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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
Research Article
Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/Mtor Signaling
Pathway in Radiation- Induced Bone Marrow Injury
Neamat H. Ahmed1*, Amina M. Medhat2, Ussama Z. Said1, Laila A. Rashed3, Abdel Rahman B. Abdel Ghaar2, Fat-
ma S.M. Moawed1 and Esraa S. A. Ahmed1
1Radiation Biology Department, National Center for Radiation Research and Technology, Atomic Energy Authority, Cairo,
Egypt.
2Biochemistry Department, Faculty of Science, Ain Shams University, Cairo, Egypt.
3Biochemistry & Molecular Biology Department, Medicine Faculty, Cairo University, Cairo, Egypt.
*Corresponding author: Neamat H. Ahmed, Radiation Biology Department, National Center for Radiation Research and Tech-
nology, Atomic Energy Authority, Egypt; Email: neamathana@ymail.com
Received Date: 01-23-2019
Accepted Date: 02-11-2019
Published Date: 02-13-2019
Copyright: © 2019 Neamat H. Ahmed
Abstract
Mesenchymal stem cells (MSCs) are a promising source for cell therapy in regenerative medi-
cine. The therapeutic properties of MSCs are related to their potentials for trans-differentiation,
Accordingly, the aim of the present study was to examine the effects of MSC on radiation-induced
bone marrow injury. It was found that exposure of rats to gamma radiation at a dose of 1.5 Gy/
-
and IL6 cytokines and decrease in GSH content in comparison with normal group. Western immu-
-
cant improvement versus irradiated group via modulation of growth factors that encounters bone
marrow suppression. In addition, exposure to radiation induces apoptosis by increasing expres-
sion of Bax gene. In contrast, treatment with MSCs decreased apoptosis through up regulation of
induced bone marrow injury.
Keywords:
Jacobs Journal of Radiation Oncology
Jacobs Publishers
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
2
Introduction
Mesenchymal stem cells (MSCs) are self-renewing
multipotent cells potentially act as a therapeutic for tissue
inhibit apoptosis, and support the growth and differentia-
tion of local stem and progenitor cells [1]. Currently, MSCs
are the most commonly used cell-based therapy because of
their regenerative effects, ease of isolation, and low immu-
nogenicity [2].
The hematopoietic system is highly sensitive to ra-
diation injury and bone marrow (BM) suppression is the
primary life-threatening injuries after exposure to radi-
ation. This indicates that the hematopoietic system is the
of total body irradiation results in bone marrow injury
and acute hematopoietic syndrome characterized by de-
pletion in the lymphocyte, granulocyte and platelet counts,
thus making the victims susceptible to infections. It also
causes mutations leading to increased incidence of de-
velopment of leukemia, as was observed among the sur-
systems upon exposure to ionizing radiation deplete the
-
branes, resulting in cell death, altered cell division, deple-
tion of stem cells, organ system dysfunction and, at high
mutations and compromising genome integrity, leading to
cell senescence and death [7]. Apoptosis is activated when
-
gene activated in multiple cancers, acts as anti-apoptotic
factor to a variety of stimuli such as radiation, hypoxia and
chemotherapy [9]. However, growing number of studies
but renders cells more sensitive to metabolic stress instead.
-
resistance [10].
Materials And Methods
Isolation of MSCs:
-
-
-
culture medium supplemented with 1% penicillin strep-
upon formation of large colonies. When large colonies de-
-
with serum-supplemented medium and were incubated in
-
ent colonies of cells were trypsinized, and counted [11].
-
-
-
-
Animals:
Male Wistar rats at 27 days of age and weighing 25
were used in this study. The animals were housed in spe-
cially designed plastic cages, ten per each, under standard
conditions of 12-h light /12-h dark cycle, normal tem-
perature, good ventilation and humidity range. The animals
were provided with a pellet concentrated diet containing all
water and food were provided ad libitum throughout the
study.
All the experimental procedures were carried out
Jacobs Publishers
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
3
“Guide for the care and use of Laboratory Animals” for the
use and welfare of experimental animals, published by the
Radiation Facility:
Whole body gamma irradiation of rats was car-
ried out using an indoor shielded Canadian Gamma Cell-
Experimental design
The animals were randomly divided into three groups each
1.
2.
-
val of 7 days for four weeks.
Gamma irradiated and treated with mesenchymal stem
rats were left for one month.
At the end of the experiment, all animals were
used for the biochemical parameters estimations. Both fe-
for the preparation of bone marrow smear, and the other
-
mations. Also part of femur was rapidly excised for histo-
pathological examination.
Hematological parameters:
Complete blood count (CBC) was determined ac-
-
rameter, automated hematology analyzer system capable of
producing several hematological parameters.
Biochemical estimations:
Levels of interleukin 6 (IL-6) and transforming
USA).
Western immunoblotting:
Bone marrow tissue proteins were extracted us-
-
room temperature for 2 h with blocking solution comprised
of 5% nonfat dried milk in 10 mM Tris-Cl, pH 7.5, 100 mM
Tween 20, membranes were incubated with the secondary
monoclonal antibodies conjugated to horseradish perox-
idase at room temperature for 2 h, and then membranes
were washed four times with the same washing buffer.
Membranes were developed and visualized by chemilumi-
nescence using Amersham detection kit according to the
-
mary and secondary antibodies were purchased from Cell
-
ric analysis of the autoradiograms using a scanning laser
Molecular Investigation:
protein (Bcl-2) and Bcl-2 associated x-protein (Bax) by
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
4
RNA Isolation and Reverse Transcription:
-
Technologies, USA) in accordance to the manufacturer’s
gel electrophoresis and stained with ethidium bromide.
performed with reverse transcriptase (Invitrogen) accord-
-
utilized in these experiments are listed in Table 1. A reac-
-
-
sis was performed at the end of the reaction. The data were
-
in triplicate in two independent experiments.
Histopathological assessment:
-
lutions of alcohol (methyl, ethyl and absolute ethyl) were
used for dehydration. Specimens were cleared in xylene and
obtained tissue sections were collected on glass slides, de-
stain for routine examination through the light electric mi-
onto charged adhesive slides and stained with mixture of
acridine orange and ethidium bromide for apoptotic exam-
ination [16].
Statistical analyses:
Results
Immunophenotyping of MSCs:
-
markers of hematopoietic cells. However, MSCs cells strong-
important cell surface markers of MSCs.
Figure 1: -
phenotyping of MSCs.
-
which is a hematopoietic stem cell marker. The grey area
represented isotype control IgG expression, while the black
line depicted the marker expression.
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
5
Hematological parameters:
It has been established that whole body exposure
to gamma radiation induced bone marrow myelosuppres-
sion and myelodysplasia which is characterized by cytope-
compared to the control. Meanwhile MSCs transplantation
-
this decrease and the platelet counts were partly recov-
ered. Similarly, data represented in
table (2) showed that
(
-
over, the lymphocytes, monocytes and neutrophils count
count. In contrast, MSCs ameliorated this effect resulting
in partial recovery of WBCs, lymphocytes, monocytes and
neutrophils.
Table 1:
Hb and Hct
Table 2:-
phils, Lymphocytes and Monocytes
Biochemical analyses:
The obtained data also showed that 1.5 Gy of gamma total
control group. Interestingly, our results showed that the
-
-
Figure 2:
-
-
Figure 3:
-
-
Legends as in Table 1
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
6
Western immunoblotting:
The results from this assay revealed markedly high-
-
-
RT-PCR for CXCR-4, BCL-2 and Bax:
-
sion levels of pro-apoptotic protein Bax along with lower
irradiated group in comparison with control . In the treat-
ments using MSCs, the pro-apoptotic Bax gene expression
BCL-2 were up regulate. Therefore, these data demonstrate
Table 3:
treated with MSCs.
Table 1:
Figure 4:
treated with MSCs.
(A)
(B)
Figure 5: Mean relative gene expression of Bcl-2 and Bax
(A) and ratio between Bax/Bcl-2 (B) of rats exposed to
Figure 6: -
Legends as in Table 1
Jacobs Publishers
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
7
Histopathology
Bone marrow aspiration smears:
The smear of control group displayed the marrow structure
-
group showed predominance of irregular and undifferenti-
ated dark blue staining cells (blast cells) accombined with
-
ation and treated with MSCs showed normal appearance of
marrow cells with different maturation stages and presence
Bone marrow biopsy sections:
The bone marrow biopsy of control group displays
the marrow structure and various component of the normal
bone marrow including adipose tissue (AT), blood sinusoid,
cellularity accompanied with the presence of a cloudlike
-
Figure 7:
-
and treated with MSCs showing the normal appearance of
marrow cells and the presence of megakaryocytes.
Figure 8:
-
treated with MSCs showing the normal appearance of mar-
row cells.
Discussion
The present study evaluated the effects of MSCs
-
even in sub-lethal doses causes drastic effect on bone mar-
row leading to bone marrow suppression, failure and even-
marrow phenotypes, and its relation to disruption of hema-
marrow supression associated with changes and signs from
whole body gamma radiation displayed a depletion of the
peripheral blood cells, suppression and impairment of bone
bone marrow haematopoietic cells, ionizing radiation re-
sults in decrease in the values of hematological pa-
rameters characterized by depletion in the lymphocyte,
erythrocytes, granulocyte and platelet counts which may
be assigned to direct damage caused by a lethal dose of ra-
Together, our results support that grafted MSCs sig-
Jacobs Publishers
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
8
-
al. who indicated the rapid recovery of leukocytes, eryth-
rocytes and thrombocytes in the MSC groups implied that
MSCs markedly enhance the recovery of haematopoiesis, al-
leviate the progenitor’s injuries by ameliorating the micro-
environment and secreted cytokines; they may also improve
play a supportive role in homing of hematopoietic stem
cells (HSCs) in bone marrow [19]. Moreover, MSCs support
long term hematopoiesis and can function as a feeder layer
maintaining HSCs in an undifferentiated state. Close contact
between mesenchymal stem cells and hematopoietic stem
cells supports the differentiation of hematopoietic stem
cells towards mature red cells. Also, MSCs are the precursor
cells of mesenchymal cell types in the hematopoietic niche,
the injuries of the hematopoietic niche caused by irradia-
tion and provided a supportive role for the hematopoietic
microenvironment that enabled the engraftment of HSCs
[20].
changes in antioxidant status, as a response to the induced
oxidative stress and production of reactive oxygen species
-
ous effects by initiating lipid peroxidation and adverse al-
terations of the cell membrane can result in a pathological
division, depletion of stem cells, organ system dysfunction
and, at high doses, death of the organism [22]. In the present
be due the interaction of the excess of hydroxyl radical,
resulting from the radiolysis of water
upon
exposure
the
phospholip-
decrease in the activity of antioxidant enzymes
and
GSH
content might result from an increase in their utilization
to
exposure
.
In the present study, therapeutic administration
of mesenchymal stem cells (MSCs) remarkably decreased
the level of lipid peroxidation and improved antioxidant ac-
-
ity, which was consistent with Lanza et al. [25] who showed
that MSCs exhibited antioxidant activity through lipid per-
oxidation prevention, increasing levels of glutathione and
superoxide dismutase, and modulating the pathways of an-
tioxidant-related protein activation. Moreover, MSCs are re-
sistant to reactive oxygen species in vitro, reduce oxidative
stress in recipient mice [26].
-
mation and cellular damage by the increasing levels of
-
rise to the development of various hematologic disorders
to hematopoietic failure and such diseases as myelodyspla-
-
ing autocrine and paracrine secretion of such growth fac-
1 is involved in normal tissue injury and plays a critical
role in the initiation, development, and persistence of ra-
level and also induces the Chemokine stromal cell-derived
-
-
migration, retention within stem cell niches, proliferation
residing at the endosteum, whereby these cells are induced
into the cell cycle to allow re-establishment of a depleted
marrow cavity resulting in recovery extracellular activation
of the latent complex by proteolytic cleavage in response to
the production of reactive oxygen species generated by ra-
diation [29].
-
the pathogenesis of the disease, due to its close relation
-
ous studies reported that radiation-induced bone marrow
depression was associated with IL-6 release. This result in-
dicated that high level of IL-6 might be harmful to the pro-
Jacobs Publishers
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
9
liferation of bone marrow stromal cells and might interfere
with the stability of the bone marrow hematopoietic micro-
Therefore, the intravenous treatment with MSCs
cytokines in serum by effectively promoting the release of
-
priate immune system disorders systemically. MSCs treat-
-
largely on the capacity of MSCs to be safely infused, selec-
array of immunosuppressive factors, cytokines, growth
factors and differentiation factors. These findings suggest
that MSCs are an effective anti-inflammatory cell-based
therapy.
-
pressed/secreted by several tissues/organs in the body, es-
-
um, whereby these cells are induced into the cell cycle to al-
low re-establishment of a depleted marrow cavity resulting
-
over production of reactive oxygen species and reduction of
-
gy of bone marrow. In agreement with our results, Georgiou
et al. illustrated that the deregulation of both CXCL12 and
-
larity following methotrexate (MTX) treatment, consistent
et al. showed a progressive depletion of LT-HSCs in the BM
been reported to limit the lifespan of HSCs, cause deleteri-
-
apoptosis leading to marked reduction in HSC repopulating
Conversely, treatment with MSC upregulate the expression
and survival of HSCs and regulation of the size to re-estab-
-
cantly enhance migration and the paracrine properties of
lung tissue, which is perhaps the fundamental mechanism
damaged tissue, which results in a poor curative effect of
Cellular apoptosis is mediated through the balance
apoptotic pathway is regulated by early translocation of
-
tected increased expression of Bax and caspase-9 in total
bone marrow cells following radiation exposure. Therefore,
these results correlate with the loss of clonogenicity of the
bone marrow cells following radiation exposure due to ei-
-
HSC senescence and/or apoptosis, due to increase in oxi-
HSCs, decreases in HSC clonogenic function and long-term
The intracellular signalling pathway from activated
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
10
thymoma viral oncogene homolog (Akt) and mechanistic
-
play a critical role in intracellular signal induction and gene
expression that involves in regulation of hematopoietic
-
-
post irradiation, correlating with prolonged pro-apoptotic
proliferative factor to a variety of stimuli such as radiation,
does not inhibit apoptosis, but renders cells more sensitive
-
-
-
self-renewal and causes HSC exhaustion in association with
biogenesis [51, 52].
of HSC apoptosis not only is one of the primary causes of
-
components of the HSC niche not only contributes to HSC
cause severe damages to HSCs and impairs their ability to
self-renew by induction of HSC apoptosis, differentiation,
and senescence and damage to the HSC niche, which may
In harmony with Mehmeti et al. the decline of an-
tioxidant enzymes and the increase of the oxidative stress
induced by irradiation may also play a crucial role in the
increased Bax expression and decreased expression of Bcl-
loss of mitochondrial integrity, increased Bax/Bcl-2 ratio
and are crucial for cytokine induced cell apoptosis. Consis-
induce apoptosis as well as increase of the apoptotic pro-
teins Bax and the decrease of the anti-apoptotic protein Bcl-
2 [55].
-
expression and lower bax expression. Also, MSCs result-
this reduce apoptosis through modulation of bax and bcl-
2 expression as well as bax/bcl-2 ratio to protect rat bone
-
garding the close relationship between free radicals, par-
the anti-apoptotic effect in this study may resulted from
the antioxidant action of MSCs as direct free radical scav-
are in agreement with Lanza et al. who showed that MSCs
prevention, increasing levels of glutathione and superoxide
dismutase, and modulating the pathways of antioxidant-re-
lated protein activation [57].
-
-
endonuclease. This implies that cells overexpressing Bcl-2
might not only survive longer, and thus have more time to
-
pair capacity. Gong et al. found that MSCs transplantation
and support the growth tissue repair after exposure to radi-
ation [1].
Conclusion
In conclusion, this study revealed that MSC transplantation
enhances hematopoiesis, repair bone marrow and improve
the peripheral blood counts and other biochemical param-
eters. Interestingly, MSCs decrease the incidence of apop-
tosis/exhaustion and senescence of bone marrow cells and
-
es mobilization of peripheral blood HSCs to re-establishing
damaged bone marrow niche. These mechanisms may con-
tribute to the MSCs therapeutic potential in the treatment
of radiation induced bone marrow injury.
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
Jacobs Publishers 11
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Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
Jacobs Publishers 12
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Jacobs Publishers 13
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
-
sion of BCL-2 in the hematopoietic system protects
-
Cite this article : Neamat H, Amina M, Ussama Z, et al. Mesenchymal Stem Cells Inhibit Apoptosis Via Modulating AKT/mTOR Signaling Pathway in Radiation- In-
duced Bone Marrow Injury. J J Rad Onc. 2018; 6(1):037.
Jacobs Publishers 14