[CANCER RESEARCH 48, 7189-7192, December 15, 1988]
Deferoxamine Inhibition of Human Neuroblastoma Viability and Proliferation
David L. Becton1 and Patty Bryles
University of Arkansas for Medical Sciences and Arkansas Children 's Hospital, Department of Pediatrics, Division of Hemalology/Oncology, Little Rock, Arkansas 72202
ABSTRACT
Patients with widespread neuroblastonta (NB) frequently have elevated
serum ferritin levels, and recently anti-NB effects of the iron chelator
deferoxamine (DFO) have been reported. We have investigated the effect
of DFO on human bone marrow NB cells from two untreated children
with Evans Stage IV disease. DFO treatment caused dose- and timedependent cytotoxicity of NB cells, with maximal killing at exposure to
50 /IM DFO for 72 h. Cytotoxicity was prevented by cotreatment with
stoichiometric amounts of iron salts and reversible by removal of DFO
or addition of iron salts within 48 h of treatment. Additionally, DFO
inhibited clonal growth of human bone marrow NB cells in methylcellulose in a time- and dose-dependent manner. These effects were also
prevented by cotreatment with iron salts. Thus, DFO has potent antitumor effects on human NB cells which appear to be related to iron
deprivation. DFO should be considered for further preclinical evaluation
as an anti-NB agent.
INTRODUCTION
Iron and ferritin appear to play an important role in the
growth and proliferation of human NB.2 Hann and coworkers
(1,2) demonstrated that patients with advanced NB frequently
have elevated serum ferritin which apparently is derived from
the NB. The relationship between ferritin, NB cells, and the
host is unknown, but previous studies have indicated an indirect
mechanism by which ferritin inhibits host immune response,
thereby enhancing tumor growth (3, 4). More recently, a direct
effect on tumor growth has been supported by the demonstra
tion that the iron chelator deferoxamine has an in vitro antiNB effect on the human NB cell lines CHP126 and CHP100
(5). We have studied the direct effect of DFO on the viability
and proliferative capacity of bone marrow NB cells obtained
from two patients with widespread NB. Our results further
support a role for iron in the growth and proliferation of NB,
and they suggest that DFO or other iron chelators should be
evaluated further for NB therapy.
MATERIALS
AND METHODS
Neuroblastoma Cells. Approximately 5 ml of heparinized bone mar
row were obtained from two patients, an 18-mo-old Black female
(Patient A) and a 15-mo-old White male (Patient B), with Evans Stage
IV NB, whose serum ferritin levels were 453 ng/ml and 592 ng/ml
(normal, <140 ng/ml), respectively. Each patient had extensive bone
marrow involvement. Informed consent was obtained according to
institutional guidelines. Heparinized marrow was diluted in TCM
(RPMI 1640; Gibco, Grand Island, NY; 10% heat-inactivated fetal calf
serum, Gibco; penicillin, 50 units/ml; and streptomycin, 20 mg/ml),
underlayed with Ficoll-Hypaque, and centrifuged at 1000 x g for 45
min. Cells at the interface were collected, washed twice in Hanks'
cytocentrifuge preparations and by neuron-specific enolase stains.
Viability of NB Cells Treated with Deferoxamine. Cells from each
patient were plated in quadruplicate in 16-mm wells at 0.5 to 1.0 x IO5
cells/well in 1.0 ml of TCM and cultured at 37°Cin 5% CO2. Various
amounts of deferoxamine mesylate (CIBA, Summit, NJ), with or with
out stoichiometric amounts of FeClj (Sigma, St. Louis, MO), were
added as indicated in "Results." Cells were removed by scraping with
a rubber policeman, and viable cells, determined by trypan blue exclu
sion, were counted daily for 5 days.
Clonal Growth of Neuroblastoma Cells. NB cells were cultured in
0.9% methylcellulose by a previously described method (6). Briefly, 1.0
x 10s NB cells were added to 1.0 ml of 0.9% methylcellulose containing
20% «-medium(Gibco), 15% PCS, and 10% human leukocyte-condi
tioned medium with or without DFO and FeCh, as described in
"Results." Cell aggregates (>20 cells) were counted at 4, 7, and 14
days. Individual NB aggregates were removed with a micropipet and
identified by Wright-Giemsa stain and by the presence of neuronspecific enolase.
RESULTS
Antineuroblastoma Effects of DFO. NB cells remained viable
in suspension culture for up to 14 days, with minimal cell death
over the first 5 days. Although proliferation did not occur,
occasional clumps of cells would adhere to the plastic surface
of the well after 2 to 3 days of culture, and thin neuritic
processes would develop, often connecting with nearby clumps
or single cells (Fig. \A ). Adherent cells could be disrupted easily
by agitation or gentle washing. Concentrations of DFO of 5 MM
or less did not affect cell viability, adherence, or morphology.
At 10 MM,minimal reductions of viability were observed after
96 h (Table 1). Additionally, there was only minimal adherence
to the culture surface, and the neuritic processes seen in control
cultures did not occur (Fig. IB). A dose of 20 MM led to a
greater than 50% reduction in viability at 96 h, and 50 MM
caused complete cell kill (Table 1). There was a slight difference
in sensitivity between the patients. The effects of viability,
morphology, and adherence were completely reversed by addi
tion of stoichiometric amounts of FeCU.
Time Course of DFO Effects. Although minimal cytotoxicity
occurred after 24 h at 50 MMDFO, significant anti-NB effects
were observed only after 48-h exposure, with about 50% cyto
toxicity at 50 MM,30% at 20 MM,and 15% at 10 MM(Fig. 2).
These values represent about one-half of eventual cell kill for
each concentration. Maximal cytotoxicity occurred at 72 h,
with only small increments over the next 48 h.
To evaluate the length of exposure to DFO necessary for
irreversible cytotoxicity, "treatment" was stopped at 24, 48, or
72 h by washing DFO from the cultures or by supplementation
with stoichiometric amounts of FeClj. At concentrations <10
MMDFO, only minimal cytotoxicity was observed even after
balanced salt solution (Gibco), and resuspended in TCM. Cell suspen
72-h exposure (data not shown). At higher concentrations, the
sions contained >80% neuroblasts in Patient A and >90% neuroblasts
cytotoxic
effects of DFO were halted if washing or FeCl3
in Patient B as confirmed by morphology from Wright-Giemsa-stained
supplementation occurred by 48 h, and no further loss of
Received 5/4/88; revised 9/16/88; accepted 9/20/88.
viability was observed (Table 2). Even with 50 MMDFO, cyto
The costs of publication of this article were defrayed in part by the payment
toxicity was reversible up to 48 h.
of page charges. This article must therefore be hereby marked advertisement in
Effect of DFO on Clonal Growth of NB Cells. Bone marrow
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed.
NB
cells grew readily in 0.9% methylcellulose supplemented
2 The abbreviations used are: NB, neuroblastoma; DFO, deferoxamine; TCM,
with human leukocyte conditioning medium; clusters of NB
tissue culture medium; PCS, fetal calf serum; PHA, phytohemagglutinin; RR,
cells were apparent within 72 h of plating, and aggregates (>20
ribonucleotide reductase.
7189
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.
DFO INHIBITION OF NEUROBLASTOMA
96
Fig. 2. Time course of DFO-induced cytotoxicity. NB cells (Patient A) were
treated with various concentrations of DFO in suspension cultures. Cell counts
were performed daily, and results are expressed as the percentage of viable cells
remaining. O, control; •¿,
50 MMDFO -I-50 MMFeCl,; É,10 MMDFO; D, 20 MM
DFO; A, 50 pM DFO.
B
Table 2 Exposure time for DFO-induced cytotoxicity
Human NB cells (1 x 10* cells/ml) were treated with various concentrations
of DFO. At the indicated times, cells were washed 2 times and replated in fresh
medium, or stoichiometric amounts of FeCI3 were added as indicated. Viable cells
were counted 96 h from the start of the experiment and are expressed as the
percentage of viable cells compared with the total number of cells cultured.
cells"TreatmentWashWashAdd
of viable
DFMO
Time(^M)
B77817964705145847139401512'
(h)0
A84756864734339738047361012Patient
7220
24244848727250
FeCl,WashAdd
Fed,WashAdd
Fig. I. Human NB bone marrow cells ( 1 x lOVml) were cultured in suspension
as described in "Materials and Methods." After 2 to 3 days in culture, many cells
became adherent and developed neuritic processes (A). Treatment with 10 >tM
DFO decreased adherence and prevented formation of neuritic processes (B).
FeCljWashAdd
242448487272%
FeCljWashAdd
FeCljWashAdd
Table I Human bone marrow NB cells cultured at 1 x ¡Ifcells/ml al various
concentrations of DFO and/or FeCk
Cells were counted after 96 h of continuous exposure and indicated as the
percentage of viable cells compared with the total number of cells cultured.
Results represent the mean of two experiments.
FeCljPatient
Mean of 2 experiments.
In some experiments, cells were exposed to various concen
trations of DFO for 1 to 48 h in suspension culture, then
washed twice, and plated in methylcellulose as above. There
A9293906233886848592Patient
B8784845821830738175
was a time- and dose-dependent effect on colony formation
(Fig. 5). No reduction in aggregates was observed at doses up
to 10 nM even for 48-h exposure. However, 48-h exposure to
20 or 50 /¿MDFO, followed by washing or FeCl3 addition,
reduced the subsequent number of aggregates at Day 14.
cellsPatient
of viable
usedDFO(MM)151020205050FeClj(MM)20502050%
Agents
DISCUSSION
We have demonstrated that DFO has potent in vitro antitucells) could be identified by Day 5 in all experiments with cells mor effects on human bone marrow NB cells. The effects are
from each patient. The number of aggregates peaked between
dose dependent, with concentrations of 10 UMbeing necessary
14 and 21 days (Fig. 3,4), and most cells were dead by Day 35. for minimal killing and 50 ¿tM
for complete cell killing (>90%).
DFO treatment caused a dose-dependent reduction in aggregate
At lower doses, DFO does not cause cell death but alters the
formation. There was no significant effect of concentrations
morphology and decreases the ability of the NB cells to adhere
less than 5 UM. At 5 or 10 n\i DFO, there were decreased
to plastic surfaces. Furthermore, the effects of DFO are time
numbers of aggregates (Fig. 4); those that did form remained
dependent, with 48 to 72 h of exposure necessary to observe
clumped and rounded, without cytoplasmic processes. At 20 cytotoxicity or alterations in morphology. Removal of DFO by
UMor greater concentrations, no aggregates were present (Figs. washing or supplementation with FeCU by 48 h prevents further
3B and 4). Addition of stoichiometric amounts of FeClj com
cytotoxicity.
pletely reversed the effect of DFO (Fig. 4).
Additionally, DFO inhibits the proliferation of NB cells in
7190
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.
DFO INHIBITION OF NEUROBLASTOMA
A '
B
5<V/M
IDFO;
Fig. 5. Effect of time of exposure to DFO on proliferation. Bone marrow NB
cells were exposed to various quantities of DFO for 12, 24, or 48 h, then washed
3 times, and cultured in 0.9% methylcellulose as described in "Materials and
Methods." Cell aggregates were counted at Day 14 of methylcellulose culture.
Columns, mean of 2 experiments; bars, higher number of aggregates between the
2 experiments.
Fig. 3. Proliferation of human NB cells in methylcellulose. Bone marrow NB
cells (1 x 105/ml) were cultured in 0.9% methylcellulose supplemented with PCS
and human leukocyte-conditioned medium, as described in "Materials and Meth
ods." Aggregates were detected by Day 5, and by Day 14 many had formed
neuritic processes which often joined with other aggregates (A). Treatment with
50 JIMDFO led to complete inhibition of aggregates (B).
l
T
50
40
30
1
ft.B
l
1
20
10
live effects were only minimally reversible after 48-h exposure
to DFO followed by washing or FeCl3 supplementation, sug
gesting that the viable cells remaining after a 48-h exposure to
DFO may lack proliferative capacity.
Recently, Blatt and Stitely demonstrated that DFO has po
tent antitumor effects on two human-derived NB cell lines (5).
Their results were similar to ours in that a dose of 15 fiM was
required for >50% cell kill, and DFO cytotoxicity was reversed
by cotreatment with ferric citrate. Additionally, they demon
strated a 50% increase in the number of cells expressing the
OK-T9 surface marker which is felt to be the transferrin recep
tor. Thus, there is evidence of iron-related anti-NB effect of
DFO in both human-derived NB cell lines and in human NB
cells.
The mechanism by which iron chelation inhibits NB cells is
uncertain. DFO blocks the proliferation of PHA- or concanavalin A-stiuui lai cd lymphocytes and of some leukemia cell lines,
apparently by preventing progression through the S phase of
the cell cycle (7-11). Lederman demonstrated significant de
creases in [3H]thymidine uptake in PHA-stimulated lympho
cytes treated with DFO, with no significant effect on RNA or
protein synthesis (7). The effect of DNA synthesis was dose
dependent, was reversed when DFO was washed from culture,
Concentration DFO (/M)
Fig. 4. Inhibition of NB aggregates by DFO. NB cells were grown in 0.9%
and was prevented by cotreatment with iron salts. Additionally,
methylcellulose, supplemented with leukocyte-conditioned medium, as described
Bornford reported that DFO decreased the growth rate and
in "Materials and Methods." DFO caused a dose-dependent reduction in the
inhibited DNA synthesis in the human leukemic line K562 ( 10).
number of aggregates counted at Day 14 in each patient. Columns, mean of two
experiments; ears, higher number of aggregates between the two experiments.
Iron is required for the enzyme RR, important in the produc
tion of deoxyribonucleotides necessary for DNA synthesis (12).
The M2 subunit of RR has a short half-life, and its regeneration
semisolid medium. The effects can be seen at doses lower than
requires the constant presence of iron (13-16). Inhibition of
those causing cytotoxicity, with slight reductions in cell aggre
M2 regeneration by iron deprivation could decrease RR activity
gates seen at 5 fiM DFO. Complete inhibition of proliferation
was observed at 20 UMor greater concentrations. The antiproand thereby impede DNA synthesis. Thus, the effect of DFO
liferative effects of DFO also appear to be related to iron on cell proliferation may be secondary to its indirect effect on
deprivation, since cotreatment with FeClj prevents the inhibi
RR activity. In fact, DFO potentiates inhibitors of RR (17),
tion of proliferation. Interestingly, however, the antiproliferaand effects similar to those caused by DFO have been observed
7191
10
20
50
»T
50)jMFeCI,
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.
DFO INHIBITION OF NEUROBLASTOMA
after treatment with hydroxyurea, a known inhibitor of RR (7,
9). The direct cytotoxicity of DFO on nonproliferating NB cells
is not explained by this mechanism, however. The association
of ferritinemia with widespread NB suggests another possible
relationship between iron and NB cells which has not been fully
elucidated. Further investigations of the anti-NB effects of DFO
should focus on this relationship, as well as on the effects of
DFO on the M2 subunit of RR in NB cells.
The demonstration of the antitumor effect of DFO on NB
cell lines and NB bone marrow cells should lead to further
preclinical and perhaps clinical evaluation. Previous investiga
tors have described tumor specificity of DFO's antiproliferative
activity, with no effect on the human diploid cell line WI 38 (1,
18). Additionally, preliminary results in our laboratory suggest
a 10-fold decrease in sensitivity to DFO in normal human bone
marrow mononuclear cells compared to human NB cells (data
not presented). Experience with DFO in patients requiring
chronic transfusion suggests that the drug can be administered
safely by various routes in relatively high doses (19). Although
drug levels are not available for routine clinical use, recently
All,un i-i al. described measuring DFO in plasma and urine by
atomic absorption spectrometry (20). A single intramuscular
dose of 10 mg of DFO/kg led to a peak plasma level of 15
¿imol/literin healthy, non-iron-overloaded controls, suggesting
that levels in the in vitro response range (20 to 50 /¿M)are
attainable. In fact, Estrov et al. have treated an infant with
refractory leukemia with i.v. DFO (in 1-0-D-arabinofuranosylcytosine) and demonstrated in vivo and in vitro responses,
without significant toxicity (21). Thus, there appears to be some
potential for use of DFO as an antitumor agent.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
ACKNOWLEDGMENTS
18.
We thank Rai Smith for typing the manuscript.
19.
REFERENCES
1. Hann. H-W. L., Levy, H. M., and Evans, A. E. Serum ferritin as a guide to
therapy in neuroblastoma. Cancer Res.. 40: 1411-1413, 1980.
2. Wantanabe. N., Niitsu. Y., Koseki, J., et al. Ferritinemia in nude mice
bearing various human carcinomas. In: F. G. Lehman (ed.), Carcioembryonic
Proteins. Vol. 1. p. 273. Amsterdam: Elsevier, North Holland. 1979.
3. Hann. H. L., Evans, A. E., Cohen, 1. J., and Leitmeyer, J. E. Biologic
20.
21.
differences between neuroblastoma Stages IV-S and IV. Measurement of
serum ferritin and E-rosette inhibition in 30 children. N. Engl. J. Med., 305:
425-429, 1981.
Matzner, Y., Hershko, C.. Polliack, A., Konjin, A. M., and Izak, G. Suppressive effect of ferritin on in vitro lymphocyte function. Br. J. Haematol.,
«.-345-353, 1979.
Blatt, J., and Stitely. S. Antineuroblastoma activity of desferoxamine in
human cell lines. Cancer Res., 47: 1749-1750, 1987.
Ito, T., Ishikawa, T., Okano, S., Hattori, T., Fujii, R., Shinozawa, T., and
Shibuya, A. Cloning of human neuroblastoma cells in methylcellulose culture.
Cancer Res., 47:4146-4149, 1987.
Lederman, H. M., Cohen. A., Lee, J. W. W., Freedman, M. H., and Gelfland,
E. W. Deferoxamine: a reversible S-phase inhibitor of human lymphocyte
proliferation. Blood, 64: 748-753. 1984.
Bowern, N., Ramshaw, P., Badenocch-Jones, P., and Doherty, P. C. Effect
of an iron-chelating agent on lymphocyte proliferation. Aust. J. Exp. Biol.
Med. Sci., 62: 743-754, 1984.
Hoffbrand, A. V., Ganeshaguru, K., Hooton, J. W. C., and Tattersall, M. H.
N. Effect of iron deficiency and desferrioxamine DNA synthesis in human
cells. Br. J. Haematol., 33: 517-526, 1970.
Humlord. A., Isaac, J., Roberts, S., Edwards, A., Young, S., and Williams,
R. The effect of desferrioxamine on transferrin receptors, the cell cycle, and
growth rates of human leukaemic cells. Biochem. J., 236: 243-249, 1986.
Forsbeck. L., Bjelkenkrantz, K., and Nilsson, K. Role of iron in the prolif
eration of the established human tumor cell lines U-937 and K-562: effects
of sin um m and a lipophilic iron chelator (PIH). Scand. J. Haematol., 37:
429-437, 1986.
Ehrenberg, A., and Reichard, P. Electrospin resonance of the iron-containing
protein B2 from ribonucleotide reducÃ-ase.J. Biol. Chem.. 247: 3485-3488,
1972.
Thelander, L., Eriksson, S., and Akerman, M. Ribonucleotide reducÃ-asefrom
calf thymus. Separation of the enzyme into two nonidentical subunits. Pro
teins Ml and M2. J. Biol. Chem., 255: 7426-7432, 1980.
Mattalino, R., Sloan, A., Plumer, E., and Klippenstein, G. Purification of
the two complimentary subunits of ribonucleotide reducÃ-asefrom calf thy
mus. Biochem. Biophys. Res. Commun., 102: 667-674, 1981.
Graslund, A., Ehrenberg, A., and Thelander, L. Characterization of the free
radical of mammalian ribonucleotide reducÃ-ase.J. Biol. Chem.. 257: 57115715, 1982.
Thelander, L., Graslund, A., and Thelander, M. Continual presence of oxygen
and iron required for mammalian ribonucleotide reduction: possible regula
tion mechanism. Biochem. Biophys. Res. Commun., 110: 859-865, 1983.
Cory, J. G., Lasater, L., and Sato, A. Effect of iron-chelating agents on
inhibitors of ribonucleotide reducÃ-ase.Biochem. Pharmacol., 30: 979-983,
1981.
Hann, H. L., Stahlhut, M. W., and London, W. T. Anti-tumor activity of an
iron chelating agent (desferoxamine) on human hepatoma cell lines. Clin.
Res., 36:495, 1988.
Modell, B., Letsky, E., Flynn, D., Reto. R., and Weatherall, D. Survival and
desferioxamine in thalassaemia major. Br. Med. J., 284: 1081-1084, 1982.
Allain, P., Mauras, Y., Chaleil. D., Simon. P., Ang, K., Cam, G., LeMignon,
L., and Simon, M. Pharmcokinetics and renal elimination of desferrioxamine
and ferrioxamine in healthy subjects and patients with haemochromatosis.
Br. J. Clin. Pharmacol., 24: 207-212, 1987.
Estrov, B., Tawa, A., Wang, X. O., Dubé,I., Sulh, H., Cohen, A., Gelfand,
E., and Freedman, M. In vitro and in vivo effects of deferoxamine in neonatal
acute leukemia. Blood, 69: 757-761, 1987.
7192
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.
Deferoxamine Inhibition of Human Neuroblastoma Viability and
Proliferation
David L. Becton and Patty Bryles
Cancer Res 1988;48:7189-7192.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/48/24_Part_1/7189
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.