1. In vitro assessment of antitumor potential of Newcastle disease virus in human cancer cells
Umesh Kumar1,2 and Sachin Kumar1*
1- Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati , India.
ABSTRACT
RESULTS
1. Genotypic and Pathotypic characterization
Newcastle disease virus (NDV) is a causative agent of highly fetal and infectious disease, affecting a broad range of avian host. Besides NDV has become a challenge for poultry industries, its oncolytic property in a broad range of cancers is an attracting feature for researchers. Although different oncolytic viruses has been proposed, NDV has advantage to being nonhuman pathogen. The oncolytic potential among different strain of NDV is varied but the molecular determinant for strain dependency to the oncolytic potential is poorly understood. In current study we isolated and characterised a novel virulent strain of NDV during an outbreak, in commercial chicken flock from India. We characterized newly isolated strain for its oncolytic property in human breast cancer cell line MCF 7. The various apoptotic assay, suggest that newly isolated strain has potential to induce apoptotic cell death in cancer cell line. The study will be a substratum for further scrutinization and use of the newly isolated strain of NDV, for virotherapy. 5’ 3’
15192 nt F HN L
NDV strain Ranchi N P M
Fig1. Complete genome sequence of NDV strains Ranchi . The number between the genes represents the intergenic sequence in the genome. The extension towards 3’ represents the leader sequence while extension to 5’ represents the trailer sequence.
ICPI
MDT
Fusion protein cleavage site sequence is 112RRQKRF117
.The pathogenicity indices, MDT (64 Hours) ICPI (1.78) is also indicate the that it is a velogenic strain.
More than 10 % genome is different than previously described strains
OBJECTIVES
2. Oncolytic characterization and assessment of antitumor potential
Fig 2. Cytopathic effect on MCF7 cell A, B, C: (negative control with mock infection, infected with 1 MOI, infected with 10 MOI respectively) 48 h post infection and D, E, F: (negative control with mock infection, infected with 1 MOI, infected with 10 MOI respectively) 72 hours post infection. A F E D B C
Fig 3. The graph represents time course, MCF7 cell survivability after NDV infection at different moi.
Fig 4. virus released from MCF-7 cells infected supernatant following infection to Newcastle disease virus strain Ranchi.
To isolate causative agent of outbreak showing symptoms of ND.
To determine the complete genome sequence of NDV isolated from outbreak.
Genotypic and pathotypic characterization of isolated virus.
Molecular characterization for oncolytic property of isolated NDV .
MATERIALS AND METHODS
Virus Isolation, purification and genome walking:
Virus was isolated from tissue sample of dead birds and identity of NDV was confirmed by HI assay. NDV was purified in chicken embryonic fibroblast cells. This isolated clone of NDV was propagated in allantoic cavity of chicken embryo. Plaque assay in BHK21 cells and alternatively by hemagglutination assay was used for virus titration. The full length genome was sequenced using genome walking.
Biological pathotyping and Phylogenetic analysis
For evaluation of pathogenicity, mean death time (MDT) and intracerebral pathogenicity index (ICPI) was used.
Phylogenetic tree of NDV strain Ranchi and 100 other full length genome sequences of NDV strains was constructed using maximum likelihood method with bootstrap value calculated for 5000 replicates.
Apoptotic analysis
The viability of cells following NDV infection was assayed by treating the cell with dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at different time point.
Chromatin condensation and nuclear fragmentation assessed by staining Hoechst is a blue fluorescence dye.
The hypodiploid cell population due to decrease in DNA content following apoptosis was assessed by flowcytometer following propidium iodide (PI) staining.
Apoptotic DNA was isolated by treating infected and mock infected cell in lysis buffer (1% NP-40 in 20mM EDTA, 50mM Tris-HCl, pH 7.5). The intact nuclei from lysed cells were removed by a standard centrifugation protocol and supernatant was collected. The DNA content of supernatant was analyzed on 2% agarose gel.
The membrane redistribution of Phosphatidylserine in MCF-7 cells following infection of NDV strain Ranchi was analyzed by annexin V and PI double-staining using a flow cytometer (FACSCalibur, USA) following the manufacturer’s protocol (Invitrogen, USA).
Immunoblot analysis for different apoptotic marker
Apoptosis specific proteins were visualized by standard, enhanced chemiluminescence HRP substrate following immunoblotting. MCF-7 cells treated with 6 nM staurospourine (Sigma, USA) were used as apositive control for apoptosis. a b
3.2%
26.7% A B
Fig 6. The flow cytometer analysis of DNA content of infected and mock-infected MCF-7 cells with moi of 1 at 48 hpi.
Fig 7. Nuclear morphology of infected and mock infected MCF-7 cells stained by Hoechst dye, 48 hpi..
Fig 5. The DNA addering pattern in MCF-7 cells infected with NDV strain Ranchi. Mock-infected MCF-7 cells (lane 1) were compared with staurospourine treated (lane 2) and infected with NDV strain Ranchi (lane 3).
1.65%
6.66%
Fig 8. The phosphatidylserine redistribution in MCF-7 cells infected with strain Ranchi is analyzed after 12 h post infection by staining the cells with fluorescein isothiocyanate-conjugated annexin V and propidium iodide double staining under flow cytometer..
Active caspase 9
β actin
42 kDa
10 kDa
Active caspase 8
32 kDa
NDV-HN
74 KDa
18 kDa
Active caspase 4
Active caspase 3
12 kDa 1
Cleaved PARP
89 KDa 2 3
Fig 9. Immunoblot analysis for different apoptotic marker. Mock-infected MCF-7 cells (lane 1) were compared with staurospourine treated (lane 2) and infected with NDV strain Ranchi (lane 3). NDV HN specific antibody was also used for confirmation of infection.
CONCLUSIONS
ACKNOWLEDGEMENTS
Isolated NDV strain is velogenic in nature which falls into newly described genotype XIII, having nt in genome.
Isolated NDV strain induce death in human breast cancer cell line MCF-7 in dose dependent and time dependent manner.
Nuclear morphology, DNA fragmentation, Phosphotidyl redistribution and other apoptotic assay suggest that the death induce by NDV is an apoptotic process not necrotic process.
Apoptosis further confirmed by presence of cleaved PARP.
Bothe extrinsic and intrinsic pathway of apoptosis play a role in cell death induce by NDV strain Ranchi.
Activation of caspase 4 suggest that there is inflammatory response also play a role in cell death.
We are thankful to Department of Biotechnology (NER-BPMC/2013/134), the Department of Science and Technology (IFA-LSBM-34), and the Board of Research on Nuclear Sciences (2012/20/37 B/06/BRNS), Government of India for there support on NDV research.
REFERENCES
Kumar U, Kumar S, Molecular characterization of an apoptotic strain of Newcastle disease virus isolated from outbreak of India. Cancer gene therapy 2015 Aug 7. doi: /cgt