Introduction
Cancer is one of the foremost health problems of today
due to its frequent incidence, high morbidity and
mortality and the duration of treatment, cost, and complications.[] Radiotherapy (RT) is also used quite
frequently besides surgery and chemotherapy in the
treatment of cancer. It is known that 50-60% of cancer
patients need RT in the course of their disease for
curative or palliative purposes.[] Patients take advantage of the benefits of RT such as not increasing and
destroying harmful cells through ionized rays during
and after cancer treatment.[,] While RT destroys
harmful cancer cells by sending rays, it also damages
healthy cells. This is the undesirable result of the treatment
applied to gain favor.[,]
There are a number of radioprotective agents used
to protect from the harmful rays of chemotherapy and
RT.[-] It has been observed that amifostine (AMF)
(WR-2721) which is a radioprotective agent reduces
the harmful effects of radiation in line with the studies
conducted.[,] As distinct from the agents used
in clinic as various cell protective agents, AMF is not
specific to a single organ, but AMF is an organic thiol
compound that can affect all organs.[-]
Mucins (MUCs) are glycoprotein structures in
the cell wall that has functions such as lubricating,
secreting, protecting, and maintaining vitality of
the cell.[] These are divided into two including
secreted and membrane-bound MUCs. The MUCs,
which create secreted gel, include MUC2, MUC5AC,
MUC5B, MUC6, MUC7, MUC8, and MUC19. The
MUCs bounded to membrane are MUC1, MUC3A,
MUC4, MUC12, MUC13, MUC15, MUC16,
MUC17, and MUC2-0 which have a single transmembrane
field and a highly cytoplasmic tail.[,]
MUC1 plays an significant role in the regulation
of many cellular features such as cell proliferation,
apoptosis, adhesion, and invasion.[] It has been
thought that the MUCs can be benefited from preventing
the damage caused by drugs used in the cancer
treatment.[] The relationship of the AMF and
MUC requires further study, especially in the treatment
of prostate cancer, which is one of the important
organs in reproductive development.
Nowadays, it is observed that the treatments such
as chemotherapy and RT damaging healthy tissues
and cells are increasing due to the increasing number
of cancer diseases. In some studies, this harm
reducing feature of the AMF has been examined.
[,] However, it is required to be able to be presented
fully and completely. This experimental study
aims to evaluate the positive or negative effects of the
AMF, which is a radioprotective agent, on MUC1 in
preventing the damage that may occur in the prostate
tissue after RT with immunohistochemical methods.
It is thought that the positive results that will be able
to be observed may contribute to the production of
drugs and the course of treatment for the cancer diseases
that are increasing day by day.
Methods
Animal Material and Care Conditions
All experimental procedures in the study were approved
by Kafkas University Animal Experiments local
ethics committee (KAÜ-HADYEK/ 2018-005).
Subject was from 12-week-old Sprague-Dawley rats
reared in Ataturk University Medical Experimental Research
and Application Center (ATADEM) randomly
selected. A total of 32 male Sprague-Dawley rats with
a body weight of 245±22.89 g were used in this study.
The rats were fed ad libitum pellets and tap water in a
room with daylight (12 h of light, 12 h of darkness).
AMF Administration
The AMF substance was administered to the rat in RT
- AMF and AMF groups intraperitoneally for 3 days at
a dose of 200 mg/kg body weight.[]
Experimental Design
Creation of the experimental groups:
1. Control group (n=8): The control group was injected
with 0.9% saline solution intraperitoneally (i.p.)
0.5 mL/day for 3 days
2. AMF group (n=8): The rats in this group were administered
only AMF intraperitoneally at a dose of
200 mg/kg for 3 days
3. RT-saline (RT-saline) group (n=8): The rats in this
group were given only RT at a total dose of 6 Gy in 3
fractions for 3 days as 2 Gy in a single fraction per day
4. RT-AMF (RT-AMF) group (n=8): The rats in this
group were administered 200 mg/kg of AMF intraperitoneally
half an hour before each daily fraction
to create prophylaxis. A daily dose of 2 Gy radiations
was applied half an hour after this administration.
These processes were repeated in the same
amount and manner for 3 days.
The dose and application of AMF were based on the
report by Gezer and Karadag-Sari.[] Lead plates were
used to shield tissues outside the irradiated area.[]
Then, the rats in the whole group were deeply
taken to anesthetize by the injection of 40 mg/kg
ketamine (Ketalar; Pfizer, Istanbul Türkiye) and 10
mg/kg xylazine (Rompun; Bayer, Istanbul, Türkiye)
and the prostate tissue samples was taken via cervical
dislocation.
Histopathological Examination
To determine the immunoreactivity of MUC1 in prostate
tissues from rats, it was detected in 10% formaldehyde
solution and it was stained with Crossman's triple
staining evaluate the overall structure of the tissue.
Immunohistochemical Examination
The streptavidin-biotin-peroxidase technique was used
to determine the immunoreactivity of MUC1 in prostate
tissues from rats. The sections in 5 µm thick were
taken from the paraffin blocks on the slides coated
with chrome alum gelatin. After deparaffinization and
rehydration procedures, the sections were shaken in
PBS (0.1 M, PH, 7.2) and incubated for 10 min in 3%
H2O2 prepared in 0.1 M PBS to prevent endogenous
peroxidase activity. After washing with PBS, heat was
applied at maximum temperature in the citrate buffer
solution in a microwave oven for 10 min to reveal the
antigens. Blocking solution A was dripped to prevent
non-specific binding. Then, the MUC1 primary antibody
(ab104978, diluted 1/50) was administered on the
sections for 1 h at room temperature and in a humid environment.
Then, broad spectrum antibody against the
strain from which the primary antibody was produced
was added on the sections and kept at room temperature
for ten minutes. After HRP, streptavidin was incubated
at room temperature for 10 min. 3,3"-Diaminobenzidine
tetrahydrochloride was used as chromogen. It was
dipped in Mayer'"s hematoxylin for 10 sec for counterstaining,
the preparations were examined under a light
microscope, and their photographs were taken. For the
purpose of determining whether the immunoreactivities
are specific to the prostate sections taken from all
groups, all procedures were kept in PBS without the addition
of primary antibody (omission control) and the
other procedures were applied the same.
The percentage of the stained cells in the sections and
the degree of staining were scored with a semi-quantitative
method in the field as criteria. The immunohistochemical
evaluations were made by looking at whether
the target cells were stained or not. Evaluation done by
two independent observers by giving values from 0 to 3
according to non-staining (-), weak (+), moderate (++),
and strong (+++) staining characteristics.[,]
Results
Histopathological Results
There was a thick fibroelastic connective tissue capsule
in the outer part of the prostate gland and this capsule
continued with the connective tissue stroma. The secretory
part of the prostate gland, which contains smooth
muscles and blood vessels in the stroma, consisted of
gland structures of various shapes. It was observed that
the epithelium of some of the gland structures was cubic
and low prismatic. It was observed that the parenchyma cells and stroma cells of the prostate tissues in the whole
group had a structurally normal appearance (Fig. 1a-d).
Fig. 1. Rat prostate tissue. (a) Control group, (b) Radiotherapy (RT)-saline group, (c) Amifostine group, (d) RT-amifostine
group. Bar: 100 µm, Triple staining.
Immunohistochemical Results
The prostate tissues taken from the control, AMF, RTsaline,
and RT-AMF groups were evaluated immunohistochemically.
It was determinde that the MUC1 immunoreactivity
in parenchyma and stroma cells of the
control group was weaker than AMF, RT-saline, and
RT- AMF groups (Fig. 2a-d).
Fig. 2. Rat prostate tissue. Mucin 1 immunoreactivity. (a) Control group, (b) Radiotherapy (RT)-saline group, (c) Amifostine
group, (d) RT+ amifostine group. Bar: 100 µm, IHC.
IHC: Immunohistochemistry.
Discussion
Although RT is a widely used treatment method in cancer
treatment, it also damages healthy tissues adjacent
to the tumor since it is not selective to tumoral tissue
and its acute and late side effects affect quality of life.
The radioprotective agents have been developed to prevent
these side effects that occur in the cancer patients
whose survival times are getting longer.[] AMF is
one of them. The radioprotectants such as AMF reduce
the efficacy of radiation in normal cells without reducing
the efficacy of radiation in tumors.[]
The effects of radiation, which is a chromosomal mutagen,
on fertility are known. For this reason, the side effects
in the reproductive system in young male patients
who are treated with RT and who live for a long time
are of particular importance.[] The MUCs are high
molecular weight glycoproteins. The MUCs are found
on the apical surface of many of the respiratory, reproductive
tracts, and gastrointestinal.[] Lubrication and
moistening, protections against epithelial, and microbial
attack are among the primary functions of the MUC1.
[] There is increasing interest in MUCs in many cancers,
including prostate cancer and the role of their expression
in prostate cancer progression is unclear.[]
Prostate cancers is leading the course of deaths from
lightning strikes in recent years. A typical feature of prostate
cancer is the presence of genetic changes that alter
the expression patterns of many molecules in prostate
epithelial cells where the disease arises. These aberrantly
expressed proteins are tumor-associated antigens.[]
It has been asserted that the MUC1 expressed on the
apical cell surface of many normal secretory epithelial
cells inhibited adhesion and promotes metastatic disease
development.[] Increased MUC1 immunoreactivity
has been observed in most breast, lung, stomach,
pancreatic, prostate, and ovarian adenocarcinomas.
[] The MUCs are accepted as important markers for
early diagnosis and treatment intended to the target
because of their abnormal and unique expression patterns during malignant progression of carcinomas.[]
The MUC1 is one of the best characterized tumor-associated
antigens. The MUC1 is important in determining
prostate cancer prognosis and has been studies as
a therapeutic target.[] Studies suggest that increased
MUC1 expression in prostate tissue will be related to
prostate cancer progression.[,,]
In a study in which DNA fragmentation was
evaluated,[2] they have formed a total of 3 groups including
12 rats in the study group and 10 in the control
group. Radiation was applied to the testicles at a
dose of 2 Gy. The rats found in the first group have
received only radiation and the rats found in the second
group have received 15 min of radiation as 200
mg/kg AMF intraperitoneally, and no treatment has
been applied to the rats in the control group. The rats
have been sacrificed for the histological evaluation
10 weeks after the radiation. They have stated that
apoptosis was low in normal seminiferous tubules in
the control group and stated that this was spontaneous apoptosis. They have stated that the AMF did not
show protection against the radiation-induced germ
cell apoptosis in rats.
The study conducted by Kokawa et al.[30] was
guiding in the use of 6 Gy ionizing radiations in
our study. In the study conducted by Kokawa et al.,
it has been determined by 2 different methods that
the fragmentation rate made peak after 9 Gy irradiations,
and they have stated that the fragmentation
decreased and necrosis increased in the subjects who
received 36.9 Gy irradiations.
In the study conducted by Gezer and Karadag-
Sari,[] a high amount of apoptotic response has
been observed in the RT-saline and RT-amiphostine
groups after 6 Gy, although four weeks passed after
the application of RT. It has been seen that there
was a statistically significant difference between the
apoptotic indices of these two study groups. It has
been concluded that the AMF administered intraperitoneally
30 min before the RT showed a protection against the apoptosis in the germ cells treated
with the RT in the rats. Studies have also reported
that MUC1 is more expressed in malignant tissues
compared to normal tissues.[,,]
Conclusion
Our study, the prostate tissues taken from the control,
AMF, RT-saline, and RT-AMF groups were also
evaluated immunohistochemically. It was seen that the
MUC1 immunoreactivity was weak in the parenchyma
and stroma cells of the control group, and excess MUC1
release was observed in the AMF, RT-saline, and RTAMF
groups. It has been seen that the MUC1 immunoreactivity
was weak in the parenchyma and stroma cells
of the control group in general, excess mucin 1 secretion
was increased in the AMF and AMF RT and RT groups,
and the lack of difference between the AMF, RT-saline
and RT-AMF groups indicates that the protective effect of the AMF varies from tissue to tissue, depending on
the dose and the number of fractions applied.
Peer-review: Externally peer-reviewed.
Conflict of Interest: All authors declared no conflict of interest.
Ethics Committee Approval: The study was approved by
the Kafkas University Animal Experiments Local Ethics
Committee (no: 2018-005, date: 26/01/2017).
Financial Support: This research was supported by the Scientific
Research Commission of the Kafkas University (Project
No: 2019-TS-60).
Authorship contributions: Concept - S.E.Y., E.K.S., A.G.;
Design - S.E.Y., E.K.S., A.G.; Supervision - E.K.S., A.G., S.E.Y.;
Funding - E.K.S., A.G., S.E.Y.; Materials - S.E.Y., E.K.S., A.G.;
Data collection and/or processing - E.K.S., A.G., S.E.Y.; Data
analysis and/or interpretation - E.K.S., A.G., S.E.Y.; Literature
search - A.G., S.E.Y.; Writing - A.G., S.E.Y.; Critical review -
E.K.S., A.G., S.E.Y.
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