Melatonin, experimental basis for a possible application in breast cancer prevention and treatment

The role of the pineal as an oncostatic gland has been studied in animal models of tumorigenesis, especially on those concerning the mammary gland. The general conclusion is that experimental manipulations activating pineal gland, or the administration of melatonin, reduce the incidence and growth rate of chemically-induced murine mammary tumors, while pinealectomy or situations which implicate a reduction of melatonin production usually stimulate mammary carcinogenesis. The direct actions of melatonin on mammary tumors have been suggested because of its ability to inhibit, at physiological doses (1nM), the in vitro proliferation of MCF-7 human breast cancer cells. In this article we review the outstanding findings related to melatonin actions on mammary which, taken together, support a possible usefulness of this indoleamine in the prevention and treatment of mammary gland malignancy.     

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.     

Inhibitory action of melatonin on a pineal antigonadotropin.

An acidic extract of bovine pineal glands, partially purified by gel chromatography, yielded two distinct fractions which inhibited compensatory ovarian hypertrophy in mice. When the fractions were tested for antigonadotropic properties in the presence of melatonin only one fraction was found to be effective. Melatonin by itself and no stimulatory effect on COH, suggesting that melatonin blocked the antigonadotropic activity of the one fraction.     

A cytotoxic, apoptotic, low-molecular weight factor from pineal gland.

Previous studies suggest that the pineal gland may play a role in tumour growth inhibition. In this respect, melatonin, as the major hormone of this gland, has been extensively studied. However, there is growing evidence for the existence of other yet unknown pineal factors that may have tumour growth inhibiting properties. Here we describe the partial purification of a highly cytotoxic low molecular weight (<400 Da) hydrophilic fraction (designated F2M3R), starting from a porcine pineal extract (PE), via methanol precipitation followed by reverse-phase HPLC. F2M3R is cytotoxic for a highly apoptosis-resistant human erythroleukemia cell line (K562) at a concentration as low as 30 microg/ml. The viability of the cells was not influenced by an identical prepared porcine pituitary extract or by melatonin. PE induces apoptosis in K562 cells as indicated by three different criteria: morphology, in situ TUNEL assay and bi-parametric FACS analysis with annexin V and propidium iodide, but does not influence the viability of stimulated peripheral blood mononuclear cells. These observations warrant further purification and validation of the cytotoxicity in a panel of different human tumour and non-malignant cells.     

Expression of transforming growth factor alpha (TGF alpha) in differentiated rat mammary tumors: estrogen induction of TGF alpha production

Primary well-differentiated dimethylbenzene alpha-anthracene (DMBA)-or nitrosomethylurea (NMU)-induced rat mammary adenocarcinomas that are estrogen dependent possess biologically active and immunoreactive transforming growth factor alpha (TGF alpha), which can be detected in a sort agar growth-promoting assay and by a specific liquid-phase competitive RIA, respectively. In contrast, tissue extracts prepared from transplantable undifferentiated DMBA-I and NMU-II rat mammary carcinomas that are estrogen independent and metastatic exhibit low or undetectable levels of TGF alpha. In addition, the primary DMBA- and NMU-induced rat mammary adenocarcinomas express a specific 4.8-kilobase TGF alpha mRNA species, whereas little or no TGF alpha mRNA can be detected in the transplantable DMBA-I and NMU-II tumors. Primary tumors synthesize type IV basement membrane collagen, whereas the transplantable tumors elaborate very little type IV collagen. Either TGF alpha or estrogens can differentially enhance the synthesis of type IV collagen by 0.5- to 4-fold over total protein synthesis in primary cultures of normal mouse mammary epithelial cells or in primary NMU-induced tumor cells, respectively. Therefore, TGF alpha could function as an estrogen-inducible autocrine growth factor for well differentiated rat mammary tumor cells by its ability to selectively regulate type IV collagen synthesis. Estrogens can modulate TGF alpha production in vivo in primary DMBA-induced rat mammary tumors, because ovariectomy results in a rapid decline (within 6 h) of TGF alpha mRNA levels. This response to estrogens can also be observed in vitro. Primary DMBA- or NMU-induced rat mammary tumor cells cultured in the presence of 17 beta-estradiol (10(-8) M) for 4 days show an increase in the level of TGF alpha mRNA over cells not treated with estrogen. This increase in TGF alpha mRNA is paralleled by a 2- to 3-fold increase in the levels of immunoreactive TGF alpha that can be detected and in the conditioned medium from estrogen-treated cells. These results suggest that TGF alpha may be an adjunct marker for those mammary tumors that are well differentiated adenocarcinomas and estrogen dependent and that estrogen-independent tumors do not constitutively produce TGF alpha or express TGF alpha mRNA.     

Light during darkness,melatonin suppression and cancer progression

Over the past few years, we have shown that the surge of melatonin in the circulation during darkness represents a potent oncostatic signal to tissue-isolated rat hepatoma 7288CTC, which is an ER+ adenocarcinoma of the liver. This oncostatic effect occurs via a melatonin receptor-mediated suppression of tumor cAMP production that leads to a suppression of the tumor uptake of linoleic acid (LA), an essential fatty acid with substantial oncogenic properties. The ability of LA to promote cancer progression is accomplished by its intracellular metabolism to 13-hydroxyoctadecadienoic acid (13-HODE) which amplifies the activity of the epidermal growth factor receptor/mitogen-activated protein kinase pathway leading to cell proliferation. By blocking tumor LA uptake, melatonin effectively blocks the production of 13-HODE and thus, markedly attenuates tumor growth. A similar effect of melatonin is observed in tissue-isolated, ER+ MCF-7 human breast cancer xenografts and nitrosomethylurea (NMU)-induced rat mammary cancers. When male rats bearing tissue-isolated hepatomas are exposed either to constant bright light (300 lux) or dim light (0.25 lux) during the dark phase of a 12L:12D photoperiod, the latency to onset was significantly reduced while the growth of tumors was markedly increased over a 4 wk period as compared with control tumors in 12L:12D-exposed rats. In constant light- and dim light during darkness-exposed rats, melatonin levels were completely suppressed while tumor growth, LA uptake and 13-HODE production were markedly increased. Similar results were obtained in constant bright light-exposed female rats bearing tissue-isolated NMU-induced mammary cancers or MCF-7 human breast cancer xenografts. To date, these studies provide the most definitive experimental evidence that light exposure during darkness increases the risk of cancer progression via elimination of the nocturnal melatonin signal and its suppression of tumor LA uptake and metabolism to 13-HODE.     

Inhibition of aromatase activity and of endocrine-responsive tumor growth by 10-propargylestr-4-ene-3, 17-dione and its 17-propionate derivative

Two androstenedione derivatives, 10-propargylestr-4-ene-3,17-dione and its 17-propionated form, were administered to normal cycling rats, and both compounds led to an inhibition of ovarian aromatase. Under in vitro conditions, only the former compound exhibited high potency as an inhibitor of rat ovarian and human placental microsomal aromatase. At 1 mg/kg/day both compounds were effective in promoting regression of 9,10-dimethyl-1,2-benzanthracene-induced mammary tumors in rats without terminating their estrous cycle. PED also inhibited growth of a human ovarian carcinoma in athymic mice. The results with the 17-propionated compound testify to the necessity of in vivo assays in screening antitumor agents. In summary, PED and its propionated derivative inhibited ovarian aromatase in vivo and inhibited the growth of hormone-responsive tumors.     

Hormonal interactions in human prostate tumor LNCaP cells

Melatonin, the hormone secreted by the pineal gland at night, has recently been found to attenuate growth and viability of benign human prostate epithelial cells. Estradiol suppressed these responses by efflecting a protein kinase C mediated inactivation of melatonin receptors. In the present study, the effects of melatonin on growth and viability of the human androgen-sensitive prostatic tumor cell line-LNCaP and the influence of estradiol on these responses were explored. Melatonin inhibited 3H-thymidine incorporation into LNCaP cells at physiological concentrations. This response decayed within 24 h. The inactivation of the response slowed down in the presence of the protein kinase C inhibitor GF-109203X. Estradiol also inhibited 3H-thymidine incorporation and its effects were additive to those of melatonin. Suppression of DNA content was observed in cells treated for 2 days with melatonin (0.1 nM); this suppression was maintained for longer periods in the presence than in the absence of estradiol. In addition, estradiol and melatonin slightly and additively decreased cell viability. These results demonstrate for the first time a direct interaction of melatonin with androgen-sensitive prostate tumor cells leading to attenuation of cell growth. They also show that unlike in benign prostate epithelial cells, estrogen attenuates LNCaP cell growth and supports rather than inactivates melatonin's action.     

Anti-Tumor Activity of Yuanhuacine by Regulating AMPK/mTOR Signaling Pathway and Actin Cytoskeleton Organization in Non-Small Cell Lung Cancer Cells

Yuanhuacine (YC), a daphnane diterpenoid from the flowers of Daphne genkwa, exhibited a potential growth inhibitory activity against human non-small cell lung cancer (NSCLC) cells. YC also suppressed the invasion and migration of lung cancer cells. However, the precise molecular mechanisms remain to be elucidated. In the present study, we report that YC significantly activated AMP-activated protein kinase (AMPK) signaling pathway and suppressed mTORC2-mediated downstream signaling pathway in H1993 human NSCLC cells. AMPK plays an important role in energy metabolism and cancer biology. Therefore, activators of AMPK signaling pathways can be applicable to the treatment of cancer. YC enhanced the expression of p-AMPKα. The co-treatment of YC and compound C (an AMPK inhibitor) or metformin (an AMPK activator) also confirmed that YC increases p-AMPKα. YC also suppressed the activation of the mammalian target of rapamycin (mTOR) expression, a downstream target of AMPK. Further study revealed that YC modulates mTORC2-associated downstream signaling pathways with a decreased expressions of p-Akt, p-protein kinase C alpha (PKCα), p-ras-related C3 botulinum toxin substrate 1 (Rac1) and filamentous actin (F-actin) that are known to activate cell growth and organize actin cytoskeleton. In addition, YC inhibited the tumor growth in H1993 cell-implanted xenograft nude mouse model. These data suggest the YC could be a potential candidate for cancer chemotherapeutic agents derived from natural products by regulating AMPK/mTORC2 signaling pathway and actin cytoskeleton organization.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Relationship Between Organization of Mammary Tumors and the Ability of Tumor Cells to Replicate Mammary Tumor Virus and to Recognize Growth-Inhibitory Contact Signals In Vitro

Mammary tumor virus (MTV) replication was confined primarily to cells organized as acini in intact mouse mammary glands. Primary mammary tumors maintained a high degree of acinar organization and cells therein continued to replicate MTV vegetatively. Nonacinar mammary cells, derived by serial transplantation of acinar tumor cells, no longer actively replicated MTV. This suggests that phenotypic differences exist among mammary epithelial cells in their ability to support virus replication, that a fundamental relationship exists between the organization of epithelium for secretion and active virus replication, and that this relationship is not altered as a primary consequence of neoplastic transformation. Mammary epithelial cells from pregnant, non-tumor-bearing, MTV-infected BALB/cfC3H mice or from acinar mammary tumors from a number of mouse strains were grown in primary monolayer cultures. Such cell cultures under the influence of insulin and cortisol exhibited the ability to organize into discrete three-dimensional structures called "domes." MTV replication in such cultures took place primarily in cells within the organized domes. Cells cultured from nonacinar tumors did not exhibit any propensity to organize into domes, nor did they replicate MTV in primary culture. This suggests that the cell organizational requirement for MTV replication observed in vivo is conserved in primary culture. Dome formation is not an effect of virus replication, as cells from uninfected BALB/c animals organized into domes in culture without concomitant MTV replication. Growth-regulating signals, exerted between contiguous cells in cultures of non-MTV-infected mammary epithelium, were not modified by the occurrence of active virus replication nor as a direct consequence of neoplastic transformation. Cells derived from nontumor BALB/cfC3H glands and from spontaneous tumors exhibited cell growth kinetics, saturation densities, and deoxyribonucleic acid synthesis kinetics nearly identical to those of noninfected normal mammary epithelium in primary culture. Cell to cell growth regulatory signals were modified in cultures of nonalveolar tumor cells wherein evidence of overgrowth is documented.     

Role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated growth by estradiol, prolactin and progesterone in human and rat mammary tumor cells: studies using TGF-alpha and EGF receptor antibodies

The biological role of transforming growth factor-alpha (TGF-alpha) in basal and hormone-stimulated proliferation of primary human and rat mammary tumor cells was studied using antibodies against TGF-alpha and its receptor. A monoclonal antibody, MAb-425 against human EGF receptor was added to in vitro soft agar, clonogenic cultures of human breast carcinoma cells under basal and estradiol(E2)-stimulated conditions. The antibody had an antagonist effect on colony growth in 4 of 10 tumors and an agonist effect in 4 (72 and 153% of control). E2-stimulated colony growth in 5 tumors (167% of control) and the antibody blocked E2-stimulation in 3 of the 5. Inhibition of E2-stimulated growth in 3 and basal growth in 4 other tumors by the EGF receptor antibody suggest that endogenously secreted TGF-alpha has a role as an autocrine/paracrine growth factor in constitutive and E2-stimulated tumor cell proliferation in a majority of human tumors. A polyclonal antibody against TGF-alpha was used to study the role of TGF-alpha in E2-, prolactin(Prl)- and progesterone(Prog)-stimulated proliferation of NMU(nitrosomethylurea)-induced rat mammary tumor cells under similar culture conditions. TGF-alpha, E2, Prl and Prog stimulated colony growth equally to 176, 187, 168 and 181% of control. The antibody produced significant and similar inhibition of TGF-alpha and E2-stimulated growth (95 and 83%). In contrast, inhibition of Prl- and Prog-stimulated growth by the antibody was only 24 and 37%. The TGF-alpha ligand antibody did not have an agonist or antagonist effect when added alone. Thus, TGF-alpha seems to be a major stimulatory growth factor mediating E2-induced tumor cell proliferation in rat mammary tumors. It is less important in Prl- and Prog-induced tumor growth and not essential for basal growth in these tumors. We conclude that TGF-alpha is a biologically important autocrine/paracrine growth factor in primary human breast cancer cell proliferation and in E2-induced rat mammary tumor growth.     

Inhibin immunoreactivity in gonadal and non-gonadal tumors

Inhibin immunoreactivity was estimated in a number of gonadal and non-gonadal tumors. Dog Sertoli cell tumors and human granulosa cell and Leydig cell tumors contained high concentrations of inhibin-like material. Levels, comparable with those in normal testes and ovaries were detected in human testicular non-seminomas and in ovarian cystadenomas, thecomas and adenofibromas. No activity was found in human testicular Sertoli/Leydig cell tumors and seminomas and in ovarian adenocarcinomas, teratomas and a dysgerminoma. Furthermore, human adrenal cortical tissue (tumor and hyperplastic adrenal) contained inhibin immunoreactivity. No activity was found in human tumors of the stomach, gut, liver, kidney, pancreas and mammary gland or in meningiomas. It is concluded that inhibin is not a good marker for specific gonadal tumors. Inhibin might have intratumor actions a growth or differentiation factor.     

PGPIPN,a Therapeutic Hexapeptide,Suppressed Human Ovarian Cancer Growth by Targeting BCL2

Bioactive peptides, either derived from nature resources or synthesized by rational design, have been demonstrated potential for therapeutic agents against numerous human diseases, including cancer. However, the mechanism of therapeutic peptides against cancer has not been well elucidated. Here we show that PGPIPN, a hexapeptide derived from bovine β-casein, inhibited the proliferation of human ovarian cancer cells line SKOV₃ as well as the primary ovarian cancer cells in vitro. Consistently, PGPIPIN also decreased tumor growth rate in xenograft ovarian cancer model mice in a dose-dependent manner. Further study demonstrated that the anti-tumor effect of PGPIPN is partially through promoting cell apoptosis by inhibiting BCL2 pathway. Thus, our study suggests that PGPIPN is a potential therapeutic agent for the treatment of ovarian cancer or other types of cancer.     

LH inhibitory properties of aqueous extracts of rat pineal glands

Crude aqueous extracts of rat pineal glands markedly inhibited the 24 and 48 hour postcastration rise of serum LH in mature male rats. After partial purification by exclusion chromatography on Sephadex G-25 the LH inhibitory activity was found to reside in a volume not coinciding with that of melatonin, indicating that some substance other than this indole may be responsible for the anti-LH property in aqueous extracts of rat pineal glands.     

Glucocorticoids stimulate the release of a viral tumor marker (MMTV gp52) while inhibiting tumor cell growth

The influence of glucocorticoid treatments on the release of mouse mammary tumor virus (MMTV) envelope antigen (gp52) has been studied in C3H mammary tumor cell cultures and compared to treatment-mediated effects on tumor cell growth. Simultaneous assessment of extracellular viral antigen levels and tumor cell growth has indicated that both are coordinately affected by glucocorticoid treatment. While gp52 release is stimulated by treatment, this effect is accompanied by an inhibition of tumor cell growth. These stimulatory and inhibitory effects are mediated by dexamethasone (DEX) in a dose-dependent fashion, and both effects are more pronounced with the synthetic glucocorticoids DEX or triamcinolone acetonide (TA). Quantitation of media gp52 levels by RIA revealed the following hierarchy of glucocorticoid enhancement: TA greater than DEX greater than prednisolone greater than hydrocortisone greater than triamcinolone. A similar order of activity was observed in terms of inhibition of cell growth. The ability of TA to enhance gp52 release was 2.4-2.7 times greater than DEX, a previously proven stimulator of MMTV expression. Cell density of B9 mammary tumor cells was reduced 73% following 72 h of 10(-8) MTA treatment while C3H Mm5mt/cl mammary tumor cells were reduced by 53%. Hormone-mediated changes in in vitro gp52 release suggest that hormones might also influence plasma levels of MMTV gp52 as a systemic marker for the presence and status of murine mammary tumors. Coordinate stimulatory and inhibitory effects suggest that glucocorticoids may play a complex role in murine mammary tumorigenesis and subsequent mammary disease.     

An endogenous protein inhibitor of DNA polymerase alpha in normal and neoplastic rat mammary tissues

Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.     

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.     

Profile of organ weights and plasma concentrations of melatonin, estradiol and progesterone during gestation and post-parturition periods in female Indian palm squirrel Funambulus pennanti

Todate, report about the role of pineal gland in maintaining the normal physiology of gestation is scanty. Present study is the first of its kind giving a detail profile of organ weights and plasma concentration of melatonin, estradiol and progesterone to suggest a possible role of pineal gland in maintaining normal physiology during gestation and post-parturition periods of female Indian palm squirrel F. pennanti. Inspite of, inverse pineal-gonadal/melatonin-steroids interrelationship in adult (non-pregnant) females, the present results study suggest a direct relationship of pineal gland activity with ovarian steroids especially during the gestation period. The inverse relationship of melatonin and ovarian steroids is again established after parturition and maintained throughout the life. Thus the pineal gland (activity as judged by its weight, biochemical contents i.e. protein and cholesterol and plasma melatonin level) maintained ovarian/uterine physiology and regulated plasma concentrations of estradiol and progesterone during gestation and post-parturition periods. It is suggested that the pineal gland and its hormone melatonin play an important role to maintain the normal physiology of gestation and the post-partum recovery in Indian palm squirrel F. pennanti.