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The Egyptian Journal of Hospital Medicine (2007) Vol., 27: 163– 175
Aspergillus fumigatus and Cylindrocarpon candidum fungi induced apoptosis
in HepG 2 cell line through activation of caspases enzymes
Osman, M.E. *, L,A,R, Sallam**, Wafaa Abdallah Ahmed***, Khttab, O.H.*,
Abo EL-Nasser, A.A* * Department of Botany and Microbiology, Faculty of science, Helwan University.
**National Research Center, Department of Microbiology and Natural Products Chemistry
Egypt.***Department of Cancer Biology, Cancer Institute, Cairo University.
Abstract
Introduction: Many investigations are now interested to discover naturally occurring
compounds, which can be used for the prevention and treatment of cancer. Most natural products which may be used as adjuvant therapy or to reduce the side effect of chemotherapy
and radiotherapy. More than 300 products obtained from microorganisms have antitumor
activities.
Results: In the study we isolated N-(3-4-Dichlorophenyl) 2-Methyl, 2,3Dihydroxypropio amide from Aspergillus fumigatus and 2.4.6. Triphenyl pyridine from Cylindrocarpon
candidum and investigate the cytotoxic effect and apoptotic effect on HepG2 cell line. The
results revealed high cytotoxic effect at the concentration of 400µg/ml for both N-(3-4-Dichlorophenyl) 2-Methyl, 2,3Dihydroxypropio amide and 2.4.6. Triphenyl pyridine and effect
is increase with time of incubation. The apoptotic effect of both products were investigated by
measurement the caspase enzymes, the results showed highest activity of caspase 3 and caspase 9. Also at concentration 400µg/ml in both products.
Conclusion: From this data we observe that two isolated product have antitumor effect
and this effect is related to the concentration of the products and incubation period. Also, the
two products induce apoptosis through increase activation of caspase 3 and caspase 9 which lead to programmed of cell death. This study need to furthermore study on experimental animal
to confirm our results.
Introduction The first line of cancer treatment is
surgery which has significantly reduced the
cancer mortality. The use of additional
treatment such as radiotherapy and chemo-therapy has resulted in no more than 5%
reduction of death (Samantha et al., 2003).
The development of tumor drug resistance during treatment is the major factor
limiting the success of chemotherapeutic
management of tumors. Such resistance may occur during primary therapy or be
acquired during subsequent treatment (Curt
et al., 1984 and Cairo et al., 1989).
Many investigations are now interested to discover naturally occurring
compounds, which can be used for the
prevention and treatment of cancer despite little understanding about their molecular
and cellular basis of action Hala et al .
(2004). Wainwright (1992) reported that
300 antitumor agents have been isolated
from microorganisms, 13% have been
obtained from fungi. Of 43 antitumor
agents obtained from fungi, 23 come from the imperfect fungi, 15 from the Basid-
iomycetes and 5 from the Ascomycetes.
Zhang et al. (1994) isolated sixteen polys-accharides from the mycelium of G. tsngac
three of them showed antitumor activity
against the solid cancer sarcoma 180/mice. Polysaccharides extracted from
Sargassum thunbergii have strong antitu-
mor effects against transplanted tumors
such as sarcoma 180 and Ehrlich solid carcinoma (Zhuang et al., 1995).
Acebal et al. (1999) isolated agroc-
helin as a new alkaloid cytotoxic substance by the fermentation of Agrob-acterium sp.
The compound was obtained from the bac-
terial cell by solvent extraction and purified
163
Aspergillus fumigatus and Cylindrocarpon candidum………
164
by silica gell chromatography. Rimpler et
al. (1996) described the ability of fungi in
the families Tricholomataceae and Polyporacea to produce antitumor activity.
The effects of a polysaccharide-
protein complex (PSPC) isolated from the
culture filtrate of T. lobayense were inves-tigated in mice injected with S-180 cells.
PSPC restored the phagocytic function of
peritoneal exudate cells (PEC) and T-cell mitogenic activity in tumor-bearing mice.
(Liu 1996).
Gary et al. (1997) purified a series of
peptide antifungal – anticancer agents kno-wn as leucinostatin A from Acremo-nium
sp. Leucinostatin A possess activity against
certain human cancer cell lines. Raha et al. (1990) found that L-Aspara-ginase from
Cylindrocarpon obtusisporum MB-10 inhi-
bits the growth of ascites fibro sarcoma and Dalton's Lymphoma tumor cells in vivo
and significantly increases the survi-val
rate of tumor bearing mice. The enzymetr-
eated normal mice become healthier and survive longer than their usual life span.
In this work we continue the
previous study on the Aspergillus fumig-atus and Cylindrocarpon candidum to emp-
hasis has been imposed for isolation, puri-
fication and identification active principles in the both extracts of the two mentioned
organisms. Furthermore we study the
cytotoxic effect of the two organisms on
HepG2 cells and trying to identify their mechanism of action.
Material and methods
Microorganisms.
Experimental fungal species
Two fungal species were used in the study:
1- Aspergillus fumigatus was obtained
during the studies curried by Mahmod
(2004)
2- Cylindrocarpon candidum was obtained
during the studies curried by Abo EL-
Nasser (2000).
Culture media. -Liquid Malt extract for Production of
antitumor agents.
This medium was described by Fang, et al. (1997). It has the following compo-
sition (g/L): malt extract, 20; peptone, l;
glucose, 20 and distilled water, 1000 ml.
Production and Extraction of antitumor
agents. Aliquots of 1L liquid malt extract
medium placed in conical flask (2L) and
autoclaved at 121 0C for 20 min. Each flask
was inoculated by 5ml spore suspension of
the experimental organism and incubated at
25 0C for 5 days. The broth media was
separated into a supernatant and mycelial
cake. The mycelial cake was extracted with
acetone and concentrated under vacuum
and the supernatant was extracted with ethyl acetate and also concentrated under
vacuum. The resulting residues were
combined and extracted with ethyl acetate to obtain a crude material. This residue was
tested for its toxicity against tumor cells.
Purification and analysis of the extracts
obtained from Aspergillus fumigates
and Cylindrocarpon candidum. 100 liters of the broth media was
separated into a supernatant and mycelial
cake. The mycelial cake was extracted with acetone and concentrated under vacuum
condition and the supernatant was extra-
cted with ethyl acetate and concentrated under vacuum condition. The resulting
residues were combined and extracted with
ethyl acetate to obtain a crude material.
The residue was fractionated on column chromatography packed with silical gell 60
G F254 for column chromatography and
eluted with ethyl acetate flowed by an increasing proportion of methanol.
Fractions were collected at 15 0C.
Osman, M.E et al
165
Fig (1) Purification of antitumor agent of Aspergillus fumigates crud.
F1 F2 F3 F4 F5 F6 F7
Extraction with acetone
Resinous residue (3.5g)
100L Culture broth
Aqueous filtrate (97L) Mycelium (225g)
separation
Extraction with EtOAc
Resinous residue (5g)
Extraction with EtOAc
Residue (7g)
Fractionation on silical gell column
by EtOAc: methanol (1:1)
All fractions were tested using HepG2 cell line
Active fraction
F5 (0.83g)
Aspergillus fumigatus and Cylindrocarpon candidum………
166
Fig (2) Purification of antitumor agent of Cylindrocarpon candidum crud.
F1 F2 F3 F4 F5
Extraction with acetone
100L Culture broth
Aqueous filtrate (98.4L) Mycelium (150g)
separation
Extraction with EtOAc
Resinous residue (3.7g) Resinous residue (1.5g)
Extraction with EtOAc
Residue (4.2g)
Fractionation on silical gell column
by EtOAc: methanol (1:1)
All fractions were tested using HepG2 cell line
Active fraction
F3 (0.5g)
Osman, M.E et al
167
Standard Protein Assay
The protein concentration in the
investigated samples was determined according to the methods of (Bradford
1976).
Cell line
Human liver carcinoma cell line
HepG2 obtained from American type culture collection.
Cell culture
HepG2 cell line was maintained and sub culture in 75 cm
2 cell culture flask
(Fisher Scientific Pittsburgh, PA). Using
10 ml of RPMI – 1640 ]Roswell park Memorial Institute medium[.
(Supplemented with 1 % (2 mM) glutamic
acid, 10 % unheated fetal bovine serum
(FBS), 100 µ/ ml penicillin and 100 µg/ ml streptomycin). All cells were grown at 37 0C in atmosphere 5% CO2 – 95 % air in a
high humidity atmosphere in water – Jacteted incubator.
Cell viability assays. 1- Trypan blue exclusion test.
0.05 % trypan blue solution was
added to cell suspension (v/v) from each
control and treated cells. Cells were examined under the light microscope. Total
cell counts and viable cell number
(survival) were determined by a standard Heamocytometer procedure. Live viable-
cells were seen colorless (impermeable to
the dye due to intact cell membrane) and dead cells were seen as blue (permeable to
dye due to disruption of cell membrane):
% live (Recovery rate) =
Count of living cells X 100
Count of total cells
2- MTT assay Cytotoxicity was measured using the
MTT ]3-(4,5-dimethyl thiazol)-2,5-
diphenyltetrazolum bromide[ cell viability assay which depend on the ability of active
mitochondria dehydrogenase enzyme of
living cells to clave the tetrazolium rings of
the yellow MTT and form a dark blue in
solution formazan crystals which is largely impermeable to cell membranes, resulting
in it accumulation within healthy cells.
Solublization of crystals, which are then
solublized. The number of viable cells is directly proportional to the level of soluble
formazan dark blue color. The extent of the
reduction of MTT was quantified by measuring the optical absorbance (OD) at
570nm (Mosmann, 1998).
Procedure HepG2 cells (0.5 X 10
5 cells/ well) in
serum. Free media were plated in fetal
bottom 96.well microtiter plate, and treated with compound for 24h at 37
0C in humi-
dified 5% CO2 atmosphere. After incuba-
tion media were removed and 40 µl MTT solution /well was added and incubated for
an 4hrs. MTT crystals were solublized by
adding 180 µl of acidified isopropanal
/well and plate was shacked at room temperature. The absorbance was read at
570 nm using ELISA reader (meter tech
960, USA). Triplicate wells were prepared for control and each individual dose and
the average was calculated. Data were
expressed as the percentage of relative viability comparison, the untreated cells
compared with control. The cytotoxicity
indicated by < 100 % relative viability.
Percentage of relative viability was calculated using the following equation:
Absorbance of treated cells /
Absorbance of control X 100. Then the half maximum inhibitory
concentration IC 50 was calculated from
the equation of the dose response curve.
3- Colorimetric Protease assays.
Caspase 3 and Caspase 9 (the
ApoTarget) The ApoTarget Colorimetric Protease
assays Catalog # KHZ0101 and KHZ0102.
Caspase 3 and caspase 9 protease assays are to be used for in vitro determination of
proteolytic activity in lysates of mamm-
alian cells (biosource International, Inc.542
Flynn Road Camarillo, California 93012, USA). Assay procedure briefly. Induce
apoptosis in cells by different
Aspergillus fumigatus and Cylindrocarpon candidum………
168
concentration of each compound. Cells
were counted and pellet 3 – 5 million cells
per sample. Resuspend cells in 50 µl of chilled cell lysis buffer and cells were
incubated on ice for 10 min. then
centrifuged and the protein concentration
was assayed by the Brad ford methods (FISI) in cytosol extract. Each cytosol was
a diluted to a concentration of 50–200µg
protein per 50 µl. cell lysis buffer. Reaction buffer ]containing DDT (dithiothreitol)[
was added to each sample. 5µl of the 4 mµ
DEVD – pNA ]amino acid sequence
composed of the chromophore, p-nitroanalinide (pNA)[ substrate (200µm
final concentration) was added and
incubated at 37 0C for 2 hrs in dark. Then
the absorbance was recorded at 405 nm
using spectrophotometer.
Results
Fractionation, purification and
Identification of the anticancer agents
from Aspergillus fumigates and
Cylindrocarpon candidum.
The culture broth of both organisms
were extracted to obtain crude antitumor active agents (figs1,2). These crude
fractions were fractionated by silica gell
column chromatography using ethyl acetate: methanol 1:1 as eluting agents.
Seven fractions and five fractions were
obtained from Aspergillus fumigates and
Cylindrocarpon candidum respectively (table 1). These different fractions were
examined for their anticancer activity
against HepG2 cell line. It was found that F5 from Aspergillus fumigates and F3 from
Cylindrocarpon candidum showed the
highest activity (fig 3). Each product was tested by Infrared
absorption spectra (IR) were measured with
FI.IR spectrophotometer. NEXUS- 460
(USA) using KB6 as standard material, Ultra violet-visible spectra (UV spectra)
were measured with Microlet Evolution 300.
UV and IR were determined in Desert Research Center (DRC). Micro analytical
unit and G.C Mass spectra were measured
with Fimmigan mat. SSQ 7000 (Thermo. Inst. Sys. Ine. U.S.A. mass spectrometer at
an-ionization Voltage 870 ev and El mode.
These measured in Ain Shams University,
Faculty of Science, Central Laboratory, to
know the structure of each product. The first products was the (N-(3-4-
Dichlorophenyl)2-Methyl 2, 3 Dihydroxyp-
ropio amide) produced from Aspergillus
fumigates it shows ג max in the UV regin at 266nm indication of substituted was being
(NH.AC) (Fig 4). In IR spectrum showed
absorption bands at 3621cm-1
OH, 3421 cm-1
NH and 1693 CO cm-1
. Mass spectra
showed M/Z 229, 149, 73 and 41 the spectra
data confirm assigned structure I.
The second product (Fig 5) was the (2.4.6. Triphenyl pyridine) produced from
Cylindrocarpon candidum. In IR spectrum
showed absorption band at 3405 cm-1 NH.
Mass spectra showed M/Z 307, 242,206,154
and 125 the spectra data confirm assigned
structure II.
Measurement of anticancer activity of
the two active fractions (DMD and TPP)
on HepG2 cell line (liver cancer). HepG2 cells were treated with graded
concentrations (25- 400 µg/ml) of DMD and
TPP for 12-24 h. and then the cell proliferation and cell viability were
monitored. The results showed that
treatment of HepG2 cells with two compounds of DMD and TPP resulted in
significant decrease viability of the cells in a
dose and time- dependent manner (12, 24 h)
as compared with control (Figs 6,7). The effect of DMD and TPP on cell proliferation
was found to remarkably inhibit the
proliferation of HepG2. The result showed that Cylindrocarpon candidu had cytotoxic
effect more than Aspergillus fumigates in
all concentration as shown in (Fig 8). Cell
proliferation was determined by the cell titer 96 TM non-radioactive cell proliferation
assay MTT. Each value is the mean of
triplicates + SD. Induction of apoptosis in HepG2 cells by DMD and TPP. The apopt-
otic effect was measured through activation
of caspase enzymes. The activation of caspase 3 and caspase 9 proteases were
measured at different concentrations of
protein contents (25-250µg) of cell treated
with graded dose of each DMD and TPP products. The results showed that highly
significant increase in activity of caspase 3
Osman, M.E et al
169
enzymes in the cell treated with DMD at
concentration (400 µM) whereas the activity
of enzyme was decreased at less concent-ration (Fig 9). The same result was obtained
when monitored the activity of caspase 9 in
HepG2 cells treated with different
concentration of DMD (Fig 10). On the
other hand, when measured the activation of
caspase 3 and caspase 9 in HepG2 cells treated with TPP was lesser than that of
DMD. The result showed dose dependent
activity of both enzymes (Figs 11,12).
Table (1): Fractionation of Aspergillus fumigatus and Cylindrocarpon candidum crude
extracts by column chromatography.
Aspergillus fumigatus
Fractions ml EtoAc: methanol
F1 100 100%EtoAc
F2 75 5% methanol
F3 150 10% methanol
F4 100 15% methanol
F5 50 20% methanol
F6 175 25% methanol
F7 100 30% methanol
Cylindrocarpon candidum
Fractions ml EtoAc : methanol
F1 75 100% EtoAc
F2 50 5% methanol
F3 100 10% methanol
F4 150 15% methanol
F5 100 20% methanol
Fig (4): Structure of active fraction of Aspergillus fumigatus.
(N-(3-4-Dichlorophenyl)2-Methyl ,2,3Dihydroxypropio amide)
OH O Cl
CH C C NH O Cl
OH CH3
Fig (4): Structure of active fraction of Aspergillus fumigatus.
F2 F3 F4 F5 F6 F7
55
60
65
70
75
80
85
90
95
100
Aspergillus fumigatus
Cylindrocarpon candidum
Sur
viva
l cel
ls (
% c
ontr
ol).
Fig(3): Effect of different fraction of Aspergillus fumigatus and
Cylindrocarpon candidum extract on cancer cells.
Aspergillus fumigatus and Cylindrocarpon candidum………
170
(N-(3-4-Dichlorophenyl)2-Methyl ,2,3Dihydroxypropio amide)
OH O
Cl
CH C C NH O Cl
OH CH3
Fig (5): Structure of active fraction of Cylindrocarpon candidum. (2.4.6. Triphenyl pyridine )
N
25 50 100 200 400
60
65
70
75
80
85
90
Fig (7): Dose response curve of TPP effect on Hep G2 cells after 12 and 24 h. of exposure
determined by trypan blue exculsion test.
after12h.
after24h.
Via
bili
ty (
contr
ol%
)
concentration of the drug (l/ml)
25 50 100 200 400
60
65
70
75
80
85
90
Fig (6): Dose response curve of DMD effect on HepG2 cells after 12 and 24 h. of exposure
determined by trypan blue exculsion test.
After12h
After24h
Via
bili
ty (
con
tro
l%)
concentration of the drug (l/ml)
Osman, M.E et al
171
100 150 200 250 300 350 400
0
10
20
30
40
50
60
DMD
TPP
Cel
l pro
lifer
atio
n (%
of c
ontro
l)
Concentration (g/ml).
Fig (8): MTTassay for determination of proliferation of HepG2 cell line
under different concentration of DMD and TPP.
0 50 100 150 200 250
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
Fig (9): The activity of Caspase 3 at different concentration
of cell protein treated with DMD
control
treate100
treate200
treate400
optical denasity o
f caspase 3
.
protin concentration (g)
0 50 100 150 200 250
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Fig (10): The activity of Caspase 9 at different concentration
of cell protein treated with DMD
control
tr100
tr200
tr400
Obtical denasity o
f caspase 9
.
protin concentration (g)
0 50 100 150 200 250
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
0.30
0.32
0.34
Fig (11):The activity of Caspase 3 at different concentration
of cell protein treated with TPP
control
tr100
tr200
tr400
OP
tical denasity o
f caspase 3
protin concentration (g)
0 50 100 150 200 250
0.00
0.05
0.10
0.15
0.20
0.25
Fig (12): The activity of Caspase 9 at different concentration
of cell protein treated with TPP
Control
Treat100
Treat200
Treat400
Obtical denasity o
f caspase 9
.
protin concentration (g)
Aspergillus fumigatus and Cylindrocarpon candidum………
172
Discussion Although, the most common fungi
causes an important source of morbidity
and mortality in immunocompromized hosts but its toxin may be useful to inhibit
the growth of other pathogenic organisms
inside the host. Some mycotoxin which
produced from fungi have cytotoxic effect on cancer cells when tested in vitro on
different cancer cell lines (Lewis et al.,
2006). Gliotoxin is a mycotoxtin produced by several moluds such as Aspergillus
fumigatus produce cytotoxic effect in liver
cancer (Stanzani et al., 2006). Many
investigations are now being carried out to discover naturally occurring compounds
and biological response modifiers (BRMs)
which can suppress and prevent many diseases including cancer (Shi et al 1992,
Samaranayake et al., 2000, Thapliyal et al.,
2002 and Samantha et al., 2003). In fact, many effective chemotherapeutic are
agents that induced programmed of cell
death (apoptosis) via activation of different
pathways (Goli et al., 2002, Kaufmann and Earnshaw 2000 and Reed, 2002) and the
caspase enzymes are the key executors of
apoptosis. In this study, we determined the role of caspases in signaling of Aspergillus
fumigates and Cylindrocarpon candidu
apoptosis in liver cancer cells ( HepG2 ) and the data revealed significant inhibition
of cell growth and proliferation in both
culture treated with N-(3-4-Dichloro-
phenyl) 2- Methyl ,2,3 Dihydroxypropio amide and 2.4.6. Triphenyl pyridine
products. This inhibition was associated
with the incubation time and the conc-entration of products. In vitro cytotoxicity
and cell proliferation were measured by
trypan blue exclusion and MMT assays.
Similar results were reported by (Leah et al., 2006), he study in vitro cytotoxic
activities of some fungal products against
different cell lines including heptoma cell line HepG2 (Saint et al., 2006).
Wright et al. (2004) demonstrated
that liver cells are able to react specifically to a fungal pathogen through increase
expression of specific receptors. Stationary
phase cultures of Aspergillus fumigates
were associated with the appearance of
typical markers of apoptosis including
elevated proteolytic enzyme activity. In these study the activities of caspase 3 and
caspase 9 were measured in HepG2 cells
after treatment with different concentr-
ations of N-(3- 4-Dichlorophenyl) 2-Methyl, 2,3 Dihydroxypropio amide and
2.4.6. Triphenyl pyridine products. The
result obtained was revealed an elevation of both enzyme activities in culture treated
with Aspergillus fumigates and Cylindro-
carpon candidu. The elevation of caspase
activity was associated with increased concentration. This result was not in
agreement with Berkova (2006), who
reported that , Aspergillus fumigates inhibit tumor necrosis factor (TNF) alpha, a
known inducer of apoptosis he also
observed that, the anti apoptotic effect of Aspergillus fumigates was associated with
a significant reduction of caspase 3. In
conclusion, invitro of anticancer activities
of the tested components; N-(3- 4-Dichlorophenyl) 2-Methyl, 2,3 Dihydroxy-
propio amide and 2.4.6. Triphenyl pyridine
which produced from Aspergillus fumig-ates and Cylindrocarpon candidum against
liver cells. Also these two products were
strong induced apoptotic effects through activation of caspase 3and caspase 9
enzymes. This study needs furthermore
confirmation in vivo study on experimental
animal module to provide accuracy of the phase one trial on the two products of
Aspergillus fumigates and Cylindrocarpon
candidu.
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175
سبيرجيللس فيويجاتس تحفيس الوركبات الوعسوله هي أ
ينوسيليدروكربوكاديد
للووت الوبرهج لخالايا الكبد السرطايه عي طريق تشيط اسيوات
الكازبيسيس
أم –** وفاء عبد هللا أبـو العال -*لطفي عبد الرؤف سالم -هحود السيد عثواى
أهاى أبو الصر الشافعى-كلثوم حسي خطاب
قسن كيوياء * -ظـاهــعـح حـــلـاى –قسن الثاخ الويكرتيلظي كليح الــعــلـم
قسن تيلظيا ** -الورعاخ الطثيعي الويكرتي الـوـركــز الـقـهــى لـلــثـحـز
ظـــاهــعـح الـقـاــرج -األرام الـوـركــز الـقـهــىللالرام
ذظمم ترواهرمما تممالواي الطثيعيمم الرممى تشثرممد تى لمما ير هعظممن احتحمماز احى
ذسمردمم الوماي الطثيعيم . كثير فى القاي العالض هي احهمرا توما فيما السمر اىاحى كعمممالض ايمممافي تلممم للرقليمممل همممي احشمممار العاثيممم للعمممالض الكيوممما العمممالض
همي الكانمماخ المقيقم لمما هركممة ذمن ىزلمما 000لقممم ظمم تى اكصممر همي . االشمعاىى
يا 04] فمي م المراسم ذمن ىمزب همركثيي وما. ذأشير سام ىلي الداليا السر اي سمثيرظيلل فيويعماذ همي ت[ يا يمركسي ترتياهيم 20هيصيل 2 –كلرفييل
ين ذممن يراسممح ذأشيرومما سمميليمركرتى كاميممم هممي[ ذرايفييممل تيريممميي 246]
يمما الكثممم السممر اث ظممما تى لمما ذممأشير سمموى ايممي ىلممى مما الدالايمما ىلممى االاالسممر اي مم ا الرممـأشير يزيممم كلومما هايخ العرىمم هايخ فرممرج ظممي الوركممة همم
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سماى هممي فرمرج الرحضمميي تيضمما ىمم مم ا الرركيممز 24كامد تىلممى يرظمح سمموي تعممم صلا الى تىلى سث للوخ الوثرهط لداليا الكثم حيس تظرخ الرانط ما هلحمظ
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