Vol.2, No.8, 857-861 (2010)
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
Openly accessible at
Potential mechanism for the effects of dexamethasone
on growth of human melanoma cells in vitro
Abdel-Moneim M. Osman1*, Omimah A. Nasseir2, Naglaa R. Ismail3
1College of Medicine, Pharmacology Department, King Abdulaziz University, Jeddah, Saudi Arabia; *Corresponding Author:
2Science college of Girsl, King Abdulziz University, Jeddah, Saudi Arabia
3 Department of Zoology, Faculty of Science, Fayoum University, Fayoum, Egypt
Received 20 March 2010; revised 7 April 2010; accepted 10 April 2010.
This study deals with the inhibitory effects of
dexamethsone on the proliferation of a human
melanoma cell line in vitro. A retarded cell pro-
liferation was observed in M-5A cells with the
presence of specific high affinity glucocorticoid
steroid receptors. There was a correlation be-
tween the number of glucococrticoid binding sites
and the reduction of cell proliferation in term of
reduced plating efficiency. Arrest or accumula-
tion of M-5A cells in G1 phase or both in G1 and
G2 phase appeared to be involved in the growth
inhibitory effect by dexamethsone. The magni-
tude and duration of cell cycle arrest up to 72
hours were dose dependent. There was a cor-
relation between the duration of the disturbance
of the cell cycle progression in M-5A cells after
dexamethsone treatment and the inhibition of
cell proliferation. Synthesis of receptor protein
was not specifically stimulated or inhibited
relative to the effect on cellular protein content
in general. This may conclude that in the mela-
noma M-5A cell, death after glucocorticoid treat-
ment is somehow related to the glucocorticoid
receptor content and to the disturbance of cell
cycle distribution.
Keywords: Melanoma Cells; Dexamethasone;
Glucocorticoid Receptors
Although malignant melanoma has not generally been
regarded a hormonally responsive neoplasm, there is
some evidence suggesting that melanoma may respond
to the hormonal condition of the host. Pregnancy was
reported to be associated with unfavorable prognosis in
stage II disease [1]. However, adrenalectomy for mela-
noma metastatic to the adrenal gland provides good pal-
liation of complete regression of distant metastatic
melanoma after bilateral adrenalectomy, suggesting a
possible role for adrenal hormones in modifying mela-
noma progression in certain patients [2]. A few reports
have been published about the moderate sensitivity of
human as well as animal melanoma to the action of glu-
cocorticoid steroid hormones. Bregman et al. [3] found
that dexamethsone at a concentration of 1 uM inhibited
the colony formation in a human melanoma cell strain
(C8146c) by 60%. Moreover, Ramirez et al. [4], showed
85% tumor rejection in mice challenged with B16
melanoma after administration of anti glucocorticoid-
induced TNF receptor family related protein. Recently,
Banciu et al. [5] reported that a glucocorticoid predniso-
lone phosphate encapsulated in long-circulating lipo-
somes exerts antitumor activity through the inhibition of
tumor angiogenesis. Therefore the aim of our study was
directed to investigate to what extent glucocorticoid in
term of dexamethasone affect human melanoma cells in
vitro. Moreover, to study whether human melanoma
cells contain glucocorticoid receptors and, if so, is there
a relation between the sensitivity to glucocorticoid and
the amount of receptors present.
This study is partly conducted in the King Fahd Medical
center, College of Medicine, King Abdulaziz University,
Jeddah, Saudi Arabia and partly in Pharmacology Unit,
National Cancer Institute, Cairo University.
2.1. Cells and Cell Culture
A human melanoma cell line M-5A was received at un-
known passage level from liquid nitrogen store National
Cancer Institute, Cairo University. Its characteristics
have been described [6].
A. M. M. Osman et al. / HEALTH 2 (2010) 857-861
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
Cells were grown in modified minimum essential Ea-
gle’s medium which penicillin (100 i.u./ml) and strep-
tomycin (100 ug/ml) had been added to the medium
contained 10% newborn calf serum inactivated by heat-
ing at 56˚C for 30 min. Cells were cultured at 37˚C in
humidified 5% CO2 atmosphere.
2.2. Evaluation of Drug Effect
To study the effect of dexamethasone on cell prolifera-
tion, a colony forming assay has been used. Exponen-
tially growing cells were trypsinized off the substratum
and counted in a hemocytometer. Sufficient numbers of
cells (500-5000) were plated in 25 cm2 Greiner flasks to
yield between 15 and 300 colonies per flaks at the time
of evaluation. The number of cells required for each ex-
periment was determined in preliminary studies. After
treatment for one hour, the incubation medium was re-
placed by fresh medium. After a time interval corres-
ponding with nearly 5 to 7 doubling times of untreated
cells, the number of colonies was counted and expressed
as percent of the controls. To asses the cytotoxic action
the per cent reduction in plating efficiency was calcu-
lated from the equation (C-T)/C × 100, where C and T
are the final numbers of colonies in control and treated
cultures, respectively.
2.3. Glucocorticoid Receptor Assay
Binding study for dexamethasone was carried out using
a whole cell assay adapted from Rosner and Cristofalo
[7] .Single cell suspensions were harvested from expo-
nentially growing cultures, transfereed in triplicate to 60
mm Falcon dishes, 1 × 106 cells per dish, and incubated
in growth medium. Twenty-four hours later, the medium
was removed and cells were washed once with PBS at
22˚C. Fresh medium was added with (3H) dexamethsone
(40 Ci/mmole, Amersham International Ltd, England) or
(3H) dexamethsone in the presence of 1000-fold excess
concentration of unlabeled dexamethsone to measure
total and nonspecific binding of dexamethasone to the
cells, respectively. The cells were incubated for 60 or 90
min in a humidified 5% CO2 atmosphere at 37˚C. Cells
were then solubilized in 2 ml of 0.5 M sodium hydroxide
at 50˚C for one hour. After cooling the cells they were
neutralized with 0.16 ml of 6 N hydrochloric acid. And
the cells transferred to scintillation vial and 15 ml of
Insta-gel scintillation liquid (Packard) was added. Ra-
dioactivity of the samples was determined in dpm in a
Packard Model 3385 scintillation counter with an aver-
age efficiency for tritium of 34%.
Specific binding of dexamethsone was calculated
from the difference between the total binding of (3H)
dexamethsone at the saturating concentration of 4 × 10-8
M dexamethasone and the nonspecific binding that oc-
curred in the presence of excess unlabeled dexamethasone.
The molar amount of specifically bound (3H) dexa-
methsone was calculated from its specific radioactivity
and the dpm/cell was measured. Conversion by Avo-
gadro’s number gave the number of specific binding
sites per cells. Protein content was determined according
to Lowry et al. [8].
2.4. Flowcytometric Analysis
Cell cycle analysis was performed using a flowcytomter
ICP II (Phywe AG. Gottingen) equipped with a sheatflow
cell essentially as described by Smets et al. [9] .Cells
were stained by 2 ml of solution which contained 2 mg
ethidium bromide, 0.8 mg Hoechst stain 33258, 0.5 g.
bovine serum albumin and 2.4 g Tris/100 ml. The per-
centage of cells with DNA per cell values corresponding
with G1, S or G2/M phase were determined by plani-
metry of the histograms.
3.1. Glucocorticoid Binding Sites
Table 1 shows the relation between the duration of in-
cubation with (3H) dexamethsone and the specific and
nonspecific binding by 106 cells. Binding of the radioac-
tive dexamethasone started rapidely as observed after 30
seconds of incubation. Maximum specific binding was
achieved after 60-90 min. Prolongation of incubation to
120 min reduced the specific binding of glucocrticoid
and increased the nonspecific binding. This decrease
appeared not to be caused by the lower dexamethasone
concentration during prolongation of the incubation (13
nM instead of 40 nm) since 13 nM must be considered
nearly saturating according to the date in Figure 1. We
have investigated the number of glucocorticoid binding
site in melanoma M-5A cells after treatment with dexa-
methsone 2.5 uM for one hour (Table 2). The number of
dexamethasone binding site per cell was determined by
the whole cell assay in control and treated culture. There
was 31% increase in number of binding sites after 24
hours. The number of binding sites remained higher than
in controls until the time that the treated cells resumed
proliferation. The increase in the number of binding
site/cells after dexamethsone treatment was dose de-
pendant (Table 3). When measured 9 days after treat-
ment with 2.5 or 12.5 uM of dexamethsone a 2.8-fold
increase was observed at the higher concentration.
Throughout these studies, it was noticed that the M-5A
melanoma cells became larger during the growth delay
induced by treatment with dexamethsone. Their total
protein content was determined in order to see whether
the increase in specific glucorticoid binding sites went
parallel to a general increase in the amount of protein per
cell. Table 4 showed a dose dependent increase in cellu-
lar protein content after dexamethsone treatment.
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Table.1. Time course of the specifically binding of (3H) dexa-
methasone by M-5A melanoma cells.
Specific binding
of (3H )
(dpm/106) × 10-3
Nonspecific binding
(% of total binding)
0.5 1 ± 0.2b 93 ± 0.6b
10 13 ± 1 79 ± 1.5
40 17 ± 1.6 73 ± 0.3
60 24 ± 0.392 64 ± 0.8
90 23 ± 1.5 63 ± 1.2
120 12 ± 0.8 77 ± 1.0
aConcentration 40 nM; bPresented are Mean ± S.E.M. of triplicate
Table 2. Specific glucocorticoid binding site in M-5A cells at
various times after treatment with dexamethasone.
Specific binding sites/cell1
× 10-3
Time after
(hr) Control Treated
Treated in
% of control
24 132 ± 2.5 173 ± 23 131
48 142 ± 4.6 189 ± 27 133
72 108 ± 1 132 ± 21 123
216 142 ± 1 189 ± 17 133
aIncubation for 60 min at 37˚C with 40 nM (3H) dexamethsone alone or
in the presence of excess unlabeled steroid dexamethsone 2.5 uM for
one hour Presented are means ± S.E.M.
Table 3. Effect of the dose of dexamethasone on the number of
specific binding sites per M-5A melanoma cells, 9 days after
Specific binding sites per cells1
× 10-3
(uM) Control Treated Treated in %
of control
2.5 142 ± 11 189 ± 17 133
12.5 131 ± 0.3 371 ± 71 283
1Incubation for 60 min at 37˚C with 40 nM (3H) dexamethsone alone
or in the presence of excess unlabeled steroid. Presented are mean ±
From recalculation of the specifically bound dexa-
methsone in fmole/mg total cellular protein, it was ob-
served that synthesis of receptor protein was not specifi-
cally stimulated or inhibited relative to the effect on cel-
lular protein content in general.
3.2. Cell Cycle Distribution after
Dexamethsone Treatment
Figure 2 shows the effect of dexamethasone treatment
on the cell cycle phase distribution of M-5A melanoma
cells at various time intervals. The pronounced effects
were observed from 10 till 96 hours after treatment of
M-5A cells for one hour with 2.5 uM dexamethasone. At
10 hours after treatment a 90% reduction of S-phase
cells was occurred (S-phase was nearly about 2% of total
cell population). Gradual restoration of S-phase com-
partment started 24 hours after treatment but after 96
hours the relative number of G2/M cell has not yet nor-
3.3. Relationship of Cell Cycle Perturbation
to Cell Survival and Proliferation
Twenty-four hours after cell incubation, dexamethsone
was supplied at various concentrations from 0.25-12.5
uM for one hour (Table 5). After treatment the cells
were washed, harvested and part of them was seeded to
determine the rate of colony formation and absolute
plating efficient (The ultimately surviving cell fraction).
A dramatically lowering of the percentage of S-phase
cells occurred at the lower concentration with decreasing
the final plating efficiency. Increasing the dexamethasone
concentration, the absolute plating efficiency attained
plateau as did the perturbation in the cell cycle phases.
Malignant melanoma is a relatively uncommon disease
comprising 1-3% of all cancers [10,11]. Melanoma has
received considerable attention from both the clinical
and investigational point of view due to marked ten-
dency to disseminate and many patients die as result of
distant metastases [12,13]. Glucocorticoid (triamcinolone)
has shown an inhibitory effect on melanoma B16/F10
cell growth [14]. So this study was directed to gain bet-
ter insight the role of glucocorticoid as modulators of
cell growth as well as the presence and characteristics of
glucocorticoid receptor in human melanoma M-5A cells.
Our studies document the presence of specific, high af-
finity glucocortcoid binding site in M-5A human mela-
noma cells. The higher number of receptor sites in our
melanoma cells are in good agreement with levels of the
65,000 to 96,000 sites per cell observed by Rosner and
Cristofalo [7] in WI-38 human fibroblasts and with high
expression of glucocorticoid receptors in human mela-
noma cells observed by Collinson et al. [2]. In our study
M-5A cells were apparently, behaved differently from
normal and leukemic lymphoblasts with regard to effect
of dexamethsone treatment on the number of binding
site/cells, since there was a dose-dependent increase in
glucocoriticoid binding sites in M-5A cells after dexa-
methsone treatment. In contrast, shipman et al. [15,16]
found decrease in the glucocorticoid receptors after treat-
ment with glucocorticoid in malignant cells from the
A. M. M. Osman et al. / HEALTH 2 (2010) 857-861
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peripheral blood of leukemic patients and in blood from
normal volunteers, respectively. That could be due to an
internal regulation of receptor levels in different respon-
sive tumor.
Cell cycle analysis of M-5A cells after dexamethasone
treatment showed depletion of S-phase 10 hours later
with accumulation of the cells in G2/M phase (Figure 2).
Moreover there was a good correlation between depletion
of S-phase which, mean arrest in G1 phase and the cyto-
toxic activity of dexamethasone (Table 5). Early study
by Smets et al. [9] has shown that prolonged glucocorti-
coid treatment of L1210 leukemic cells potentiated cy-
tolysis by accumulation of the cells in the early G1 phase.
So, the overall proposed that the antiproliferative effect
of dexamethsone against growth of M-5A cells could be
due to G1 arrest which enhanced the probability of tran-
sit to the G0 compartment in which dexamethasone me-
diated cytolysis would occur. Such effect may be medi-
Table 4. Specific glucocorticoid binding in relation to protein
content of M-5A cells, 9 days after treatment with dexameth-
sone for one hour.
asone Con-
× 10-3
binding/ mg
0 131 59 362 ± 55d 601 ± 92d,e
2.5 172 60 554 ± 16 515 ± 15e
12.5 300 61 1000 ± 59 489 ± 29e
a Incubation for 60 min at 37˚C with 40 nM (3H) dexamethsone alone
or in the presence of excess unlabeled steroid dexamethsone 2.5,12.5
uM for one hour; bAverage number obtained from triplicate for total
and nonspecific binding, respectively; cMean data from two sister
flasks; dresented are mean ± S.E.M.; eNot significantly different.
Table 5. Relationship between cell cycle phase distribution
proliferation rate and plating efficiency of M-5A cells after
treatment with various concentration of dexamethasone.
Cell cycle distribution 24b
hours after treatment
(uM) G1% S% G2+M%
No. of visi-
ble colonies
8 days after
(% of con-
(% of
0 63 ± 4 21 ± 4 16 ± 6100 ± 7 100 ± 4
0.25 66 ± 5 10 ± 2 24 ± 771 ± 7 96 ± 4
1.25 63 ± 3 7 ± 1 30 ± 451 ± 2 78 ± 4
6.25 66 ± 2 7 ± 3 27 ± 135 ± 4 60 ± 2
12.5 62 ± 11 7 ± 3 31 ± 820 ± 1 56 ± 2
aDexamethsone was supplied for one hour and 24 hours later the cells
were collected as described in the materials and methods; bPresented
are mean ± S.E.M. of two separate experiment, 3 flasks for each ex-
Figure 1. Binding of (3H) dexamethasone by M-5A melanoma
cells (dpm/106 cells) at different drug concentration. Average
data (M ± S.E.M.) from four experiment.Incubations were for
60 mininutes at 37°C. • total binding, º nonspecific binding, ◊
specific binding.
Figure 2. Effect of dexamethsone treatment (2.5 uM for 60
min) on cell cycle phase distribution of M-5A cells at the indi-
cated hours post-treatment. X-axis, channel number (relative
DNA/cell); Y-axis, cell/channel (× 10-4). Channels 45-120 (S
and G2/M) amplified 4-fold. Relative compositions are given
in the figure.
A. M. M. Osman et al. / HEALTH 2 (2010) 857-861
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/
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