Newcastle disease virus (NDV), an avian paramyxovirus, has a potential oncolytic effect that may be of significance in the treatment of a variety of cancer diseases. An attenuated lentogenic isolate of NDV (HUJ) demonstrated a selective cytopathic effect upon a panel of human and mouse lung tumor cells, as compared to human nontumorigenic lung cells. The virus-selective oncolytic effect is apoptosis dependent, and related to higher levels of viral transcription, translation and progeny virus formation. Furthermore, NDV-HUJ oncolytic activity is directed in-cis and not through induction of cytokines, that may act in-trans on neighboring cells. Development of primary lung tumors and of the consequent metastasis in mice inoculated with mouse lung tumor cells 3LL-D122 was decreased following treatment with NDV-HUJ. The preferential killing of the tumor cells is not due to a deficiency in the interferon (IFN) system, as expression of the IFN-β gene, in the infected cells, is properly induced. Moreover, pretreatment with IFN effectively protected the tumor cells from the virus oncolytic effect. We conclude therefore, that NDV-HUJ should have a significant benefit in the treatment of lung cancer as well as other malignancies.
Lung cancer is a principal malignant disease worldwide, with annual incidence and total death on the rise. The prognosis of nonsmall cell lung cancer (NSCLC) following treatment by surgery, radiotherapy and/or chemotherapy is rather poor, with 5-year survival rates of 5–14%.1, 2 Therefore, there is an urgent need for new measures of treatment. Newcastle disease virus (NDV), an avian paramyxovirus, has been evaluated in experimental models and in some clinical studies as an anti cancer agent and some therapeutic benefits have been reported.3, 4, 5 Two major approaches were undertaken in these studies: first, the virus was applied to directly infect and kill the tumor cells.4, 6, 7, 8 Second, the virus was used as an immunomodulatory agent by infecting autologous cells ex vivo, followed by injection of the ‘xenogenized’ cells to induce immune surveillance against the tumor.9, 10, 11, 12 The virus may induce an oncolytic effect either directly, through apoptosis or necrosis of the infected cells, or, indirectly, through induction of cytokines such as tumor-necrosis factor (TNF) and interleukin-1 (IL1), which would then act in-trans to cause tumor cell death.13, 14 Several studies have attributed the selective oncolytic activity of some RNA viruses, including vesicular stomatitis virus (VSV) and NDV, to defects in the interferon (IFN) system, observed in many tumor cell lines.15, 16, 17, 18 As NDV is very sensitive to IFN, its replication was found to be inhibited in the IFN competent normal cells but not in the tumor cells that failed to develop a proper antiviral state. However, many tumor cells do not display defects in the IFN system yet still are selectively destroyed by NDV.19 Consequently, little is known of the molecular mechanisms responsible for the preferential lysis of tumor cells.20
We recently reported the application of a lentogenic isolate of NDV-HUJ in a clinical trial with patients suffering from glioblastoma multiforme (GBM). Toxicity of this attenuated virus was minimal, and one patient achieved a complete response.8 We describe in the present work a detailed characterization of this lentogenic NDV isolate in comparison with a mesogenic virus (NDV-MTH-68) that had been applied extensively in clinical trials.6, 7 NDV may be significant in the treatment of lung carcinomas, as its natural tropism is to the lung.21 Therefore, in this study we examined the oncolytic effect of NDV-HUJ on NSCLC, both in vitro and in a mouse model. We have studied the replication cycle of the virus in both nontumorigenic and cancer human lung cells, and elucidated the mechanisms underlining the selective oncolytic activity of NDV.
Materials and methods
NDV B1 (Hitchner) strain22 was purchased from the American Type Culture Collection (ATCC; B-1 V-188). The virus was propagated regularly since 1971 in the allantoic cavity of 10- to 11-day-old embryonated eggs. NDV-HUJ isolate (Hebrew University, Jerusalem, Israel) was derived from the B1 strain in 2002 through repeated end point limited dilutions and subcloning in fertilized-SPF eggs. NDV-HUJ was deposited in the ATCC under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of patent procedure (Patent Deposit Designation PTA 7970) on 1 November 2006 and approved on 9 April 2007. Patent no. US 7223389B2, containing the sequences was issued on 29 May 2007. The virus was also deposited at the European Collection of Cell Cultures (ECACC), Ref. No 0607240. NDV-MTH-68 strain was propagated in fertilized-SPF eggs. Viruses grown in eggs were purified on sucrose gradients (20–60%, w/w) using standard methods.6, 23
The following lung carcinoma cell lines of murine or human origin were used:
CRL-5891—human adenocarcinoma, non-small cell lung cancer (NCI-H-1734). CRL-5875—human adenocarcinoma, non-small cells lung cancer (NCI-H-156). Human lung carcinoma lines, A549 (ACC-107)24 and human nontumorigenic lung cells MRC5 (CCL-171),25 were obtained from the ATCC. DF-1 chicken fibroblast cell line was obtained from E. Bachrach, Tel Aviv University. MRC5 cells were grown in Dulbecco's modified Eagle's medium and A549 cell in Eagle's minimal essential medium, both media containing 10% fetal calf serum (FCS), glutamine (2 mM), penicillin and streptomycin. Cultures were incubated at 37 °C in 5% CO2 incubator. Cell monolayer cultures were infected for 1 h, at the multiplicities indicated in the legends to the figures, excess virus was removed by washing twice and fresh media with 2% FCS, and without trypsin, was added for a further incubation at 37 °C, in 5% CO2 incubator. Murine cells: 3LL-D122 Lewis lung carcinoma cells, a highly tumorigenic clone (kindly provided by Prof Lea Eisenbach, The Weizmann Institute of Science, Israel). The cells were tested for their sensitivity to the virus in vitro and in vivo in a syngeneic mouse model.26
Analysis to NDV surface proteins in infected cells
Cells (2 × 106) at 48 h postinfection were collected, washed in phosphate-buffered saline (PBS) and incubated with anti-NDV chicken serum (at 1:400 dilution) for 1 h on ice. Cells were washed twice with PBS and incubated with goat antichicken immunoglobulin-fluorescein isothiocyanate (IgG-FITC)-conjugated 1:200 dilution (Jackson, Nest Grove, PA, USA, catalogue no. 703-096-155) on ice for 30 min. After incubation, cells were washed with PBS, and stained with 0.5 μg ml−1 propidium iodide (PI, Sigma, St Louis, MO, USA), an indicator for cell mortality. The relative levels of surface NDV antigens were assessed by fluorescence-activated cell sorting (FACS) analysis (FITC Green stain FL1 and PI Red stain FL3 channels in the flow cytometer FACSort; Becton Dickinson, Franklin Lakes, NJ, USA). Data were analysed using CellQuest software (Becton Dickinson).
Cells (2 × 106) at 48 h postinfection were harvested, washed with PBS and fixed in 70% ethanol. Cells were washed twice with PBS and incubated in 1 ml of PBS containing 50 μg ml−1 PI (Sigma) and 200 μg ml−1 RNase A (Sigma) at 37 °C for 10 min. The stained cells were analysed for red fluorescence (FL2-A) and data were analysed using CellQuest software (Becton Dickinson).
Caspase-3 enzyme activity assay
Activation of caspase-3 enzyme was tested using CaspACE assay System (Promega, Madison, WI, USA). Cells (3 × 106) were infected with NDV-HUJ at multiplicity of infection (MOI)=10 for 40 h. Before infection, cells, as indicated, were treated with recombinant human IFN-β 100 μ ml−1 (Betaferon) for 16 h. Control cultures were treated, as indicated, with etoposide (ETO) (30 μM) for 40 h. Cell were harvested, washed with PBS and extracted in cell lysis buffer (100 μl). Protein extracts (50 μg) were taken for caspase-3 activity measurements. Reaction mixtures (100 μl) contained, caspase assay buffer (Promega), HEPES 100 mM, pH 7.5, sucrose 10%, CHAPS 0.1%, dimethylsulphoxide (2 μl), DTT 2 mM and the substrate AC-DEVD-pNA 0.2 mM. Reactions were incubated at 37 °C for 4 h and free product pNA was monitored by a spectrophotometer at 405 nm. All reactions were linear with respect to time and the amount of enzyme added. Averages of triplicate reactions (±s.d.) are presented.
Total RNA was isolated from cells (6 × 106) at different times post-NDV infection using the RNeasy RNA isolation kit (Qiagen, Hamburg, Germany). To distinguish between the minus (−) and plus (+) strands of NDV RNA, we used a two-step reverse transcription (RT)–PCR. For the synthesis of cDNA, identical amounts of RNA (1 μg) were used as template for AMV reverse transcriptase (Promega). RT reaction was carried out to specifically identify the (−) strand genomic NDV RNA, with a reverse primer to the NDV leader (−) RNA sequence (Leader Start 5′-IndexTermACCAAACAGAGAATCGGTGAG-3′), and to specifically identify the (+) strand, antigenomic NDV RNA, a reverse primer to the trailer (+) RNA sequence was used (trailer end: 5′-IndexTermGTCTCATTCACCAAATC-3′) from the NDV L gene. PCR amplification of the specific cDNAs was carried out in 50 μl containing, Taq DNA polymerase, PCR buffer, deoxynucleotide triphosphates mix (Sigma), 1 μM forward and reverse primers and 50 ng cDNA. For a PCR amplification of the (−) NDV cDNA we used the forward primer Leader Start and the reverse primer Leader End (5′-IndexTermGCCAACAAACCACTCAGGCA-3′) from the NDV NP gene to produce a dsDNA fragment of 229 bp. For a PCR amplification of the (+) NDV cDNA we used the forward primer Trailer End and the reverse primer Trailer Start (5′-IndexTermGTACTTGACTCGTGCTCAAC-3′) to produce a dsDNA fragment of 329 bp. In parallel, RT–PCR of cell β-actin mRNA was carried out as a control, on all RNA samples, using primers of two different exons, to prevent amplification of cell DNA contamination. Actin forward (5′-IndexTermCCAACCGTGAAAAGATGACC-3′) and actin reverse (5′-IndexTermGCTGTGGTGGTGAAGCTGTA-3′) primers were used to produce DNA of 270 bp. RT–PCR was also carried out to IFN-β mRNA using the following primers: IFN-β forward (5′-IndexTermACATCCCTGAGGAGATTAAGCA-3′) and IFN-β reverse (5′-IndexTermGCCAGGAGGTTCTCAACAATAG-3′). The IFN primers produced a DNA of 150 bp. DNA products were collected after 20, 25, 30 and 35 of PCR cycles and results presented from the linear range of the amplification, after 25 or 30 cycles. Quantitative analysis of the DNA fragments was done by ImageMaster VDS-CL scanner (Amersham Pharmacia, Piscataway, NJ, USA) using the TINA20 software. Relative NDV RNA values were calculated by dividing the DNA band density derived from viral RNA to that derived from the actin RNA, in each sample.
NDV infectivity (TCID50)
Cells were infected with NDV-HUJ at MOI=10 for 1 h, excess virus was removed and cell cultures were washed twice with PBS. The cultures were further incubated at 37 °C in a medium containing 2% FCS and medium aliquots (2 ml) were removed at 1, 24 and 48 h postinfection for assay of progeny virus production. Virus titer in the medium was determined by infection of permissive DF1 avian cells with and without trypsin (3 μg ml−1; Sigma, catalogue no. T-0134) to activate the F0 viral protein. After 72 h of incubation the presence of virus was determined by the microtiter hemagglutination assay,27 using suspension of 0.5% chicken red blood cells.
Mice tumor models
Subcutaneous tumors and metastasis in mice models. Groups of four C57/BL mice, 6- to 7-week old, were inoculated subcutaneously (s.c.) to the mouse flank with 3LL-D122 cells (1 × 106 per 0.2 ml) to develop primary tumor as well as lung metastases. About 20 days post-tumor cells implantation when tumors in the flank reached a volume of 200 mm3, mice were treated either by NDV (109 EID50 per dose) or PBS twice a week, following anesthesia (chloral hydrate 4%, 0.14 ml per mouse). Tumor volume was calculated on the basis of the formula V=(L × W2)/2 (L, length; W, width).28 Some tumors were excised, weighed and tissue sections were prepared for histological examination. Following injection of tumor cells to the flank and treatment with NDV-HUJ, lung metastasis and local primary tumors in the flank were removed and virus presence in these tissues was analysed by inoculation of tumor extracts into the allantoic cavity of embryonated eggs.
Intrathoracic (i.t.) tumors: group of 10 C57/BL mice, 6- to 7-week old, were inoculated i.t. with 3LL-D122 cells (1 × 106 per 75 μl) for primary tumors formation, following anesthesia (chloral hydrate 4% 0.14 ml per mouse). Four mice were killed at different times after cell implantation to observe for lung tumors. At 17 days post-cell implantation, tumor formation was observed and treatment began with NDV (109 EID50 per dose) or PBS, by i.v. administration twice a week. Two mice from each group were killed after NDV treatments for histological immune staining. Survival curves (Figure 7B) represent the remaining four mice per group.
Human tumor implantation: CD-1 nude mice (eight per group) were injected s.c. with human PC3-CxCr4 cells (107).29 The mice were treated i.v. with PBS or with NDV-HUJ (0.4 × 109 EID50 per dose) 14 days post-tumor cells implantation once a week for 4 weeks. Tumor volume (mm3) was determined during 41 days post-tumor cells implantation. At the end of the experiment all mice were alive but due to stress signs in the control PBS-treated mice and according to ethics restrictions the experiment was terminated.
All in vivo experiments were done under ethics restrictions (Ethics committee research no. MD 88.40–4).
Formalin-fixed, paraffin-embedded tissue samples were cut into 4 μm sections, deparaffinized and incubated for 1 h at room temperature with rabbit anti-NDV serum 1:1000. Positive signal was detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (goat antirabbit IgG; DAKOCytomation, Glostrup, Denmark) and DAB (DAKOCytomation) substrate. Contra-staining was done with hematoxylin (DAKOCytomation).
Pathogenicity and biological indices of NDV-HUJ and NDV-MTH-68 isolates
The NDV-HUJ was isolated by repeated subcloning of the B1 strain in the allantoic cavity of embryonated eggs (Materials and methods). Virulence of the NDV-HUJ virus was compared with that of a well-known oncolytic isolate NDV-MTH6 using different in vivo and in vitro assays (Table 1). The intracerebral pathogenicity index (ICPI) was determined by intracranial injection of 1-day-old chicks with the virus, and disease development and death were scored for 8 days (Table 1, see footnote a). Mean death time (MDT) was determined by injection of the virus into the allantoic cavity of embryonated eggs and the time that each embryo was observed dead was recorded (Table 1, see footnote b). These validated virulence parameters indicated that NDV-HUJ is not pathogenic to chicken as compared to the pathogenic NDV-MTH virus. On the basis of these parameters, NDV-HUJ appears to be an attenuated lentogenic virus, whereas NDV-MTH is a virulent mesogenic strain. Sequence analysis of genes encoding for the precursor surface proteins (HN and F) of the two NDV strains, indicated multiple amino-acid differences. It is of interest that the cleavage site between the F1 and F2 subunits contains multibasic amino-acid residues in NDV-MTH strain and only one basic amino acid in the NDV-HUJ strain (Table 1, see footnotes c and d). This sequence difference explains NDV-HUJ dependence on trypsin supplementation for multicycle infection in mammalian cells that do not express a protease with adequate specificity (Table 3). We also compared several additional biological features, such as neuraminidase enzyme and hemagglutination activities, of the two NDV strains that may determine the pathogenicity and oncolytic activity of these viruses (Supplementary Table 1). The neuraminidase enzyme activity, hemagglutination activity and infectivity of NDV-HUJ appear to be thermolabile compared to NDV-MTH. Although NDV-HUJ lost the above-mentioned activities within 5 min of incubation at 56 °C, NDV-MTH retained the activities for 15–30 min. The thermostability of the fusion (F) activity, as well as the substrate affinity (Km) of the neuraminidase in both NDV strains is similar.
Preferential killing of tumor cells by NDV-HUJ
Several lung tumor cells were tested for the oncolytic activity of NDV-HUJ, the mouse cells 3LL-D122 and the human lines CRL5891, CRL5875 and A549, cultivated from human NSCLC. All tumor cell lines were found sensitive to NDV-HUJ (Table 2). To compare the consequence of NDV infection in tumor and nontumorigenic lung cells, we applied the MRC5 cells, derived from normal embryonic human lung. The latter cell is a nontumorigenic diploid lung cell, as it cannot grow in severe combined immunodeficient mice and in soft agar. The MRC5 lung cell was selected as a control, as normal pneumocyte–epithelial cell line is not available.
Cells were infected with NDV-HUJ for 48 h and stained with antibodies against NDV for infection and with PI for cell death (Figure 1). The results indicated that although most A549 tumor cells (97%) were infected by the virus, some of the nontumorigenic cells MRC5 (18%) remain uninfected, probably because this cell is less permissive for NDV infection (Figures 4 and 5; Table 3). Indeed, killing of the infected A549 tumor cells was much more extensive (45%) as compared to MRC5 cells (14%). One possible explanation for the preferential killing by NDV-HUJ is that the IFN system30 is defective in the tumor cells and thus the virus replicates more extensively in these cells as compared with the nontumorigenic cells. To examine this possibility, cells were pretreated with IFN-β for 16 h to activate the IFN system and subsequently infected with NDV-HUJ. The results presented in Figure 1 clearly indicate that pretreatment of A549 tumor cells with IFN-β reduced the oncolytic activity of the virus from 45% in the untreated cells to 12% in the IFN-treated cells. Interestingly, the antiviral effect of IFN was lower in the nontumorigenic MRC5 cells as compared to the tumor cells. Moreover, cytotoxicity of the virus increased with longer times of infection, at 72 h postinfection the fraction of dead A549 tumor cells was about 70%, although no significant increase in toxic effect was observed in the nontumorgenic MRC5 cells. However, at higher MOI (above 10) no increase in cytotoxicity was seen (data not shown). We conclude, therefore, that the preferential oncolytic activity of NDV-HUJ cannot be explained only by defects in the IFN response machinery of the tumor cells.
NDV-HUJ induces a selective apoptosis in lung tumor cells
To investigate whether the cytopathic effect of NDV-HUJ is due to apoptosis, we first analysed cellular DNA degradation, a hallmark of apoptosis, using FACS analysis (Figure 2). The fraction of cells in sub-G1 phase of cell cycle indicates apoptotic cells.31 Infection of nontumorigenic MRC5 cells by NDV-HUJ did not change the fraction of apoptotic cells (3% in mock and in infected cells). However, infection of the A549 tumor cells resulted in a marked increase in the fraction of apoptotic cells (from 1% in mock to 31% in the infected cultures). Interestingly, pretreatment with IFN-β protected the tumor cells from the oncolytic effect of the virus (decreased the fraction of apoptotic cells from about 31% without IFN to 5% with IFN). No significant effect of IFN on the MRC5 cells was noticed, as apoptosis is anyhow very low in the infected cells (Figure 2).
To further investigate the consequences of NDV-HUJ infection on the two cells, we analysed caspase-3 activation, a critical protease in the apoptotic cascade. Infection with NDV-HUJ activated the caspase-3 enzyme in both types of cells, yet the increased enzyme-specific activity in the A549 tumor cells was significantly higher than in the nontumorigenic MRC5 cells (Figure 3). It should be noted that constitutive caspase-3 activity was consistently higher in the mock nontumorigenic MRC5 cells, as compared to the mock A549 tumor cells, as expected.18
Two positive controls were included in the experiment: (i) ETO, a well-known cytotoxic drug,32, 33 caused, as expected, a stronger activation of caspase-3 in dividing nontumorigenic cells as compared to the tumor cells; (ii) Addition of caspase-3-specific inhibitor, Z-VAD-FMK, to the ETO-treated cells, blocked the enzymatic activity, indicating that indeed this assay specifically measures caspase-3 enzyme in the cell extracts (data not shown). Taken together, these results indicate that although both the virus and ETO-induced apoptosis, the relative specificity of these agents toward the two cells appears to be at variance. IFN pretreatment of the cultures did not change much the caspase-3 activity in the infected nontumorigenic cells, yet, IFN decreased activation of caspase-3 in the infected A549 tumor cells by 30% (P-value <0.05). Taken together, the results shown in Figures 2 and 3 indicate that NDV-HUJ causes a selective apoptotic effect to the tumor cells and this effect is not due to a deficiency in the IFN response system of the tumor cells.
Virus replication in tumor and nontumorigenic lung cells
To determine whether the selective oncolytic activity of NDV-HUJ is due to preferential replication of the virus in the tumor cells, we compared kinetics and extent of viral RNA synthesis in the tumor and nontumorigenic cells. Productive infection of avian cells with NDV, a negative strand RNA virus, results within a few hours, in the transcription of subgenomic (+) strand mRNAs on the parental genomic minus (−) RNA template. Later on, genome size (+) RNA is produced to serve as a template for progeny genomic (−) RNA replication.34
Total RNA was isolated from cells at different times postinfection and the amount of the two viral RNA strands was determined by RT–PCR. Primers for the RT–PCR were designed to detect the full size minus and plus genomic RNAs, by using sequences from the leader and trailer regions of the corresponding viral RNA (Materials and methods). In parallel, RT–PCR of cell actin mRNA was carried out on the same RNA preparations as a control (Figure 4). Amounts of the viral RNA were calculated in relation to the level of actin mRNA in the cell (Figure 4). As expected, low levels of genomic NDV (−) RNA were found at 1 h postinfection in both cells, probably representing the parental virus genomic (−) RNA, in the cytoplasm. In contrast, no (+) NDV RNA was detected at 1 h postinfection. Transcription of (+) RNA was evident, beginning at 7 h (data not shown) and reached a plateau at 24 h postinfection in both the tumor and nontumorigenic cells. Replication of progeny genomic (−) RNA was observed at 16–24 h postinfection in both the tumor and nontumorigenic cells. Levels of (–) RNA and (+) RNA transcription, at 24 h postinfection, were about two fold higher in the A549 tumor cells as compared to the nontumorigenic cells MRC5 (Figure 4).
In previous experiments (Figures 1, 2, 3), the antiviral activity of IFN-β was measured indirectly through its effect on induction of apoptotic cascades following infection. To further analyse this point, the antiviral activity of IFN was tested directly on viral RNA transcription and replication. Pretreatment of the two cells with IFN for 16 h before infection resulted in significant inhibition (∼40%, P-value <0.05) of (+) RNA synthesis in the nontumorigenic MRC5 cells treated with IFN compared to the untreated MRC5 cells. However, no inhibition of the (–) RNA transcription in these cells was demonstrated. In the A549 tumor cells there was no apparent inhibition of (−) RNA and (+) RNA synthesis.
Preferential NDV-HUJ protein synthesis in the tumor cells
To further investigate the cascade of viral replication in the nontumorigenic and tumor cells, viral protein synthesis was analysed (Figure 5). Cells were infected with NDV-HUJ for 48 h, stained for surface viral antigens with anti-NDV serum and analysed by FACS (FL1). The results indicate that viral protein synthesis in the tumor cells is much higher than in nontumorigenic cells. Extent of NDV antibody staining, as calculated from the FACS, FL1, suggests a 10- to 100-fold higher level of intracellular viral proteins in the tumor as compared to nontumorigenic cells. Infected cell populations were resolved into two fractions based on the extent of antibody staining (Figure 5, FL1). The dead cell fraction appears to display a higher level of viral proteins, as compared to live population and thus a shift to the right of the logarithmic x axis (see also Figure 1). Interestingly, although pretreatment with IFN strongly inhibited viral protein synthesis in the tumor cells, it had no effect on protein synthesis in the nontumorigenic cells. Taken together, it appears that the major antiviral effect of IFN in the tumor cells is at the translation level rather than the transcription level of the virus (compare Figures 4 and 5).
Progeny virion particles produced by the human cells
The results presented in Figures 4 and 5 clearly indicate the synthesis of genomic (−) RNA and of viral proteins needed for the assembly of progeny virus in the human cells. To directly analyse for virus production, medium harvested at different times postinfection was analysed for the presence of infectious progeny virus, using a permissive chicken cell line DF1 as indicator. To differentiate between infectious and noninfectious virions in the medium, trypsin was added during titration of progeny virus on DF1 cells. Trypsin activates the fusion protein precursor (F0) and thus progeny NDV infectivity (Table 1). Without trypsin, no infectious progeny virus was detected in any of the medium samples (data not shown). To avoid potential carryover of residual parental virus in the tested medium, cells were extensively washed after 1 h of adsorption and an aliquot of the added medium was tested for the presence of infectious virus. The results presented in Table 3 indicate no virus at 1 h postinfection of the tumor cells. At 24 h postinfection, low titers of progeny virus were detected in the medium of both nontumorigenic and tumor cells. At 48 h postinfection, titer of virus produced by the tumor cells was consistently 10-fold higher than by the nontumorigenic cells. In conclusion, these results indicate completion of the replication cycle to produce low levels of noninfectious progeny virions by both the tumor and the nontumorigenic cells in culture. Pretreatment of the cultures with IFN-β resulted in inhibition of virus production in the tumor cells but not in the nontumorigenic cells. This inhibition is consistent with the suppression of viral protein synthesis in the tumor cells pretreated by IFN (Figure 5).
The IFN system in the A549 tumor cells is functional
To gain a better understanding of the involvement of the IFN system in the oncolytic effect of NDV we analysed for the induction and response to IFN in the tumor cells.
The results presented in Figures 1, 2, 3 and 5 and Table 3 indicate that pretreatment with IFN before infection decreases both the oncolytic effect and the amount of virus produced in the tumor cells. This protection indicates that the IFN response system in tumor cells is fully functional. It is possible, however that induction of the IFN genes in the infected tumor cells is defective. To resolve this issue, the transcription of IFN-β gene was analysed in the A549 tumor cells following 24 h of infection. The results shown indicate extensive transcription of IFN-β mRNA following infection (Supplementary Figure 1). Treatment with IFN-β before infection somewhat decreased the induced levels of IFN-β mRNA but levels were still higher than in the control mock cells. Taken together, the results indicate that both the IFN induction and IFN response system are functional in the tumor cells.
The oncolytic activity is dependent on NDV-HUJ replication
The finding that NDV-HUJ displays a selective cytopathic effect in the tumor cell cultures could be a direct consequence of viral replication to induce the intrinsic apoptotic cascade. Alternatively, the selective cytopathic effect could be due to secretion of toxic cytokines from the infected cells, which could cause in-trans apoptosis of neighboring uninfected cells, by the extrinsic apoptotic cascade. To differentiate between these possibilities, tumor cell cultures (A549) were infected with increasing viral multiplicities (MOI) and the correlation of infected cells (FL1, staining for NDV proteins) to dead apoptotic cells (FL3, PI staining) was assessed by FACS. The results presented in Figure 6a show that with increasing MOI, a higher percentage of cells are infected, as expected (lower right panel). Notwithstanding, although the percentage of infected cells that are apoptotic increase with the MOI (5.7–61.2%, upper right panel), the percentage of noninfected apoptotic cells remains low independent of MOI (0.4–5.1%, upper left panel). This would suggest a direct effect in-cis of the virus on the apoptotic cascade. To validate this observation, conditioned medium was collected from both uninfected and infected A549 cells, at 48 h postinfection, and subjected to ultracentrifugation to remove any virus from the medium (Materials and methods). The virus free conditioned media was subsequently added to fresh A549 cell cultures and the cells were analysed for apoptosis (Figure 6b). If indeed NDV-HUJ induces apoptosis in-trans, one would expect increased apoptosis upon addition of the conditioned medium from the infected cultures. The results clearly indicate no induction of apoptosis in-trans by the conditioned medium of infected cultures (lower panel).
Effect of NDV-HUJ on mouse tumor models
To assess the oncolytic effect of the virus in vivo, a syngeneic mouse (C57/BL) model of lung cancer was established. Mice were injected with tumor cells (3LL-D122) either s.c. in the flank to develop primary tumor, as well as lung metastases (Figure 7A), or i.t. to establish a primary lung tumor (Figure 7B). Following s.c. inoculation of cells into the flank mice were treated with NDV-HUJ by s.c. injections, when the tumor volume was about 200 mm3 (Figure 7A). Lungs were observed for metastasis by direct observation after 30–37 days (when mice demonstrated stress symptoms). Mice treated with the virus developed less metastasis as compared to the control group. Survival rate of the NDV-HUJ-infected mice were higher than the PBS-treated mice and at day 37 when all the mock-treated mice died, some of the NDV-treated mice were still alive and metastasis free. Moreover, NDV-HUJ treatment prevented tumor volume expansion compared to the PBS-treated group of mice (Figure 7A). Immunohistochemistry staining indicated that mice treated with the virus had improved lung tumor structure to a spongier like normal tissue (compare Figures 7C(c–d)). Virus injected s.c. reached the lung and infected tumor cells, as shown by immunohistochemistry staining of lung tissue sections with antibodies to NDV (Figure 7C(c–d)). Furthermore, we were able to isolate infectious virus from excised tissues of the flank primary tumor and from the lungs, 4 days after injection of the virus. Virus was detected by injection of tumor crude extracts to eggs, as described in Materials and methods.
In a second set of experiments i.t. mouse model was established and effect of NDV-HUJ on the primary lung tumor was investigated. Appearance of primary tumors in the lungs was determined at different time intervals. When tumors were first observed in the lungs (17 days after cell implantation) the mice were i.v. treated with NDV-HUJ or with PBS. The NDV-HUJ treatment delayed mice death significantly compared with the control group (34 days vs 20 days post-tumor implantation, P-value <0.05; Figure 7B).
Immunohistochemistry staining of lung tissue sections from the i.t.-injected group indicated a positive viral infection even 4 days after i.v. injection of the virus (Figures 7C(j–l)). Some background weak staining (Figure 7C(d and h)) in a tissue of mock infected mouse, compared to the NDV antigen positive staining (Figure 7C(f, j and l)), is due to the use of rabbit polyclonal anti serum to NDV.
To further evaluate the potential oncolytic activity of NDV-HUJ, it was also tested in a prostate cancer model. Nude mice were inoculated subcutaneously with human prostate cancer PC-3 cells.35 Tumors developed in all mice within 21–30 days. Fourteen days post-tumor implantation, mice were divided into two groups: a group treated i.v. with NDV-HUJ and a control group was injected with PBS. The results demonstrate that NDV-HUJ treatment decreased tumor volume significantly (P-value <0.05) as compared to the mock-treated group (Figure 8). Furthermore, although 47% of the viral-treated animals were free of tumors, all mice in the control group exhibited tumor growth at the end of the experiment.
Although several clinical trials have indicated the potential application of NDV as an oncolytic agent, little information is available as to the mechanisms that facilitate the preferential killing of tumor cells. Several studies have suggested a correlation between the virulence of a particular NDV strain, as measured in chicken, and its capacity to replicate in mammalian cells and consequently to induce an oncolytic effect.4, 34 NDV virulence in avian is associated, at least in part, with presence of basic amino acids at the cleavage site sequence of the F0 precursor protein.36, 37 A cluster of basic amino acids at this site (RRQRR/F), as found in the F0 of the mesogenic MTH virus, is compatible with efficient cleavage by trypsin-like cell proteases. Cleavage of the F0 activates its fusion activity and, consequently, infectivity of the progeny virus (Table 1). However, application of virulent NDV strains (mesogenic or velogenic) for the treatment of cancer patients is problematic due to environmental biohazard related to potential virus spread and pathogenicity. We describe in the present work an attenuated (lentogenic) NDV isolate (HUJ) that expresses a fusion protein (F0) with a cleavage site (GRQGR/L) that is not compatible with certain cellular secreted protease activation. Yet, this attenuated virus exhibits a selective oncolytic activity to tumor cells both in culture and in vivo (Table 2 and Figures 1, 2, 7 and 8), similar to the more virulent (mesogenic) NDV- MTH (Table 1). The oncolytic effect of MTH and HUJI was tested on Daudi cells in vitro both virus causes similar (50 and 70%) mortality of the infected tumor cells (48 and 72 h, respectively) following infection. Moreover both strains of the virus reduced total cell number in the infected samples comparing to the mock sample. (unpublished data). The oncolytic activity of NDV-HUJI virus was a consequence of a selective induction of apoptosis in the tumor cells. Induction of the apoptotic cascade was evident by measuring directly for cell DNA fragmentation and by assay of caspase-3 enzyme activation (Figures 2 and 3). Apoptotic activity of another NDV lentogenic strain (rLaSota) in a variety of tumor cells was demonstrated previously but no comparison to nontumorigenic cells was shown.3 Interestingly, when the apoptotic activities of NDV and ETO, a standard anti cancer drug, were compared, the two agents appear to function in a different mode. Although proliferating nontumorigenic cells were highly sensitive to ETO, as compared to tumor cells, they were less sensitive to NDV. This phenomenon may be explained in several ways: first cytotoxic drugs, such as ETO, are known to be more potent on dividing cells as compared with resting cells, and thus in the in vivo setting they preferentially kill the tumor cells.32 In the experiments described in this work, cultures of the nontumorigenic and tumor cultures were subconfluent proliferating cells and thus the preferential cytotoxic activity of ETO on the tumor was relatively reduced.28 NDV on the other hand, preferentially kills tumor cells relative to the nontumorigenic cells, irrespective of their proliferation state. Second, a major hallmark of tumor cells is related to the induction of anti apoptotic genes (IAPs) that effectively reduce the killing activity of cytotoxic drugs.19 In contrast, the oncolytic activity of NDV appears to bypass this barrier for effective and selective oncolytic therapy.
Oncolytic activity of a virus may theoretically be facilitated either by the ability of the virus to replicate better in the tumor as compared to nontumorigenic cells or due to a selective interaction of a viral gene product with a cellular component, specific to the tumor cells, that induces apoptosis.16, 38 To test for the first possibility, we compared viral replication in tumor and nontumorigenic cells of human lung origin. Levels of both RNA strands (+ and −) increased at 7–24 h postinfection and steady state levels of both RNA strands were relatively higher in the tumor cells as compared to the nontumorigenic cells (Figure 4). In addition, since infection was performed without the addition of trypsin, differences in levels of viral proteins were much more pronounced; approximately 10- to 100-fold higher in the tumor as compared to the nontumorigenic cells (Figure 5). However the progeny virus secreted from the infected cells is not infectious and no infectious virus was found in the media of both cells when titrated in the absence of supplemented trypsin, When titration was performed in the presence of trypsin production of infectious progeny virus is relatively low in both cells, yet it is about a log higher in the tumor cells at 48 h postinfection (Table 3). These observations suggest a partial block at the level of viral assembly in both cultured human cells.
Several studies have shown that tumor cells have defects in the IFN response system and therefore, cannot develop an antiviral state upon infection with viruses like VSV.15, 16 Such a defect results in higher viral replication in the tumor cells and consequently to the selective oncolysis.17 NDV, an avian virus, may represent a different example of oncolytic virus as its V gene product, that is known to counteract IFN response in avian cells, is completely inactive in mammalian cells.39 Notwithstanding, the oncolytic effect of NDV was shown related to a deficient IFN system in one study15 and conversely shown independent of the IFN system in yet another study.19 To further investigate the role of the IFN system in the oncolytic mechanism of NDV-HUJ, we pretreated the cells with recombinant IFN-β and tested for replication and the oncolytic activities of the virus. The results clearly indicate that the tumor cells were sensitive to IFN and the oncolytic activity of NDV was suppressed after pretreatment with IFN (Figures 1, 2, 3). In concurrence, the replication cycle of NDV was also suppressed in tumor cells pretreated with IFN. Interestingly, whereas synthesis of plus and minus strands RNA were marginally suppressed in the tumor cells, protein synthesis was most effectively inhibited in the IFN-treated tumor cells (Figures 4 and 5). It should be noted, however, that plus strand synthesis in the MRC5 cells was strongly suppressed by IFN. Taken together these results suggest that the major effect of IFN in the infected mammalian cells is at the level of viral protein translation as was shown in infected avian cells.40 However, it could still be argued that induction of the IFN gene, in response to NDV infection, is deficient in the A549 cells and thus viral replication is not blocked. To address this issue, induction of IFN-β gene upon NDV infection was directly analysed in the tumor cells. As shown in Supplementary Figure 1, IFN-β mRNA levels increased due to NDV infection above the basal level of the mock uninfected sample. Taken together, these results indicate that both induction and antiviral response to IFN is not deficient in these tumor cells. Indeed the results demonstrate that the apoptotic cascade induced during infection of the tumor cells is more dominant than the anti viral pathway induced by IFN. The IFN secreted from the infected tumor cell could only protect other neighboring cells which yet have not been infected like the IFN pre-treated cells (Figure 1 and 2).
Two hypotheses have been forwarded to explain the preferential killing of tumor cells by the virus. NDV may induce a cytopathic effect indirectly by stimulating secretion of cytokines, such as TNF and IL1, to affect in-trans the uninfected cells in the tissue,13, 14, or directly, by killing in-cis the infected cells. Our data support direct killing of the infected cells, as the oncolytic effect was directly dependent on the MOI and moreover, medium harvested from the infected cells showed no oncolytic activity (Figure 6).
Several clinical studies were reported using different NDV strains. Treatment with NDV-MTH strain improved survival, weight gain, and reduction of disease relative to control groups.6, 7 The MTH is a potent IFN inducer41 and the virus causes apoptosis independent of P53 expression.42 The NDV isolate PV701 showed minimal toxicity in patients and objective tumor shrinkage at high viral doses.43, 44
The anti tumor activity of NDV-HUJ was also demonstrated in mouse models of primary and metastasis lung tumors. NDV treatment effectively inhibited primary tumor volume extending and the formation of lung metastasis (Figure 7A). Moreover, NDV-HUJ treatment delayed death of the mice significantly (although the control group lived an average of 20 days, the NDV-HUJ-treated mice survived for 34 days; P-value <0.05; Figure 7B). It should be noted that the virus reached the lung through both routes of inoculation, i.v. and s.c., as demonstrated by extensive immune staining of lung tumor tissue for viral antigens and by isolation of infectious virus from the lung.
Although the virus appears to undergo one cycle of replication in cultured cell lines, in the in vivo mouse models the tumor tissues exhibited extensive NDV-positive immune staining, suggesting multiple cycles of replication in the lung (Figure 7C). In addition, infectious virus could be isolated from the tumor tissues, even 4 days following virus injection. This would suggest the presence of serine type proteases, that are known to be secreted by Clara cells in the lung.45
The virus also exhibited a therapeutic effect on human prostate tumors transplanted in nude mice. Mice treated with NDV-HUJ had a decreased tumor volume compared to the PBS-treated groups and 47% of the NDV-treated mice were tumor free at the end of the experiment (Figure 8). Infectious virus was also isolated in GBM patients treated with NDV-HUJ, from brain tumor tissue biopsy and from tumor cystic fluid at 3–4 days after the last dosage, and from blood after 9 days, indicating a prolonged persistence of the virus in the treated patients.8
In conclusion, we describe in this work an attenuated NDV isolate with a selective oncolytic activity, demonstrated in cell cultures and in mice models. The oncolytic activity is related to a greater extent of viral replication, as well as to selective apoptotic activity in tumor cells.
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This work was supported by grant from the Philip Morris US and international external research program and by a grant of the European commission, program number 6. The Cinigene Network of excellence. The ICPI assay was performed by Dr I Samina, The Veterinary Institute, Bet Dagan, Israel.
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Yaacov, B., Elihaoo, E., lazar, I. et al. Selective oncolytic effect of an attenuated Newcastle disease virus (NDV-HUJ) in lung tumors. Cancer Gene Ther 15, 795–807 (2008). https://doi.org/10.1038/cgt.2008.31
- NDV—Newcastle disease virus
- lung cancer
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