[CANCER RESEARCH 33, 2830-2833,
November 1973]
Reversal by Melatonin of the Effect of Pinealectomy on Tumor
Growth1
Ali A. H. EI-Domeiri and Tapas K. Das Gupta
Division of Surgical Oncology, Department of Surgery, Abraham Lincoln School of Medicine and Cook County Hospital, Chicago, Illinois 60680
SUMMARY
Pinealectomy is known to cause accelerated growth of
transplanted melanoma in hamsters. The precise mechanisms
involved in this action have not yet been identified. Since the
pineal is the only organ that produces melatonin, this
investigation was undertaken to determine the effect of this
indole in the same tumor model. Administration of 0.1 mg of
exogenous melatonin i.p. daily for 3 weeks did not influence
the growth rate of tumors at 3- and 6-week intervals in intact
animals. Administration of the same dosage of melatonin to
pinealectomized animals, however, abolished the accelerating
effect of pinealectomy on the growth of melanoma implants.
Therefore it is concluded that the effect of pinealectomy on
tumor growth is due to a deficiency in endogenous melatonin.
INTRODUCTION
There is convincing evidence that removal of the pineal
organ results in increased incidence and growth of certain
induced and transplanted tumors in rodents (1, 3,4, 14). The
mammalian pineal organ is rich in biogenic amines, namely
serotonin, noradrenalin, and histamine. A characteristic indole,
melatonin, is highly localized in the pineal organ (13).
Melatonin is related to and can be synthesized from serotonin
through the action of hydroxyindole-0-methyltransferase,
an
enzyme found only in pineal tissue (2).
This study was undertaken to investigate the effect of
administration of small doses of exogenous melatonin on the
growth of transplanted melanoma in both intact and pinealec
tomized Syrian hamsters.
MATERIALS
AND METHODS
A series of 3 experiments was conducted using a total of
188 male Syrian hamsters (Mesocricetus auratus), 4 to 6 weeks
old and weighing between 40 and 65 g. The animals were kept
in plastic disposable cages, 2 to a cage, at a room temperature
of 74—78°F with approximately 40% relative humidity and
under normal day-night lighting conditions. All animals were
fed Rockland complete diet and given water ad libitum.
Tumor implants consisted of a cell suspension of hamster
malignant melanoma, first described by Fortner and Allen (7)
'This study was supported by USPHS Grant CA-11211-04 from the
National Cancer Institute, NIH, Bethesda, Md. 20014.
Received April 27, 1973; accepted August 20, 1973.
2830
as a spontaneously growing neoplasm in the Syrian hamster.
The tumor was maintained in a hamster colony in our
laboratory by transplants at 3-month intervals into the s.c.
tissue. Hamster melanoma grows rapidly and gives rise to
widespread métastaseswithin 2 to 4 months.
Under sterile conditions a section of tumor measuring about
1 ml was removed from the donor animal, placed in a sterile
Petri dish, and minced in 10 ml of sterile 0.9% NaCl solution.
After continuous mincing for 15 minutes the mixture was
passed through a No. 150 wire mesh screen, using the Tracer
Laboratory (Waltham, Mass.) precipitation apparatus. The
resulting cell suspension was diluted with sterile 0.9% NaCl
solution to obtain a count of 5 X 10s cells per 0.1 ml. The cell
suspension for each experiment was prepared fresh and the
proportion of viable cells was evaluated by the trypan blue
exclusion test. The percentage of viable cells in the various
suspensions ranged between 95 and 99%.
Melatonin (5-methoxy-jV-acetyltryptamin)
was obtained in a
pure form from the Regis Chemical Co., Chicago, 111.
(Compound 370215) as a powder consisting of white crystals.
This powder was dissolved in absolute ethanol, then diluted
with distilled water to obtain a concentration of 0.1 mg of
melatonin in 0.1 ml of 5% ethanol. Because of the tendency of
melatonin to precipitate in a mixture of alcohol and water this
solution was prepared immediately before injection. Animals
in the treated groups received 0.1 ml of the melatonin solution
whereas animals in the control groups received either 0.1 ml of
0.9% NaCl solution or 5% ethanol. Injections were given i.p.
daily for 21 days. The animals were killed the 21st, 28th, or
42nd day after tumor inoculation. During these intervals the
tumors remained localized and were easily dissected from the
s.c. tissue; after dissection they were washed with 0.9% NaCl
solution and dried. Tumor from each animal was weighed with
a Torsion balance dial-type Model DLT-2 (Torsion Balance
Co., Clifton, N. J.) with a capacity of 120 g and sensitivity of
2 mg. The method of pinealectomy and sham operation used
in this study has been described in detail by Das Gupta and
Terz (4). In summary, animals were anesthetized using ether in
a bell jar and the anesthesia was maintained by ether mask
during the operation. The head was shaved and prepared and a
2-cm longitudinal incision was made on the vertex. The
periosteum was then elevated and a rectangular area of the
skull was exposed. A bone flap was raised and the dura mater
was incised to expose the pineal organ lying under the
confluence of the venous sinuses. Bleeding was controlled by
pressure and the pineal organ was then removed by cutting the
stalk. In sham-operated animals, all the steps of the operation
CANCER RESEARCH
VOL. 33
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Melatonin and Effect of Pinealectomy on Tumor Growth
were carried out except removal of the pineal organ.
The 1st experiment was designed to study the effect of
pinealectomy on melanoma implants; the 2nd and 3rd
experiments were done to evaluate the effect of melatonin on
the growth of the tumor in the intact and in the pinealectomized hamsters.
days. A number of animals in each group was killed on the
21st day and the rest on the 42nd day after inoculation. The
tumor weight in the pinealectomized animals that received
melatonin did not differ significantly from that in the intact
animals that received 0.9% NaCl solution (p > 0.05) (Table 3).
DISCUSSION
EXPERIMENTS
AND RESULTS
Experiment 1. Sixty-nine hamsters were randomly divided
into 3 groups. Animals in Group 1 had pinealectomies, those
in Group 2 had sham operations, and those in Group 3 were
kept intact. A week later all animals were inoculated with 0.1
ml of tumor suspension and then received 0.1 ml of 0.9% NaCl
solution daily for 21 days. Approximately one-half of the
animals in each group were killed on the 21st day and the
remaining animals on the 42nd day after inoculation. The
mean tumor weight in the pinealectomized animals was
significantly larger (p < 0.05) than in the intact and the
sham-operated controls (Table 1).
Experiment 2. Sixty-four hamsters were inoculated with
tumor suspension and then randomly divided into 3 groups.
Group 1 received 0.1 ml of 5% ethanol containing 0.1 mg of
melatonin, Group 2 received 0.1 ml of 5% ethanol, and Group
3 received 0.1 ml of 0.9% NaCl solution for 21 days.
Approximately one-half the animals in each group were killed
at 28 days and the remaining animals at 42 days. The
difference between the mean tumor weight among the groups
was not significant (p > 0.05) (Table 2).
Experiment 3. Fifty-five hamsters were divided into 2
groups; animals in Group 1 were pinealectomized, and in
Group 2 they were left intact. One week later, all animals were
inoculated with 0.1 ml of tumor suspension. Each animal in
Group 1 received 0.1 ml of the melatonin solution and those
in Group 2 received 0.1 ml of 0.9% NaCl solution daily for 21
An earlier report (4) indicated that removal of the pineal
organ resulted in an increase in the growth and spread of
transplanted melanoma in adult hamsters. The result of the 1st
experiment in our study confirmed this finding. The mean
tumor weight in the pinealectomized animals was significantly
higher than in the intact and the sham-operated hamsters. With
experience, pinealectomy (4) in adult hamsters becomes a
relatively simple procedure and results in low morbidity and
mortality. Following operation, the animals completely re
cover within 1 week, resume normal activity, and steadily gain
weight. Statistical analysis of the body weight of hypophysectomized and intact hamsters that did not receive tumor
implants revealed that the variances were homogeneous in the
Table 3
Effect of 0.1 mg melatonin on the growth of melanoma
in pinealectomized hamsters
Pinealectomy +
melatoninDuration0.1 mg
(days)21
of
animals1
42No.
tumor
wt ±S.E.
(g)0.677
and
solutionNo.
0.9% NaCl
tumor
wt ±S.E.
of
animals10 (g)0.642
±0.072
±0.134
1
16Mean 6.401 ±0.953Intact 18Mean 6.018 ±0.808
a Difference between mean values not significant (p > 0.05).
Table 1
Effect of pinealectomy on the growth of melanoma"
Pinealectomy
tumorwt±
S.E.(g)0.560
Duration(days)2142No.
ofanimals1213Mean
Sham operated
tumorwt
S.E.(g)0.248
±
ofanimals1112Mean
±0.0717.145
±1.681No.
±0.10211.390±
1.095No.
Intact
tumorwt
S.E.(g)0.297
±
ofanimals1011Mean
±0.0615.189±
0.960
" Difference between the means is significant (p < 0.05).
Table 2
Effect of 0.1 mg melatonin on the growth of melanoma in the intact hamster"
0.1 mg melatonin
tumorwt
S.E.(g)1.273
±
Duration(days)2842No.
ofanimals1112Mean
±0.2076.098
±0.928No.
5% ethanol
tumorwt
S.E.(g)1.412±
±
ofanimals1011Mean
0.1387.404
±0.750No.
0.9% NaCl solution
tumorwt
S.E.(g)1.244
±
ofanimals1010Mean
0.1795.255
±
±0.982
0 Difference between mean values is not significant (p < 0.05).
NOVEMBER
1973
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2831
Ali A. H. El-Domeiri and Tapas K. Das Gupta
2 groups at 4- and 6-week intervals. Therefore, the increase in
tumor weight in pinealectomized animals is believed to be
directly related to the absence of the pineal organ and not due
to a generalized deterioration in the condition of the animal
following the operation. The use of tumors from separate
donor animals to make cell suspensions for an individual
experiment and the inherent variability in the number of cells
as well as the difference in the ratio of viable cells in the
various suspensions, we believe, account for the difference in
tumor volume in the comparable groups in the different
experiments. Thus, only the difference between the means in
the individual experiments was considered in interpretation of
the results.
Because of the consistent effect of pinealectomy on
transplanted melanoma in hamsters, the same experimental
model was chosen to study the effect of melatonin on the
growth of this tumor in intact as well as pinealectomized
animals. The administration
of 0.1 mg melatonin daily
following tumor inoculation did not appear to influence the
growth of tumors in intact animals. The mean tumor weight in
the melatonin-treated
group was not significantly different
(/>>0.05) from that in animals receiving either 5% ethanol or
0.9% NaCl solution. However, when the same dose of
melatonin was administered to pinealectomized animals no
accelerating effect of pinealectomy on tumor growth was
detected. We also evaluated the effect of administration of
increasingly large doses of melatonin to the intact and the
pinealectomized hamsters (6). No change in the growth rate of
melanoma implants was noted in the intact animals that
received up to 4 mg melatonin daily and in pinealectomized
animals; the only demonstrable effect was absence of the
accelerated tumor growth. Other workers, however, reported
that administration
of pineal extract or melatonin may
influence tumor growth in the intact rats and mice. Tapp and
Blumfield (16) reported that there were incidences of
inhibition of the growth of some chemically induced tumors in
these animals following administration
of pineal extract.
Hamilton (11), in another study, noted an increase in the yield
of chemically induced mammary adenocarcinomata in relation
to fibroadenomata
in female rats receiving 0.1 mg of
melatonin daily.
The reversal of the effect of pinealectomy by a small dose
of exogenous melatonin strongly suggests that the loss of
endogenous melatonin is the factor responsible for the
acceleration of tumor growth. The rapidity with which this
effect is produced and the small dose required to correct it
indicates that the hamster pineal produces a minute amount of
endogenous melatonin and that the available reserve in the
animal body is either extremely small or nonexistent.
There is considerable evidence that the mammalian pineal
influences the functions of the brain, the endocrine glands,
and the gonads (10). Available data indicate that the pineal
organ exerts an inhibitory
effect on the gonads (5).
Pinealectomy produces an increase in the weight of the ovaries
and testicles in rodents (15). This effect is due to an increase
in the production of pituitary follicle-stimulating hormone and
luteinizing hormone following pinealectomy (8). In contrast,
administration of exogenous melatonin to pinealectomized
animals results in a decrease in the production of folliclestimulating hormone and luteinizing hormone and inhibits
2832
the stimulating effect of pinealectomy on the gonads (9, 15).
There is experimental evidence to suggest that these effects are
mediated by melatonin, which influences centers in the
hypothalamus and brain stem (17).
In contrast the precise mechanism by which endogenous
melatonin produces its restraining effect on tumor growth is
not yet clear. Although existing evidence indicates that an
increase in the level of melanophore-stimulating
hormone is
produced by pinealectomy and that melatonin administration
produces the reverse effect (12), there is no direct evidence to
indicate that the enhancing effect of pinealectomy on hamster
melanoma is related to changes in the level of circulating
melanophore-stimulating
hormone. It is conceivable that the
same tumor model may be used to evaluate the effect of
hypophysectomy on the growth of melanoma. The diverse and
complex hormonal changes that follow removal of the
pituitary gland, however, pose a difficult problem if correla
tion between changes in tumor growth and an individual
hormone is contemplated.
The results of this study strongly suggest that the
accelerating effect of pinealectomy on tumor growth is due to
deficiency in endogenous melatonin. It has been demonstrated
that other biological effects produced by melatonin are due to
its action as a mediator on the hypothalamohypophyseal
axis.
Further investigation is required to determine whether similar
mechanisms are involved in producing a restraining effect on
tumor growth.
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Melatonin and Effect of Pinealectomy on Tumor Growth
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NOVEMBER
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1973
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2833
Reversal by Melatonin of the Effect of Pinealectomy on Tumor
Growth
Ali A. H. El-Domeiri and Tapas K. Das Gupta
Cancer Res 1973;33:2830-2833.
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