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Melatonin inhibition of MCF-7 human breast-cancer cells growth: influence of cell proliferation rate
ELSFVlFR
Cancer
Letters
93 (1995)
207-212
Melatonin inhibition of MCF-7 human breast-cancer cells growth: influence of cell proliferation rate S. Cos*, E.J. Sinchez-Barcelb Department of Physiology and Pharmacology, School of Medicine, University of Cantabria. Cardenal Herrera Orr‘r ,s& 3901 I Santander. Spain
Received28 March 1995;accepted24 April 1995
-
__.-
Abstract
We have studied whether the cell proliferation rate modifies the inhibitory actions of melatonin on MCF-7 cell growth. The proliferative rate of cells was altered by plating them at different densities (5 x lo4 to 100 x IO4 cells/dish) in media with low charcoal-stripped serum concentrations. In this way, population doubling time ranged from 33 h (for density = 100 x lo4 cells/dish) to 75 h (for density = 5 x IO4 cells/dish). Mekttonin ( 10-9M) only inhibited fast proliferating MCF-7 cells, increasing their cell doubling time, and did not signiticantly modify the length of doublingtime in the cultureswith low proliferation rate, in which doubiing time was already long. These data clearly show that thereis a direct relation between proliferative rate of ceils and melatonin inhibitory actions on MCF-7 cells. Keywords: Melatonin; Pineal gland; MCF-7 cell; Cell growth; Breast cancer
1. InWtim
The role of the pineal as an oncostatic gland has been shown in much research done on animal models of tumorigenesis, especially on those concerning the mammary gland [2,6,7,13,20,211. Thus, the experimental manipulations enhancing pineal gland actions, or the administration of the pineal hormone melatonin, reduces the incidence and growth rate of chemically induced mammary tumors in rats, while pinealectomy usually stimulates mammary carcinogenesis [2,4,6,13, Correspondingauthor, Tel.: +34 42 201988; Fax: +34 42 201903. l
0304-3835/95/$09.50 0 SSDl 0304-3835(95)0381
1995 Elsevier I-A
Science
Ireland
Ltd.
All rights
20,211. The mechanisms by which melatonin reducesthe development of mammary tumors are still unknown. Some of the proposed explanations are based on the melatonin-induced changes in circulating concentrations of some of the pituitary and gonadal hormones that control tumoral growth [2,6,20]. The immunomodulatory role of melatonin [ 181as well as its actions as an endogenous hydroxyl radical scavenger[5,19] could also be involved in its antitumoral actions. However, as well as these indirect actions of melatonin on mammary tumor growth, some direct effects of this indoleamine on mammary tissue have been suggestedfrom research in vitro made on MCF-7 human breast cancer cells [2.6,8.14]. In vitro, reserved
208
S. Cos. E.J. Sbnchez-Barrel6 / Cancer Letters 93 (1995) 207-212
physiological concentrations of melatonin have a direct antiproliferative effect on MCF-7 human breast cancer cells in culture [16]. This inhibitory effect depends on the presence of serum [3,10], thus indicating that the indole is somehow interacting with somemitogens present in serum. Melatonin inhibits MCF-7 cell growth by blocking or delaying the cell progression from GdGr to the DNA synthetic phase of the cell cycle [14]. Morphological researchalso demonstratesthat melatonin delays the entry of MCF-7 cells into mitosis, allowing the cells to achieve a greater differentiation [lS]. The factors capable of modifying the susceptibility of MCF-7 cells to the growth-inhibitory effect of melatonin have been widely studied. It is common knowledge that there are certain factors which influence this susceptibility, such as: the prevailing culture conditions [3,9]; the melatonin concentration [3,9]; the pattern of application of the hormone [12]; and the presence of hormonal and/or growth factors in the culture medium [3,4,10,14]. Thus, we considered it worthwhile to study whether host factors, such as cell proliferation rate, influenced the antiproliferation response to melatonin. 2. Materials and methods 2.1. Cell culture MCF-7 human breast cancer cells (kindly donated by Dr. D.E. Blask of the Mary Imogene BassettHospital ResearchInstitute, Cooperstown, NY) were maintained as monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Chemical Co., St. Louis, MO) supplemented with 5% fetal bovine serum (FBS) (Biological Industries, Israel), penicillin (20 units/ml) and streptomycin (20 &ml) (Sigma Chemical Co., St. Louis, MO), at 37°C in a humid atmosphere containing 5% COz. Before each experiment, MCF-7 cells were harvested with 0.25% trypsin in phosphate-buffered solution (5 min incubation at 37”(Y),from confluent monolayer cultures in T-l SO stock flasks, and were passedrepeatedly through a 25gauge needle to produce a single cell suspension.
2.2. Obtaining cell cultures with different prohferalion rates Cells from several stock flasks were pooled, and cell counts and viability (trypan blue exclusion test) were determined. This pooled cell suspension was diluted in DMEM supplementedwith 5% FBS stripped with dextran-coated charcoal (csFBS), in order to obtain cellular densities of 5 x 104, 10 x 104, 20 x 104, 30 x 104, SOx lo4 or 100 x lo4 cells/ml. One-ml aliquots from each of these final dilutions were added to 60 x 15mm culture plates with 4 ml of DMEM + 5% csFBS. Every 24 h, in lots of six plates, cells were harvested from plates and cell counts were determined. The population doubling time was calculated from growth curves to give evidence that different rates of proliferation had been achieved. 2.3. Melatonin actions Pooled cells from stock flasks were suspendedin DMEM supplementedwith 5% csFBS, in order to obtain cellular densities of 5 x 104, 10 x 104, 20 x 104, 30 x 104, SO x lo4 or 100 x lo4 cells/ml. One-ml aliquots from each of these final dilutions were added to 60 x 1S-mmculture plates with 4 ml of DMEM + 5% csFBS. After MCF-7 cells had firmly attached to the dishes (4 h), media were aspirated and replaced for fresh DMEM + 5% csFBS containing either 10M9M melatonin (Sigma Chemical Co., Dorset, UK) or the melatonin diluent ethanol (final ethanol concentration per plate 0.0001%). After 24, 48, 72 or 96 h of incubation, cells were harvested from plates and cell counts were determined. The population doubling time was calculated from growth curves. Experiments were performed with six dishesfor each experimental condition. The results represent data from three different experiments. 2.4. Statistical analysis Regression studies were performed to establish the relationship betweenpopulation doubling time and number of plated cells. The effects of melatonin on cell proliferation were processed by twoway analysis of variance (factors: time and treatment) followed by the Student-Newman-Keuls test for determining the differences between group means (controls vs. melatonin treatment). Differ-
S. Cos, E.J. Scinchez-Barceld
/ Cancer
Letters
93 ( 1995)
207-212
209
6.50, Y = - 0.0261 R = 0.967
CJ?
X + 6.708
5.00--
20
30
40
Doubling
50
60
time
70
80
I
90
(hours)
Fig. 1. Relationship between the number of plated cells and the doubling time. Cells were plated at different densities (5 x IO4 to 100 X IO4 cells/plate) in DMEM with charcoal-stripped FBS. The amount of cells was estimated after 24,48,72 and 96 h of growth. The doubling time was calculated from growth curves and plotted against the log of the number of plated cells.
encesin doubling time between controls and melatonin treated cells were analyzed by Student’s t-test. 3. Results
By plating MCF-7 cells at different densities (from 5 x IO4to 100 x lo4 cells/dish) in DMEM with 5% csFBS different rates of cell proliferation were obtained. Fig. 1 shows the highly significant inverse correlation between the number of plated cells and the doubling time in these MCF-7 cells cultured in media with stripped serum. The doubling time ranged from 32.6 h for the lowest cell density (5 x IO4 cells/dish), to 74.5 h for the highest cell density tested (100 x lo4 cells/dish). The antiprolifkrative effectsof low9M melatonin on these MCF-7 cell cultures were clearly dependent on the rate of cellular growth (or, i.e, of the cell density) (Fig. 2). Thus, for cell densities of 20 x lo4 cells/plate or lower, which corresponded to lower proliferation rates,melatonin had virtually no effects. However, for ceil densities of 30 x IO4 cells/plate or higher, melatonin significantly inhibited cell proliferation. If we analyzed the melatonin effectsafter 4 days of culture, we observedthat this pineal hormone significantly reduced cell proliferation in those cultures of higher rate of prolifera-
[.', c,: 'j, ‘i
Jr
,.'
Fig. 2. A&proliferative effects of low9 M melatonin on MCF7 ceils with different rates of growth. Difftrent growth rates were obtained by plating MCF-7 cells at difberent densities in DMEM with charcoal-stripped FBS (see Fig. I) Differences between means are: “P < 0.05; bP < 0.01.
tion (31% in 30 x 104;37% in 50 x 104;and 46% in 100 x IO4cells/plate density) but not in those of lower proliferative rate (7% in 5 x 10J; 2.5% in
S. Cos, E.J. Stinchez-Barceld /Cancer Letters 93 (1995) 207-212
210 A : f?
1001
-
CONTROL
90
1
80
.E ;
60 50
.&
40
-2 ;g
30 70 20 /
5
Number
20 of plated
30 cells
50
per dish
100 (x 104)
Fig. 3. Effects of 10m9M melatonin on population doubling time of MCF-7 cells growing at different proliferative rates obtained by plating the cells at different densities in DMEM with charcoal-stripped FBS (see Fig. 1). Differences between means are: “P < 0.05; bP < 0.01; ‘P c 0.0005; and dP < 0.0001 vs. controls.
10 x 104;and 2% in 20 x 104;differencesvs. controls were not significant). Fig. 3 shows how melatonin influences doubling time in cells growing at different rates of proliferation. Melatonin increased cell doubling time in those plates with higher rates of proliferation (and consequently lower doubling time). The maximun effect appeared in cells plated at 100 x lo4 cells/dish in which doubling time changed from 32.6 h in control to 85.5 h in melatonin treated cells. Melatonin did not significantly modify the length of the doubling time in cultures with low proliferation rate, in which doubling time was already long. 4. Discussion There is evidence that the growth of MCF-7 cells, a cellular line derived from a pleural effusion of a patient with metastatic breast cancer [22], can be inhibited by the presence of melatonin in the culture medium [3,9]. The aim of the present work was to study the influence of the rate of cellular growth on the antiproliferative response to melatonin. To obtain different rates of cell proliferation, MCF-7 cells were plated at different densities in a culture medium with charcoal-stripped serum. In
these culture media with partial depletion of growth nutrients, the rate of cell proliferation is clearly dependent on cell density, as demonstrated by the negative correlation between doubling time and cell density (doubling time decreaseswith the increase of cell density). The method used to obtain different proliferative rates of MCF-7 cells was described by Jakesz et al. (1984) [ 171.To explain why low-density cells grow at lower proliferation rates than those plated at higher densities, Jakeszet al. proposed that MCF-7 cells could releasesomegrowth factors into the culture medium which stimulate their own growth. This explanation is supported by a great deal of research which shows that medium conditioned by MCF-7 cells for several days, and added to freshly plated cells, significantly increases the growth rate and the [ 3H]thymidine incorporation [ 11,231. In our experiment, the antiproliferative effects of melatonin were thus studied on cells with low proliferative rates (doubling time 74.5 h) as well as on rapidly proliferating cells (doubling time 32.6 h). The antiproliferative power of melatonin showed a good correlation with the proliferation rate of the MCF-7 cells. This effect was highest in those cultures with lower doubling time. There was, however, a clear cut difference between melatonin actions at low (5 x lo4 to 20 x 104) or at high (30 x lo4 to 100 x 104) cell densities. Thus, while melatonin reduced MCF-7 cell proliferation only by 2-7% for cell densities between 5 x lo4 and 20 x lo4 cells/plate, the inhibition of proliferation due to melatonin ranged from 31 to 46% for cell densities between 30 x lo4 to 100 x lo4 cells/plate. Melatonin increases population doubling time in those cultures with high proliferative rate (and consequently low doubling time), but did not modify the length of the doubling time of slowly proliferating cells, which is already large. The changesof the population doubling time, could be explained by the fact that melatonin increases MCF-7 cell cycle time (Cos and Sanchez-Barcelo, unpublished data). Since stripping of fetal bovine serum with dextran-coated charcoal removes the steroid hormones as well as most of the lactogenic hormones [l], the cell proliferation in media with this stripped serum should be mainly dependent on the autocrine growth stimulator-y factors secreted
S. Cos. E.J. Sinchez-Barcelb
/ Cancer
by the cells [23]. The antiproliferative effects of melatonin in these circumstances support the hypothesis that this indolamine may inhibit the action and/or the release of autocrine stimulatory factors from MCF-7 cells [l 11. We can conclude from these experiments, that the rate of growth of MCF-7 cells strongly affects the antiproliferative action of physiological doses of melatonin on these human tumoral cells. There is a direct relation between the proliferative rate of the cells and melatonin inhibitory actions. The more the cells are stimulated the more powerful are the melatonin inhibitory actions. Acknowledgements
This work has been supported by grants from the Spanish DGICYT (PB92-0831) and Foundation Ram6n Areces. References
Ill B&was, R. and Vonderhaar, B.K. (1987) Role of Serum in the prolactin responsivenessof MCF-7 human breast cancer cells in long-term tissue culture. Cancer Res., 47, 3509-3514. 121Blask, D.E. (1984) The pineal: an oncostatic gland? In: The Pineal Gland, pp. 253-284, Editor: R.J. Reiter. Raven Press. New York. I31 Blask, D.E. and Hill, SM. (1986) Effects of melatonin on cancer: studies on MCF-7 human breast cancer cells in culture. J. Neural Transm. Suppl., 21, 433-449. [41 Blask, D.E. and Hill, SM. (1988) Melatonin and cancer: basic and clinical aspects.In: Melatonin Clinical Perspectives, pp. 128-173. Editors: A. Miles, D.R.S. Philbrick and C. Thompson. Oxford University Press,New York. VI Blask. D.E. and Wilson, S.T. (1994) Melatonin and oncostatic signal transduction: evidence for a novel mechanism involving glutathione and nitric oxide. In: Advances in Pineal Research. Vol. 8, pp. 465-471. Editors: M. Moller and P. Pevet. John Libbey, London. 161Blask, D.E., Hill, SM., Pelletier, D.B., Anderson, J.M. and Lemus-Wilson, A. (1989) Melatonin: an anticancer hormone of the pineal gland. In: Advances in Pineal Research,Vol. 3, pp. 259-263. Editors: R.J. Reiter and S.F. Pang. John Libbey, London. [71 Bask, D.E., Cos, S., Hill, S.M., Bums, D.M.. LemusWilson, A. and Grosso, D.S. (1991) Melatonin action on oncogenesis.In: Role of Melatonin and Pineal Peptides in Neuroimmunomodulation, pp. 233-240. Editors: F. Fraschini and R.J. Reiter. Plenum Press, New York. WI Blask, D.E., Lemus-Wilson, A., Wilson, S.T. and Cos, S. (1993) Neurohormonal modulation of cancer growth by
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pineal melatonin. In: Melatonin and the Pmeal Giandfrom Basic Scienceto Clinical Application, pp. 303-3 i 0. Editors: Y. Touitou, J. Arendt and P Pevet. Elsevier Science Publishers, Amsterdam. 191 Cos, S. and Blask, D.E. (1990) Effects of the pineal hormone melatonin on the anchorage-independent growth of human breast cancer cells (MCF-7) in 3 c!onogenic culture system. Cancer Lett.. 50, 115~-119 1101 Cos, S. and Blask, D.E. (1991) Effects of melaronm on growth factors activity in MCF-7 human breast cancer cells. In: Advances in Pineal Research. Vol. i. pp. 365-367. Editors: J. Arendt and P Pevcl frrhn L~bbey, London. 1111 Cos, S. and Blask, D.E. (1994) Melatonm modulates growth factor activity in MCF-7 human beast cancer cells. J. Pineal Res., 17, 25-32. 1121Cos, S. and Sgnchez-Barcelb,E.J. (1994) Dd~erencesbetween pulsatile or continuous exposure to meiatomn on MCF-7 human breast cancer cell proliferation. Cancer Lett.. 85. 105-109. (131 Cos, S., GariJo. F.. Corral, J.. Mediavilla. M.D and Sgnchez-Barcelb,E.J. (1989) Histopathologic features of DMBA-induced mammary tumors in blind-underfed, blind-anosmic and blind cold-exposed rats: Influence of the pineal gland. J. Pineal Res., 6. 22I - 271. 1141 Cos. S., Blask, D.E., Lemus-Wilson, A. and H111,A.B. ( 1991)Effects of melatonin on the cell cycle kinetics and ‘estrogen-rescue’of MCF-7 human breast cancer cells in culture. J. Pineal Res., 10, 36-43 (151 Crespo, D., FemBndez-Viadero. 0.. Verduga, R., Ovrjero, V. and Cos, S. (1994)Interaction between melatonin and estradiol on morphological and morphometric lestures of MCF-7 human breast cancer cells .I. Pineal Rrs.. 16. 215-222. 1161Hill. S.M. and Blask, D.E. (1988) Effects of the pmeal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCC-?i in culture. Cancer Res.. 48, 6121-61X [I71 Jakesz, R.. Smith, C.A., Aitken, S., Hug. K., Schuette. W.. Shackney. S. and Lippman M. (1984) influence of cell proliferation and cell phaseon expression of estrogen receptor in MCF-7 breast cancer cells. Cancer Res. 44. 619-625. [IsI Maestroni, G.J.M. (1993) The immunoneuroendocrinc role of melatonin. J. Pineal Res.. 14, I-- 10. 1191 Poeggeler.B., Reiter, R.J., Tang, D.X.. Chcn. L.D and Manchester, L.C. (1993) Melatonin. hydroxyl radicalmediated oxidative damage and aging: rt hypothesis. J. Pineal Res., 14. 151-168. 1201Sanchez-Barcelo, E.J., Cos, S. and MedIavilla, M.D. (1988)Influence of pineal gland function on the initiation and growth of hormonedependent breast rumors. Possible mechanisms.In: The Pineal Gland and Cancer. pp. 221-232. Editors: D. Gupta. A. Attanasioand R.J. Reiter. Brain Research Promotion, Tiibingen [211Shah, P.N., Mhatre. M.C. and Kotharr. L.S. (1984) Effect of melatonin on mammary carcinogeneslsin intact
212
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and pinealectomized rats in varying photoperiods. Cancer Res., 44, 3403-3407. 1221 Soule, H.D., Vaquez, J., Long, A., Albert, S. and Brennan, M.J. (1973) A human cell line from a pleural effusion derived from a breast carcinoma. J. Nat). Cancer Inst., 51, 1409-1412.
1231 Vignon. F., Deroq, D., Chambon, M. and Rochefort, H. (1983)Les prottines oestrogeno-induites skrttees par les celhdes mammaires can&reuses humaines MCF-7 stimulent leur proliferation. CR Acad. Sci. (Paris), 296, 151-157.
Cancer
Letters
93 (1995)
207-212
Melatonin inhibition of MCF-7 human breast-cancer cells growth: influence of cell proliferation rate S. Cos*, E.J. Sinchez-Barcelb Department of Physiology and Pharmacology, School of Medicine, University of Cantabria. Cardenal Herrera Orr‘r ,s& 3901 I Santander. Spain
Received28 March 1995;accepted24 April 1995
-
__.-
Abstract
We have studied whether the cell proliferation rate modifies the inhibitory actions of melatonin on MCF-7 cell growth. The proliferative rate of cells was altered by plating them at different densities (5 x lo4 to 100 x IO4 cells/dish) in media with low charcoal-stripped serum concentrations. In this way, population doubling time ranged from 33 h (for density = 100 x lo4 cells/dish) to 75 h (for density = 5 x IO4 cells/dish). Mekttonin ( 10-9M) only inhibited fast proliferating MCF-7 cells, increasing their cell doubling time, and did not signiticantly modify the length of doublingtime in the cultureswith low proliferation rate, in which doubiing time was already long. These data clearly show that thereis a direct relation between proliferative rate of ceils and melatonin inhibitory actions on MCF-7 cells. Keywords: Melatonin; Pineal gland; MCF-7 cell; Cell growth; Breast cancer
1. InWtim
The role of the pineal as an oncostatic gland has been shown in much research done on animal models of tumorigenesis, especially on those concerning the mammary gland [2,6,7,13,20,211. Thus, the experimental manipulations enhancing pineal gland actions, or the administration of the pineal hormone melatonin, reduces the incidence and growth rate of chemically induced mammary tumors in rats, while pinealectomy usually stimulates mammary carcinogenesis [2,4,6,13, Correspondingauthor, Tel.: +34 42 201988; Fax: +34 42 201903. l
0304-3835/95/$09.50 0 SSDl 0304-3835(95)0381
1995 Elsevier I-A
Science
Ireland
Ltd.
All rights
20,211. The mechanisms by which melatonin reducesthe development of mammary tumors are still unknown. Some of the proposed explanations are based on the melatonin-induced changes in circulating concentrations of some of the pituitary and gonadal hormones that control tumoral growth [2,6,20]. The immunomodulatory role of melatonin [ 181as well as its actions as an endogenous hydroxyl radical scavenger[5,19] could also be involved in its antitumoral actions. However, as well as these indirect actions of melatonin on mammary tumor growth, some direct effects of this indoleamine on mammary tissue have been suggestedfrom research in vitro made on MCF-7 human breast cancer cells [2.6,8.14]. In vitro, reserved
208
S. Cos. E.J. Sbnchez-Barrel6 / Cancer Letters 93 (1995) 207-212
physiological concentrations of melatonin have a direct antiproliferative effect on MCF-7 human breast cancer cells in culture [16]. This inhibitory effect depends on the presence of serum [3,10], thus indicating that the indole is somehow interacting with somemitogens present in serum. Melatonin inhibits MCF-7 cell growth by blocking or delaying the cell progression from GdGr to the DNA synthetic phase of the cell cycle [14]. Morphological researchalso demonstratesthat melatonin delays the entry of MCF-7 cells into mitosis, allowing the cells to achieve a greater differentiation [lS]. The factors capable of modifying the susceptibility of MCF-7 cells to the growth-inhibitory effect of melatonin have been widely studied. It is common knowledge that there are certain factors which influence this susceptibility, such as: the prevailing culture conditions [3,9]; the melatonin concentration [3,9]; the pattern of application of the hormone [12]; and the presence of hormonal and/or growth factors in the culture medium [3,4,10,14]. Thus, we considered it worthwhile to study whether host factors, such as cell proliferation rate, influenced the antiproliferation response to melatonin. 2. Materials and methods 2.1. Cell culture MCF-7 human breast cancer cells (kindly donated by Dr. D.E. Blask of the Mary Imogene BassettHospital ResearchInstitute, Cooperstown, NY) were maintained as monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Chemical Co., St. Louis, MO) supplemented with 5% fetal bovine serum (FBS) (Biological Industries, Israel), penicillin (20 units/ml) and streptomycin (20 &ml) (Sigma Chemical Co., St. Louis, MO), at 37°C in a humid atmosphere containing 5% COz. Before each experiment, MCF-7 cells were harvested with 0.25% trypsin in phosphate-buffered solution (5 min incubation at 37”(Y),from confluent monolayer cultures in T-l SO stock flasks, and were passedrepeatedly through a 25gauge needle to produce a single cell suspension.
2.2. Obtaining cell cultures with different prohferalion rates Cells from several stock flasks were pooled, and cell counts and viability (trypan blue exclusion test) were determined. This pooled cell suspension was diluted in DMEM supplementedwith 5% FBS stripped with dextran-coated charcoal (csFBS), in order to obtain cellular densities of 5 x 104, 10 x 104, 20 x 104, 30 x 104, SOx lo4 or 100 x lo4 cells/ml. One-ml aliquots from each of these final dilutions were added to 60 x 15mm culture plates with 4 ml of DMEM + 5% csFBS. Every 24 h, in lots of six plates, cells were harvested from plates and cell counts were determined. The population doubling time was calculated from growth curves to give evidence that different rates of proliferation had been achieved. 2.3. Melatonin actions Pooled cells from stock flasks were suspendedin DMEM supplementedwith 5% csFBS, in order to obtain cellular densities of 5 x 104, 10 x 104, 20 x 104, 30 x 104, SO x lo4 or 100 x lo4 cells/ml. One-ml aliquots from each of these final dilutions were added to 60 x 1S-mmculture plates with 4 ml of DMEM + 5% csFBS. After MCF-7 cells had firmly attached to the dishes (4 h), media were aspirated and replaced for fresh DMEM + 5% csFBS containing either 10M9M melatonin (Sigma Chemical Co., Dorset, UK) or the melatonin diluent ethanol (final ethanol concentration per plate 0.0001%). After 24, 48, 72 or 96 h of incubation, cells were harvested from plates and cell counts were determined. The population doubling time was calculated from growth curves. Experiments were performed with six dishesfor each experimental condition. The results represent data from three different experiments. 2.4. Statistical analysis Regression studies were performed to establish the relationship betweenpopulation doubling time and number of plated cells. The effects of melatonin on cell proliferation were processed by twoway analysis of variance (factors: time and treatment) followed by the Student-Newman-Keuls test for determining the differences between group means (controls vs. melatonin treatment). Differ-
S. Cos, E.J. Scinchez-Barceld
/ Cancer
Letters
93 ( 1995)
207-212
209
6.50, Y = - 0.0261 R = 0.967
CJ?
X + 6.708
5.00--
20
30
40
Doubling
50
60
time
70
80
I
90
(hours)
Fig. 1. Relationship between the number of plated cells and the doubling time. Cells were plated at different densities (5 x IO4 to 100 X IO4 cells/plate) in DMEM with charcoal-stripped FBS. The amount of cells was estimated after 24,48,72 and 96 h of growth. The doubling time was calculated from growth curves and plotted against the log of the number of plated cells.
encesin doubling time between controls and melatonin treated cells were analyzed by Student’s t-test. 3. Results
By plating MCF-7 cells at different densities (from 5 x IO4to 100 x lo4 cells/dish) in DMEM with 5% csFBS different rates of cell proliferation were obtained. Fig. 1 shows the highly significant inverse correlation between the number of plated cells and the doubling time in these MCF-7 cells cultured in media with stripped serum. The doubling time ranged from 32.6 h for the lowest cell density (5 x IO4 cells/dish), to 74.5 h for the highest cell density tested (100 x lo4 cells/dish). The antiprolifkrative effectsof low9M melatonin on these MCF-7 cell cultures were clearly dependent on the rate of cellular growth (or, i.e, of the cell density) (Fig. 2). Thus, for cell densities of 20 x lo4 cells/plate or lower, which corresponded to lower proliferation rates,melatonin had virtually no effects. However, for ceil densities of 30 x IO4 cells/plate or higher, melatonin significantly inhibited cell proliferation. If we analyzed the melatonin effectsafter 4 days of culture, we observedthat this pineal hormone significantly reduced cell proliferation in those cultures of higher rate of prolifera-
[.', c,: 'j, ‘i
Jr
,.'
Fig. 2. A&proliferative effects of low9 M melatonin on MCF7 ceils with different rates of growth. Difftrent growth rates were obtained by plating MCF-7 cells at difberent densities in DMEM with charcoal-stripped FBS (see Fig. I) Differences between means are: “P < 0.05; bP < 0.01.
tion (31% in 30 x 104;37% in 50 x 104;and 46% in 100 x IO4cells/plate density) but not in those of lower proliferative rate (7% in 5 x 10J; 2.5% in
S. Cos, E.J. Stinchez-Barceld /Cancer Letters 93 (1995) 207-212
210 A : f?
1001
-
CONTROL
90
1
80
.E ;
60 50
.&
40
-2 ;g
30 70 20 /
5
Number
20 of plated
30 cells
50
per dish
100 (x 104)
Fig. 3. Effects of 10m9M melatonin on population doubling time of MCF-7 cells growing at different proliferative rates obtained by plating the cells at different densities in DMEM with charcoal-stripped FBS (see Fig. 1). Differences between means are: “P < 0.05; bP < 0.01; ‘P c 0.0005; and dP < 0.0001 vs. controls.
10 x 104;and 2% in 20 x 104;differencesvs. controls were not significant). Fig. 3 shows how melatonin influences doubling time in cells growing at different rates of proliferation. Melatonin increased cell doubling time in those plates with higher rates of proliferation (and consequently lower doubling time). The maximun effect appeared in cells plated at 100 x lo4 cells/dish in which doubling time changed from 32.6 h in control to 85.5 h in melatonin treated cells. Melatonin did not significantly modify the length of the doubling time in cultures with low proliferation rate, in which doubling time was already long. 4. Discussion There is evidence that the growth of MCF-7 cells, a cellular line derived from a pleural effusion of a patient with metastatic breast cancer [22], can be inhibited by the presence of melatonin in the culture medium [3,9]. The aim of the present work was to study the influence of the rate of cellular growth on the antiproliferative response to melatonin. To obtain different rates of cell proliferation, MCF-7 cells were plated at different densities in a culture medium with charcoal-stripped serum. In
these culture media with partial depletion of growth nutrients, the rate of cell proliferation is clearly dependent on cell density, as demonstrated by the negative correlation between doubling time and cell density (doubling time decreaseswith the increase of cell density). The method used to obtain different proliferative rates of MCF-7 cells was described by Jakesz et al. (1984) [ 171.To explain why low-density cells grow at lower proliferation rates than those plated at higher densities, Jakeszet al. proposed that MCF-7 cells could releasesomegrowth factors into the culture medium which stimulate their own growth. This explanation is supported by a great deal of research which shows that medium conditioned by MCF-7 cells for several days, and added to freshly plated cells, significantly increases the growth rate and the [ 3H]thymidine incorporation [ 11,231. In our experiment, the antiproliferative effects of melatonin were thus studied on cells with low proliferative rates (doubling time 74.5 h) as well as on rapidly proliferating cells (doubling time 32.6 h). The antiproliferative power of melatonin showed a good correlation with the proliferation rate of the MCF-7 cells. This effect was highest in those cultures with lower doubling time. There was, however, a clear cut difference between melatonin actions at low (5 x lo4 to 20 x 104) or at high (30 x lo4 to 100 x 104) cell densities. Thus, while melatonin reduced MCF-7 cell proliferation only by 2-7% for cell densities between 5 x lo4 and 20 x lo4 cells/plate, the inhibition of proliferation due to melatonin ranged from 31 to 46% for cell densities between 30 x lo4 to 100 x lo4 cells/plate. Melatonin increases population doubling time in those cultures with high proliferative rate (and consequently low doubling time), but did not modify the length of the doubling time of slowly proliferating cells, which is already large. The changesof the population doubling time, could be explained by the fact that melatonin increases MCF-7 cell cycle time (Cos and Sanchez-Barcelo, unpublished data). Since stripping of fetal bovine serum with dextran-coated charcoal removes the steroid hormones as well as most of the lactogenic hormones [l], the cell proliferation in media with this stripped serum should be mainly dependent on the autocrine growth stimulator-y factors secreted
S. Cos. E.J. Sinchez-Barcelb
/ Cancer
by the cells [23]. The antiproliferative effects of melatonin in these circumstances support the hypothesis that this indolamine may inhibit the action and/or the release of autocrine stimulatory factors from MCF-7 cells [l 11. We can conclude from these experiments, that the rate of growth of MCF-7 cells strongly affects the antiproliferative action of physiological doses of melatonin on these human tumoral cells. There is a direct relation between the proliferative rate of the cells and melatonin inhibitory actions. The more the cells are stimulated the more powerful are the melatonin inhibitory actions. Acknowledgements
This work has been supported by grants from the Spanish DGICYT (PB92-0831) and Foundation Ram6n Areces. References
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