ORIGINAL PAPER

Cytolytic Effects and Apoptosis Induction of Newcastle DiseaseVirus Strain AF2240 on Anaplastic Astrocytoma Brain TumorCell 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

� Springer Science+Business Media, LLC 2011

Abstract Newcastle disease virus (NDV) is a member of

genus Avulavirus within the family Paramyxoviridae.

Interest of using NDV as an anticancer agent has arisen from

its ability to kill tumor cells with limited toxicity to normal

cells. In this investigation, the cytotolytic properties of NDV

strain AF2240 were evaluated on brain tumor cell line,

anaplastic astrocytoma (U-87MG), by using MTT assay.

Cytological observations were studied using fluorescence

microscopy and transmission electron microscopy to

show the apoptogenic features of NDV on U-87MG. DNA

laddering in agarose gel electrophoresis and terminal

deoxyribonucleotide transferase-mediated dUTP-X nick

end-labeling staining assay confirmed that the mode of cell

death was by apoptosis. However, analysis of the cellular

DNA content by flowcytometery showed that there was a

loss of treated U-87MG cells in all cell cycle phases (G1, S

and G2/M) accompanied with increasing in sub-G1 region

(apoptosis peak). Early apoptosis was observed 6 h post-

inoculation by annexin-V flow-cytometry method. It could

be concluded that NDV strain AF2240 is a potent antitumor

agent that induce apoptosis and its cytotoxicity increasing

while increasing of time and virus titer.

Keywords NDV strain AF2240 � Cytotoxicity �Apoptosis � Flow-cytometry � Anaplastic astrocytoma

Background

Brain tumor is formed by abnormal and uncontrolled cell

division; it is abnormal growth of tissue found inside the

skull. It can be dangerous because of the importance of the

brain and the limited amount of space inside the skull.

Most primary brain tumors do not metastasize systemically

but spread locally through extensions of infiltrating tumor

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

adults [42]. Surgery; radiation and chemotherapy are the

current treatments for brain tumor [18, 24, 26, 40]. New-

castle disease virus (NDV) was first isolated in 1926 in

Newcastle, England, in domestic chickens where it caused

a severe disease known as Newcastle disease. ND is a

disease of poultry that affecting the alimentary and respi-

ratory tract as well as the central nervous system [9].

Exposure to humans however, results in mild conjunctivi-

tis, laryngitis and influenza-like symptoms [11]. The NDV

showed promise in clinical trials as an antineoplastic agent.

It is one of the nonengineered oncolytic viruses, which has

a long history as a broad-spectrum oncolytic agent that can

destroy tumor cells and stimulate the immune system

[12, 19]. Many preclinical studies indicate that NDV rep-

licates in human cancer cells but not in normal cells, some

R. Ali � K. Yusoff

Faculty of Biotechnology and Bimolecular Sciences,

Universiti Putra Malaysia, Serdang, Malaysia

A. Ideris � A. R. Omar

Faculty of Veterinary Medicine, Universiti Putra Malaysia,

Serdang, Malaysia

A. M. Alabsi � A. M. Ali (&)

Faculty of Agricultures and Biotechnology, Universiti Sultan

Zainal Abidin, Kuala Terengganu, Malaysia

e-mail: ailali7673@yahoo.com

A. M. Alabsi

e-mail: aied@unisza.edu.my

R. Saif-Ali

Faculty of Medicine, Universiti Malaya,

Kuala Lumpur, Malaysia

123

Neurochem Res (2011) 36:2051–2062

DOI 10.1007/s11064-011-0529-8

types of NDV are able to directly kill certain types of

cancer cells and NDV–infected cancer cells can cause the

immune system to respond in different ways [34]. Early

success with the viral vaccine in humans was first reported

in the United States in 1980 [32]. Interest in the use of

NDV as an anticancer agent has arisen from the ability of

the virus to selectively kill human tumor cells with limited

toxicity to normal cells. Many strains of NDV (73-T,

MH68, Italian, Ulester, Rokin, PV701 and HUJ) have been

shown to exhibit an oncolytic activity [12, 36]. In addition,

the oncolytic effects of six Malaysian strains of NDV

(AF2240, 01/C, Ijuk, S, F, and V4) have also been studied

on several tumor cell lines [34, 36]. The goal of most

cancer therapy is to reduce the number of tumor cells and

to prevent their further accumulation (decrease the rate of

cell proliferation). Therefore, in this study the effects of

NDV strain AF2240 on the proliferation and the morpho-

logical changes of brain tumor cell line, anaplastic astro-

cytoma (U-87MG), was tested in vitro.

Materials and Methods

Propagation and Purification of NDV Strain AF2240

NDV was propagated in allantoic fluid of 9–11 days-old

embryonated chicken eggs at 37�C for 48 h. The allantoic

fluid was harvested and the presence of virus was con-

firmed by the haemaglutination test [2]. NDV strains

AF2240 purified as previously described [4, 47].

Cells and Cell Culture

Human brain tumor cell line U-87MG was obtained from

American Type Culture Collection (ATCC, VA, USA).

HCN-2 and 3T3, normal cell lines, kindly provided by Prof.

Dr. Abdul Manaf Ali, Faculty of Biotechnology and

Molecular Biology, University Putra Malaysia (UPM) were

used as control. All cell lines were grown as a monolayer in

25 cm2 tissue culture flasks (NunclonTM, Denmark) at 37�C

in an atmosphere of 5% CO2. MEM medium supplemented

with 10% fetal calf serum, 1% antibiotics and three addi-

tional supplements (2 mM Glutamine, 1 mM Sodium

Pyruvate, and 1% nonessential amino acid) was used for

U-87MG. Normal cell lines were grown in DMEM medium.

MTT Cytotoxicity Assay

U-87MG cells were trypsinized and counted using hemo-

cytometer then were seeded in 96-well micro plate at

3 9 105 cells/ml and then incubated at 37�C in 5% CO2 to

allow cells attachment. The medium was removed and

replaced with fresh medium containing test virus at various

concentrations (double dilution) and were incubated at

37�C, 5% CO2 for 72 h. Each concentration was assayed in

triplicates (n = 3). Seventy-two hours later, 20 ll of MTT

(5 mg/ml) solution was added to each well and then the plate

was further incubated for 4 h. All remaining supernatant

were removed and 150 ll of DMSO was added to dissolve

the formed crystal formazan. MTT assay reading was per-

formed using ELISA plate reader (Biotek EL340, USA).

Quantification of Apoptosis Using Propidium Iodide

and Acridine Orange Double staining

Cells were quantified using propidium iodide (PI) and

acridine-orange (AO) double staining according to standard

procedures and examine under fluorescence microscope

(Lieca attached with Q-Floro Software) [17, 30]. U-87MG

cells were seeded in six-well plate and incubated at 37�C in

5% CO2 atmosphere. Twenty-four hours later, the medium

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

incubation period, detached cells in the medium were

collected and added back to trypsinised adherent cells. The

cell suspensions were washed with PBS and then incubated

with 5 ll of acridine orange (10 lg/ml) and 5 ll propidium

iodide (10 lg/ml) at a ratio of 1:1 in 1 ml of cells and

recentrifuged at 1,000 rpm/5 min. After centrifuge, super-

natant was removed leaving 50 ll of remaining supernatant

with pellet. The pellet was resuspended and 10 ll of cell

suspension was pipetted on slide before putting on cover

slip. Within 30 min, the slide was analyzed using fluores-

cent microscope (Leica, Germany). Each experiment was

assayed three times (n = 3). Viable, apoptotic and necrotic

cells was quantified in a population of 200 cells. In apop-

totic bodies, blebbing of plasma membrane and conden-

sation of chromatin were seen. In contrast, necrotic cells

fluoresced red after propidium iodide staining with tiny

fraction of chromatin dispersed around the nucleus. This

assay provides a useful quantitative evaluation and was

done three times (n = 3).

Transmission Unltrastructural Effects of NDV Strains

AF2240 on U-87MG Cells (TEM)

U-87MG brain tumor cells were treated with NDV at IC50

concentration and incubated for 24, 48, and 72 h at 37�C.

The cultured cells were harvested using trypsin and cen-

trifuged for 10 min at 1,500 rpm. The pellets were fixed in

4% (v/v) glutaraldehyde in 0.1 M coccadylate buffer (pH

7.4) for 4 h at 4�C. The fixed cells were centrifuged, and

the pellets were blocked in serum which was later fixed

in glutaraldeyde overnight at 4�C. The specimens were

washed in three changes of sodium coccadylate buffer

2052 Neurochem Res (2011) 36:2051–2062

123

(pH 7.4) for 10 min each, postfixed in 1% osmium tetraoxide

at 4�C. The specimens were then washed in three changes of

sodium coccadylate buffer (pH 7.4) for 10 min each and

dehydrated with a graded series of acetone (35, 50, 75, 95,

and 100%). The cells were then infiltrated with acetone and

resin and embedded with 100% resin in beam capsule, and

left to polymerize at 60�C for 48 h. The area of interest in the

embedded cells resin block was chosen using the toulidine

blue staining and later examined using light microscope. The

selected area was cut in ulltrathin sections using ultrami-

crotome. The sections were placed into a grid and stained

with uranyl acetate for 10 min followed by 50% filtered

acetone, and finally stained using lead which was then

washed twice with distilled water. The stained samples were

then viewed under transmission electron microscopy (Phil-

lips, Eindhoven, The Netherlands).

DNA Fragmentation Assay

Cells at a concentration of 5 9 106 cells/ml were seeded

into six-well plate (NunclonTM, Denmark) in 2 ml culture

medium with a concentration of IC50 value of virus. Some

wells were left with no virus to be used as a control. After

the 72 h of incubation, detached cells in the medium were

collected and added back to trypsinised adherent cells. The

cells were spun down at 1,000 rpm for 10 min. The

supernatant was discarded and the pellet was washed with

PBS twice. The DNA extraction from treated and untreated

cells was carried out according to protocol of a kit for

Blood and Cultured Cells from QIAGEN.

TUNEL Assay

The cells treated with concentration of IC50 value of virus

value were grown on Lab-Tek Chamber slides and the

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

processed in the apoptosis detection assay. Each experi-

ment was assayed three times (n = 3). The TUNEL Assay

was carried out using a kit for Apoptosis Detection from

Promega, USA.

Analysis of Cellular DNA Content Using Propidium

Iodide

Cells at a concentration of 5 9 106 cells/ml of U-87MG

cell line was seeded into six-well plate in 2 ml culture

medium with a concentration of IC50 value of virus and

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

control. After the incubation period, the cultured cells were

harvested using trypsin and centrifuged for 10 min at

1,000 rpm at room temperature. Cell pellets were fixed by

adding 500 ll of 80% cold ethanol and kept for at least 2 h

at -20�C. Cells were pelleted at 1,000 rpm for 10 min and

the ethanol was discarded. The cell pellet was washed with

1 ml (PBS/sodium azide) twice. The pellet was resus-

pended with 1 ml of (PBS ? 0.1% triton X-100 ?10 mm

EDTA ? 50 lg/ml RNase ? 2 lg/ml Propidium iodide)

followed by incubated for � to 1 h at 4�C. Finally, samples

were placed in 12 9 75 Falcon tubes and the cell cycle was

analyzed by flow cytometer (Beckman Coulter, USA).

Each experiment was assayed three times (n = 3).

Flow Cytometry (Annexin V/PI Double Staining)

Cells at a concentration of 5 9 106 cells/ml of U-87MG

cell line was seeded into six-well plate in 2 ml culture

medium with a concentration of IC50 value of virus and

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

control. After the incubation period, the cultured cells were

harvested using trypsin and centrifuged for 10 min at

1,000 rpm. The early apoptosis detection for treated and

untreated cells was carried out using Annexin V & Apo

2.7-PE kit (Clontech Laboratories, Inc., USA). Each

experiment was assayed three times (n = 3).

Statistical Analysis

Data was expressed as mean ± SD. Statistical analysis was

performed with Student’s t-test for data from MTT cyto-

toxicity assay, AO/PI staining assay, TUNEL and, flow

cytometry. Differences were considered significant at

P \ 0.001.

Results

Cytotolytic Effects of NDV on Normal Cells and Brain

Tumor Cells

The cytotoxicity of NDV on U87 cells was investigated

using MTT assay after 72 h treatment (Figs. 1, 4). NDV

was clearly found to exert antiproliferative effects toward

U87 brain tumor cells. The IC50 value, which is the con-

centration required for 50% growth inhibition, is deter-

mined to be 52 ± 1.1. Comparatively, NDV showed no

significant cytolytic effect at the same titre used in the

brain cell line toward HCN-2 and 3T3, normal cell lines

(Figs. 2, 3).

Phase Contrast Microscope

The morphological changes of U-87MG cells after treat-

ment with NDV strain AF2240 were observed under Phase

Neurochem Res (2011) 36:2051–2062 2053

123

Contrast Microscope. The cells were treated with the IC50

value of the virus showed morphological changes including

rounding up of nuclei, shrinkage or decreased of nuclear

diameters and condensation of chromatin occurred. Fur-

thermore the cells in culture lost contact with adjacent cells

(Fig. 5).

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

observed with a green intact nuclear structure blebbing and

nuclear margination were noticed. Differential scoring of

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-

chondria in treated cells were ruptured and condensed

(Fig. 7c). The evidences which suggested that the apopto-

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

of the cellular membrane occurred at 48 and 72 h post-

inoculation indicating of late stage of apoptosis. At 72 h

post-inoculation secondary necrosis was observed and

some apoptotic bodies eventually degenerated (Fig. 7).

Effects of NDV on Brain Tumor Cell (U-87MG) DNA

Fragmentation

Fragmentation of chromosomal DNA is the biological

hallmark of apoptosis, and can be detected by a ladder

Fig. 1 Cytolytic effects of NDV strain AF2240 on U-87MG cell line.

The IC50 value is the concentration required for 50% growth

inhibition. Fifty percent of cell viability (IC50) was obtained at virus

titer of 52 ± 1.76 HAU/ml

Fig. 2 Cytolytic effect of NDV AF2240 strain on HCN-2 cell line.

The IC50 value was not obtained because the cell reduction was not

effected by same virus titer

Fig. 3 Cytolytic effect of NDV AF2240 strain on 3T3 cell line. The

IC50 value was not obtained because the cell reduction was not

effected by same virus titer

2054 Neurochem Res (2011) 36:2051–2062

123

formation on gel electrophoresis. The DNA ladder assay is

generally accepted as specific for apoptosis because it

detects oligonucleosomal cleavage rather than artificial

DNA cleavage or necrosis. The DNA of U-87MG cells

treated for 24, 48 and 72 h with NDV strain AF2240 were

extracted and analyzed by agarose gel electrophoresis

(Fig. 8). Untreated U-87MG cells contained only high-

molecular-weight genomic DNA. Whereas DNA extracted

from U-87MG at 24, 48 and 72 h post-inoculation showed

the characteristic pattern of nucleosomal laddering specific

to apoptosis which was visible as faint bands on the gel

(Fig. 8). NDV strain AF2240 produced DNA fragments of

lower molecular weight consisting of multimers of

180–200 bp on U-87MG cell line.

Apoptotic (U-87MG) Cells Detection by TUNEL Assay

After Treatment of NDV

Apoptotic cells were detected and localization by green

fluorescence (FITC-12-dUTP) on a red background [pro-

pidium iodide (PI)] as observed by fluorescence micros-

copy, as described above. Untreated U-87MG cells were

shown in red because of the staining with propidium

iodide. Red staining is indicating viable cells which contain

intact genomic DNA. Whereas treated U-87MG cells were

shown the TUNEL? nuclei appeared as yellow to green

fluorescence. The treated cells containing multiple DNA

breaks at 24, 48 and 72 h post-inoculation were stained

yellow to green fluorescence indicating apoptosis (Fig. 9).

The analysis of many different slides revealed that the

green staining was generally more pronounced in apoptotic

cells treated with the NDV strain AF2240 according to

time (Fig. 9e).

Effects of NDV on Cell Cycle Distribution

The state of U-87MG cells was monitored by flow

cytometry after propidium iodide staining nuclei. DNA

histogram showed that NDV strain AF2240 did not induce

cell cycle arrest in any specific phase (Fig. 10). In

untreated U-87MG cells, the G1, S, and G2/M populations

represented 51.14, 31.10, and 14.63%, respectively

(Table 1). The treatment resulted in a loss of U-87MG cells

in all three phases of the cycle accompanied with a large

increase in the sub-G1 region (Table 1). NDV induced a

concentration- and time-dependent increase in the propor-

tion of sub-G1 population. Apoptosis peak (sub-G1) was

found in untreated cells with small percentage for about

3.14%. The sub-G1 population in U-87MG cells treated

with NDV strain AF2240 were 10.29 and 19.45% for 24

A

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0.6

0.8

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D

CONTROL IC 50 IC 25

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55

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%ce

ll p

op

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0 20 40 60 80 1 2 3

1 2 3 1 2 3

days

non-viable viablenon-viable viable

non-viable viableFig. 4 MTT Proliferation assay

for various virus titers (IC50 and

IC25) against U-87MG brain

tumor cell line at 24, 48 and

72 h post-inoculation. a The

growth rates decreased in the

treated cells as compared with

the untreated cells whereas

inoculation with a higher titter

of virus (IC50) decreased the

growth rate morethan low titre

(IC25). 1 The percentage of

viable and non-viable U-87MG

cells population of untreated

cells (control). 2 The percentage

of viable and non-viable

U-87MG cells population

treated with IC25 value of NDV

AF2240 titer. 3 The percentage

of viable and non-viable

U-87MG cells population

treated with IC50 value of NDV

AF2240 titer

Neurochem Res (2011) 36:2051–2062 2055

123

and 48 h post-inoculation, respectively (Fig. 10). This

virus-induced cell cycle perturbation concurred with the

results of the TUNEL assay to suggest that brain tumor

U-87MG cell lines undergo apoptosis more extensively

with increasing in time.

Early Apoptosis Detection on U-87MG Cells After

Treatment with NDV Strain AF2240 by Flow

Cytometry (Annexin V/PI Double Staining)

Apoptotic cells exclude all dyes which are in use for cell

viability assays, such as PI, while necrotic cells do not. In

cells with a damaged cell membrane PI induces a red

fluorescence on the DNA, whilst it is excluded by cells

with a preserved cytoplasm membrane. Hence during the

initial phase of apoptosis, the cells are still able to exclude

PI and therefore do not show any red fluorescence signal,

similar to that of living cells. Figure 11 showed the results

of Annexin V/PI flow cytometry of U-87MG cells after

treatment with IC50 value of NDV strain AF2240. The

lower left quadrant of the cytograms shows the viable cells,

which excluded PI and were negative for Annexin V

binding. The upper right quadrant represents the non-via-

ble, necrotic cells, positive for Annexin V binding and

showing PI uptake. The lower right quadrant represents the

Fig. 5 a Phase contrast microscope examination of untreated

U-87MG cells and b phase contrast microscope examination of

U-87MG cells treated with NDV AF2240 (IC50, 52 HAU/ml) after

72 h. Untreated cells showed normal structure without prominent

apoptosis and necrosis. The virus caused the cells to lose contact with

adjacent cells, rounding up of nuclei and cell membrane. The virus

caused the cells to lose contact with adjacent cells, and cell membrane

blebbing (arrow). (magnification 2009). c Fluorescence microscopy

examination of Untreated U-87MG cells (control) and d fluorescence

microscopy examination of U-87 MG cells treated with NDV AF2240

IC50, 52 HAU/ml after 72 h. Viable cells are uniformly green with

round nucleus (N), The apoptotic cells are green with condensed

chromatin (C), nuclear fragmentation (F), and cell membrane

blebbing (arrow) (magnification 4009) (Color figure online)

0

10

20

30

40

50

60

70

80

90

100

110

control 24 hrs 48 hrs 72 hrs

time (hours)

cells

per

cen

tag

e (%

)

viable

apoptoic

necrotic

Fig. 6 Percentages of viable, apoptotic, and necrotic cells after NDV

strain AF2240 treatment using fluorescence microscopy. U-87MG

cell death via apoptosis increased significantly (*P \ 0.001) in time-

dependent manner. However, no significant (P [ 0.001) difference

was observed in the cell count of necrosis

2056 Neurochem Res (2011) 36:2051–2062

123

apoptotic cells, Annexin V positive and PI negative,

demonstrating Annexin V binding and cytoplasmic mem-

brane integrity (Fig. 11). The Annexin V ?/PI - apoptotic

cell population for U-87MG cell line increased from

0.05 ± 1.94% in untreated cells, to 4.26 ± 2.97% in

treated cells at 6 h post-infection.

Discussion

Oncolytic viruses are viruses that infect and replicate in

cancer cells, destroying these harmful cells and leaving

normal cells largely unaffected. Like all viruses, oncolytic

viruses seek to penetrate a host cell and ‘‘trick’’ it into

replicating more of the virus until ultimately, it bursts.

NDV is an oncolytic virus with the ability to induce tumor

lysis through different mechanisms [41]. The results of

current study reveal that NDV strain AF2240 possesses

promising antiproliferative properties against U-87MG cell

line. MTT assay was carried out to determine the titer of

the virus that cause 50% cell reduction. The IC50 values for

cytolytic effect of NDV strain AF2240 on U-87MG cell

lines was 52 HAU/ml. However, no significant reduction in

cell viability was observed in treated HCN-2 and 3T3

normal cell lines treated with NDV strain AF2240; there-

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]

found that NDV strain 73-T killed human and rat neuro-

blastoma but not normal fibroblast. Another study showed

that the NDV appears to replicate and kill tumor cells

selectively better than normal human cells [37]. Further-

more, a study by Meyyappan [29] reported that NDV strain

AF2240 induced cytolytic effect on the MCF-7 and MDA-

231 breast cancer cell lines, with IC50 values of 64 and

4 HAU/ml, respectively, and IC50 values of NDV strain

V4-UPM on the MCF-7 and MDA-231 breast cancer cell

lines were 128 and 96 HAU/ml, respectively. Wali [45]

stated that NDV strains F and Ijuk have cytolytic effet on

the MCF-7 and MDA-231 breast cancer cell lines. Another

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)

Neurochem Res (2011) 36:2051–2062 2057

123

studies by Zawawi [48] and Alabsi [1] stated that NDV

strain V4-UPM and AF2240 has cytolytic effect on leu-

kemia cell lines in vitro and in vivo. In conclusion, the

NDV AF2240 is an effective anti-cancer agent in brain

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

one of the morphological changes occurred because of the

digesting of the protein structures, which conform the

cytoskeleton, by specialized peptidases (known as caspas-

es) that have been activated inside the cell [22]. Another

morphological change was that cells in culture lost contact

with adjacent cells, during which time any specialized

surface structures such as microvilli disappeared and con-

volutions began to form [22]. Transmission electron

microscopy could reveal the changes of cell ultrastructure

6 5 4 3 2 1

Fig. 8 Effects of NDV strain AF2240 on DNA fragmentation. DNA

extracted from U-87MG cell line at various time periods. Lane 1:

marker, Lane 2: untreated cells, Lane 3: cells treated with treated with

NDV for 72 h, Lane 4: cells treated with NDV for 12 h, Lane 5: cells

treated with NDV for 24 h and Lane 6: cells treated with NDV for

48 h

%

E Treated cells

Normal cells 97% 89 96% apoptotic cells 3% %2 4%

Untreated cells

Normal cells % % %apoptotic cells 45% %55 69%

C D

A B

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.

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

untreated cells (Color figure

online)

2058 Neurochem Res (2011) 36:2051–2062

123

during the apoptotic process [21]. Intracellular and plasma

membrane structural modifications have been widely rec-

ognized as crucial factors involved in cell injury and death.

Changes in nuclear morphology and in organelle structure

as well as specific phenomena at the cell surface, namely

surface smoothing and surface blebbing, are often consid-

ered as markers associated with cell pathology [25].

Morphological features of apoptosis were also observed

by using fluorescent microscopy (AO/PI method). Chro-

matin condensation, nuclear shrinkage and formation of

apoptotic bodies can easily be observed under fluorescence

microscopy, after appropriate staining of nuclei with DNA-

specific fluorochromes [6]. The AO/PI viability assay is a

fluorometric cell viability assay. AO is a membrane-

permeable cationic dye that binds to nucleic acids of viable

cells and at low concentrations it causes a green fluores-

cence. PI is impermeable to intact membranes but readily

penetrates the membranes of nonviable cells and binds to

DNA or RNA, causing orange fluorescence. When AO and

PI are used simultaneously, viable cells stained green

fluorescence under dark field fluorescence microscopy,

while nonviable cells stained fluorescence orange [27].

The cell viability results showed an obvious decrease of

living cells in treated U-87MG cells. When viewed under

fluorescence microscopy, it was observed that untreated

cells had round intact nuclei and stained green indicating

viable cells whereas the treated cells stained green and

exhibited features of apoptotic cells, which had irregular

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

a untreated U-87MG and b U-87MG cells at 6 h post-inoculation

of IC50 value of NDV strain AF 2240. The lower left quadrants of

each panel (Rl) show the viable cells, which exclude PI and are

negative for Annexin V binding. The upper right quadrants (R2)

contain the non-viable, necrotic cells, positive for Annexin V binding

and for PI uptake. The lower right quadrants (R3) represent the

apoptotic cells, Annexin V positive and PI negative. The Annexin

V ?/PI - apoptotic cell population for U-87MG cell line increased

from 0.05 ± 1.94% in untreated cells, to 4.26 ± 2.97%

Neurochem Res (2011) 36:2051–2062 2059

123

shaped nucleus, shrinkage, condensed chromatin and cell

membrane blebbing [5]. Quantification of apoptosis with

differential scoring of treated and control cells revealed a

significant (P \ 0.05) difference in the number of apop-

totic cells in a time-dependent manner but with insignifi-

cant difference between numbers of necrosis positive cells

in control and treated cells, concluding that NDV strain

AF2240, did not induce necrotic effects. Fluorescence

microscopy examination confirmed the onset of apoptosis

features. These morphological criteria that implicate

apoptotic cell death were further confirmed by transmission

electron microscopy and transmission electron microscopy.

TEM has been considered a milestone of the research in

the field of apoptosis. By means of TEM the results in this

study showed that untreated brain tumor U-87MG cells

showed no changes and exhibited intact nucleus with clear

cytoplasm while after treatment of U-87MG cells with

NDV strain AF2240, some cells showed typical morpho-

logic changes of apoptosis, including shrinkage and

membrane blebbing. Other changes occur within the

nucleus during apoptotic death such as the appearance of

dense, crescent-shaped chromatin aggregates which line

nuclear membrane. Later, the nucleolus disintegrates;

nuclear membrane develops deep invaginations and, ulti-

mately, the nucleus fragmente into dense granular particles

(apoptotic bodies) which eventually degenerated to ‘‘sec-

ondary necrosis’’ [38]. It is thought that the nuclear chan-

ges are due to activation of endogenous nuclease(s) which

cleaves DNA into oligonucleosomal fragments [35, 38].

In addition the terminal deoxynucleotidyl transferase

(TdT)-mediated dUTP-fluorescein nick-end labeling

(TUNEL) assay was also conducted to confirm the results

of morphological features of the oncolytic effect of NDV

on the U-87MG brain cell line. TUNEL staining is the

standard technique for detection of apoptosis because it

permits visualization of DNA cleavage which can be

visualized and quantities directly by fluorescence micros-

copy [14, 15, 39, 46].

This test showed that NDV strain AF2240 caused a

significant increase in the percentage of apoptotic cells on

U-87MG brain cell line and the percentage of apoptotic

cells increased with increasing of time, in which apoptotic

cells were more abounded at longer durations of treatment.

In the cell populations of control in both cell lines, cells

were having intact DNA or low undetectable levels of

fragmentation but treated cells were TUNEL positive and

apoptotic cells increased correlated with the time of

treatment.

DNA ladder assay provides a sensitive assay for the

detection of DNA fragmentation, but this method is qual-

itative rather than quantitative [44]. In this study we

reported that NDV strain AF2240 stimulate DNA frag-

mentation characteristic of apoptosis in U-87MG cell line

at 24, 48 and 72 h post-inoculation. The DNA ladder assay

can be decreased in samples where the number of apoptotic

cells is low [28] and during the apoptotic process genomic

DNA is cleaved in more consequent steps, first cleavage

into fragments with length 300 kbp is followed by addi-

tional fragmentation into 50 kbp and thereafter to oligo-

nucleosomal DNA fragments [35]. It was also reported by

[6] that DNA laddering is not detectable in adherent cells

but is characteristic of the floating cells regardless of

treatment. Even with enhancement of the fragmented DNA

in the sample, DNA ladder formation is observed only

when the extent of oligonucleosomal cleavage is promi-

nent. Internucleosomal cleavage of DNA is likely to be in

the later phase of apoptotic process [7, 16, 35]. In most cell

types, the biochemical characteristics of apoptotic response

include activation of endogenous calcium and magnesium

dependent endonucleases, leading to fragmentation of the

chromosomal DNA. Initially, the DNA fragments are large

(50–300 kb) but are later digested to oligonucleosomal size

(multimers of 180–200 bp). The formation of this distinct

DNA ladder is considered to be a biochemical hallmark of

apoptosis.

Further confirmation of the mode of cell death was

carried by flow cytometric analysis of cell cycle. It mea-

sures apoptotic changes in cells by staining with DNA dyes

[43]. This method is useful for quantitative estimates of the

fractions of cells in the different phases of the cell cycle

[13]. Untreated and treated U-87MG brain cell line were

evaluated for apoptosis by measuring the amount of

apoptotic cells using of DNA flow cytometry (FCM). The

apoptotic cells with degraded DNA were represented in so-

called ‘‘sub-G1’’ peaks on DNA histograms. Apoptotic

cells, due to a change in membrane permeability, showed

an increased up-take of the vital dye, PI, compared to live

cells [33, 43].

Figures and tables reveal that there was a loss of treated

U-87MG cells in all three phases of the cycle (G1, S and

G2/M) accompanied with a large increase in the sub-G1

region (apoptosis peak) in the fluorescence histograms.

Concluding that NDV strain AF2240 caused an increasing

in the percentage of sub-G1 region which increased with

increasing of time and did not induce specific cell cycle

arrest in specific phase.

Annexin binding assay is a method permits the detection

of the early phases of apoptosis before the loss of cell

membrane integrity [3, 44]. This is based on the phenom-

enon that phosphatidylserine (PS) is exposed at the outer

membrane of the cell during apoptosis and on the ability of

annexin V to bind to PS with high affinity [10]. Phospha-

tidylserine (PS) only exists in the cytoplasm side of

cell plasma membrane, and externalization of PS occurs in

the early stage of apoptosis. Annexin-V could specifi-

cally conjugate to the PS to detect the apoptotic cells [21].

2060 Neurochem Res (2011) 36:2051–2062

123

The analysis of the treated U-87MG cells by annexin V

versus PI, revealed four populations: live cells, apoptotic

cells, late apoptotic cells and permeabilized cells. In this

experiment, we observed increasing in the percentage of

target cells in the annexin positive–PI negative (early

apoptosis) quadrant at 6 h post-inoculation.

These results complies with the other studies [1, 29, 45,

48], which stated that the effectiveness of NDV strains

included NDV strain AF2240 as oncolytic agent was found

on breast cancer cell lines and leukemia cell lines and the

nature of cell death caused by this virus was characterized

as apoptosis. Therefore these results show that NDV strain

AF2240 was capable to induce apoptosis on brain tumor

cells, anaplastic astrocytoma (U-87MG), in vitro.

To put these results together, we believe that the

induction of apoptosis play a role in inhibiting brain tumor

cells when treat with NDV strain AF2240 and that cyto-

toxicity increasing while increasing the titer of the virus.

This discovery may provide a theoretical basis for this type

of treatment. The mechanism of the virus infection is still

unclear. NDV would have a bright future in the treatment

of tumors and further work may lead to relative antitumor

agents to be used in clinical settings.

Acknowledgments This research was funded in part by the

National Cancer Council (MAKNA), Malaysia. The authors also

acknowledge additional support from Universiti Putra Malaysia

(UPM), Serdang, Malaysia.

References

1. Alabsi A (2008) Effects of newcastle disease virus strains

AF2240 and V4-UPM on cytolysis and apoptosis of leukemia cell

lines. Doctor of philosophy Thesis, Universiti Putra Malaysia,

Malaysia

2. Alexander DJ (1988) Newcastle disease diagnosis. In: Alexander

DJ (ed) Newcastle disease. Kluwer Acadimic Publishers, Neth-

erlands, pp 147–160

3. Aubry J, Blaecke A, Lecoanet-Henchoz S, Jeannin P, Herbault N,

Caron G, Moine V, Bonnefoy J (1999) Annexin V used for

measuring apoptosis in the early events of cellular cytotoxicity.

Cytometry 37:197–204

4. Chambers P, Samson ACR (1980) A new structural protein for

Newcastle disease virus. J Gen Virol 50:155–166

5. Chan KM, Rajab NF, Ishak MHA, Ali AM, Yusoff K,

Din LB, Inayat-Hussain SH (2006) Goniothalamin induces apop-

tosis in vascular smooth muscle cells. Chemico-Biol Interact

159:129–140

6. Clarke RG, Lund EK, Johnson IT, Pinder AC (2000) Apoptosis

can be detected in attached colonic adenocarcinoma HT29 cells

using Annexin V binding, but not by TUNEL assay or sub-G0

DNA content. Cytometry 39:141–150

7. Cohen GM, Sun XM, Snowden RT, Dinsdale D, Skilleter DN

(1992) Key morphological features of apoptosis may occur in the

absence of internucleosomal DNA fragmentation. Biochem J

286:331–334

8. Doonan F, Cotter TG (2008) Morphological assessment of

apoptosis. Methods 44:200–204

9. Doyle TM (1927) A hitherto unrecorded disease of fowls due to a

filter passing virus. J Comp Pathol 40:144–169

10. Engeland MV, Nieland LJW, Ramaekers FCS, Schutte B,

Reutelingsperger CPM (1998) Annexin V-Affinity assay: a

review on an apoptosis detection system based on phosphatidyl-

serine exposure. Cytom 31:1–9

11. Fenner F, Bachmann P, Gibbs E, Murphy F, Studdert M, White D

(1987) Veterinary Virol. Academic Press, Orlando

12. Freeman AI, Zakay-Rones Z, Gomori JM, Linetsky E, Rasooly L,

Greenbaum E, Rozenman-Yair S, Panet A, Libson E, Irving CS,

Galun E, Siegal T (2006) Phase I/II trial of intravenous NDV-

HUJ oncolytic virus in recurrent glioblastoma multiforme. Mol

Ther 13:221–228

13. Fried J, Perez AG, Clarkson BD (1976) Flow cytofluorometric

analysis of cell cycle distributions using propidium iodide,

Properties of the method and mathematical analysis of the data.

J Cell Biol 71:172–181

14. Gao N, Keane MJ, Ong T, Ye J, Miller WE, Wallace WE (2001)

Effects of phospholipid surfactant on apoptosis induction by

respirable quartz and kaolin in NR8383 rat pulmonary. Toxicol

Appl Pharmacol 175:217–225

15. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of

programmed cell death in situ via specific labeling of nuclear

DNA fragmentation. J Cell Biol 119:493–501

16. Gooch JL, Yee D (1999) Strain-specific differences in formation

of apoptotic DNA ladders in MCF-7 breast cancer cells. Cancer

Lett 144:31–37

17. Guan T, Qin F, Du J, Geng L, Zhang Y, Li M (2007) AICAR inhibits

proliferation and induced S-phase arrest, and promotes apoptosis in

CaSki cells. Acta Pharmacologica Sinica 28:1984–1990

18. Henson JW (2005) Glioblastoma multiforme and anaplastic gli-

oma: a patient guide. http://brain.mgh.harvard.edu/PatientGuide.

htm. Accessed on 7 Feb

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

(2007) Induction of apoptosis and cell-cycle arrest in human

colon cancer cells by meclizine. Food Chem Toxicol 45:

935–944

21. Lin W, Li D, Chen Q, Lu H (2004) Clinical and experimental

study of oxaliplatin in treating human gastric carcinoma. World J

Gastroenterol 19:2911–2915

22. Liu C, Xu HY, Liu (2001) Induction of caspase-dependent

apoptosis in cultured cells by the avian coronavirus infectious

bronchitis virus. Am Soc Microbiol 75:6402–6409

23. Lorence RM, Richard KW, Katubig BB, Reyes HM, Phuangsab

A, Mitchell BR, Cascino CJ, Walter RJ, Peeple ME (1994)

Complete regression of human neuroblastoma xenographts in

athimic mice after local Newcastle disease virus therapy. Natl

Cancer Inst 86:1228–1233

24. Maher EA, Furnari FB, Bachoo RM, Rowitch DH, Louis DN,

Cavenee WK, DePinho RA (2001) Malignant glioma: genetics

and biology of a grave matter. Genes Dev 15:1311–1333

25. Malorni W, Fais S, Fiorentini C (1998) Morphological aspects of

apoptosis. In: Cossarizza A, Boraschi D (eds) Apoptosis: labo-

ratory manual of experimental methods. Purde University, West

Hafayette.

26. Mangiardi JR, Kane H (2003) A,B,C’s of brain tumors from their

biology to their treatment. http://drshortz.com/brain%20tumor.

htm. Accessed on 27 March

27. Mascotti K, McCullough J, Burger SR (2000) HPC viability

measurement: trypan blue versus acridine orange and propidium

iodide. Transfusion 40:693–696

28. Matalova E, Panova A (2002) Detection of apoptotic DNA ladder

in pig leukocytes and its precision using LM-PCR (Ligation

Neurochem Res (2011) 36:2051–2062 2061

123

Mediated Polymerase Chain Reaction). J Univ Vet Pharm Sci

71:163–168

29. Meyyappan M (2003) Oncolytic effect of Newcastle disease virus

on the MCF-7 and MDA-231 breast cancer cell lines. Master

Thesis, Universiti Putra Malaysia, Malaysia

30. Mishell BB, Shiiqi SM, Henry C (1980) Selected methods. In:

Mishell BB, Shiiqi SM (eds) Cellular immunology. Freeman, San

Francisco, pp 21–22

31. Muneesh T, Vishva MD (1995) Fas- and tumor necrosis factor-

induced apoptosis is inhibited by the poxvirus crmA gene prod-

uct. Am Soc Biochem Mol Biol 270:3255–3260

32. Nelson NJ (1999) Scientific Interest in Newcastle disease virus is

reviving. J Natl Cancer Inst 91:1708–1710

33. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C

(1991) A rapid and simple method for measuring thymocyte

apoptosis by propidium iodide staining and flow cytometry.

J Immunol Methods 139:271–279

34. Niederhuber JE (2006) Questions and answers about Newcastle

disease virus. http://www.cancer.gov/cancertopics/pdq/cam/NDV/

Patient/page2. Accessed 16 March

35. Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA,

Wakeling AE, Walker PR, Sikorska M (1993) Apoptotic death in

epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments

prior to or in the absence of internucleosomal fragmentation.

EMBO J 12:3679–3684

36. Omar A, Aini I, Ali A, Othman F, Yusoff K, Abdullah JM, Wali H,

Zawawi M, Meyyappan N (2003) An overview on the development

of Newcastle disease virus as an anti-cancer therapy. Malays J Med

Sci 10:4–12

37. Reichard KW, Lorence RM, Cascino CJ, Peeples ME, Walter RJ,

Fernando MB, Reyes HM, Greager JA (1992) Newcastle disease

virus selectively kills human tumor cells. J Surg Res 52:448–453

38. Sho E, Sho M, Singh TM, Xu C, Zarins CK, Masuda H (2001)

Blood flow decrease induces apoptosis of endothelial cells in

previously dilated arteries resulting from chronic high blood flow.

Arterioscler Thromb Vasc Biol 21:1139–1145

39. Stahelin BJ, Marti U, Solioz M, Zimmermann H, Reichen J

(1998) False positive staining in the TUNEL assay to detect

apoptosis in liver and intestine is caused by endogenous nucleases

and inhibited by diethyl pyrocarbonate. J Clin Pathol 51:204–208

40. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B,

Taphoorn MJB, Belanger K, Brandes AA, Marosi C, Bogdahn U,

Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A,

Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO (2005)

Radiotherapy plus concomitant and adjuvant temozolomide for

glioblastoma. N Engl J Med 352:987–996

41. Szeberenyi J, Fabian Z, Torocsik B, Kiss K, Csatary LK (2003)

Newcastle disease virus-induced apoptosis in PC12 pheochro-

mocytoma cells. Am J Therap 10:282–288

42. Tatter SB (2005) The new WHO Classification of Tumors

affecting the Central Nervous System. http://neurosurgery.mgh.

harvard.edu/newwhobt.htm. Accessed on 11 May

43. Telford WG, King LE, Fraker PJ (1994) Rapid quantitation of

apoptosis in pure and heterogeneous cell populations using flow

cytometry. J Immunol Methods 172:1–16

44. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C

(1995) A novel assay for apoptosis flow cytometric detection of

phosphatidylserine early apoptotic cells using fluorescein labeled

expression on Annexin V. J Immunol Methods 184:39–51

45. Wali HSM (2003) The effects of Newcastle disease virus (NDV) on

breast cancer cell lines. Master Thesis, Universiti Putra Malaysia,

Malaysia

46. Yasuhara S, Zhu Y, Matsui T, Tipirneni N, Yasuhara Y, Kaneki

M, Rosenzweig A, Jeevendra Martyn JA (2003) Comparison of

Comet assay, electron microscopy, and flow cytometry for

detection of apoptosis. J Histochem Cytochem 51:873–885

47. Yusoff K, Tan WS, Lau CH, Ng BK, Ibrahim AL (1996) Sequence

of the haemagglutinin-neuraminadase gene of the Newcastle dis-

ease virus oral vaccine strain V4 (UPM). Avian Pathol 25:837–844

48. Zawawi M (2007) Cytolytic effect of Newcastle disease virus

strain V4 (UPM) on leukemic cell lines CEM-SS and HL60.

Master Thesis, Universiti Putra Malaysia, Malaysia

2062 Neurochem Res (2011) 36:2051–2062

123