian cancer, endometrial cancer, liver cancer, and osteosarcomas.

The Antiproliferative Actions of Melatonin in Human Breast Cancer Cells Are Receptor Mediated

Numerous groups have demonstrated that melatonin can bind and activate the MT1 and MT2 G protein-coupled melatonin receptors in a variety of tissues. For example, Brydon et al8 reported that melatonin binds and activates the MT1 and MT2 receptors in cells of the hamster pars tuberalis stimulating their binding to and activation of various G proteins including Gai2, Gai3, Gaq, and Ga11. Collins et al9 and Yuan et al10 have reported that the growth-inhibitory effects of melatonin in breast cancer cells can be reversed by MT1 and MT2 melatonin antagonists and that overexpression of the MT1 receptor in human breast cancer cells can significantly enhance both the in vitro and in vivo inhibitory response of tumor cells to melatonin (Figure 1). Furthermore, Ram et al11 reported that the MT2 is not expressed in our MCF-7 breast cancer cells, whereas Lai et al12 demonstrated that the activated MT1 receptor, but not the MT2 receptor, is able to mediate the antiproliferative actions of melatonin in our breast cancer cells and that the activated MT1 receptor couples to Gai2, Gai3, Gaq, and

Figure 1. Melatonin effects on breast cancer cells MT1 overexpression. Effects of the melatonin receptor antagonist S20928 on melatonin-mediated growth inhibition of MCF-7 cells. Various concentrations of the melatonin receptor antagonist S20928 were used to antagonize the growth inhibitory effects of melatonin on (A) parental MCF-7 and (B) MT1-transfected MCF-7 breast cancer cells (n = 3 independent experiments). MLT = melatonin (10-9 M); S = Servier 20928 (10-5 to 10-9 M); a: P < .05 vs control; b: P < .05 vs 10-9 M MLT treatment



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Ga11 proteins. Lai et al13 further demonstrated using confo-cal microscopy that the MT1 receptor is localized to the MCF-7 cell membrane and that some MT1 receptors colo-calize with caveolin-1, a key protein in caveolea (lipid rafts), which are membrane-associated signaling platforms (Figure 2). Immunohistochemical analysis of 50 paraffin blocks of breast tumor biopsies demonstrated a positive correlation between MT1 and ERa expression; however, the sample size was insufficient to allow for a meaningful correlation between the MT1 melatonin receptor and any other prognostic makers in breast cancer.

Modulation of ER and Other Nuclear Receptors in Breast Cancer Cells by Melatonin

Interplay between the melatonin and estrogen-signaling pathways has long been suspected; early on we observed that melatonin could suppress the estrogen-induced prolif-eration of human breast cancer cells.14 This observation was followed-up by a report by Molis et al15 that melatonin could suppress the expression of ERa at the mRNA levels. A more detailed analysis by Ram et al11 found that melato-nin could suppress estrogen-induced transcriptional activity of the ERa, downregulating the expression of a number of

mitogenic proteins and pathways including the antiapop-totic protein Bcl-2, while inducing the expression of growth-inhibitory and apoptotic pathways including TGF-b and Bax. We have demonstrated that the inhibitory actions of melatonin on ERa transcriptional activity are mediated via activation of Gai2 signaling12 and that suppression of cAMP/PKA signaling results in a decreased phosphoryla-tion on the PKA-sensitive S263 site on the ERa (Figure 3). Del Rio et al16 confirmed our observations that melatonin can modulate ERa transcriptional activity and further reported that calmodulin (CaM) was involved in this pro-cess. We believe that these observations are not inconsistent, as we have demonstrated that the PKA pathway can impact Ca++/CaM activity in various tissues.17

In addition to suppressing ERa transcriptional activity, melatonin, via different G proteins, can also modulate the transactivation of a number of members of the steroid hor-mone/nuclear receptor superfamily. Kiefer et al18 have demonstrated that melatonin can suppress the transcrip-tional activity of the glucocorticoid receptor (GR) while enhancing the transcriptional activation of the retinoic acid receptor alpha (RARa) in human breast cancer cells in response to their ligands, via activation of Gai2 and Gaq pro-teins, respectively. Dai et al,19 from our laboratory, have also demonstrated that melatonin can suppress the tran-scriptional activity of the retinoic acid–related orphan receptor alpha (RORa) in human breast cancer cells. In addition, we have recently found (unpublished results) that melatonin can potentiate the transcriptional activity of the retinoic X receptor alpha (RXRa) and the vitamin D3 recep-tor (VDR) in response to the specific ligand. Melatonin,

Figure 2. Confocal analysis of MT1 receptor intracellular localization. (A) MCF-7 breast cancer cells and HEK293 embryonic kidney cells were grown on cover slips and incubated with the MT1 antibody followed by Alexa-488-conjugated goat antirabbit IgG (green). Membrane and cytoplasmic MT1 is observed in MCF-7 cells but not in HEK293 cells. (B) MCF-7 breast cancer cells were grown on cover slips and were incubated with the MT1 antibody (1:500) and or CAV-1 antibody (1:100) in complete culture medium. Cav-1 and MT1 were revealed by incubating cells incubated with the Cav-1 and MT1 antibodies and then with the Alexa Fluor 546 (red)- or Alexa Fluor 488 (green)-conjugated secondary antibodies, respectively, under nonpermiabilized conditions. All immunofluorescence samples were observed using a Laser Scan Confocal microscope

Figure 3. ERa PKA phosphorylation site suppressed by MLT. (A) ERE-luciferase reporter assay in MCF-7 human breast tumor cells treated with diluent, 10-8 M melatonin (Mlt), 10-8 M 17-b-estradiol (E2), or pretreated for 5 minutes with Mlt followed by E2. (B, Top) Schematic representation of the location of ERa phosphorylation sites and the kinases that have been shown to be responsible for phosphorylation at those sites. (B, Below) Western blot analysis of lysates prepared from MCF-7 cells incubated with 10-8 M E2, 10-7 MLT, or combination with MLT pretreatment followed by E2 using antibodies specific for ERa, ERa P-S236, and GAPDH as a loading control




however, does not modulate all nuclear receptors as we have not seen any effect on ERb transcriptional activity in human breast cancer or when ERb is expressed in HEK293 embryonic kidney cells.12 Although we have demonstrated that melatonin via various G proteins can modulate a number of nuclear receptors, and that melatonin administra-tion can diminish the phosphorylation of the ERa at the PKA-sensitive S263 (Figure 3), we still do not know if melatonin’s effects are mediated via direct changes in phos-phorylation of the nuclear receptor, regulation of coactivator (eg, CaM, SRC1, etc) or corepressor phosphorylation, or both. Regardless, these data clearly demonstrate the ability of melatonin, via signal transduction pathways, to affect gene expression in human breast cancer cells.

Repression of Breast Cancer Cell Invasion/Metastasis by MelatoninSeveral early correlative studies observed that the plasma level of melatonin is significantly reduced in cancer patients with metastatic disease when compared with those without metastases.20,21 In 1998, Cos et al22 proposed that melatonin plays an inhibitory role in regulating breast tumor invasion and metastasis. In this in vitro study, physiologic concentra-tions of melatonin (10-9 M) were reported to significantly reduce the invasive capacity of MCF-7 human breast cancer cell as measured by Falcon invasion chamber assays, a modified Boyden chamber assay to block 17-b-estradiol (E2) induced MCF-7 cell invasion. Unfortunately, it is gen-erally viewed that MCF-7 human breast tumor cells are poorly to noninvasive, which brings into question the report of melatonin’s anti-invasive actions reported by Cos et al.22

To evaluate the potential anti-invasive/antimetastatic actions of melatonin, we employed 3 clones of MCF-7 cells with demonstrated high metastatic potential including the MCF-7/6 clone derived by serial passages in nude mice, MCF-7Her2.1 cells stably transformed with and overexpressing the Her2-neu/c-erbB2 construct, and MCF-7CXCR4 cells stably transformed with and overexpressing the CXCR4 cytokine G protein–coupled receptor. The Her2/neu (c-erbB2) receptor and the CXCR4 receptors are well described as increasing the metastatic and invasive actions of breast cancer cells when expressed at high levels,23 and in the case of the CXCR4 receptor, enhancing metastatic homing to bone.24 As shown in Figure 4, the invasive capacity of these clones was significantly greater than those of parental MCF-7 cells with MCF-7/6 < MCF-7CXCR4 < MCF-7Her2.1 cells. Treatment of MCF-7Her2.1 cells and the other 2 invasive clones with melatonin (10-8 or 10-9 M) resulted in significant suppression (60% to 85% decrease) of cell invasion using a transwell assay system and matrigel-covered inserts. The anti-invasive response to melatonin was enhanced by overexpression of the MT1

receptor and inhibited by administration of luzindole, an MT1/MT2 melatonin receptor antagonist.

Invasion and metastasis in breast cancer is driven in many cases by enhanced activity of the p38 MAPK signaling pathway leading to elevated expression of the matrix metal-loproteinases MMP2 and MMP9.25 Mao et al26 has demonstrated that each of these metastatic MCF-7 clones has enhanced expression of the phosphorylated p38 and MMP2 and MMP9 and that melatonin administration can block p38 phosphorylation and subsequent MMP2 and MMP9 expres-sion thereby, suppressing breast cancer cell invasion. Though we know these actions are MT1 receptor mediated, we do not yet know the downstream signaling pathway involved from the MT1 receptor to p38. We suspect, however, that Rho may be a key player in this process. Follow-up studies are cur-rently underway using xenographs of these metastatic clones to look at melatonin’s actions on metastasis in vivo.

The Role of Melatonin and the MT1 Receptor in the Normal Mammary GlandConsiderable data have accumulated demonstrating that the pineal hormone melatonin plays an important role in the etiology of breast cancer development and progression. However, little is known with regard to the effects of mela-tonin on the development, growth, and function of the normal breast. To determine if melatonin, via its G-protein coupled receptor MT1, affects the development, growth, and function of the mouse mammary gland, we generated an MMTV-MT1 conditional mammary gland knock-in (KI) transgenic mouse (MT1-mKI). Real-time PCR and Western blotting analyses demonstrate MT1 and Flag (MT1-Flag tagged) expression within mammary glands of pubescent virgin MT1 transgenic female mice and a significant increase in MT1 expression in pregnant MT1 transgenic mice. Analysis of mammary gland whole mounts from 3 breeder lines of MT1 transgenic mice demonstrate reduced ductal branching, ductal growth, and TEB formation within the mammary gland of nulliparous MT1-mKI transgenic mice (Figure 5). Elevated levels of MT1 expression in pregnant and lactating female MT1 transgenic mice are associated with a dramatic reduction in lobulo-alveolar development. A significant reduction in body weight of suckling pups from MT1 transgenic dams is also observed. However, body weight differences between pups of trans-genic and control dams is lost after weaning, supporting the concept that impaired lactation in MT1 transgenic mothers leads to decreased pup weights. Furthermore, enhanced expression of MT1 in MT1-mKI transgenic pubescent mice significantly suppresses ERa expression in mammary gland epithelium. These data demonstrate that melatonin, via its MT1 receptor, regulates lobulo-alveolar develop-ment and, thus, mammary gland development in mice.



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Circadian Disruption, Clock, Melatonin, and Breast Cancer

Life is a rhythmic phenomenon and many, if not most, bio-logic and physiologic activities oscillate with time. Circadian (meaning about 24-hours) rhythms are innate components that overlay our physiology, metabolism, behavior, and health, but are not completely understood. The basic function of the internal central circadian clock, located in the brain within the suprachiasmatic nuclei (SCN) of the hypothalamus is to temporally organize our body’s functions into predictable rhythms that are synchro-nized by external time cues provided by the environmental light/dark cycle (ie, photoperiod).2 Dramatic changes in

Western lifestyles, such as night shift work, have significant affected our circadian rhythms and subsequently society and health due to the circadian clock’s inability to quickly or fully adjust, resulting in acute or chronic circadian dis-ruption.27,28 The pineal hormone melatonin, the most stable and reliable output and biomarker of the central circadian clock, is synthesized at night (during darkness) and sup-pressed during daytime in response to light. Circadian disruption, by exposure to light at night (LAN; eg, night shift work) can dramatically suppress nighttime production of melatonin, diminishing or abolishing its multifaceted effects on organismal and cellular physiology. Unfortu-nately, the occurrence of gastroenterological disorders, obesity, diabetes, cardiovascular disease, infertility, and

Figure 4. Characterization of melatonin responsiveness and MT1 receptor expression of a panel of human breast cancer cells with various invasive potential. (A) The relative invasive potential of MCF-7, MCF-7/AZ, MCF-7/6, and MCF-7/Her2.1 breast cancer cells. Cells were plated onto matrigel invasion chambers at 5 × 104 cells/mL and incubated for 4 days. The number of invading cells in 5 random fields was counted microscopically at 100× magnification. Data are presented as fold of control (MCF-7, 100%). (B) Effects of melatonin on the invasive potential of MCF-7/Her2.1 cells. MCF-7/Her2.1 cells were plated onto matrigel-invasion chambers after being serum starved for 24 hours and incubated in the medium containing melatonin (Mlt, 10-9 M) or diluent (Control, 0.00001% ethanol) for 4 and 6 days. Data are represented as percentage of control (100%). *, P < .05 versus diluent-treated cells, n = 3. (C) Effect of MT1 receptor overexpression on melatonin-regulated invasive potential of MCF-7/6 cells. MCF-7/6 breast cancer cells were transiently transfected with either pcDNA3.1 empty vector (V) or pcDNA3.1-MT1 plasmid (MT1) or sham transfected (P). Twenty-four hours following transfection, cells were plated onto matrigel-invasion chambers and treated with melatonin (Mlt, 10-9 M) or diluent (Control, 0.00001% ethanol) for 4 days. Data are presented as percentage of the sham-transfected diluent-treated control (100%). An RT-PCR analysis of MT1 expression is shown to demonstrate the overexpression of MT1 in pcDNA3.1-MT1-transfected cells. (D) Effect of Luzindole on melatonin-regulated invasive potential of MCF-7/6 cells. MCF-7/6 breast cancer cells were serum starved and plated onto matrigel invasion chambers and treated with diluent (Control, 0.00001% ethanol), melatonin (Mlt, 10-9 M), Luzindole (LZD, 10-8 M), or Luzindole for 15 minutes followed by melatonin (10-9 M) treatment (LZD + Mlt), for 2 days. Data are presented as percentage of ethanol-treated control (100%). *, P < .05 versus vehicle-treated control cells; **, P < .05 versus melatonin-treated cells, n = 3. For invasion assays in A, B, C, and D, the number of invading cells in 5 random fields was counted microscopically at 100× magnification. The percentage of invasive cells was calculated as described previously. One-way ANOVA followed by Student–Newman–Kuel’s post hoc test analyses was used to determine statistically significant differences in the percentage of invasive cells among different treatment groups




Figure 5. MT1 expression suppresses mammary gland development in mice. (Top) Western blot analysis of MT1 transgene expression. Total cellular protein was isolated from the number 4 inguinal mammary gland of developmentally staged MT1-KI female transgenic mice. Seventy-five micrograms of total protein from each sample was separated by 12% PAGE and subjected to Western blot analysis using an anti–flag specific antibody. (A) Elevated expression of MT1 in mammary gland inhibits ductal branching and terminal end bud (TEB) formation at puberty. Mice were sacrificed at age 4, 8, or 12 weeks. The number 4 inguinal mammary gland was excised and spread onto a glass microscope slide, fixed in acidic ethanol, and processed using the whole-mount procedure and stained with carmine. MT1 expression inhibits TEB formation at 4 and 8 weeks of age; the ductal branching observed in MT1 transgenic mice was less extensive than nontransgenic mice. The inhibitory effects of MT1 on TEB formation and ductal branching are statistically significant. (A, Lower panel) Data are expressed as numbers per mm2 ± standard error of mean. (B) Mammary gland expression of transgene MT1 inhibits lobulo-alveolar development. Transgenic and control mice were sacrificed at days 14 and 18 of pregnancy and day 1 of lactation. Mammary tissue from nontransgenic control MT1-KI mice showed suppressed lobulo-alveolar development at day 14 of pregnancy with ducts bearing few lateral and terminal alveoli. At day 18 of pregnancy and lactation large regions of mammary tissue exhibited sparse lobulo-alveolar expansion in the MT1 transgenic glands. (C) MT1 transgenic mice exhibit defects in mammary epithelial proliferation. BrdU incorporation was determined by immunohistochemistry of paraffin-embedded mammary glands. When compared with nontransgenic mammary glands at the same developmental time points, a significant reduction in BrdU-positive immunoreactivity in epithelial cells was observed in mammary tissue from MT1 transgenic mice at puberty 8 weeks through day 18 of pregnancy. No proliferation is observed in late stages of lactation. (D) Elevated expression of the MT1 transgene reduces the expression of the whey acid milk protein (WAP) in mammary gland. WAP expression was determined by immunohistochemistry of paraffin-embedded mammary glands. When compared with nontransgenic mammary glands at the same developmental time points, a significant reduction in WAP-positive immunoreactivity in epithelial cells was observed in mammary tissue from MT1 transgenic mice at puberty 8 weeks through day 18 of pregnancy

some forms of cancer, particularly breast cancer, have been found to be significantly elevated in night shift workers, who comprise at least 20% of our workforce.29 With the

characterization and understanding of how the “circadian clock” works in both the SCN and in cells of peripheral tissues, the interplay between the circadian clock and



Hill et al 343

melatonin, the developing role of circadian disruption, the circadian clock, and the result of suppressed melatonin pro-duction in the etiology of breast cancer, it is essential to now develop and characterize animal models (eg, the female mouse) that will demonstrate the organismal, sys-temic, cellular, and molecular results of disruption of circadian rhythms, the “circadian clock,” and melatonin on human health and, in particular, breast cancer.

Melatonin: A Biomarker of Regulation/Disruption and an Inhibitor of CancerThe SCN transmits environmental photoperiodic informa-tion to the pineal gland, via a multisynaptic pathway, to regulate melatonin synthesis. Melatonin can be reliably measured directly in blood and indirectly through its metab-olite in urine. Melatonin exhibits a robust and highly consistent circadian rhythm with nighttime (dark phase) synthesis and secretion into the bloodstream (peak 2-3 am) and low daytime (light phase) levels with light inhibiting daytime pineal melatonin production.30 Furthermore, pineal melatonin production is suppressed by LAN, resulting in lower melatonin levels in night shift workers.

Regulation of the Circadian System by LightThe nerve pathway propagating light signals extend from the retina into the paired SCN of the anterior hypothala-mus.2 The SCN are key components of the internal

“biological clock,” or central circadian pacemaker, that regulates daily rhythms. As shown in Figure 6, the “clock” mechanism that operates in both the SCN and peripheral cells (including cancer cells) consists of interacting positive and negative feedback loops that regulate the transcription of the clock genes, 12 of which have been identified.31 The Clock and BMAL1 genes participate in a positive feedback loop, whereas period (Per) and cryptochrome (Cry) genes are involved in a negative feedback loop. A heterodimer of CLOCK and BMAL1 proteins rhythmically activates the transcription of clock genes Per1, Per2, Per3, Cry1, Cry2, and Rev-erba, and also activate the downstream clock- controlled genes (CCGs). The rhythmic expression of BMAL1 is generated via its transcriptional activation by RORa and transcriptional repression by Rev-erba.32 We have recently reported33 that elevated expression of RORa1 can enhance BMAL1 transcription and expression in MCF-10A human breast epithelial cells and MCF-7 breast cancer cells and that melatonin blunts the transcriptional activation of the RORa1 blocking its induction of BMAL1 expression. Thus, melatonin, via its MT1 receptor, can directly suppress an important element of the clock mechanism in peripheral tissues, specifically breast epithelial and cancer cells.

Circadian/Clock Genes and Their Roles in TumorigenesisA connection between cancer development and the circa-dian cycle has been demonstrated in hormone-related cancers including breast and prostate cancers through the studies of pilots, flight attendants, and night shift workers who are more likely to have disrupted circadian cycles due to their abnormal work hours.29 Approximately 15% to 20% of all mammalian genes are CCGs, indicating extensive cir-cadian gene regulation.34 Recent findings suggest that some CCGs function as tumor suppressors at the systemic, cellu-lar, and molecular levels due to their involvement in cell proliferation, apoptosis (p53, Bax), cell cycle control (ChK2), and DNA damage response (p53, BRCA1 & 2, Ku70).35 A recent study showed that disruption of the Per2 gene is associated with tumor development in mice.36 We have recently reported that expression of the clock protein (Per2) is lost in human breast cancer, whereas its reintro-duction increases p53 expression and induces apoptosis.33

Recently, the antiaging protein Silencing Information Regulator Two family member, SIRT1, has been linked with the molecular circadian clock and cancer.37,38 SIRT1 is a NAD+-dependant class III histone deacetylase (HDAC) that affects a broad spectrum of cellular processes including gene silencing, DNA repair, apoptosis, cellular metabolism, cellular senescence, and aging. SIRT1 has been reported to bind directly to the CLOCK/BMAL1 heterodimer, promot-ing deacetylation and degradation of PER2 (a tumor

Figure 6. The molecular components of the clock in human breast cancer cells




suppressor that interacts with BRCA1). SIRT1 also sup-presses the transcription or activity of a number of genes including p53, FOXOA1 & 3, and PGC-1a. This associa-tion involves suppression of DNA-repair enzymes including p53, BRCA1 & 2, and Ku70. Thus, SIRT1 may be a focal point through which the circadian clock influences cancer

development (Figure 6). Given that we have found that melatonin can regulate the expression of BMAL1, via mod-ulation of RORa transcriptional activity, and that SIRT1 is associated with BMAL1, we asked whether melatonin is able to modulate the expression of SIRT1 in human breast cancer cells. As shown in Figure 7, SIRT1 is expressed at high levels in MCF-7 and MDA-MB-231 human breast cancer cell lines as well as human umbilical vascular endo-thelial cells. Administration of melatonin (10-8 M) following transfection of an MT1-receptor expression construct into MCF-7 cells dramatically suppressed SIRT1 expression.

Model of Melatonin’s Antineoplastic Actions in Breast CancerFrom our studies and those described in the literature, partic-ularly the work of the Blask laboratory elucidating the actions of melatonin on the in vivo growth of mammary tumors and its role in suppressing the fatty acid linoleic acid/13-HODE signaling pathway, we have developed a model of melato-nin’s action on breast cancer initiation, promotion, and progression. Our studies in the MT1-mKI mice (Figure 5) clearly demonstrate that melatonin, via its MT1 receptor, can repress the proliferation of mammary epithelial cells and

Figure 8. The points at which melatonin, via the MT1 receptor, affects circadian disruption to modulate the initiation, promotion, and progression, phases of breast cancer development

Figure 7. Melatonin inhibition of SIRT1 expression. Inhibition of SIRT1 expression in MCF-7 human breast cancer and human umbilical vascular endothelial cells in response to MT1 overexpression and melatonin (10-8 M) administration for 36 hours



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slow/suppress the development of the mouse mammary gland. Given that we have demonstrated that melatonin can modulate components of the biological clock (BMAL1 and SIRT1) in breast epithelial and cancer cells, that PER2 and SIRT1 can modulate DNA repair and apoptotic pathways, and that melatonin, via the MT1 receptor, can enhance RARa transcriptional activity significantly preventing NMU-induced mammary tumor development in rats, we have outlined in this model (Figure 8) the currently known path-ways by which melatonin can suppress the initiation of breast cancer. These pathways include upregulation of PER2 expres-sion by inhibition of SIRT1 leading to enhanced expression and activity of the DNA repair/apoptotic proteins BRCA1 & 2, p53, Ku70 and the apoptotic-associated factors FOXOA3, estrogen-related receptor alpha (ERRa), and Bax. Melatonin, via its potentiation of RARa signaling, can potentially stimu-late differentiation of breast tumor cells making them sensitive to apoptotic mechanisms. In addition to suppressing tumor initiation, melatonin can block or suppress the promo-tional and progressional phases of cancer development by (a) inhibiting the uptake of linoleic acid and its conversion to 13-HODE, decreasing the mitogenic MAPK/ERK signaling pathway; (b) suppressing/blocking the mitogenic estrogen signaling pathway, repressing ERa expression and transcrip-tional activity; and (c) potentiating the transcriptional activity of the RARa, RXRa, and VDR to stimulate their growth-suppressive and even apoptotic-activating mechanisms. Finally, melatonin can further modulate tumor progression by inhibiting the phosphorylation and activation of the p38 MAPK pathway, a key pathway in the invasion/metastatic process of breast cancer cells. These studies clearly demon-strate that melatonin, via its MT1 receptor, can suppress the development of cancer via a broad spectrum of mechanisms that span all phases of cancer development and that suppres-sion of melatonin production by circadian disruption will have significant and dire consequences on cancer initiation, cancer promotion, and cancer progression.

Declaration of Conflicting Interests

The author declared no potential conflicts of interests with respect to the authorship and/or publication of this article.

Funding

The author disclosed receipt of the following financial support for the research and/or authorship of this article:

This work was supported by NIH grant CA-054152-14 and Army DOD grant to SMH.

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Molecular Mechanisms of Melatonin Anticancer Effects

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