Potential mechanism for the effects of dexamethasone on growth of human melanoma cells in vitro

doi:10.4236/health.2010.28129

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Vol.2, No.8, 857-861 (2010)

doi:10.4236/health.2010.28129

HEALTH

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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:

moneimosman@hotmail.com

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.

ABSTRACT

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

1. INTRODUCTION

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.

2. MATERIALS AND METHODS

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

858

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. RESULTS

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.

A. M. M. Osman et al. / HEALTH 2 (2010) 857-861

859

Table.1. Time course of the specifically binding of (3H) dexa-

methasone by M-5A melanoma cells.

Incubation

time

(min)

Specific binding

of (3H )

dexamethasonea

(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

flasks

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

Treatment

(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

treatment.

Specific binding sites per cells1

× 10-3

Dexamethsone

concentration

(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 ±

S.E.M.

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-

malized.

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.

4. DISCUSSION

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

860

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.

Dexameth-

asone Con-

centration

(uM)

Specific

binding

sites/cella,b

× 10-3

Nonspe-

cific/total

specific

bindinga,b

(%)

Total

cellular

proteina,c

(ug/106

cells)

Specific

binding/ mg

cellular

protein

(fmol)

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

Dosea

(uM) G1% S% G2+M%

No. of visi-

ble colonies

8 days after

treatment

(% of con-

trol)

Final

plating

efficiency

(% of

control)

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-

periment.

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

861

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