Samuel Cos

Melatonin increases p53 and p21WAFl expression in MCF-7 human breast cancer cells in vitro

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.

Melatonin and Mammary Pathological Growth

In this article we review the state of the art on the role of the pineal gland and melatonin in mammary cancer tumorigenesis in vivo as well as in vitro. The former hypothesis of a possible role of the pineal gland in mammary cancer development was based on the evidence that the pineal, via its main secretory product, melatonin, downregulates some of the pituitary and gonadal hormones which control mammary gland development and are also responsible for the growth of hormone-dependent mammary tumors. Furthermore, melatonin could act directly on tumoral cells, thereby influencing their proliferative rate. Other possible origins of melatonins antitumoral actions could be found in its antioxidant or immunoenhancing properties. The working hypotheses of most experiments were that the activation of the pineal gland, or the administration of melatonin, should give rise to antitumoral behavior; conversely, suppression of the pineal gland or melatonin deficits should stimulate mammary tumorigenesis. From in vivo studies on animal models of tumorigenesis, the general conclusion is that experimental manipulations activating the pineal gland, or the administration of melatonin, enlarge the latency and reduce the incidence and growth rate of chemically induced mammary tumors, while pinealectomy usually has the opposite effects. The direct actions of melatonin on mammary tumors have been suggested because of its ability to inhibit, at physiological doses (1 nM), the in vitro proliferation and invasiveness of MCF-7 human breast cancer cells. The fact that most studies have been performed on two models, chemically induced mammary adenocarcinoma in rats (in vivo studies) and the cell tumor line MCF-7 (in vitro studies), makes the generalization of the results somewhat difficult. However, the characteristics of these actions, comprising different aspects of tumor biology such as initiation, proliferation, and metastasis, as well as the doses (physiological range) at which the effect is accomplished, give special value to these findings. On the strength of these data, the small number of clinical studies focusing on the possible therapeutic value of melatonin on breast cancer is surprising.

EFFECTS OF MELATONIN ON THE CELL CYCLE KINETICS AND ESTROGEN-RESCUE OF MCF-7 HUMAN BREAST CANCER CELLS IN CULTURE

Cos S, Blask DE, Lemus‐Wilson A, Hill AB. Effects of melatonin on the cell cycle kinetics and “estrogen‐rescue of MCF‐7 human breast cancer cells in culture. J Pineal Res 1991:10:3642.

Melatonin modulates aromatase activity in MCF-7 human breast cancer cells

Abstract:  Most of the current knowledge about the mechanisms by which melatonin inhibits the growth of breast cancer cells point to an interaction of melatonin with estrogen‐responsive pathways, thus behaving as an antiestrogenic hormone. However, a possible effect of melatonin on the local synthesis of estrogens had not been examined. The objective of this work was to study whether melatonin may modify the aromatase activity in MCF‐7 breast cancer cells thus modulating the local estrogen biosynthesis. In MCF‐7 cells cultured with testosterone in estradiol‐free media, melatonin (1 nm) counteracts the testosterone‐induced cell proliferation dependent on the local biosynthesis of estrogens from testosterone by the aromatase activity of the cells. We found that melatonin reduces the aromatase activity (measured by the tritiated water release assay) of MCF‐7 cells both at basal conditions and when aromatase activity was stimulated by cAMP or cortisol. The greatest inhibition of the aromatase activity was obtained with 1 nm melatonin, the same concentration that gives the highest antiproliferative and anti‐invasive effects of MCF‐7 cells. Finally, by RT‐PCR, we found that melatonin downregulates aromatase expression at the transcriptional level in the MCF‐7 cells. We conclude that melatonin, at physiological concentrations, decreases aromatase activity and expression in MCF‐7 cells. This aromatase inhibitory effect of melatonin, together with its already known antiestrogenic properties interacting with the estrogen‐receptor, makes this indoleamine an interesting tool to be considered in the prevention and treatment of hormone‐dependent mammary neoplasias.

Melatonin inhibits the growth of DMBA-induced mammary tumors by decreasing the local biosynthesis of estrogens through the modulation of aromatase activity

Melatonin inhibits the growth of breast cancer cells by interacting with estrogen‐responsive pathways, thus behaving as an antiestrogenic hormone. Recently, we described that melatonin reduces aromatase expression and activity in MCF‐7 human breast cancer cells, thus modulating the local estrogen biosynthesis. To investigate the in vivo aromatase‐inhibitory properties of melatonin in our current study, this indoleamine was administered to rats bearing DMBA‐induced mammary tumors, ovariectomized (ovx) and treated with testosterone. In these castrated animals, the growth of the estrogen‐sensitive mammary tumors depends on the local aromatization of testosterone to estrogens. Ovariectomy significantly reduced the size of the tumors while the administration of testosterone to ovx animals stimulated tumor growth, an effect that was suppressed by administration of melatonin or the aromatase inhibitor aminoglutethimide. Uterine weight of ovx rats, which depends on the local synthesis of estrogens, was increased by testosterone, except in those animals that were also treated with melatonin or aminoglutethimide. The growth‐stimulatory effects of testosterone on the uterus and tumors depend exclusively on locally formed estrogens, since no changes in serum estradiol were appreciated in testosterone‐treated rats. Tumors from animals treated with melatonin had lower microsomal aromatase activity than tumors of animals from other groups, and incubation with melatonin decreased the aromatase activity of microsomal fractions of tumors. Animals treated with melatonin had the same survival probability as the castrated animals and significantly higher survival probability than the uncastrated. We conclude that melatonin could exert its antitumoral effects on hormone‐dependent mammary tumors by inhibiting the aromatase activity of the tumoral tissue.

Melatonin modulates growth factor activity in MCF-7 human breast cancer cells

Cos S, Blask DE. Melatonin modulates growth factor activity in MCF‐7 human breast cancer cells. J. Pineal Res. 1994: 17: 25–32.

Does melatonin induce apoptosis in MCF-7 human breast cancer cells in vitro?

Melatonin inhibits proliferation of the estrogen‐responsive MCF‐7 human breast cancer cells. The objective of this work was to assess whether melatonin not only regulates MCF‐7 cell proliferation but also induces apoptosis. In this experiment we used 1,25‐dihydroxycholecalciferol (D3) as a positive control because it inhibits MCF‐7 cell proliferation and induces apoptosis. MCF‐7 cells were cultured with either 1 nM melatonin, 100 nM D3 or its diluent to determine their effects on cell proliferation, cell viability, cell‐cycle phase distribution, population of apoptotic cells, and expression of p53, p21WAF1, bcl‐2, bcl‐XL and bax proteins. After 24 or 48 hr of incubation, both melatonin and D3‐treatment significantly decreased the number of viable cells in relation to the controls, although no differences in cell viability were observed between the treatments. The incidence of apoptosis, measured as the population of cells falling in the sub‐G1 region of the DNA histogram, or by the TUNEL reaction, was similar in melatonin‐treated and control cells whereas, as expected, apoptosis was higher among cells treated with D3 than in controls. The expression of p53 and p21WAF1 proteins significantly increased after 24 or 48 hr of incubation with either melatonin or D3. No significant changes in bcl‐2, bcl‐XL and bax mRNAs were detected after treatment with melatonin whereas in D3‐treated cells, a significant drop in bcl‐XL was observed. These data support the hypothesis that melatonin reduces MCF‐7 cell proliferation by modulating cell‐cycle length through the control of the p53–p21 pathway, but without clearly inducing apoptosis.

Melatonin inhibits dna synthesis in mcf-7 human breast cancer cells in vitro

The aim of the present work was to study whether physiological doses of melatonin (1nM) modified DNA synthesis in MCF-7 human breast cancer cells. Exponentially growing MCF-7 cells were incubated for 24 h with thymidine (2mM) for blocking mitosis and synchronizing the cell division cycle. Synchronization was assessed by a flow cytometry study which showed that after release from excess thymidine, 82.3% of the cells were in phase G1. Lots of these synchronized cells were pulsed for 1h with [3H]deoxythymidine ([3H]dThy) or [3H]dThy + melatonin, at 0,3,6,9,12,15 or 24 h from the release of the mitotic arrest. The exposition of these synchronized MCF-7 cells to melatonin for only 1h, significantly inhibited [3H]dThy incorporation when it was at 6 or 9 h. after release from mitotic block, at a time when DNA precursor incorporation was the highest and the number of cells in S phase was maximum. We conclude that, at least in part, melatonin antiproliferative effects on MCF-7 cells could be mediated by a reduction of DNA synthesis.

Melatonin inhibits both ERα activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium

Abstract:  Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen‐responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti‐estrogens suggesting that the activity of these compounds is mediated by ERα. The aims of this work were to test whether melatonin inhibits Cd‐induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd‐induced ERα transactivation. We show that melatonin prevents the Cd‐induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ERα‐ and ERβ‐mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd‐induced ERα‐mediated transcription in both estrogen response elements (ERE)‐ and AP1‐containing promoters, whereas ERβ‐mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ERα‐(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ERα at both ERE‐luc and AP1‐luc sites. We can infer from these experiments that melatonin regulates Cd‐induced transcription in both ERE‐ and AP1 pathways. These results also reinforce the hypothesis of the anti‐estrogenic properties of melatonin as a valuable tool in breast cancer therapies.

Modulation of the length of the cell cycle time of MCF-7 human breast cancer cells by melatonin

It has been shown that melatonin has a direct inhibitory effect on the proliferation of MCF-7 human breast cancer cells in culture. In the present work, we studied whether the length of the cell cycle of MCF-7 cells in increased by melatonin. In MCF-7 cells partially synchronized and labelled with [3H]thymidine, melatonin (10(-9)M), added to the culture medium, shifted the period of the labeling index rhythm from 20.36 hours to 23.48 hours. The fact that melatonin significantly increased (p<0.005) the duration of the cell cycle of human breast cancer cells, support the notion that this hormone exerts part of its antitumor effect through a cell-cycle-specific mechanism by delaying the entry of MCF-7 cells into mitosis.