Neurochem Res (2011) 36:2051 2062
DOI 10.1007/s11064-011-0529-8
ORIGINAL PAPER
Cytolytic Effects and Apoptosis Induction of Newcastle Disease
Virus Strain AF2240 on Anaplastic Astrocytoma Brain Tumor
Cell Line
" " "
Rola Ali Aied M. Alabsi Abdul Manaf Ali
" "
Aini Ideris Abdul Rahman Omar
"
Khatijah Yusoff Riyadh Saif-Ali
Accepted: 3 June 2011 / Published online: 14 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Newcastle disease virus (NDV) is a member of inoculation by annexin-V flow-cytometry method. It could
genus Avulavirus within the family Paramyxoviridae. be concluded that NDV strain AF2240 is a potent antitumor
Interest of using NDV as an anticancer agent has arisen from agent that induce apoptosis and its cytotoxicity increasing
its ability to kill tumor cells with limited toxicity to normal while increasing of time and virus titer.
cells. In this investigation, the cytotolytic properties of NDV
strain AF2240 were evaluated on brain tumor cell line, Keywords NDV strain AF2240 Cytotoxicity
anaplastic astrocytoma (U-87MG), by using MTT assay. Apoptosis Flow-cytometry Anaplastic astrocytoma
Cytological observations were studied using fluorescence
microscopy and transmission electron microscopy to
show the apoptogenic features of NDV on U-87MG. DNA Background
laddering in agarose gel electrophoresis and terminal
deoxyribonucleotide transferase-mediated dUTP-X nick Brain tumor is formed by abnormal and uncontrolled cell
end-labeling staining assay confirmed that the mode of cell division; it is abnormal growth of tissue found inside the
death was by apoptosis. However, analysis of the cellular skull. It can be dangerous because of the importance of the
DNA content by flowcytometery showed that there was a brain and the limited amount of space inside the skull.
loss of treated U-87MG cells in all cell cycle phases (G1, S Most primary brain tumors do not metastasize systemically
and G2/M) accompanied with increasing in sub-G1 region but spread locally through extensions of infiltrating tumor
(apoptosis peak). Early apoptosis was observed 6 h post- cells in normal brain. Grade III astrocytoma, anaplastic
astrocytoma, is commonly spread to surrounding brain
tissue and it is the most common primary brain tumor in
R. Ali K. Yusoff adults [42]. Surgery; radiation and chemotherapy are the
Faculty of Biotechnology and Bimolecular Sciences,
current treatments for brain tumor [18, 24, 26, 40]. New-
Universiti Putra Malaysia, Serdang, Malaysia
castle disease virus (NDV) was first isolated in 1926 in
Newcastle, England, in domestic chickens where it caused
A. Ideris A. R. Omar
Faculty of Veterinary Medicine, Universiti Putra Malaysia, a severe disease known as Newcastle disease. ND is a
Serdang, Malaysia
disease of poultry that affecting the alimentary and respi-
ratory tract as well as the central nervous system [9].
A. M. Alabsi A. M. Ali (&)
Exposure to humans however, results in mild conjunctivi-
Faculty of Agricultures and Biotechnology, Universiti Sultan
Zainal Abidin, Kuala Terengganu, Malaysia tis, laryngitis and influenza-like symptoms [11]. The NDV
e-mail: [e-mail]
showed promise in clinical trials as an antineoplastic agent.
A. M. Alabsi It is one of the nonengineered oncolytic viruses, which has
e-mail: [e-mail]
a long history as a broad-spectrum oncolytic agent that can
destroy tumor cells and stimulate the immune system
R. Saif-Ali
[12, 19]. Many preclinical studies indicate that NDV rep-
Faculty of Medicine, Universiti Malaya,
Kuala Lumpur, Malaysia licates in human cancer cells but not in normal cells, some
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2052 Neurochem Res (2011) 36:2051 2062
types of NDV are able to directly kill certain types of concentrations (double dilution) and were incubated at
cancer cells and NDV infected cancer cells can cause the 37°C, 5% CO2 for 72 h. Each concentration was assayed in
immune system to respond in different ways [34]. Early triplicates (n = 3). Seventy-two hours later, 20 ll of MTT
success with the viral vaccine in humans was first reported (5 mg/ml) solution was added to each well and then the plate
in the United States in 1980 [32]. Interest in the use of was further incubated for 4 h. All remaining supernatant
NDV as an anticancer agent has arisen from the ability of were removed and 150 ll of DMSO was added to dissolve
the virus to selectively kill human tumor cells with limited the formed crystal formazan. MTT assay reading was per-
toxicity to normal cells. Many strains of NDV (73-T, formed using ELISA plate reader (Biotek EL340, USA).
MH68, Italian, Ulester, Rokin, PV701 and HUJ) have been
shown to exhibit an oncolytic activity [12, 36]. In addition, Quantification of Apoptosis Using Propidium Iodide
the oncolytic effects of six Malaysian strains of NDV and Acridine Orange Double staining
(AF2240, 01/C, Ijuk, S, F, and V4) have also been studied
on several tumor cell lines [34, 36]. The goal of most Cells were quantified using propidium iodide (PI) and
cancer therapy is to reduce the number of tumor cells and acridine-orange (AO) double staining according to standard
to prevent their further accumulation (decrease the rate of procedures and examine under fluorescence microscope
cell proliferation). Therefore, in this study the effects of (Lieca attached with Q-Floro Software) [17, 30]. U-87MG
NDV strain AF2240 on the proliferation and the morpho- cells were seeded in six-well plate and incubated at 37°Cin
logical changes of brain tumor cell line, anaplastic astro- 5% CO2 atmosphere. Twenty-four hours later, the medium
cytoma (U-87MG), was tested in vitro. in each well was removed and replaced with the virus at
IC50 concentration dissolved in medium and incubated at
37°C in 5%CO2 atmosphere for 24, 48, and 72 h. After
Materials and Methods incubation period, detached cells in the medium were
collected and added back to trypsinised adherent cells. The
Propagation and Purification of NDV Strain AF2240 cell suspensions were washed with PBS and then incubated
with 5 ll of acridine orange (10 lg/ml) and 5 ll propidium
NDV was propagated in allantoic fluid of 9 11 days-old iodide (10 lg/ml) at a ratio of 1:1 in 1 ml of cells and
embryonated chicken eggs at 37°C for 48 h. The allantoic recentrifuged at 1,000 rpm/5 min. After centrifuge, super-
fluid was harvested and the presence of virus was con- natant was removed leaving 50 ll of remaining supernatant
firmed by the haemaglutination test [2]. NDV strains with pellet. The pellet was resuspended and 10 ll of cell
AF2240 purified as previously described [4, 47]. suspension was pipetted on slide before putting on cover
slip. Within 30 min, the slide was analyzed using fluores-
Cells and Cell Culture cent microscope (Leica, Germany). Each experiment was
assayed three times (n = 3). Viable, apoptotic and necrotic
Human brain tumor cell line U-87MG was obtained from cells was quantified in a population of 200 cells. In apop-
American Type Culture Collection (ATCC, VA, USA). totic bodies, blebbing of plasma membrane and conden-
HCN-2 and 3T3, normal cell lines, kindly provided by Prof. sation of chromatin were seen. In contrast, necrotic cells
Dr. Abdul Manaf Ali, Faculty of Biotechnology and fluoresced red after propidium iodide staining with tiny
Molecular Biology, University Putra Malaysia (UPM) were fraction of chromatin dispersed around the nucleus. This
used as control. All cell lines were grown as a monolayer in assay provides a useful quantitative evaluation and was
25 cm2 tissue culture flasks (NunclonTM, Denmark) at 37°C done three times (n = 3).
in an atmosphere of 5% CO2. MEM medium supplemented
with 10% fetal calf serum, 1% antibiotics and three addi- Transmission Unltrastructural Effects of NDV Strains
tional supplements (2 mM Glutamine, 1 mM Sodium AF2240 on U-87MG Cells (TEM)
Pyruvate, and 1% nonessential amino acid) was used for
U-87MG. Normal cell lines were grown in DMEM medium. U-87MG brain tumor cells were treated with NDV at IC50
concentration and incubated for 24, 48, and 72 h at 37°C.
MTT Cytotoxicity Assay The cultured cells were harvested using trypsin and cen-
trifuged for 10 min at 1,500 rpm. The pellets were fixed in
U-87MG cells were trypsinized and counted using hemo- 4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer (pH
cytometer then were seeded in 96-well micro plate at 7.4) for 4 h at 4°C. The fixed cells were centrifuged, and
3 9 105 cells/ml and then incubated at 37°Cin 5%CO2 to the pellets were blocked in serum which was later fixed
allow cells attachment. The medium was removed and in glutaraldeyde overnight at 4°C. The specimens were
replaced with fresh medium containing test virus at various washed in three changes of sodium coccadylate buffer
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Neurochem Res (2011) 36:2051 2062 2053
(pH 7.4) for 10 min each, postfixed in 1% osmium tetraoxide adding 500 ll of 80% cold ethanol and kept for at least 2 h
at 4°C. The specimens were then washed in three changes of at -20°C. Cells were pelleted at 1,000 rpm for 10 min and
sodium coccadylate buffer (pH 7.4) for 10 min each and the ethanol was discarded. The cell pellet was washed with
dehydrated with a graded series of acetone (35, 50, 75, 95, 1 ml (PBS/sodium azide) twice. The pellet was resus-
and 100%). The cells were then infiltrated with acetone and pended with 1 ml of (PBS ? 0.1% triton X-100 ?10 mm
resin and embedded with 100% resin in beam capsule, and EDTA ? 50 lg/ml RNase ? 2 lg/ml Propidium iodide)
left to polymerize at 60°C for 48 h. The area of interest in the followed by incubated for Ä™ to 1 h at 4°C. Finally, samples
embedded cells resin block was chosen using the toulidine were placed in 12 9 75 Falcon tubes and the cell cycle was
blue staining and later examined using light microscope. The analyzed by flow cytometer (Beckman Coulter, USA).
selected area was cut in ulltrathin sections using ultrami- Each experiment was assayed three times (n = 3).
crotome. The sections were placed into a grid and stained
with uranyl acetate for 10 min followed by 50% filtered Flow Cytometry (Annexin V/PI Double Staining)
acetone, and finally stained using lead which was then
washed twice with distilled water. The stained samples were Cells at a concentration of 5 9 106 cells/ml of U-87MG
then viewed under transmission electron microscopy (Phil- cell line was seeded into six-well plate in 2 ml culture
lips, Eindhoven, The Netherlands). medium with a concentration of IC50 value of virus and
were incubated at 37°C in an atmosphere of 5% CO2 for
DNA Fragmentation Assay 72 h. Some wells were left with no virus to be used as a
control. After the incubation period, the cultured cells were
Cells at a concentration of 5 9 106 cells/ml were seeded harvested using trypsin and centrifuged for 10 min at
into six-well plate (NunclonTM, Denmark) in 2 ml culture 1,000 rpm. The early apoptosis detection for treated and
medium with a concentration of IC50 value of virus. Some untreated cells was carried out using Annexin V & Apo
wells were left with no virus to be used as a control. After 2.7-PE kit (Clontech Laboratories, Inc., USA). Each
the 72 h of incubation, detached cells in the medium were experiment was assayed three times (n = 3).
collected and added back to trypsinised adherent cells. The
cells were spun down at 1,000 rpm for 10 min. The Statistical Analysis
supernatant was discarded and the pellet was washed with
PBS twice. The DNA extraction from treated and untreated Data was expressed as mean Ä… SD. Statistical analysis was
cells was carried out according to protocol of a kit for performed with Student s t-test for data from MTT cyto-
Blood and Cultured Cells from QIAGEN. toxicity assay, AO/PI staining assay, TUNEL and, flow
cytometry. Differences were considered significant at
TUNEL Assay P \ 0.001.
The cells treated with concentration of IC50 value of virus
value were grown on Lab-Tek Chamber slides and the Results
slides were incubated at 37°C in an atmosphere of 5% CO2.
The slides were washed with PBS after 24, 48 and 72 h and Cytotolytic Effects of NDV on Normal Cells and Brain
processed in the apoptosis detection assay. Each experi- Tumor Cells
ment was assayed three times (n = 3). The TUNEL Assay
was carried out using a kit for Apoptosis Detection from The cytotoxicity of NDV on U87 cells was investigated
Promega, USA. using MTT assay after 72 h treatment (Figs. 1, 4). NDV
was clearly found to exert antiproliferative effects toward
Analysis of Cellular DNA Content Using Propidium U87 brain tumor cells. The IC50 value, which is the con-
Iodide centration required for 50% growth inhibition, is deter-
mined to be 52 Ä… 1.1. Comparatively, NDV showed no
Cells at a concentration of 5 9 106 cells/ml of U-87MG significant cytolytic effect at the same titre used in the
cell line was seeded into six-well plate in 2 ml culture brain cell line toward HCN-2 and 3T3, normal cell lines
medium with a concentration of IC50 value of virus and (Figs. 2, 3).
were incubated at 37°C in an atmosphere of 5% CO2 for
72 h. Some wells were left with no virus to be used as a Phase Contrast Microscope
control. After the incubation period, the cultured cells were
harvested using trypsin and centrifuged for 10 min at The morphological changes of U-87MG cells after treat-
1,000 rpm at room temperature. Cell pellets were fixed by ment with NDV strain AF2240 were observed under Phase
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2054 Neurochem Res (2011) 36:2051 2062
Quantification of Apoptosis Using Propidium Iodide
and Acridine Orange Double Staining
Apoptotic, necrotic, and viable U-87MG cells were scored
under fluorescence microscope. This is also included the
control cells (untreated) whereby, 200 cells were randomly
and differentially counted. This study revealed that NDV
triggered morphological features that relates to apoptosis in
a time-dependent manner (Fig. 5). Whereby, early apoptosis
is obvious by intercalated AO. In several of such cases, the
fluorescent bright-green color could be seen in treated
U-87MG cells only. In contrast, untreated cells were
Fig. 1 Cytolytic effects of NDV strain AF2240 on U-87MG cell line.
The IC50 value is the concentration required for 50% growth
observed with a green intact nuclear structure blebbing and
inhibition. Fifty percent of cell viability (IC50) was obtained at virus
nuclear margination were noticed. Differential scoring of
titer of 52 Ä… 1.76 HAU/ml
treated U-87MG cells (200 cells population) showed that
there is a statistical significant (P \ 0.001) difference in
apoptosis positive cells, which indicates clearly that NDV
has a time-dependent apoptogenic effect (Fig. 6).
Effects of NDV on Brain Tumor Cell (U-87MG) Using
Transmission Electron Microscopy
Treated cells showed death corresponded very well to the
cross sectional classical signs of apoptosis: cell shrinkage,
increased cellular granularity, the formation of apoptotic
bodies, and dilated nuclear membranes (Fig. 7). Mito-
Fig. 2 Cytolytic effect of NDV AF2240 strain on HCN-2 cell line.
chondria in treated cells were ruptured and condensed
The IC50 value was not obtained because the cell reduction was not
(Fig. 7c). The evidences which suggested that the apopto-
effected by same virus titer
genic effect of NDV on U-87MG cells is time-dependent
manner were obtained through observation of dynamic
micrographs images using transmission electron micros-
copy. Features of early stage of apoptosis was observed at
24 h post-inoculation such as, cell shrinkage, chromatin
condensation in dense masses under the nuclear membrane
and margination along the inner nuclear membrane, and
compaction of the cytoplasm with development of vacuoles
in the cytoplasm. At 48 h post-inoculation, the cells broke
up into discrete fragments to form apoptotic bodies and
nucleus fragmentation was observed. At 24 h post-inocu-
lation, cytoplasmic organelles such as intact mitochondria
can still be observed whereas at 48 h post-inoculation
numerous mitochondria were found in the centre of the
apoptotic cells. Membrane blebbing without disintegration
Fig. 3 Cytolytic effect of NDV AF2240 strain on 3T3 cell line. The
IC50 value was not obtained because the cell reduction was not
of the cellular membrane occurred at 48 and 72 h post-
effected by same virus titer
inoculation indicating of late stage of apoptosis. At 72 h
post-inoculation secondary necrosis was observed and
some apoptotic bodies eventually degenerated (Fig. 7).
Contrast Microscope. The cells were treated with the IC50
value of the virus showed morphological changes including Effects of NDV on Brain Tumor Cell (U-87MG) DNA
rounding up of nuclei, shrinkage or decreased of nuclear Fragmentation
diameters and condensation of chromatin occurred. Fur-
thermore the cells in culture lost contact with adjacent cells Fragmentation of chromosomal DNA is the biological
(Fig. 5). hallmark of apoptosis, and can be detected by a ladder
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Neurochem Res (2011) 36:2051 2062 2055
Fig. 4 MTT Proliferation assay
non-viable viable
A 1
for various virus titers (IC50 and
1.2 3 2
4
100
IC25) against U-87MG brain
CONTROL IC 50 IC 25
tumor cell line at 24, 48 and 90
1
72 h post-inoculation. a The 80
growth rates decreased in the
70
0.8
treated cells as compared with
60
the untreated cells whereas 0.6
97 98
96
50
inoculation with a higher titter
40
0.4
of virus (IC50) decreased the
30
growth rate morethan low titre
20
0.2
(IC25). 1 The percentage of
10
viable and non-viable U-87MG
0
0
cells population of untreated
0 20 40 60 80
1 2 3
cells (control). 2 The percentage
hours
days
of viable and non-viable
U-87MG cells population
non-viable viable
treated with IC25 value of NDV non-viable viable
2 3
AF2240 titer. 3 The percentage
100
100
of viable and non-viable
39
U-87MG cells population
43
80
49 45
80
53
treated with IC50 value of NDV 55
AF2240 titer
60
60
40
40
61
57
55
51
47
45
20
20
0 0
1 2 3 1 2 3
days days
formation on gel electrophoresis. The DNA ladder assay is shown the TUNEL? nuclei appeared as yellow to green
generally accepted as specific for apoptosis because it fluorescence. The treated cells containing multiple DNA
detects oligonucleosomal cleavage rather than artificial breaks at 24, 48 and 72 h post-inoculation were stained
DNA cleavage or necrosis. The DNA of U-87MG cells yellow to green fluorescence indicating apoptosis (Fig. 9).
treated for 24, 48 and 72 h with NDV strain AF2240 were The analysis of many different slides revealed that the
extracted and analyzed by agarose gel electrophoresis green staining was generally more pronounced in apoptotic
(Fig. 8). Untreated U-87MG cells contained only high- cells treated with the NDV strain AF2240 according to
molecular-weight genomic DNA. Whereas DNA extracted time (Fig. 9e).
from U-87MG at 24, 48 and 72 h post-inoculation showed
the characteristic pattern of nucleosomal laddering specific Effects of NDV on Cell Cycle Distribution
to apoptosis which was visible as faint bands on the gel
(Fig. 8). NDV strain AF2240 produced DNA fragments of The state of U-87MG cells was monitored by flow
lower molecular weight consisting of multimers of cytometry after propidium iodide staining nuclei. DNA
180 200 bp on U-87MG cell line. histogram showed that NDV strain AF2240 did not induce
cell cycle arrest in any specific phase (Fig. 10). In
Apoptotic (U-87MG) Cells Detection by TUNEL Assay untreated U-87MG cells, the G1, S, and G2/M populations
After Treatment of NDV represented 51.14, 31.10, and 14.63%, respectively
(Table 1). The treatment resulted in a loss of U-87MG cells
Apoptotic cells were detected and localization by green in all three phases of the cycle accompanied with a large
fluorescence (FITC-12-dUTP) on a red background [pro- increase in the sub-G1 region (Table 1). NDV induced a
pidium iodide (PI)] as observed by fluorescence micros- concentration- and time-dependent increase in the propor-
copy, as described above. Untreated U-87MG cells were tion of sub-G1 population. Apoptosis peak (sub-G1) was
shown in red because of the staining with propidium found in untreated cells with small percentage for about
iodide. Red staining is indicating viable cells which contain 3.14%. The sub-G1 population in U-87MG cells treated
intact genomic DNA. Whereas treated U-87MG cells were with NDV strain AF2240 were 10.29 and 19.45% for 24
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OD
% cell population
%cell poplulation
%cell population
2056 Neurochem Res (2011) 36:2051 2062
Fig. 5 a Phase contrast microscope examination of untreated blebbing (arrow). (magnification 2009). c Fluorescence microscopy
U-87MG cells and b phase contrast microscope examination of examination of Untreated U-87MG cells (control) and d fluorescence
U-87MG cells treated with NDV AF2240 (IC50, 52 HAU/ml) after microscopy examination of U-87 MG cells treated with NDV AF2240
72 h. Untreated cells showed normal structure without prominent IC50, 52 HAU/ml after 72 h. Viable cells are uniformly green with
apoptosis and necrosis. The virus caused the cells to lose contact with round nucleus (N), The apoptotic cells are green with condensed
adjacent cells, rounding up of nuclei and cell membrane. The virus chromatin (C), nuclear fragmentation (F), and cell membrane
caused the cells to lose contact with adjacent cells, and cell membrane blebbing (arrow) (magnification 4009) (Color figure online)
110 U-87MG cell lines undergo apoptosis more extensively
100
with increasing in time.
viable
90
apoptoic
80
necrotic
Early Apoptosis Detection on U-87MG Cells After
70
Treatment with NDV Strain AF2240 by Flow
60
Cytometry (Annexin V/PI Double Staining)
50
40
Apoptotic cells exclude all dyes which are in use for cell
30
viability assays, such as PI, while necrotic cells do not. In
20
cells with a damaged cell membrane PI induces a red
10
0 fluorescence on the DNA, whilst it is excluded by cells
control 24 hrs 48 hrs 72 hrs
with a preserved cytoplasm membrane. Hence during the
time (hours)
initial phase of apoptosis, the cells are still able to exclude
PI and therefore do not show any red fluorescence signal,
Fig. 6 Percentages of viable, apoptotic, and necrotic cells after NDV
strain AF2240 treatment using fluorescence microscopy. U-87MG similar to that of living cells. Figure 11 showed the results
cell death via apoptosis increased significantly (*P \ 0.001) in time-
of Annexin V/PI flow cytometry of U-87MG cells after
dependent manner. However, no significant (P [ 0.001) difference
treatment with IC50 value of NDV strain AF2240. The
was observed in the cell count of necrosis
lower left quadrant of the cytograms shows the viable cells,
which excluded PI and were negative for Annexin V
and 48 h post-inoculation, respectively (Fig. 10). This binding. The upper right quadrant represents the non-via-
virus-induced cell cycle perturbation concurred with the ble, necrotic cells, positive for Annexin V binding and
results of the TUNEL assay to suggest that brain tumor showing PI uptake. The lower right quadrant represents the
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cells percentage (%)
Neurochem Res (2011) 36:2051 2062 2057
Fig. 7 Transmission electron
micrographs of U-87MG cell
line cells at various stages of
apoptosis a untreated U-87MG
cell (control) with intact nucleus
(n) mitochondria (m) and clear
cytoplasm (magnification
8,0009). b Chromatin
condensed at the nuclear
periphery (arrow)
(magnification 12,0009).
c Apoptotic cell containing
nuclear fragments (arrow)
(magnification 8,0009).
d Membrane blebbing indicated
by arrow (magnification
8,0009). e Cell with obvious
vacuolization and numerous
formation of apoptotic bodies
indicated by arrow
(magnification 12,0009)
apoptotic cells, Annexin V positive and PI negative, the virus that cause 50% cell reduction. The IC50 values for
demonstrating Annexin V binding and cytoplasmic mem- cytolytic effect of NDV strain AF2240 on U-87MG cell
brane integrity (Fig. 11). The Annexin V ?/PI - apoptotic lines was 52 HAU/ml. However, no significant reduction in
cell population for U-87MG cell line increased from cell viability was observed in treated HCN-2 and 3T3
0.05 Ä… 1.94% in untreated cells, to 4.26 Ä… 2.97% in normal cell lines treated with NDV strain AF2240; there-
treated cells at 6 h post-infection. fore all investigations regarding the apoptogenic property
were carried on U-87MG. This result complies with the
previous studies, a study reported by Lorence et al. [23]
Discussion found that NDV strain 73-T killed human and rat neuro-
blastoma but not normal fibroblast. Another study showed
Oncolytic viruses are viruses that infect and replicate in that the NDV appears to replicate and kill tumor cells
cancer cells, destroying these harmful cells and leaving selectively better than normal human cells [37]. Further-
normal cells largely unaffected. Like all viruses, oncolytic more, a study by Meyyappan [29] reported that NDV strain
viruses seek to penetrate a host cell and   trick  it into AF2240 induced cytolytic effect on the MCF-7 and MDA-
replicating more of the virus until ultimately, it bursts. 231 breast cancer cell lines, with IC50 values of 64 and
NDV is an oncolytic virus with the ability to induce tumor 4 HAU/ml, respectively, and IC50 values of NDV strain
lysis through different mechanisms [41]. The results of V4-UPM on the MCF-7 and MDA-231 breast cancer cell
current study reveal that NDV strain AF2240 possesses lines were 128 and 96 HAU/ml, respectively. Wali [45]
promising antiproliferative properties against U-87MG cell stated that NDV strains F and Ijuk have cytolytic effet on
line. MTT assay was carried out to determine the titer of the MCF-7 and MDA-231 breast cancer cell lines. Another
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2058 Neurochem Res (2011) 36:2051 2062
6 5 4 3 2 1 tumor cell lines in vitro. It has a great potential to be an
effective anticancer due to the ability to kill cancer cells
and not normal cells.
Cell death can occur by two distinct processes: apoptosis
and necrosis [31]. In this study, we had confirmed that the
cell death caused by NDV strain AF2240 infection on brain
tumor cell line, anaplastic astrocytoma (U-87MG), occur-
red through apoptosis. Apoptotic cells share a number of
common features such as cell shrinkage, nuclear conden-
sation, membrane blebbing, chromatin cleavage, and for-
mation of pyknotic bodies of condensed chromatin [8, 20].
These distinctive morphological features form the basis of
some of the most widely used techniques for the identifi-
cation and quantification of apoptosis, and thus morpho-
logic description using Phase Contrast microscopy,
fluorescence microscopy or electron microscopy remains
one of the best ways to define apoptosis [8]. Phase Contrast
Microscopy was used to observe the unstained cells
changing within its media after inoculation of NDV to
determine apoptotic features. Rounding up of nuclei was
Fig. 8 Effects of NDV strain AF2240 on DNA fragmentation. DNA
one of the morphological changes occurred because of the
extracted from U-87MG cell line at various time periods. Lane 1:
marker, Lane 2: untreated cells, Lane 3: cells treated with treated with digesting of the protein structures, which conform the
NDV for 72 h, Lane 4: cells treated with NDV for 12 h, Lane 5: cells
cytoskeleton, by specialized peptidases (known as caspas-
treated with NDV for 24 h and Lane 6: cells treated with NDV for
es) that have been activated inside the cell [22]. Another
48 h
morphological change was that cells in culture lost contact
studies by Zawawi [48] and Alabsi [1] stated that NDV with adjacent cells, during which time any specialized
strain V4-UPM and AF2240 has cytolytic effect on leu- surface structures such as microvilli disappeared and con-
kemia cell lines in vitro and in vivo. In conclusion, the volutions began to form [22]. Transmission electron
NDV AF2240 is an effective anti-cancer agent in brain microscopy could reveal the changes of cell ultrastructure
Fig. 9 (TUNEL) assay.
a Untreated cells. b TUNEL-
stained U-87MG cells at 24
post-inoculation. c TUNEL-
stained U-87MG cells at 48
post-inoculation. d TUNEL-
stained U-87MG cells at 72
post-inoculation. Cells were
double-stained with fluorescein-
12-dUTP and propidium iodide. A B
Normal cells show red nuclei,
whereas apoptotic cells show
yellow to green nuclei (arrows)
(magnification 4009).
e Percentage of apoptotic
U-87MG cells. Cells revealed
that apoptotic cells generally
increasing after treatment with
the NDV strain AF2240
according to time compare with
CD
untreated cells (Color figure
online)
E
Treated Normal cells
97% 98% 96%
cells
apoptotic cells
3% 2% 4%
Untreated Normal cells
% % %
cells
apoptotic cells
45% 55% 69%
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Neurochem Res (2011) 36:2051 2062 2059
Fig. 10 Cell cycle (DNA
content) flow cytometer
histograms of U-87MG brain
tumor cell lines treated with
IC50 value of NDV strain
AF2240 a untreated cells
(control) b cells treated with
NDV after 24 h c cells treated
with NDV after 48 h. Shows
increasing in percentage of sub-
G1 (apoptotic) cell population
(broken DNA)
Table 1 Percentage of U-87MG cells in different cell cycle phase after treatment with virus
U-87MG cells Cell cycle phase
Sub-G1 (%) G1 (%) S (%) G2/M (%)
Untreated cells (control) 3.14 51.14 31.10 14.63
Cells treated after 24 h 10.29 49.63 29.94 10.14
Cells treated after 48 h 19.45 47.78 25.70 8.06
Fig. 11 Contour diagram of Annexin V/PI flow cytometry contain the non-viable, necrotic cells, positive for Annexin V binding
a untreated U-87MG and b U-87MG cells at 6 h post-inoculation and for PI uptake. The lower right quadrants (R3) represent the
of IC50 value of NDV strain AF 2240. The lower left quadrants of apoptotic cells, Annexin V positive and PI negative. The Annexin
each panel (Rl) show the viable cells, which exclude PI and are V ?/PI - apoptotic cell population for U-87MG cell line increased
negative for Annexin V binding. The upper right quadrants (R2) from 0.05 Ä… 1.94% in untreated cells, to 4.26 Ä… 2.97%
during the apoptotic process [21]. Intracellular and plasma permeable cationic dye that binds to nucleic acids of viable
membrane structural modifications have been widely rec- cells and at low concentrations it causes a green fluores-
ognized as crucial factors involved in cell injury and death. cence. PI is impermeable to intact membranes but readily
Changes in nuclear morphology and in organelle structure penetrates the membranes of nonviable cells and binds to
as well as specific phenomena at the cell surface, namely DNA or RNA, causing orange fluorescence. When AO and
surface smoothing and surface blebbing, are often consid- PI are used simultaneously, viable cells stained green
ered as markers associated with cell pathology [25]. fluorescence under dark field fluorescence microscopy,
Morphological features of apoptosis were also observed while nonviable cells stained fluorescence orange [27].
by using fluorescent microscopy (AO/PI method). Chro- The cell viability results showed an obvious decrease of
matin condensation, nuclear shrinkage and formation of living cells in treated U-87MG cells. When viewed under
apoptotic bodies can easily be observed under fluorescence fluorescence microscopy, it was observed that untreated
microscopy, after appropriate staining of nuclei with DNA- cells had round intact nuclei and stained green indicating
specific fluorochromes [6]. The AO/PI viability assay is a viable cells whereas the treated cells stained green and
fluorometric cell viability assay. AO is a membrane- exhibited features of apoptotic cells, which had irregular
123
2060 Neurochem Res (2011) 36:2051 2062
shaped nucleus, shrinkage, condensed chromatin and cell at 24, 48 and 72 h post-inoculation. The DNA ladder assay
membrane blebbing [5]. Quantification of apoptosis with can be decreased in samples where the number of apoptotic
differential scoring of treated and control cells revealed a cells is low [28] and during the apoptotic process genomic
significant (P \ 0.05) difference in the number of apop- DNA is cleaved in more consequent steps, first cleavage
totic cells in a time-dependent manner but with insignifi- into fragments with length 300 kbp is followed by addi-
cant difference between numbers of necrosis positive cells tional fragmentation into 50 kbp and thereafter to oligo-
in control and treated cells, concluding that NDV strain nucleosomal DNA fragments [35]. It was also reported by
AF2240, did not induce necrotic effects. Fluorescence [6] that DNA laddering is not detectable in adherent cells
microscopy examination confirmed the onset of apoptosis but is characteristic of the floating cells regardless of
features. These morphological criteria that implicate treatment. Even with enhancement of the fragmented DNA
apoptotic cell death were further confirmed by transmission in the sample, DNA ladder formation is observed only
electron microscopy and transmission electron microscopy. when the extent of oligonucleosomal cleavage is promi-
TEM has been considered a milestone of the research in nent. Internucleosomal cleavage of DNA is likely to be in
the field of apoptosis. By means of TEM the results in this the later phase of apoptotic process [7, 16, 35]. In most cell
study showed that untreated brain tumor U-87MG cells types, the biochemical characteristics of apoptotic response
showed no changes and exhibited intact nucleus with clear include activation of endogenous calcium and magnesium
cytoplasm while after treatment of U-87MG cells with dependent endonucleases, leading to fragmentation of the
NDV strain AF2240, some cells showed typical morpho- chromosomal DNA. Initially, the DNA fragments are large
logic changes of apoptosis, including shrinkage and (50 300 kb) but are later digested to oligonucleosomal size
membrane blebbing. Other changes occur within the (multimers of 180 200 bp). The formation of this distinct
nucleus during apoptotic death such as the appearance of DNA ladder is considered to be a biochemical hallmark of
dense, crescent-shaped chromatin aggregates which line apoptosis.
nuclear membrane. Later, the nucleolus disintegrates; Further confirmation of the mode of cell death was
nuclear membrane develops deep invaginations and, ulti- carried by flow cytometric analysis of cell cycle. It mea-
mately, the nucleus fragmente into dense granular particles sures apoptotic changes in cells by staining with DNA dyes
(apoptotic bodies) which eventually degenerated to   sec- [43]. This method is useful for quantitative estimates of the
ondary necrosis  [38]. It is thought that the nuclear chan- fractions of cells in the different phases of the cell cycle
ges are due to activation of endogenous nuclease(s) which [13]. Untreated and treated U-87MG brain cell line were
cleaves DNA into oligonucleosomal fragments [35, 38]. evaluated for apoptosis by measuring the amount of
In addition the terminal deoxynucleotidyl transferase apoptotic cells using of DNA flow cytometry (FCM). The
(TdT)-mediated dUTP-fluorescein nick-end labeling apoptotic cells with degraded DNA were represented in so-
(TUNEL) assay was also conducted to confirm the results called   sub-G1  peaks on DNA histograms. Apoptotic
of morphological features of the oncolytic effect of NDV cells, due to a change in membrane permeability, showed
on the U-87MG brain cell line. TUNEL staining is the an increased up-take of the vital dye, PI, compared to live
standard technique for detection of apoptosis because it cells [33, 43].
permits visualization of DNA cleavage which can be Figures and tables reveal that there was a loss of treated
visualized and quantities directly by fluorescence micros- U-87MG cells in all three phases of the cycle (G1, S and
copy [14, 15, 39, 46]. G2/M) accompanied with a large increase in the sub-G1
This test showed that NDV strain AF2240 caused a region (apoptosis peak) in the fluorescence histograms.
significant increase in the percentage of apoptotic cells on Concluding that NDV strain AF2240 caused an increasing
U-87MG brain cell line and the percentage of apoptotic in the percentage of sub-G1 region which increased with
cells increased with increasing of time, in which apoptotic increasing of time and did not induce specific cell cycle
cells were more abounded at longer durations of treatment. arrest in specific phase.
In the cell populations of control in both cell lines, cells Annexin binding assay is a method permits the detection
were having intact DNA or low undetectable levels of of the early phases of apoptosis before the loss of cell
fragmentation but treated cells were TUNEL positive and membrane integrity [3, 44]. This is based on the phenom-
apoptotic cells increased correlated with the time of enon that phosphatidylserine (PS) is exposed at the outer
treatment. membrane of the cell during apoptosis and on the ability of
DNA ladder assay provides a sensitive assay for the annexin V to bind to PS with high affinity [10]. Phospha-
detection of DNA fragmentation, but this method is qual- tidylserine (PS) only exists in the cytoplasm side of
itative rather than quantitative [44]. In this study we cell plasma membrane, and externalization of PS occurs in
reported that NDV strain AF2240 stimulate DNA frag- the early stage of apoptosis. Annexin-V could specifi-
mentation characteristic of apoptosis in U-87MG cell line cally conjugate to the PS to detect the apoptotic cells [21].
123
Neurochem Res (2011) 36:2051 2062 2061
9. Doyle TM (1927) A hitherto unrecorded disease of fowls due to a
The analysis of the treated U-87MG cells by annexin V
filter passing virus. J Comp Pathol 40:144 169
versus PI, revealed four populations: live cells, apoptotic
10. Engeland MV, Nieland LJW, Ramaekers FCS, Schutte B,
cells, late apoptotic cells and permeabilized cells. In this
Reutelingsperger CPM (1998) Annexin V-Affinity assay: a
experiment, we observed increasing in the percentage of
review on an apoptosis detection system based on phosphatidyl-
serine exposure. Cytom 31:1 9
target cells in the annexin positive PI negative (early
11. Fenner F, Bachmann P, Gibbs E, Murphy F, Studdert M, White D
apoptosis) quadrant at 6 h post-inoculation.
(1987) Veterinary Virol. Academic Press, Orlando
These results complies with the other studies [1, 29, 45,
12. Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L,
48], which stated that the effectiveness of NDV strains
Greenbaum E, Rozenman-Yair S, Panet A, Libson E, Irving CS,
Galun E, Siegal T (2006) Phase I/II trial of intravenous NDV-
included NDV strain AF2240 as oncolytic agent was found
HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol
on breast cancer cell lines and leukemia cell lines and the
Ther 13:221 228
nature of cell death caused by this virus was characterized
13. Fried J, Perez AG, Clarkson BD (1976) Flow cytofluorometric
as apoptosis. Therefore these results show that NDV strain
analysis of cell cycle distributions using propidium iodide,
Properties of the method and mathematical analysis of the data.
AF2240 was capable to induce apoptosis on brain tumor
J Cell Biol 71:172 181
cells, anaplastic astrocytoma (U-87MG), in vitro.
14. Gao N, Keane MJ, Ong T, Ye J, Miller WE, Wallace WE (2001)
To put these results together, we believe that the
Effects of phospholipid surfactant on apoptosis induction by
induction of apoptosis play a role in inhibiting brain tumor
respirable quartz and kaolin in NR8383 rat pulmonary. Toxicol
cells when treat with NDV strain AF2240 and that cyto- Appl Pharmacol 175:217 225
15. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of
toxicity increasing while increasing the titer of the virus.
programmed cell death in situ via specific labeling of nuclear
This discovery may provide a theoretical basis for this type
DNA fragmentation. J Cell Biol 119:493 501
of treatment. The mechanism of the virus infection is still
16. Gooch JL, Yee D (1999) Strain-specific differences in formation
of apoptotic DNA ladders in MCF-7 breast cancer cells. Cancer
unclear. NDV would have a bright future in the treatment
Lett 144:31 37
of tumors and further work may lead to relative antitumor
17. Guan T, Qin F, Du J, Geng L, Zhang Y, Li M (2007) AICAR inhibits
agents to be used in clinical settings.
proliferation and induced S-phase arrest, and promotes apoptosis in
CaSki cells. Acta Pharmacologica Sinica 28:1984 1990
Acknowledgments This research was funded in part by the
18. Henson JW (2005) Glioblastoma multiforme and anaplastic gli-
National Cancer Council (MAKNA), Malaysia. The authors also
oma: a patient guide. http://brain.mgh.harvard.edu/PatientGuide.
acknowledge additional support from Universiti Putra Malaysia
htm. Accessed on 7 Feb
(UPM), Serdang, Malaysia.
19. Kirn D, Martuza RL, Zwiebel J (2001) Replication-selective
virotherapy for cancer: biological principles, risk management
and future directions. Nat Med 7:781 787
20. Lin JC, Ho YS, Lee JJ, Liu CL, Yang TL, Wu CH
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