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Hassan et al. Iraqi Journal of Science, 2020, Vol. 61, No. 2, pp: 285-294
DOI: 10.24996/ijs.2020.61.2.6
_____________________________
*Email: abdsarah55@gmail.com 285
In Vitro Oncolytic activity of non-virulent Newcastle Disease Virus LaSota Strain
against Mouse mammary adenocarcinoma
Sarah A.H. Hassan*1, Aida B. Allawe1, Ahmed Majeed Al-Shammari2
1University of Baghdad, Department of Microbiology, College of Veterinary Medicine, Iraq
2 Mustansiriyah University, Iraqi Center for Cancer and Medical Genetics Research, Experimental Therapy
Department, Baghdad Iraq
Received: 23/7/ 2019 Accepted: 28/ 8/2019
Abstract
Newcastle disease virus (NDV) is a wide-spectrum anti-tumor agent. The
oncolytic selectivity of NDV, a family of Paramyxoviridae, depends on the
differential type of inducing different death pathways. This work was conducted to
further understand the oncolytic effect of LaSota strain. A mouse breast cancer
model (Murine mammary adenocarcinoma cell line AMN3) was used in this study.
Methyl Thiazolyl Tetrazolium (MTT) viability assay tested different NDV
multiplicity of infection (MOI) values on mouse mammary adenocarcinoma cells
incubated for 72 hours post-infection. The IC50 values and anti-tumor activity of
LaSota strain against AMN3 cell line were determined. Following Hematoxylin and
Eosin Stain, we examined the morphological modifications of IC50 along with 10
MOI values of NDV. The induction of NDV apoptosis in AMN3 cells was
investigated using the technique of staining acridine orange and propidium iodide
(AO / PI). Immunocytochemistry assay was performed using anti-NDV mAbs and
caspases 8 and 9 to study NDV replication and apoptosis induction mechanisms.
The lentogenic LaSota NDV strain, a live vaccine, demonstrated the oncolytic effect
on mammary cancer cells of the AMN3 mouse and showed that LaSota strain
triggered a dose-dependent increase in infected cells’ apoptosis relative to untreated
mammary cancer cells. The immunocytochemistry study showed that NDV infected
cells were positive for virus infection and that caspase9 in mouse mammary cancer
cells after LaSota strain infection was significantly enhanced compared to caspase 8.
In conclusions, NDV LaSota strain had oncolytic effects by destroying tumor cells
and triggering the intrinsic apoptosis pathways in mouse mammary cancer cells.
However, the mechanisms of the in vivo anti-tumor activity need to be better
understood.
Keywords. NDV-Newcastle disease virus; Lentogenic NDV; Apoptosis; Breast
cancer cells; Oncolytic therapy; MTT assay.
ISSN: 0067-2904
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286
NDV
(oncolytic)
AMN3MTT
IC50LaSota
AMN3
IC5010 MOI NDV
AMN3
AO / PImAbsNDV
LaSota NDV
AMN3LaSota
NDV
LaSota
LaSota
Introduction
Cancer is the leading cause of death globally [1]. Several factors are known to lead to the increased
cancer incidence, such as environmental pollution [2]. Lately, virotherapy, which means using viruses
as therapy for cancer, is recognized to have possible clinical applications for cancer treatment, as
many viruses demonstrated anti-tumor properties with the power to suppress cancer cells without
harming normal cells [3]. Newcastle disease virus (NDV) is a member of the new genus Avulavirus
within the family Paramyxoviridae [4]. NDV is one of such oncolytic viruses which are presently
being used in modern preclinical and clinical studies [5]. Csatary was the first to report arrested
development of aggressive metastatic colon tumor in a Hungarian farm following an outbreak of NDV
in 1971, indicating a correlativity with NDV infection [6]. Interest in the utilization of the anti-tumor
NDV to kill tumor cells was due to its specifications in targeting tumor cells through three different
mechanisms, cytolysis secondary to virus replication, apoptosis induction, and antigenic modification
to the surfaces of tumor cells to make them more recognizable by the immune system [7]. It is also
suggested that NDV may work as an anti-angiogenic agent [8]. Recently, it was found to suppress
cancer cells through inhibition of the glycolysis pathway to enhance the anti-tumor activity [9]. Based
on their pathogenicity in chickens NDV strains are classified into three types; velogenic, mesogenic,
and lentogenic. The vaccination of poultry was satisfactory with the lentogenic or non-virulent
attenuated strains such as NDV Hitchner-B1, Ulster, and LaSota, while the velogenic and mesogenic
strains are considered as threats to agriculture. Therefore, lentogenic NDV strains have been employed
widely as oncolytic viruses [10, 11, 12]. Moreover, the lentogenic strain LaSota was shown to enhance
the conventional chemotherapies such as methotrexate and 5-Flurouracel [13, 14]. Rituximab and
doxorubicin combination with NDV can work as anti-hematological cancer therapies [15]. Retinoic
acid synergizes with attenuated NDV against digestive system tumors [16]. Bacterial hyaluronidase
enhances attenuated the NDV potency in a mouse mammary tumor model [17].
Therefore, it is necessary to understand the mechanism behind virus replication in mouse tumor
models and its efficiency as a model for human cancer treatment. The current study was designed to
study the lentogenic LaSota NDV strain as an oncolytic agent against mouse mammary
Hassan et al. Iraqi Journal of Science, 2020, Vol. 61, No.2, pp: 285-294
287
adenocarcinoma cells and to evaluate this activity through different parameters to facilitate further
experiments that aim to develop NDV as an anti-tumor agent in preclinical experiments.
Materials and Methods
Cell Line
Murine mammary adenocarcinoma cell line AMN3 was grown in RPMI-1640 medium (US
Biological, USA) supplied with 10% fetal bovine serum (FBS) (Capricorn Scientific, Germany), 100
units/ml penicillin, and 100 µg/ML streptomycin and incubated at 37 ºC. The AMN3 cell line was
provided by the Cell Bank Unit, Experimental Therapy Department, Iraqi Center for Cancer and
Medical Genetic Research (ICCMGR), Mustansiriyah University, Baghdad, Iraq. The AMN3 cell line
was sub-cultured every three days, or when the cultures made 80% confluence. The cell line was
tested regularly for mycoplasma contamination by the cell bank unit of the ICCMGR.
LaSota strain
LaSota strain, a lentogenic virulent strain of NDV used as lyophilized allantoic fluids (live
vaccine), was obtained from Al Kindi for Production of Veterinary Vaccines and Drugs (IKLV)
(Baghdad, Iraq). The virus was propagated in embryonated chicken eggs and tittered on Vero-slam
cells for TCID50 determination and MOI calculation according to a standard procedure [18].
Cytotoxicity MTT assay
The MTT (Methyl Thiazolyl Tetrazolium) assay was carried on in order to assess the viability of
the AMN3 cell line after infection with NDV LaSota strain. AMN3 cell line was seeded at 10,000
cells/well at a total volume of 100 μl in growth medium. It reached a 70-80% confluent monolayer.
AMN3 cell line was infected with LaSota NDV for different multiplicity of infection (MOI) values
(0.1, 0.5, 1, 3, 5, 10, 15 and 20), whereas control wells remained untreated, aspiring only to the media
and exchanging with free serum media. Viability of the cells was calculated after 72 hours of
incubation. The old media from each well was removed, then 100 μl of MTT solution were added into
each well, followed by incubation for 2 hours at 37˚C in the dark. The MTT solution was removedand
50 μl of DMSO (Dimethyl Sulphoxide) (Santa Cruz Biotechnology, USA) were added to each well,
followed by 37˚C re-incubation for 30 min. MTT was measured using an ELISA microplate reader
(FLUOstar OPTIA Reader, Germany) and the absorbance was read at 584 nm wavelengths.
Percentage of cytotoxicity was calculated as (C- T)/C * 100, where C is the mean optical density of
control wells, and T is the optical density of infected wells. The assay was performed in triplicate.
Percent inhibition of tumor growth versus MOI graphs of the LaSota strain was obtained and the
concentration causing half the maximum inhibitory (IC50) was determined [19].
Cytopathological changes
The AMN3 cell line was cultured on coverslips (50,000 cells/cover) at a total volume of 2 ml in
their serum culture medium. When the cells reached 70-80% confluent monolayer in multi-well tissue
culture plates (6-wells), they were exposed to the dose of IC50 and to 10 MOI of LaSota strain in
serum free media. The old culture media were removed and the cells were incubated for 2h to allow
the virus to infect the cells, then the media were aspirated and exchanged with new free serum media.
The untreated control cells were prepared similarly without virus inoculum. Then the cells were
incubated for 48 hrs. at 37˚C with a moisturized atmosphere containing 5% CO2. The infected cells
were examined under an inverted light microscope to record the morphological alterations. After
removing the suspension medium, the cells fixed with 10% formaldehyde were diluted in PBS for 10
min and stained with H and E stain. Finally, all slides were mounted with DPX and photographed by
inverted microscope (Leica Microsystems, Germany) at 400x.
Apoptosis assay
The AMN3 cell line was cultured in a 96 well plate at a total volume of 100 μl with a density of
10,000 cells/well in the growth medium until 70-80% confluent monolayer was formed. Cells were
exposed to the virus dose of IC50 and to 10 MOI values in serum free media for 2 hrs. After that the
old media were exchanged with new serum free media. The AMN3 control was prepared similarly
without virus inoculum. The cells were incubated for 48 hours at 37˚C with 5% CO2 and humidity. At
the end point (48h), the medium was discarded, cells were stained using 1µL AO (acridine orange) and
1µL PI (propidium iodide) double staining at a ratio of 1:1 in 1 ml of PBS at a total volume of 50 μl/
well for 20 seconds. Double staining was aspirated directly from the wells. The morphological
changes of viable and apoptotic cells were observed and photographed by an inverted fluorescent
microscope (Leica Microsystems, Germany).
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Immunocytochemistry
As described for cytopathological changes, the cells were cultured on coverslips and treated with
the NDV dose of IC50 and 10 MOI values, whereas another group was left as control without virus
infection. The cells were incubated at 37˚C for 48 hours under a moisturized atmosphere with 5%
CO2. Later, cells were fixed with cold acetone for 2 min. Cells in 3% H2O2 and 1% fetal bovine
albumin solution in PBS were blocked for 10 minutes. The cells were incubated with multiple primary
antibodies (anti-NDV, anti-caspase8 and anti-caspase9diluted at 1:50 for 1-1.5 hours at room
temperature. After washing with PBS, the cells were incubated with biotinylated mouse secondary
antibody (diluted as 1:100) for 1 hour. After washing, the substrate DAB was prepared freshly in DAB
buffer and sufficient drops of freshly prepared DAB substrate mixture were applied to cover the cell
section. The slides were incubated in the dark for 20 minutes at room temperature. After that, the
plates were washed with PBS and counterstained with hematoxylin stain for 5-10sec. They were then
washed with distilled water and with PBS and immediately mounted using DPX on glass slides. The
examination was performed using light microscopy.
Quantitative image analysis
The digital pictures of immunocytochemistry were used for quantitative analysis for the
hematoxylin - DAB stained slides and taken with a Leica inverted microscope provided with a camera
(Leica Microsystems, Germany). Three dissimilar staining zones of ICC images of each slide were
analyzed in this study. Firstly, color, de-convolution assay was used for un-mixing with the DAB and
hematoxylin stained areas, leaving a free image [20]. In addition, two other images were obtained; The
first image is the hematoxylin stain and the second one is the DAB image, where the DAB image was
quantified. The numerate of pixels of a specific intensity measure vs. their respective intensity was
elevated utilizing "Fiji" version of ImageJ from <http: //Fiji. sc> [21]. We changed the intensity
numbers in the data window to Optical Density (OD) numbers with the following formula: OD = log
(max intensity/Mean intensity), where max intensity = 255 for 8-bit images.
Statistical analysis
Data were shown as mean ± SD for all measurements of triplicate observations. For MTT and ICC
experiments, n = 3 images were used. One-way analysis of variance (ANOVA) multiple comparison
was done to show variations among groups. The statistical significance analyses were done utilizing
(GraphPad Prism version 7.0 for Windows, GraphPad Software, San Diego, CA, USA) to estimate the
cytotoxicity and to detect the apoptotic impact of LaSota strain on AMN3 cell line in vitro. p < 0.05
was regarded as statistically significant.
Results
In vitro anti-tumoral activity
To determine the inhibitory effect of LaSota strain in vitro, we inoculated different MOI (0.1, 0.5,
1, 3, 5, 10,15, 20 MOI) of LaSota strain on monolayers of Murine mammary adenocarcinoma cell line
AMN3. After 72 hours, the IC50 and tumor growth inhibition (GI) were determined (Table-1, Figure-
1). The anti-tumor behavior of LaSota strain showed that the increase in the dose of LaSota strain led
to increasing the inhibitory effect of the virus on AMN3 cancer cells.
The cytotoxicity at the dose of 20 MOI LaSota strain was 57.5 % with an estimated IC50 of 8.0
MOI after an exposure period of 72 hrs.
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289
Figure 1- In vitro antitumor activity of LaSota strain. (A) IC50 of LaSota strain in AMN3 cell line (B)
The AMN3 cell line (10,000 cells per well) were transferred in 96-well flat plates and LaSota strain
(0.1,0.5,1, 3, 5,10,15,20 MOI) was added in triplicates. After 72 h, tumor growth inhibition was
measured by the evaluation of MTT. For calculation of % tumor growth inhibition (GI), the untreated
cells were set to zero.
Table 1- The growth inhibition% of LaSota strain on AMN3 cell line after 72 hours post exposure to
ND virus.
MOI of LaSota strain
Value of growth inhibition (M ± SD) %
20
57.57 ± 8.42
15
50.97 ± 10.36
10
48.28 ± 9.15
5
36.59 ± 4.33
3
32.73 ± 4.54
1
25.28 ± 6.02
0.5
18.85 ± 6.45
0.1
17.05 ± 4.41
Cytopathology and detection of apoptosis
The light inverted fluorescent microscope was utilized to observe the morphological alterations
and the ratios of viable, necrotic and apoptotic cells in the population of AMN3 cell line exposed to
the dose of IC50 and 10 MOI doses of NDV LaSota strain for 48h, compared to untreated AMN3 cell
line. Under the light microscope, the unstained breast cancer cells were observed and showed round
and shrinking cells. We also noticed an increase in cell granulation. In addition, the changes in the
unstained culture showed large empty plaque spaces between cells (Figure-2 A2-A3) compared with
the control cells that did not show any change for the same duration (Figure-2 A1). Whereas under the
light microscope, the H&E stained AMN3 cell line showed t many cytopathological changes which
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290
included vacuolation, cell degeneration, condensation of nucleus, and cells detachment (Figure-2 B2,
B3), compared with the untreated cells Figure-(2B1). Furthermore, inverted fluorescent microscopy
examination utilizing PI and AO was conducted; the selective permeability property of the entire
plasma membrane of viable cells permitted the entry of AO stain, whereas they were impermeable to
PI staining. The nuclei of viable cells were stained with green when observed under inverted
fluorescence microscope, whereas the plasma membrane of necrotic cells was no longer intact which
allowed PI to enter and make the cells appear red. The viable cells could be distinguished from
apoptotic cells based on morphological deviations such as smaller cells size and fluoresced orange or
red color. In addition, an increase in green to red shift was noticed in apoptotic LaSota strain-infected
AMN3 cells (Figure-2C2-C3) after 48hrs , as compared with untreated control (viable green cell)
(Figure-2 C1). This green to red shift in fluorescence was more at the treatment with the 10 MOI doses
than IC50 of LaSota strain; however, 10 MOI of LaSota strain induced more effect (apoptosis,
compared to the IC50 dose.
Figure 2-AMN3cell line treated for 48hrs with IC50 or10 MOI doses of LaSota strain afters; 400x. (A)
In cross microscope, unstained cell showing the rounded cell (orange arrow) and vacuole degeneration
(yellow arrow). (B) In H &E staining, a treated cell showing vacuole degeneration (yellow arrow),
hypertrophy, and condensation of nuclei (red arrow) compared to an untreated control cell. (C) In
acridine orange and propidium iodide staining, a treated cell showed green, viable cells (blue arrow)
and red apoptosis cells (white arrow) viewed under a fluorescence microscope.
Detection of NDV and Caspase 8 and 9 proteins expression
This assay was performed to identify the mechanism of apoptosis induction and determine the
exact in vitro pathway for apoptosis of LaSota-treated AMN3 cell line after 48h post inoculation.
Immunocytochemistry analysis showed that all tumor cells infected with LaSota strain were positive
for the expression of ND and caspase 8 and 9 proteins Figures-(3A2-3, B2-3 and C2-3). Antigens in
the infected cells were stained in brown, while no brown staining was observed in the positive control
(uninfected cells) that were not exposed to LaSota strain but exposed to both primary and secondary
antibodies Figures-(3A1, B1 and C1) (Nuclei were stained blue by counterstain).
Digital Image Scoring
Further analysis of ICC stained images x revealed a significant (P<0.05) increase in optical density
related to protein expression of anti-NDV and caspase9 in LaSota strain -Infected AMN3 breast
cancer cell line as compared to the optical density of caspase8 as well as to positive control, as judged
Hassan et al. Iraqi Journal of Science, 2020, Vol. 61, No.2, pp: 285-294
291
by Digital Image Scoring using ImageJ program analysis of LaSota strain (10 MOI and IC50)-infected
AMN3 cell line after 48 h post inoculation. Figure-3D shows demonstrative zones and pixel intensity
analysis of ICC images. The analysis showed that the three proteins (anti-NDV, Caspase8, and
Caspase9) were significantly expressed when compared to control and not stained cells (positive
control), using ANOVA one-way multiple comparison test (Figure-3D1andD3). However, significant
(P<0.05 ( increases in caspase9 activity and ND antibody in ND-infected AMN3 breast cancer cell
were recorded as compared to caspase8 expression. Further, caspase8 show no significant difference
(P<0.05) in optical density of expression after IC50 treatment Figure-3 D2, when compared with
caspase9. These results indicated decreased caspase-8 expression and, in contrast, increased caspase-9
expression, which is considered as an interesting result reflecting that Caspase9 have an important
effect in LaSota strain-stimulated cell death. Furthermore, the increase in the optical density was
correlated with the dose of LaSota strain (Table-2).
Table 2-The optical densities of anti-NDV, Caspase8, and Caspase9 (M ± SD) of IC50 and 10 MOI
doses of LaSota strain on AMN3 cell line after 48 hours post-exposure
MOI of NDV
The optical density of protein expression (M ± SD)
Anti NDV
Caspase8
Caspase9
LaSota strain
10MOI
0.556 ± 0.038
0.521 ± 0.007
0.563 ± 0.029
LaSota strain
IC50
0.510 ± 0.014
0.499 ± 0.059
0.534 ± 0.050
Positive control
0.426 ± 0.022
0.421 ± 0.026
0.427 ± 0.024
Figure 3- The results of immunocytochemistry on AMN3 cell line treated with IC50 and 10 MOI
doses of LaSota strain after 48h of exposure to LaSota strain. Cancer cells take the brown cytoplasm
stain (white arrow ) (DAB stain 400x) compared to untreated control cancer cells which were
negative to DAB stain (black arrow) (DAB stain 400x) using the primary antibody markers (A) ND
antibody (B) caspase8 (C) caspas9 . (D) Digital Image Scoring showing significant proteins
expression when stained with relative mAbs against the markers, as analyzed by ImageJ program.
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Data were analyzed by ANOVA one-way multiple comparison test. Differences were considered
significant at P≤0.05.
Discussion
In the current study, we used a lentogenic avirulent vaccine strain of NDV to evaluate oncolytic
activities in our mouse model. The use of pathogenic replicative NDV strains, such as mesogenic or
velogenic strains, for the treatment of cancer patients was reported to be problematic due to
environmental biohazards associated with the pathogenicity of the virus and the potential viral spread.
The attenuated NDV strains, such as HUJ, Ulster, and Hitchner-B1, selectively infect cancer cells and
do not cause side effects in patients [22]. Besides, the NDV is a safe virus for the treatment of humans;
its RNA genome is stable with the absence of genetic recombination, lack of antigenic drift, and lack
of noticed human to human transmission [23].
The overall results from our study showed that NDV LaSota strain is effective against mouse
mammary adenocarcinoma, in which the virus accomplishes its effect through cytolysis and apoptosis.
In addition, the MTT assay was conducted to evaluate the effects of LaSota strains on cell viability of
Murine mammary adenocarcinoma cell line (AMN3). The assay helped to find the inhibitory
concentration (IC50) value of the virus that causes 50% death of the tumor cells. The IC50 value
obtained was 8.0MOI, while growth inhibition was 57.5 % at 20 MOI LaSota strain. These results
manifest that the virus induced an efficient killing of 50% of the AMN3, compared to untreated
AMN3 cell, demonstrating its potent oncolytic effects. Elankumaran et al. [24] used 14 cell lines from
a spectrum of malignancies which were infected with nonpathogenic NDV (rLaSota V.F) and rBC-
Edit viruses. The NDV showed a wider spectrum of cytotoxicity with a very low MOI. This could be
due to the absence of expression of the V protein. The morphological alterations seen in the treated
AMN3 cancer cells were confirmed by the cytopathological effects observed using H and E stain.
Inverted microscopic analysis of the AMN3 cells treated with LaSota strain exhibitd round and
shrinkage cells, which resulted in large, empty, plaque spaces between cells, while the untreated cells
did not show any changes for the same duration. As shown by a previous study, HeLa cell-adapted
NDV was used at passage number 5 and cytopathological impacts were identified by morphological
changes which included cell shrinkage, rounding, detachment from the monolayer, and formation of
giant cells (syncytium). Parental NDV (unadapted to HeLa cells)-infected HeLa cells appeared normal
in gross morphology [25]. We further supported the effect of LaSota strain on tumor proliferation
through induction of apoptosis and examining the PI/AO-stained cells under fluorescence microscopic.
The results showed that LaSota strain increased the number of apoptotic cells in the infected AMN3
cancer cells in a dose-dependent manner. The viable cells could be distinguished from apoptotic cells
based on morphological deviations such as the smaller cell size and the fluoresced orange or red, while
the increase in green to red shift was noticed in LaSota strain-infected AMN3 cell line. On the other
hand, the untreated AMN3 control cells demonstrated an increase in the viable green cells after 48
hours of treatment, demonstrating apoptosis features. This result proves the fact that the virus has an
oncolytic effect on cancer cells. Al-Shamery et al. [26] revealedthat an Iraqi strain of NDV had in
vitro effects on inducing apoptosis in AMN3, while the in vivo study found that the endoplasmic
reticulum pathway is the dominant pathway for NDV in inducing apoptosis. The ability of the NDV to
kill cancer cells has also been studied on many types of human cell lines, such as MDA-231and MCF-
7, using six different vaccines of NDV strains [27]. These lentogenic strain have proven to be
relatively effective in inhibiting tumor growth through apoptosis triggering.
Thus, the current study manifested that the LaSota strain of the virus possesses an in vitro
anticancer activity on AMN3 cancer cells. The immunocytochemistry assay was conducted to detect
protein expression. This assay was used to study the exact pathway of apoptosis by observing the
expressed caspase8 and 9 proteins and to examine the relativity of NDV infection by using anti-NDV
mAb on infected AMN3 cancer cells after 48h of LaSota strain inoculation. The results showed
increases in the expression levels of the three protein in AMN3 cancer cells infected with LaSota
strain, while non-treated cells were used for comparison. The results showed a decrease in the
expression of caspase-8, as compared to an increase in caspase-9, which is an interesting result. This
suggests that caspase-9 is necessary in mammary adenocarcinoma cells for NDV-induced apoptosis
through mitochondrial pathways. Finally, the immunocytochemistry assay results were analyzed by an
image J program; the results showed significant expression of all proteins when compared with LaSota
strain non-treated cells, except that the IC50 did not affect caspas8 expression significantly (P<0.05).
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Further, caspase8 showed less significant difference (P<0.05) in optical density of protein expression
when compared with caspase9 . Our work is in agreement with the study of Humadai, T.J [28] who
indicated a significant increase in the average percentage of cell expression of caspase 9 in the NDV
exposed group, which implied that NDV had a powerful effect on inducing intrinsic (mitochondrial)
apoptotic pathways [29]. Furthermore, our findings are in agreement with those of a previous study on
cells of mouse fibroblasts (3T3) and human colon adenocarcinoma (HT-29) cells [30].
Finally, we indicate that the LaSota strain showed a remarkable antitumor activity against AMN3
mouse mammary adenocarcinoma cells, which was induced through cytolysis and apoptosis via
intrinsic (mitochondrial) pathway. This effect was more dictated by the increased percentage of
caspase-9 expression as compared to that of caspase8.
Acknowledgments
The authors would like to extend their thanks to the Department of Experimental Therapeutics,
Iraqi Center for Cancer and Medical Genetic Research, Mustansiriyah University, Baghdad, Iraq. We
also like to thank the specialist Dr. Aida Bara Allawe, for valuable guidance during research work and
Dr. Ahmed M. Al-Shammari for the use of their laboratory facilities.
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