Effect of the synthetic pineal peptide epitalon on spontaneous carcinogenesis in female C3H/He mice

Abstract

The potential preventive effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on spontaneous tumorigenesis in mice was studied. One-year-old female C3H/He mice were kept for 6.5 months under standard conditions. Epitalon was injected at a dose of 0.1 microg, 5 times a week. Long-term exposure to Epitalon in small doses did not show any toxic effect. Treatment with Epitalon decreased the number of tumor-bearing mice with malignant tumors and prevented the development of metastases. Spontaneous tumors of the reproductive organs (mammary glands and ovaries) were predominant in both groups of mice (control and experimental). The mammary gland tumors were different variants of invasive ductal carcinomas. In the ovaries, granulosa-cell tumors were found. Tumors were in the minority in other organs and had benign characteristics. In control mice, metastases were found in 3 out of 9 tumor-bearing mice, all of them being from tumors of the reproductive organs. Treatment with Epitalon slowed down the development of metastases from spontaneous tumors, and no metastases were found in the experimental mice. These data highlight the antimetastatic effect of Epitalon as part of its oncostatic properties.

Full text

Abstract. The potential preventive effect of the synthetic
pineal peptide Epitalon
®
(Ala-Glu-Asp-Gly) on spontaneous
tumorigenesis in mice was studied. One-year-old female
C3H/He mice were kept for 6.5 months under standard
conditions. Epitalon was injected at a dose of 0.1 Ìg, 5 times
a week. Long-term exposure to Epitalon in small doses did
not show any toxic effect. Treatment with Epitalon decreased
the number of tumor-bearing mice with malignant tumors and
prevented the development of metastases. Spontaneous tumors
of the reproductive organs (mammary glands and ovaries)
were predominant in both groups of mice (control and
experimental). The mammary gland tumors were different
variants of invasive ductal carcinomas. In the ovaries,
granulosa-cell tumors were found. Tumors were in the
minority in other organs and had benign characteristics. In
control mice, metastases were found in 3 out of 9 tumor-
bearing mice, all of them being from tumors of the
reproductive organs. Treatment with Epitalon slowed down
the development of metastases from spontaneous tumors, and
no metastases were found in the experimental mice. These
data highlight the antimetastatic effect of Epitalon as part of
its oncostatic properties.
Pineal hormones affect the activity of the female
reproductive organs and play a significant role in the
general protection against different foreign factors,
including carcinogens. The pineal hormone melatonin and
the pineal peptide preparation Epithalamin have been
shown to inhibit the development of spontaneous and
chemically-induced neoplasms (1,2), especially of
hormone-dependent mammary tumors (3-5). Epitalon
®
,
the synthetic derivative of Epithalamin, has also been
found to reduce the incidence of spontaneous tumors in
inbred CBA and HER-2/neu transgenic mice, as well as in
Swiss-derived SHR mice, and of chemically-induced colon
tumors in rats (6-10).
The effect of the synthetic peptide Epitalon on
spontaneous carcinogenesis in inbred C3H/He mice was
studied. The findings add to our understanding of the role
of pineal hormones in the pathogenesis of cancer.
Materials and Methods
Chemicals. The tetrapeptide Epitalon (Ala-Glu-Asp-Gly) was
synthesized on the basis of the amino acid sequence of Epithalamin
by G.I. Grigoriev, St. Petersburg Institute of Bioregulation and
Gerontology, Russia.
Animals. Twice-bearded one-year-old female C3H/He mice
(n=117, Harlan Labs., Jerusalem, Israel) were used in the
experiment. The animals were kept, 5 per polypropylene cage,
under a standard 12-h light/12-h darkness regimen at 21Æ to 23ÆC.
Standard laboratory chow and tap water were available ad libitum.
Tumorigenic experiment. The animals were divided into 2 groups:
control (n=56) and experimental (n=61). Mice in the control
group received sterile saline at a dose of 0.2 ml/mouse,
subcutaneously (s.c.) 5 days a week. Mice in the experimental
group received Epitalon s.c. on the same days at a dose of 0.1 Ìg
dissolved in 0.2 ml saline/mouse. The experiment was terminated
6.5 months after the first injection of Epitalon, and the mice were
killed by cervical dislocation.
Scoring and analysis. All the animals were checked visually 5
days a week and weighed monthly throughout the experimental
period. They were autopsied after 6.5 months. The position and
size of each tumor were recorded. All tumors were fixed in 4%
neutral formaldehyde and, after routine histological processing,
were embedded in paraffin. Sections (3-Ìm-thick) through the
253
Correspondence to: Professor I. Zusman, Koret School of
Veterinary Medicine, Faculty of Agricultural, Food and
Environmental Quality Sciences, The Hebrew University of
Jerusalem, Rehovot 76100, Israel. e-mail: zusmani@agri.huji.ac.il
Key Words: Epitalon, mice, pineal peptides, reproductive organs,
carcinogenesis.
in vivo 20: 253-258 (2006)
Effect of the Synthetic Pineal Peptide Epitalon on
Spontaneous Carcinogenesis in Female C3H/He Mice
GEORGE KOSSOY
1
, VLADIMIR N. ANISIMOV
2
, HERZEL BEN-HUR
3
,
NADJA KOSSOY
1
and ITSHAK ZUSMAN
1
1
Koret School of Veterinary Medicine, Faculty of Agricultural, Food and Environmental Quality Sciences,
The Hebrew University of Jerusalem, Rehovot, Israel;
2
Department of Carcinogenesis and Oncogerontology, NN Petrov Research Institute of Oncology, St.Petersburg, Russia;
3
Laboratory of Experimental Medicine, Park Rabin, Rehovot, Israel
0258-851X/2006 $2.00+.40

middle part of each tumor were stained with hematoxylin and
eosin (H&E). All data were analyzed statistically by the
Student's t-test.
Results
Long-term exposure to Epitalon in small doses did not show
any toxic effects, and no mortalities were recorded. During
the experiment, no changes were found in the weight of the
experimental mice versus that of their control counterparts.
Although the experiment (6.5 months) was not long
enough to determine the effect of Epitalon on spontaneous
tumorigenesis, its results are expected to be of interest to
experimental oncologists.
Treatment with Epitalon decreased the number of
tumor-bearing mice with malignant tumors and prevented
the development of metastases (Table I). Spontaneous
tumors of the reproductive organs (mammary glands and
ovaries) were predominant in both groups of mice (control
and experimental) (Figures 1A, 2A). Mammary-gland
tumors presented different variants of invasive ductal
carcinomas, while ovarian tumors were of the granulosa-
cell type. Tumors in the other organs were in the minority
and had benign characteristics: trichofolliculomas and
trichoepitheliomas in the skin, and adenomas in the lungs
and liver.
In control mice, metastases were found in 3 out of 9
tumor-bearing mice, all of them being from tumors of the
reproductive organs (Table I). In 2 mice, metastases of
mammary tumors were found in the lungs (Figure 1A,B), and
in 1 mice, a metastasis of an ovarian tumor was found in the
neighboring fat tissues (Figure 2A,B). No metastasized
tumors were found in the experimental mice.
Discussion
The aim of the present study was to evaluate the effect of
the synthetic pineal peptide Epitalon on spontaneous
tumorigenesis in female C3H/He mice. Spontaneous
mammary tumors and their metastases in C3H mice have
been described previously (11). Among spontaneous
ovarian tumors in these mice, granulosa-cell tumors
predominate (12). In transgenic FVB/N mice carrying the
breast cancer gene HER-2/neu, Epitalon prolonged the
average lifetime of animals without neoplasms by 34.2%,
decelerated the development of age-related disturbances in
reproductive activity and suppressed the formation of
neoplasms (7). The peptide decreased the incidence of
breast adenocarcinomas, lung metastases and multiple
tumors, increased the number of mice without breast
tumors, and prolonged the lifetime of mice with breast
tumors by 1.4-fold, compared to their control counterparts.
In outbred Swiss-derived SHR mice, Epitalon increased the
maximum lifespan by 12.3% in comparison with the control
group (8). Although Epitalon treatment did not influence
the total incidence of spontaneous tumors, it inhibited the
development of leukemia by 6-fold relative to the control
group. In tumor-bearing LIO rats exposed to 1,2-
dimethylhydrazine, Epitalon at a daily dose of 1 Ìg/rat
throughout the course of the experiment significantly
decreased the number of tumors in the colon compared to
the controls, and inhibited the development of tumors in
the jejunum and ileum (9). Moreover, Epitalon inhibited
the mitotic activity of both the epithelial cells adjacent to
tumors and the tumor cells themselves when the treatment
was given throughout the experiment (10). In parallel, a
high level of apoptosis was seen in this group of rats.
Melatonin or Epitalon treatment was followed by longer
mean and maximum survivals in 10% of the last survivors
among senescence-accelerated mice (SAM) (13). It should
be noted that the anticarcinogenic effect of Epitalon was
most effective when it was administered throughout the
experiment, i.e., at both the initiation and promotion stages
of carcinogenesis (6-8).
The antitumorigenic effects of Epitalon and melatonin are
manifested in their inhibition of cell proliferation (4,14).
Among the different mechanisms that have been described
to date governing the effects of these pineal components (3),
their anti-invasive and antimetastatic activities have been
least studied, despite the opinion that these parameters are
an important part of the oncostatic action of these
components (15). It has been shown, for example, that
melatonin as a supplementary treatment to chemotherapy
promotes the 5-year survival rate of non-small cell lung
cancer patients with metastases (16).
in vivo 20: 253-258 (2006)
254
Table I. Incidence, localization and type of tumors in mice treated and
not treated with Epitalon.
Parameters studied Saline Epitalon
Total number of mice 56 61
Number of tumor-bearing mice 9 (16.1%) 7 (11.5%)
Number of malignant tumor-bearing mice 6 (10.7%) 3 (4.9%)
Total number of tumors 9 7
Number of metastasizing tumors 3 -
Localization and type of tumors:
Mammary gland: invasive ductal carcinoma 5 (2)
a
3
Ovaries: granulosa-cell tumor 1 (1)
a
1
Skin: trichofolliculoma 2 -
trichoepithelioma - 2
Lungs: adenoma 1 -
Liver: hepatoma - 1
a
In parentheses, the number of mice with metastases.

This study showed that Epitalon decreased the number of
tumor-bearing mice with malignant tumors and prevented
the metastasis of spontaneous tumors from the mouse
reproductive organs. The antitumor effect of Epitalon
appeared to be similar to those of its natural analogs (3).
Acknowledgements
This work was supported in part by grant #00-04.48481 from the
Russian Foundation for Basic Reseach and by grant #039-6119
from the Israeli Government.
Kossoy et al: Epitalon and Cancer
255
Figure 1. Tumor of the mammary gland. H&E. x80. A, Invasive ductal carcinoma. B, Metastasized tumor in the lungs.

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256
Figure 2. Tumor of the ovary. H&E. A, x800. B, x200. A, Granulosa-cell tumor. Note invasive lodgement of tumor cells in a blood vessel (arrow). B,
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Received December 16, 2005
Accepted February 3, 2006
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