Technology in Cancer Research and Treatment ISSN 1533-0346 2014 June 30. Epub ahead of print.
TNF-related Apoptosis-inducing Ligand Delivered by rNDV is a Novel Agent for Cancer Gene Therapy
Fu-Liang Bai, Ph.D. Hui Tian, M.S. Yin-Hang Yu, Ph.D. Jie-Chao Yin, Ph.D. Gui-Ping Ren, Ph.D. Bing Zhou, M.S. De-Shan Li, Ph.D.*
www.tcrt.org DOI: 10.7785/tcrt.2012.500446 Recombinant Newcastle disease virus (rNDV) as antitumor agent has been shown to be effective for cancer therapy. And TNF-related apoptosis-inducing ligand (TRAIL) also has been demonstrated potentially cancer-therapeutic effects. In this study, we constructed TRAIL delivered by rNDV (rNDV-TRAIL) and investigated whether TRAIL would generate the potential synergistic therapeutic effects with rNDV for cancer therapy. In vitro experiments indicated that TRAIL expressed by rNDV demonstrated good biological activity. TRAIL significantly enhanced inducing apoptosis of rNDV in death receptor expression cancer cell lines. Experiments in malignant melanoma-bearing mice demonstrated that expression of TRAIL delivered by rNDV significantly inhibited the tumor growth and prolonged the survival of treated animals compared to control. In conclusion, oncolytic capacity of rNDV was augmented by TRAIL and the inherent anti-neoplastic properties of NDV were enhanced by the introduction of therapeutic TRAIL gene.
Department of Biopharmaceutical Teaching and Research, College of Life Science, Northeast Agricultural University, Mucai street 59, Xiangfang District, Harbin 150030, China
Key words: Antitumor drug; Cancer gene therapy; Cancer therapy; Recombinant Newcastle disease virus; TNF-related apoptosis-inducing ligand.
Introduction NDV excels as an ideal antitumor agent when genetically engineered viruses were tested for human cancer therapy. It has been reported that reverse genetics technology could enhance the oncolytic virotherapy of NDV. However, most of these NDV strains are mesogenic, and such highly infectious strains are problematic in clinical use due to possible unintentional release of highly infectious viruses into the environment, which affect almost all species of domestic and wild birds (1-5). The lentogenic strain of NDV is seldom used in cancer therapy, because it is considered to be non-lytic and thus less likely to have direct cytolytic effects. But in terms of safety, it may be a better candidate in future clinical use. The lentogenic strain of NDV is pathogenic free for man and poultry as antineoplastic drug in large scale manufactures (6). In this study, we investigated the anti-tumor capacity of lentogenic strain of NDV in vitro and evaluated its therapeutic effects in xenograph mice in vivo. Abbreviations: rNDV: Recombinant Newcastle Disease Virus; NDV: Newcastle Disease Virus; TRAIL: TNF-related Apoptosis-inducing Ligand; rNDV-TRAIL: TRAIL Delivered by rNDV; HN: Hemagglutinin-neuraminidase; L: RNA-dependent RNA Polymerase; TNF: Tumor Necrosis F actor; EGFP: Enhance Green Fluorescent Protein; TCID50: Tissue Culture Infectious Dose 50; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal Bovine Serum; SPF: Specific Pathogen Free; IMDM: Iscove’s Modified Dulbecco’s Medium; PFU: Plaque Forming Unit; PBS: Phosphate Buffer Saline; ELISA: Enzyme Linked Immunosorbent Assay; GS: Gene Start; GE: Gene End; HA: Hemagglutination; MOI: Multiplicity of Infection.
*Corresponding author: De-Shan Li, Ph.D. Phone: 186-451-59190645 E-mail: [email protected]
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The TRAIL receptors are attractively therapeutic targets in cancer research, which can induce tumor cell apoptosis and have minimal toxicity to normal tissues. Therefore, TRAIL is a promising anti-cancer agent due to its ability to lyse tumors by binding with TRAIL receptors (7). TRAIL induces apoptosis in established tumor cell lines but not in transformed normal cells, which is a potential anti-cancer drug do to its selectivity. TRAIL induces apoptosis by expression of “death-inducing” and “decoy” receptors on cells. And apoptosis induced by TRAIL or other tumor necrosis factor (TNF) members may be regulated by inhibitory proteins that bind to Fas-associated death domain or other proteins in the caspase pathway (8). Previous studies have proved that TRAIL is an efficient oncolytic agent for cancer therapy (9-11). In addition, previous papers studying oncolytic viruses armed with TRAIL gene for cancer treatment had demonstrated therapeutic effects (12-15). However, there was no report about rNDV armed with TRAIL. Though TRAIL was induced on human monocytes after NDV stimulation and that TRAIL mediated the tumoricidal activity of NDV-stimulated human monocytes (16), its expression was not enough to reach effective therapeutic level. In this study, we demonstrated the synergistic effects between NDV and TRAIL. Genetically-engineered Newcastle disease virus delivering TRAIL gene as a potential drug candidate significantly enhanced therapeutic effects for malignant melanoma. Our data also demonstrated that rNDV-TRAIL greatly inhibited the tumor growth in B16-F10-bearing mice. Notably, we demonstrated that rNDV-TRAIL prolonged the survival of tumor-bearing mice. These results indicated that rNDV-TRAIL could be potentially applied as an enhanced oncolytic agent in clinic for melanoma patients. Materials and Methods
with Dulbecco’s modified Eagle medium (DMEM; BioWhittaker, Inc., Walkersville, MD, USA) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) and 1% chicken allantoic fluid. Mouse malignant melanoma cells (B16-F10 cells) were a kind gift from Dr. Jiahuai Han (XiaMen University, China). BHK-21, CT-26, HepG2 and A549 cells were purchased from ATCC (Rockville, MD, USA). BHK-21 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 1% nonessential amino acids and 1% sodium pyruvate. B16-F10 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mmol/L), 1% penicillin/streptomycin, 1% nonessential amino acids and 1% sodium pyruvate. CT-26 cells were maintained in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin. DF-1 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were grown at 37C under 5% CO2. All cell cultures were regularly tested for mycoplasma contamination. Construction of the rNDV’s cDNA Clones Containing the Transcription Cassettes of TRAIL Gene The NDV LaSota strain was used as a backbone for construction of rNDV cDNA clones. The TRAIL gene flanked by NDV gene end and gene star was cloned into pMD18-T vector (NEB) by PCR using the sense primer (59-gttaacttaagaaaaaatac gggtagaaccgccaccatggagacagacacactcctgct-39), and antisense primer (59-gttaactaagccaactaaaaaggccccgaaa-39). The resulting plasmid was named pMD-TRAIL. Viral genome and TRAIL gene was connected by gene star and gene end. The resulting plasmid was named pMD-TRAIL. The TRAIL gene was subcloned into the HN-L gene junction of rNDV at the Pme1 site to generate the recombinant plasmids pBl-rNDV-TRAIL. All cloning sequences were verified by sequencing.
Ethics Statement Rescue of Recombinant Viruses This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Chinese Association For Laboratory Animal Sciences (CALAS), Animal Health Products, Committee on the Ethics of Animal Experiments Defence Research and Development China and Animal Experiments of the University of Northeast Agricultural (approval number: SCXK-2012-0002). All surgery and euthanasia were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Rescue of rNDV and rNDV-TRAIL were performed as previously described (17). In brief, BHK-21 cells in six-well plates were co-transfected using Lipofectamine 2000 with 0.5 μg pBL-N, 0.25 μg pBL-P, 0.1 μg pBL-L and 1 μg of the recombinant plasmids pBl-rNDV and pBl-rNDV-TRAIL (DNA:Lipofectamine::2:3). Three days after transfection recombinant viruses were harvested from the supernatant of the transfected cells. Recombinant viruses were grown in 9-day-old embryonated SPF chicken eggs and purified as described previously (18). The rescued viruses were termed rNDV and rNDV-TRAIL.
Cell Lines, Viruses and Other Reagents MTT Cytotoxicity Assays rNDV and rNDV-TRAIL were grown in embryonated specificpathogen-free (SPF) eggs. All viruses were propagated in DF-1 chicken fibroblasts cells (ATCC, Manassas, VA, USA)
HepG2, B16-F10, A549 and CT-26 cells were infected with viruses at 0.01 MOI or TRAIL in 96-well plates for 24, 48,
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TRAIL-delivered by NDV for Cancer Gene Therapy 72 and 96 hours in triplicate for each condition and PBS was added instead of the virus as a control. At each time point, 20 μl of MTT (5 mg/ml) in PBS solution was added to each well, the plate was further incubated for 4 hours. Most of the medium was removed and 100 μl of DMSO was added into the wells to soluble the crystals. Finally the OD was measured by a BIO-RAD (ELISA) reader at wavelength of 450 nm. ELISA HepG2, B16-F10, A549 and CT-26 cells were infected with rNDV or rNDV-TRAIL at a MOI of 0.1 and the infected supernatants were harvested 48 hours later. The supernatants were diluted 1:10, 1:50, 1:250 and analyzed TRAIL expression by ELISA for presence of TRAIL protein using Quantikine M kit (R&D systems). Analysis of Apoptosis using Reverse Transcription and Quantitative Real-time PCR Total RNA from xenografts was extracted using the Trizol reagent method. 1 ml of TRIZOL (Invitrogen, Osaka, Japan) was used for every 50-100 mg of spleen tissue and homogenized in an RNase free environment. Chloroform was then added (200 μl for each 1 ml TRIZOL) and the samples were centrifuged at high speed for 15 minutes at 48C. The aqueous layer was then transferred into a new tube and RNA was precipitated with iso-propanol followed by one wash using 70% ethanol. The RNA precipitate was then dissolved in 10-15 μl of RNAse free water and analyzed for quantity and quality using a spectrophotometer. Total RNA was reverse transcribed using the Gene Amp kit (Applied Biosystems; ABI, Foster City, CA, USA). 20 ng of the resulting cDNA was then used in the real-time PCR step. Six genes were tested by real-time PCR including. All real-time PCR assays were performed in triplicate in a 96-well plate using the 7900 Sequence Detector System (ABI) according to the manufacturer’s protocol. Data analysis was performed using the Sequence Detector System (SDS) software (ABI) and the results were expressed as foldchange in relative mRNA expression level, calculated using the ΔCt method with β-actin (ACTB) as the reference gene and the non-treated cells as baseline. Induction of Apoptosis in H22 and B16-F10 Cells HepG2 and B16-F10 cells were grown at 378C with 5% CO2 until 70% to 80% confluence was reached. Apoptosis was assessed by incubation these cells with rNDV-TRAIL or rNDV for 48 hours. After incubation with the recombinant viruses, as described above, the cells were trypsinized and collected. The cells were then washed in cold PBS, adjusted to 1 3 106 cells/ml with PBS, labeled with Annexin V-FITC and PI (Annexin V-FITC Kit, BD, San Diego, CA, USA),
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and analyzed with a FACScan flow cytometer (BD, San Jose, CA, USA). The treatments were performed in triplicate, and the percentage of labeled cells undergoing apoptosis in each group was determined and calculated. Animal Experiments Six to eight week-old C57BL/6 mice were purchased from the West China Experimental Animal Center of Sichuan University (Sichuan, China). Mice were permitted 1 week to acclimate to their environment before manipulation. All surgical procedures were completed in accordance with the guidelines on the care and use of laboratory animals for research purposes by the West China Hospital Cancer Center’s Animal Care and Use Committee. Mice were inoculated with 5 3 107 B16-F10 melanoma cells in the right footpad. Primary tumors usually became palpable on day 5-6 and with an average diameter of 6-8 mm. On day 7, the tumor-bearing mice in two modes were randomly and respectively assigned into 6 groups and each group contained 16 mice. Each mouse in virus-treated group received 5 3 107 pfu rNDV and rNDVTRAIL intratumoral injection on day 7, 9, 11, 13 and 15 with a total of 5 times. The mice in the control groups received normal saline, serving as injection control. The details of the treatment were described previously (19). Tumor dimensions were measured with calipers every 2 day with a total of 10 times. The tumor volumes were calculated according to the following formula: length 3 width2 3 0.52. Results Generation of rNDV-TRAIL and Production of TRAIL Proteins. To generate recombinant Newcastle disease viruses, we followed the design principle described in previous (20), and constructed two recombinant Newcastle disease viruses, one consisting of the extracellular domain of human TRAIL (AA95-281), a second consisting of the EGFP. The foreign gene was located between HN and L of NDV genome (Figure 1A). All recombinant viruses were grown to high titers in embryonated chicken eggs. The growth characteristics of the recombinant NDV viruses were examined in a single-step growth cycle in DF-1 cell lines (Figure 1B). The kinetics of replication of the recombinant viruses (MOI 5 0.5) in B16-F10 showed that insertion of the foreign gene resulted in a slightly delay in the onset of replication (Figure 1C). However, after 72 hours, virus titers reached to the same level as the parental LaSota strain. To determine the expression of TRAIL gene, A549, CT-26, B16-F10 and HepG2 tumor cells were infected with the rNDV or rNDV-TRAIL at MOI of 0.1, and the supernatants were collected at 96 hours post-infection. The TRAIL protein
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Figure 1: Construction of rNDV-TRAIL, growth characteristics and foreign genes express in the recombinant virus-infected tumor cells. (A) Construction of the therapeutic rNDV vectors inserting TRAIL gene. GE: gene end; GS: gene start. (B) Comparison of viral growth kinetics. (C) Growth kinetics of recombinant Newcastle disease viruses in B16-F10 cells. (D) Expression of the TRAIL gene in tumor cell lines infected with rNDV-TRAIL. Experiments were repeated three times. One-way ANOVA revealed a significant effect. **P 0.01, vs. control.
Figure 2: Biological activity of TRAIL expressed by rNDV-TRAIL. (A-D) Tumor cytotoxicity of the TRAIL inhibited the tumor cells growth. All the values are the mean and SEM of triplicate samples. ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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TRAIL-delivered by NDV for Cancer Gene Therapy was detected by ELISA using anti-TRAIL-specific antibody. The result showed that all tumor cells infected with rNDVTRAIL expressed the desired gene products, while no foreign genes were detected in the tumor cells infected with rNDV (Figure 1D). We concluded that TRAIL gene was expressed in rNDV-TRAIL-infected tumor cells. Identification of Biological Activity of TRAIL Expression in rNDV-TRAIL-infected Tumor Cells After confirmation expression of TRAIL in rNDV-TRAILinfected tumor cells, biological activity of TRAIL expression in rNDV-TRAIL-infected tumor cells need to identified. To address the bioactivity of TRAIL expressed by rNDVTRAIL, in vitro, we first analyzed TRAIL-R2 expression on HepG2, B16-F10, A549 and CT-26 cells (Figure 2A). In order to identify biological activity of the TRAIL expressed by rNDV-TRAIL, we ruled out that replicating virus in the supernatant used UV inactivation to avoid virus interference. The TRAIL protein (TRAIL, 100 ng/ml) was then added directly into the culturing tumor cells as commercial TRAIL as control. As shown in Figure 2B-2E, the cytotoxicity of the TRAIL for tumor cells was efficient at the appointed time. The results exhibited that TRAIL expressed by rNDV could obviously inhibited HepG2, B16-F10, A549 and CT-26 cells growth after 72 hours post infection.
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Real-time PCR Analyzed TRAIL-induced Tumor Cells Apoptosis TRAIL induces apoptosis of tumor cells by binding to death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. These receptors possess an intracellular death domain, which triggers the activation of the caspase signaling cascade with or without the involvement of mitochondria after receptor-ligand association (21). Previous study has identified TRAIL induced apoptosis by both mitochondrial pathway and extrinsic apoptotic pathway (22). BAX, BCL-2 and caspase-9 are TRAIL-induced apoptosis gene of mitochondrial pathway and caspase-8 is TRAIL-induced apoptosis gene of extrinsic apoptotic pathway. Fas-L and caspase-3 are shared with both mitochondrial pathway and extrinsic apoptotic pathway (21). To confirm the mechanism of apoptosis induced by rNDV and TRAIL, we investigated the relationship between rNDV-TRAIL and apoptosis-related genes after studied the expression of TRAIL-R2 in tumor cells in experiment. Several expressing level of mRNA related to TRAIL-induced apoptosis were analyzed, including, BAX, BCL-2, Fas-L, caspase-8, caspase-3 and caspase-9. The mRNA from cDNA of detergent-lysed tumor cells was analyzed using a Real-Time PCR at different time. As shown in Figure 3A-3F, apoptosis-related genes were increased in tumor cells after infected with rNDV-TRAIL (MOI 5 0.5) compared to rNDV-infected group (MOI 5 0.5).
Figure 3: NDV inserted TRAIL enhanced induce-tumor apoptosis potency. (A-F) Regression analyzed the rNDV-TRAIL-induced tumor cell lines apoptosis. Expression of each gene was calculated relative to the expression of housekeeping gene, β-actin and the results are expressed as the n-fold difference relative to β-actin. All the values are the mean and SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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Figure 4: TRAIL enhanced NDV inhibition of tumor cells growth. (A-D) rNDV-TRAIL enhanced the anti-tumor potency in vitro. Student’s paired twotailed t-test revealed a significant effect. *P 0.05, **P 0.01, vs. control. (E) The rNDV-TRAIL enhanced the ability to induce apoptosis of tumor cells. X axis: cells labeled Annexin V-FITC. Y axis: cells labeled PI. All the values are the mean and SEM of triplicate samples. Student’s paired two-tailed t-test revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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Anti-tumor Effects of rNDV were Enhanced though Inserting TRAIL Gene As anti-cancer drugs, the cytotoxic effects of agent for malignant tumor cells are important in cancer therapy. Therefore, we investigated the anti-tumor potential of rNDV-TRAIL in tumor cell lines with rNDV as control. The tumor cell lines HepG2, B16-F10, A549 and CT-26 were infected with rNDV-TRAIL or rNDV at an MOI of 0.01. Cytotoxicity was assessed by MTT assay potency at 24, 48, 72 and 96 hours post infection. The result in Figure 4A-4D suggested that the inserting TRAIL greatly enhanced the cytotoxic effects of rNDVL for tumor cells, which inhibited the growth of tumor cells in vitro compared with control. Also, apoptotic rates for B16-F10 and H22 cells treated with the rNDV and rNDVTRAIL were detected by apoptosis assay using flow cytometry. As shown in Figure 4E, TRAIL enhanced potency of rNDV to induce early (Q4) and late (Q2) stage apoptosis of B16-F10 and HepG2 cells. Xenograph Mice Treated by rNDV-TRAIL Inhibited Tumor Growth In Vivo and Prolonged the Survival Time of Animals After confirmation of the success for TRAIL gene expression and biological activity in vitro, we examined the anti-cancer efficacy of rNDV-TRAIL in melanoma model. None of the animals exhibited significant weight loss over the study period (data not shown) during treatment. The most common side effect was development of localized swelling at the injection site, which subsided over the next few days after the last inoculation. We randomly selected 8/16 mice in each group to minor the tumor size. As shown in Figure 5A, the tumor size in the virus-treated groups was significantly smaller than that in the PBS-treated group. Furthermore, the tumor size of rNDV- TRAIL-treated mice was smaller than rNDV-treated mice, which is statistically significant (Student’s paired two-tailed t-test revealed a significant effect, **P 0.01) compared with rNDV-treated group. As shown in Figure 5B, over the next 120 days, remaining 6/8 animals in the rNDV group and 2/8 animals in the rNDV-TRAIL group in footpad melanoma developed significant tumors and needed to be sacrificed. The remaining animals in each group either completely cleared or had persistent pigmented nodules that did not change in size. The survival rate results suggested that rNDV-TRAIL will be an ideal drug for melanoma therapy, which enhances oncolytic ability by combining oncolytic TRAIL gene. TRAIL Expressed in Tumors and rNDV-TRAIL Only Infected Tumor Cells not Normal Cells Also, we detected the expression of foreign genes in tumors by ELISA. As shown in Figure 6A, the productions of foreign
Figure 5: rNDV-TRAIL effectively suppressed tumor growth and prolongs animal survival in melanoma model. (A) Short-term tumor growth in B16-F10 melanoma-bearing mice treated with rNDV, rNDV-TRAIL with PBS as control (n 5 8 in each group). (B) Survival of the B16-F10 melanoma model animals in 120 days period after treatment with the recombinant viruses. The tumor-bearing mice were sacrificed when the tumor volume developed to a significant size (diameter 18 mm). All the values are the mean and SEM of triplicate samples. Student’s paired two-tailed t-test revealed a significant effect, *P 0.05, **P 0.01.
genes were only detected in the tumors of rNDV-infected mice and were not detectable in serum of mice (data not shown). We further confirmed that the recombinant NDV viruses selectively replicate in tumor cells. As shown in Figure 6B, enhanced green fluorescent protein expression could be detected in B16-F10 melanoma cells infected by rNDV-EGFP, but not in MCF-10A normal cells, suggesting that the recombinant NDV retained tumor-selectivity. After confirm the safety of rNDV-TRAIL for human cells, we detected rNDV-TRAIL in organization of mice including heart, liver, brain, spleen and tumors. No HA titer was occurred in organization of mice, but rNDV-TRAIL only replicated in tumor (data not shown). These phenomena further tested that lentogenic NDV is a safe antitumor drug for human use. In addition, we observed spleens of mice treated with rNDV-TRAIL were stimulated and enlarged (data not shown). The reason for this phenomenon may be due to tumor cell lysed by TRAIL, and inclusions stimulated the immune system in mice.
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Bai et al. mesogenic NDVs have been forbidden in many countries for its high pathogenicity for chicken. Notably, previous study has reported that mesogenic strain was 1555 times stronger than lentogenic strain in killing human normal cells (25). The lentogenic NDVs are seldom used in cancer therapy for they are considered to be non-lytic and thus less likely to have direct cytolytic effects (6). However, the innately targeted lentogenic NDV strains may have meaningful potential in treating cancers. And our previous study has confirmed the therapeutic effects of lentogenic NDV for cancers (26). Based on this, avirulent strains LoSota may be a better candidate in future clinical use. It was used as a vaccine for prevention of NDV worldwide. Thus, the safety of LaSota for humans and animals has been well clarified (25). TRAIL is a member of the TNF superfamily, interacts with its functional death receptors and induces apoptosis in a wide range of cancer cell types (10). Therefore, TRAIL has been considered as an attractive agent for cancer therapy. However, TRAIL appears to be unable to induce apoptosis in normal cells while the physiological role of the apoptosisinducing TRAIL in cancer therapy is not enough (27-29). Here we provide the first demonstration that TRAIL delivered by rNDV can selectively suppress growth of melanoma without side effect to melanoma-bearing mice. Furthermore, our data indicate that TRAIL is an efficient drug for cancer therapy, which is consistent with previous study (30).
Figure 6: TRAIL only expressed in tumors and rNDV only replicated in tumor cells. (A) Tumor homogenates was analyzed in melanoma models. (B) rNDV-TRAIL retains tumor-selective cytotoxicity. A total of 106 cells/well were placed in a six-well plate. Non-tumor cells MCF-10A and tumor cells B16-F10 were infected with rNDN-EGFP at an MOI of 0.1. The infected cells were examined with fluorescent microcopy at 96 hours post-infection. All the values are the mean and SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
Discussion and Conclusion NDV is a highly contagious viral disease, which infects almost all species of domestic and wild birds, especially the mesogenic and velogenic strains. Currently, naturally occurring and recombinant NDV viruses have been shown to be effective oncolytic agents against human cancers (1, 2, 23, 24). The mesogenic NDVs and some lentogenic NDVs with the modified F protein as oncolytic agents are widely used in cancers virotherapy. But such highly infectious strains of NDV (mesogenic) are problematic in clinical use for the possible unintentional release of pathogen in the environment. Mesogenic strains of NDV may cause losses of poultry industries. Eggs production of chicken without maternal antibody will drop when they are infected with mesogenic NDV. In addition,
In this study, we proceed to investigate the anti-tumor potential of rNDV-TRAIL based on the antineoplastic potency of NDV and TRAIL. We also tested its curative effects in melanoma model. The results of this study demonstrated that rNDV and TRAIL were interacting partners in cancer therapy, which opened a new horizon for cancer therapy. Although expression of a function gene by rNDV is not somewhat surprising, we have to particularly stress the efficiency of rNDV-TRAIL in aggressive cancer suppression. Importantly, therapeutic effects in the melanoma tumor model revealed that TRAIL delivered by NDV significantly inhibited tumor growth and prolonged animals’ survival. Previous study reported that increasing expression of oncolytic protein was beneficial to inhibit tumor growth (31). However, high concentration of TRAIL had side effect to animals and human beings (32). Therefore, the appropriate concentration of TRAIL would be better in human cancer therapy. In this study, expression level of TRAIL delivered by rNDV was delivered and had no potential toxicity for cancer patients, suggesting that rNDVTRAIL will be an optimal agent for cancer therapy. In summary, rNDV-TRAIL is thought to exert its tumor suppressor and therapy role by regulating tumor apoptosis. This study provides significant evidences supporting the antineoplastic effects of rNDV-TRAIL and suggests that rNDVTRAIL has potency in treatment of melanoma. Based on the
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TRAIL-delivered by NDV for Cancer Gene Therapy oncolytic therapy of the TRAIL and anti-tumor properties of rLaSota, rNDV-TRAIL has potential to be used for clinical application in melanoma patients. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments I would like to thank support of Medical Research Council. This work is supported by National Natural Science Foundation of China (NSFC, Grant No. 31200121), Heilongjiang Funds for Young Scientists (QC2012C099) and Northeast Agriculture University Funds for Ph.D. (2012RCB43). References 1. Cassel, W. A., Murray, D. R. Treatment of stage II malignant melanoma patients with a Newcastle disease virus oncolysate. Natural Immunity and Cell Growth Regulation 7, 351-352 (1988). PMID: 3065642 2. Steiner, H. H., Bonsanto, M. M., Beckhove, P., Brysch, M., Geletneky, K., Ahmadi, R., Schuele-Freyer, R., Kremer, P., Ranaie, G., Matejic, D., Bauer, H., Kiessling, M., Kunze, S., Schirrmacher, V., HeroldMende, C. Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol 22, 4272-4281 (2004). DOI: 10.1200/JCO.2004.09.038 3. Bateman, A. R., Harrington, K. J., Kottke, T., Ahmed, A., Melcher, A. A., Gough, M. J., Linardakis, E., Riddle, D., Dietz, A., Lohse, C. M., Strome, S., Peterson, T., Simari, R., Vile, R. G. Viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells. Cancer Res 62, 6566-6578 (2002). PMID: 12438252 4. Vigil, A., Park, M. S., Martinez, O., Chua, M. A., Xiao, S., Cros, J. F., Martinez-Sobrido, L., Woo, S. L., Garcia-Sastre, A. Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus. Cancer Research 67, 8285-8292 (2007). DOI: 10.1158/00085472.CAN-07-1025 5. Vigil, A., Martinez, O., Chua, M. A., Garcia-Sastre, A. Recombinant Newcastle disease virus as a vaccine vector for cancer therapy. Molecular Therapy: the Journal of the American Society of Gene Therapy 16, 1883-1890 (2008). DOI: 10.1038/mt.2008.181 6. Krishnamurthy, S., Takimoto, T., Scroggs, R. A., Portner, A. Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines. Journal of Virology 80, 5145-5155 (2006). DOI: 10.1128/JVI.02618-05 7. Grosse-Wilde, A., Voloshanenko, O., Bailey, S. L., Longton, G. M., Schaefer, U., Csernok, A. I., Schutz, G., Greiner, E. F., Kemp, C. J., Walczak, H. TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development. J Clin Invest 118, 100-110 (2008). DOI: 10.1172/JCI33061 8. Zhang, X. D., Franco, A. V., Nguyen, T., Gray, C. P., Hersey, P. Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. J Immunol 164, 3961-3970 (2000). PMID: 10754286 9. Ivanov, V. N., Bhoumik, A., Ronai, Z. Death receptors and melanoma resistance to apoptosis. Oncogene 22, 152-161 (2003). DOI: 10.1038/ sj.onc.1206456 10. Zhang, X. D., Franco, A., Myers, K., Gray, C., Nguyen, T., Hersey, P. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Research 59, 2747-2753 (1999). PMID: 10364001
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11. Qiu, B., Sun, X., Zhang, D., Wang, Y., Tao, J., Ou, S. TRAIL and paclitaxel synergize to kill U87 cells and U87-derived stem-like cells in vitro. Int J Mol Sci 13, 9142-9156 (2012). DOI: 10.3390/ijms13079142 12. Sova, P., Ren, X. W., Ni, S., Bernt, K. M., Mi, J., Kiviat, N., Lieber, A. A tumor-targeted and conditionally replicating oncolytic adenovirus vector expressing TRAIL for treatment of liver metastases. Mol Ther 9, 496-509 (2004). DOI: 10.1016/j.ymthe.2003.12.008 13. Burroughs, K. D., Kayda, D. B., Sakhuja, K., Hudson, Y., Jakubczak, J., Bristol, J. A., Ennist, D., Hallenbeck, P., Kaleko, M., Connelly, S. Potentiation of oncolytic adenoviral vector efficacy with gutless vectors encoding GMCSF or TRAIL. Cancer Gene Ther 11, 92-102 (2004). DOI: 10.1038/sj.cgt.7700660 14. Ziauddin, M. F., Guo, Z. S., O’Malley, M. E., Austin, F., Popovic, P. J., Kavanagh, M. A., Li, J., Sathaiah, M., Thirunavukarasu, P., Fang, B., Lee, Y. J., Bartlett, D. L. TRAIL gene-armed oncolytic poxvirus and oxaliplatin can work synergistically against colorectal cancer. Gene Ther 17, 550-559 (2010). DOI: 10.1038/gt.2010.5 15. Tamura, K., Wakimoto, H., Agarwal, A. S., Rabkin, S. D., Bhere, D., Martuza, R. L., Kuroda, T., Kasmieh, R., Shah, K. Multimechanistic tumor targeted oncolytic virus overcomes resistance in brain tumors. Mol Ther 21, 68-77 (2013). DOI: 10.1038/mt.2012.175 16. Washburn, B., Weigand, M. A., Grosse-Wilde, A., Janke, M., Stahl, H., Rieser, E., Sprick, M. R., Schirrmacher, V., Walczak, H. TNFrelated apoptosis-inducing ligand mediates tumoricidal activity of human monocytes stimulated by Newcastle disease virus. J Immunol 170, 1814-1821 (2003). PMID: 12574346 17. Krishnamurthy, S., Huang, Z., Samal, S. K. Recovery of a virulent strain of newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation. Virology 278, 168-182 (2000). DOI: 10.1006/viro.2000.0618 18. Huang, Z., Krishnamurthy, S., Panda, A., Samal, S. K. Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist. Journal of Virology 77, 8676-8685 (2003). PMID: 12885886 19. He, Q. M., Wei, Y. Q., Tian, L., Zhao, X., Su, J. M., Yang, L., Lu, Y., Kan, B., Lou, Y. Y., Huang, M. J., Xiao, F., Liu, J. Y., Hu, B., Luo, F., Jiang, Y., Wen, Y. J., Deng, H. X., Li, J., Niu, T., Yang, J. L. Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice. J Biol Chem 278, 21831-21836 (2003). DOI: 10.1074/jbc.M300880200 20. Kolakofsky, D., Pelet, T., Garcin, D., Hausmann, S., Curran, J., Roux, L. Paramyxovirus RNA synthesis and the requirement for hexamer genome length: the rule of six revisited. J Virol 72, 891-899 (1998). PMID: 9444980 21. Suliman, A., Lam, A., Datta, R., Srivastava, R. K. Intracellular mechanisms of TRAIL: apoptosis through mitochondrial-dependent and -independent pathways. Oncogene 20, 2122-2133 (2001). DOI: 10.1038/sj.onc.1204282 22. Srivastava, R. K. Intracellular mechanisms of TRAIL and its role in cancer therapy. Molecular Cell Biology Research Communications 4, 67-75 (2000). DOI: 10.1006/mcbr.2001.0265 23. Cassel, W. A., Murray, D. R. A ten-year follow-up on stage II malignant melanoma patients treated postsurgically with Newcastle disease virus oncolysate. Medical Oncology and Tumor Pharmacotherapy 9, 169-1671 (1992). PMID: 1342060 24. Karcher, J., Dyckhoff, G., Beckhove, P., Reisser, C., Brysch, M., Ziouta, Y., Helmke, B. H., Weidauer, H., Schirrmacher, V., HeroldMende, C. Antitumor vaccination in patients with head and neck squamous cell carcinomas with autologous virus-modified tumor cells. Cancer Res 64, 8057-8061 (2004). DOI: 10.1158/0008-5472. CAN-04-1545 25. Walter, R. J., Attar, B. M., Rafiq, A., Delimata, M., Tejaswi, S. Two avirulent, lentogenic strains of Newcastle disease virus are cytotoxic for some human pancreatic tumor lines in vitro. Journal of the Pancreas 13, 502-513 (2012). DOI: 10.6092/1590-8577/977
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26. Bai, F., Niu, Z., Tian, H., Li, S., Lv, Z., Zhang, T., Ren, G., Li, D. Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy. Immunology Letters 159, 36-46 (2014). DOI: 10.1016/j.imlet.2014.02.009 27. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673-682 (1995). PMID: 8777713 28. Griffith, T. S., Fialkov, J. M., Scott, D. L., Azuhata, T., Williams, R. D., Wall, N. R., Altieri, D. C., Sandler, A. D. Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma. Cancer Res 62, 3093-3099 (2002). PMID: 12036919
29. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., Ashkenazi, A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271, 1268712690 (1996). PMID: 8663110 30. Stolfi, C., Pallone, F., Monteleone, G. Molecular targets of TRAIL- sensitizing agents in colorectal cancer. International Journal of Molecular Sciences 13, 7886-7901 (2012). DOI: 10.3390/ijms13077886 31. Tanaka, S., Sugimachi, K., Shirabe, K., Shimada, M., Wands, J. R. Expression and antitumor effects of TRAIL in human cholangiocarcinoma. Hepatology 32, 523-527 (2000). DOI: 10.1053/jhep.2000.9716 32. Hymowitz, S. G., Christinger, H. W., Fuh, G., Ultsch, M., O’Connell, M., Kelley, R. F., Ashkenazi, A., de Vos, A. M. Triggering cell death: the crystal structure of Apo2L/TRAIL in a complex with death receptor 5. Molecular Cell 4, 563-571 (1999). PMID: 10549288 Received: December 3, 2013; Revised: April 7, 2014; Accepted: April 14, 2014
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TNF-related Apoptosis-inducing Ligand Delivered by rNDV is a Novel Agent for Cancer Gene Therapy
Fu-Liang Bai, Ph.D. Hui Tian, M.S. Yin-Hang Yu, Ph.D. Jie-Chao Yin, Ph.D. Gui-Ping Ren, Ph.D. Bing Zhou, M.S. De-Shan Li, Ph.D.*
www.tcrt.org DOI: 10.7785/tcrt.2012.500446 Recombinant Newcastle disease virus (rNDV) as antitumor agent has been shown to be effective for cancer therapy. And TNF-related apoptosis-inducing ligand (TRAIL) also has been demonstrated potentially cancer-therapeutic effects. In this study, we constructed TRAIL delivered by rNDV (rNDV-TRAIL) and investigated whether TRAIL would generate the potential synergistic therapeutic effects with rNDV for cancer therapy. In vitro experiments indicated that TRAIL expressed by rNDV demonstrated good biological activity. TRAIL significantly enhanced inducing apoptosis of rNDV in death receptor expression cancer cell lines. Experiments in malignant melanoma-bearing mice demonstrated that expression of TRAIL delivered by rNDV significantly inhibited the tumor growth and prolonged the survival of treated animals compared to control. In conclusion, oncolytic capacity of rNDV was augmented by TRAIL and the inherent anti-neoplastic properties of NDV were enhanced by the introduction of therapeutic TRAIL gene.
Department of Biopharmaceutical Teaching and Research, College of Life Science, Northeast Agricultural University, Mucai street 59, Xiangfang District, Harbin 150030, China
Key words: Antitumor drug; Cancer gene therapy; Cancer therapy; Recombinant Newcastle disease virus; TNF-related apoptosis-inducing ligand.
Introduction NDV excels as an ideal antitumor agent when genetically engineered viruses were tested for human cancer therapy. It has been reported that reverse genetics technology could enhance the oncolytic virotherapy of NDV. However, most of these NDV strains are mesogenic, and such highly infectious strains are problematic in clinical use due to possible unintentional release of highly infectious viruses into the environment, which affect almost all species of domestic and wild birds (1-5). The lentogenic strain of NDV is seldom used in cancer therapy, because it is considered to be non-lytic and thus less likely to have direct cytolytic effects. But in terms of safety, it may be a better candidate in future clinical use. The lentogenic strain of NDV is pathogenic free for man and poultry as antineoplastic drug in large scale manufactures (6). In this study, we investigated the anti-tumor capacity of lentogenic strain of NDV in vitro and evaluated its therapeutic effects in xenograph mice in vivo. Abbreviations: rNDV: Recombinant Newcastle Disease Virus; NDV: Newcastle Disease Virus; TRAIL: TNF-related Apoptosis-inducing Ligand; rNDV-TRAIL: TRAIL Delivered by rNDV; HN: Hemagglutinin-neuraminidase; L: RNA-dependent RNA Polymerase; TNF: Tumor Necrosis F actor; EGFP: Enhance Green Fluorescent Protein; TCID50: Tissue Culture Infectious Dose 50; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Fetal Bovine Serum; SPF: Specific Pathogen Free; IMDM: Iscove’s Modified Dulbecco’s Medium; PFU: Plaque Forming Unit; PBS: Phosphate Buffer Saline; ELISA: Enzyme Linked Immunosorbent Assay; GS: Gene Start; GE: Gene End; HA: Hemagglutination; MOI: Multiplicity of Infection.
*Corresponding author: De-Shan Li, Ph.D. Phone: 186-451-59190645 E-mail: [email protected]
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The TRAIL receptors are attractively therapeutic targets in cancer research, which can induce tumor cell apoptosis and have minimal toxicity to normal tissues. Therefore, TRAIL is a promising anti-cancer agent due to its ability to lyse tumors by binding with TRAIL receptors (7). TRAIL induces apoptosis in established tumor cell lines but not in transformed normal cells, which is a potential anti-cancer drug do to its selectivity. TRAIL induces apoptosis by expression of “death-inducing” and “decoy” receptors on cells. And apoptosis induced by TRAIL or other tumor necrosis factor (TNF) members may be regulated by inhibitory proteins that bind to Fas-associated death domain or other proteins in the caspase pathway (8). Previous studies have proved that TRAIL is an efficient oncolytic agent for cancer therapy (9-11). In addition, previous papers studying oncolytic viruses armed with TRAIL gene for cancer treatment had demonstrated therapeutic effects (12-15). However, there was no report about rNDV armed with TRAIL. Though TRAIL was induced on human monocytes after NDV stimulation and that TRAIL mediated the tumoricidal activity of NDV-stimulated human monocytes (16), its expression was not enough to reach effective therapeutic level. In this study, we demonstrated the synergistic effects between NDV and TRAIL. Genetically-engineered Newcastle disease virus delivering TRAIL gene as a potential drug candidate significantly enhanced therapeutic effects for malignant melanoma. Our data also demonstrated that rNDV-TRAIL greatly inhibited the tumor growth in B16-F10-bearing mice. Notably, we demonstrated that rNDV-TRAIL prolonged the survival of tumor-bearing mice. These results indicated that rNDV-TRAIL could be potentially applied as an enhanced oncolytic agent in clinic for melanoma patients. Materials and Methods
with Dulbecco’s modified Eagle medium (DMEM; BioWhittaker, Inc., Walkersville, MD, USA) containing 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA) and 1% chicken allantoic fluid. Mouse malignant melanoma cells (B16-F10 cells) were a kind gift from Dr. Jiahuai Han (XiaMen University, China). BHK-21, CT-26, HepG2 and A549 cells were purchased from ATCC (Rockville, MD, USA). BHK-21 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin, 1% nonessential amino acids and 1% sodium pyruvate. B16-F10 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mmol/L), 1% penicillin/streptomycin, 1% nonessential amino acids and 1% sodium pyruvate. CT-26 cells were maintained in DMEM/F12 with 10% FBS, 1% penicillin/streptomycin. DF-1 cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were grown at 37C under 5% CO2. All cell cultures were regularly tested for mycoplasma contamination. Construction of the rNDV’s cDNA Clones Containing the Transcription Cassettes of TRAIL Gene The NDV LaSota strain was used as a backbone for construction of rNDV cDNA clones. The TRAIL gene flanked by NDV gene end and gene star was cloned into pMD18-T vector (NEB) by PCR using the sense primer (59-gttaacttaagaaaaaatac gggtagaaccgccaccatggagacagacacactcctgct-39), and antisense primer (59-gttaactaagccaactaaaaaggccccgaaa-39). The resulting plasmid was named pMD-TRAIL. Viral genome and TRAIL gene was connected by gene star and gene end. The resulting plasmid was named pMD-TRAIL. The TRAIL gene was subcloned into the HN-L gene junction of rNDV at the Pme1 site to generate the recombinant plasmids pBl-rNDV-TRAIL. All cloning sequences were verified by sequencing.
Ethics Statement Rescue of Recombinant Viruses This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Chinese Association For Laboratory Animal Sciences (CALAS), Animal Health Products, Committee on the Ethics of Animal Experiments Defence Research and Development China and Animal Experiments of the University of Northeast Agricultural (approval number: SCXK-2012-0002). All surgery and euthanasia were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Rescue of rNDV and rNDV-TRAIL were performed as previously described (17). In brief, BHK-21 cells in six-well plates were co-transfected using Lipofectamine 2000 with 0.5 μg pBL-N, 0.25 μg pBL-P, 0.1 μg pBL-L and 1 μg of the recombinant plasmids pBl-rNDV and pBl-rNDV-TRAIL (DNA:Lipofectamine::2:3). Three days after transfection recombinant viruses were harvested from the supernatant of the transfected cells. Recombinant viruses were grown in 9-day-old embryonated SPF chicken eggs and purified as described previously (18). The rescued viruses were termed rNDV and rNDV-TRAIL.
Cell Lines, Viruses and Other Reagents MTT Cytotoxicity Assays rNDV and rNDV-TRAIL were grown in embryonated specificpathogen-free (SPF) eggs. All viruses were propagated in DF-1 chicken fibroblasts cells (ATCC, Manassas, VA, USA)
HepG2, B16-F10, A549 and CT-26 cells were infected with viruses at 0.01 MOI or TRAIL in 96-well plates for 24, 48,
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TRAIL-delivered by NDV for Cancer Gene Therapy 72 and 96 hours in triplicate for each condition and PBS was added instead of the virus as a control. At each time point, 20 μl of MTT (5 mg/ml) in PBS solution was added to each well, the plate was further incubated for 4 hours. Most of the medium was removed and 100 μl of DMSO was added into the wells to soluble the crystals. Finally the OD was measured by a BIO-RAD (ELISA) reader at wavelength of 450 nm. ELISA HepG2, B16-F10, A549 and CT-26 cells were infected with rNDV or rNDV-TRAIL at a MOI of 0.1 and the infected supernatants were harvested 48 hours later. The supernatants were diluted 1:10, 1:50, 1:250 and analyzed TRAIL expression by ELISA for presence of TRAIL protein using Quantikine M kit (R&D systems). Analysis of Apoptosis using Reverse Transcription and Quantitative Real-time PCR Total RNA from xenografts was extracted using the Trizol reagent method. 1 ml of TRIZOL (Invitrogen, Osaka, Japan) was used for every 50-100 mg of spleen tissue and homogenized in an RNase free environment. Chloroform was then added (200 μl for each 1 ml TRIZOL) and the samples were centrifuged at high speed for 15 minutes at 48C. The aqueous layer was then transferred into a new tube and RNA was precipitated with iso-propanol followed by one wash using 70% ethanol. The RNA precipitate was then dissolved in 10-15 μl of RNAse free water and analyzed for quantity and quality using a spectrophotometer. Total RNA was reverse transcribed using the Gene Amp kit (Applied Biosystems; ABI, Foster City, CA, USA). 20 ng of the resulting cDNA was then used in the real-time PCR step. Six genes were tested by real-time PCR including. All real-time PCR assays were performed in triplicate in a 96-well plate using the 7900 Sequence Detector System (ABI) according to the manufacturer’s protocol. Data analysis was performed using the Sequence Detector System (SDS) software (ABI) and the results were expressed as foldchange in relative mRNA expression level, calculated using the ΔCt method with β-actin (ACTB) as the reference gene and the non-treated cells as baseline. Induction of Apoptosis in H22 and B16-F10 Cells HepG2 and B16-F10 cells were grown at 378C with 5% CO2 until 70% to 80% confluence was reached. Apoptosis was assessed by incubation these cells with rNDV-TRAIL or rNDV for 48 hours. After incubation with the recombinant viruses, as described above, the cells were trypsinized and collected. The cells were then washed in cold PBS, adjusted to 1 3 106 cells/ml with PBS, labeled with Annexin V-FITC and PI (Annexin V-FITC Kit, BD, San Diego, CA, USA),
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and analyzed with a FACScan flow cytometer (BD, San Jose, CA, USA). The treatments were performed in triplicate, and the percentage of labeled cells undergoing apoptosis in each group was determined and calculated. Animal Experiments Six to eight week-old C57BL/6 mice were purchased from the West China Experimental Animal Center of Sichuan University (Sichuan, China). Mice were permitted 1 week to acclimate to their environment before manipulation. All surgical procedures were completed in accordance with the guidelines on the care and use of laboratory animals for research purposes by the West China Hospital Cancer Center’s Animal Care and Use Committee. Mice were inoculated with 5 3 107 B16-F10 melanoma cells in the right footpad. Primary tumors usually became palpable on day 5-6 and with an average diameter of 6-8 mm. On day 7, the tumor-bearing mice in two modes were randomly and respectively assigned into 6 groups and each group contained 16 mice. Each mouse in virus-treated group received 5 3 107 pfu rNDV and rNDVTRAIL intratumoral injection on day 7, 9, 11, 13 and 15 with a total of 5 times. The mice in the control groups received normal saline, serving as injection control. The details of the treatment were described previously (19). Tumor dimensions were measured with calipers every 2 day with a total of 10 times. The tumor volumes were calculated according to the following formula: length 3 width2 3 0.52. Results Generation of rNDV-TRAIL and Production of TRAIL Proteins. To generate recombinant Newcastle disease viruses, we followed the design principle described in previous (20), and constructed two recombinant Newcastle disease viruses, one consisting of the extracellular domain of human TRAIL (AA95-281), a second consisting of the EGFP. The foreign gene was located between HN and L of NDV genome (Figure 1A). All recombinant viruses were grown to high titers in embryonated chicken eggs. The growth characteristics of the recombinant NDV viruses were examined in a single-step growth cycle in DF-1 cell lines (Figure 1B). The kinetics of replication of the recombinant viruses (MOI 5 0.5) in B16-F10 showed that insertion of the foreign gene resulted in a slightly delay in the onset of replication (Figure 1C). However, after 72 hours, virus titers reached to the same level as the parental LaSota strain. To determine the expression of TRAIL gene, A549, CT-26, B16-F10 and HepG2 tumor cells were infected with the rNDV or rNDV-TRAIL at MOI of 0.1, and the supernatants were collected at 96 hours post-infection. The TRAIL protein
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Figure 1: Construction of rNDV-TRAIL, growth characteristics and foreign genes express in the recombinant virus-infected tumor cells. (A) Construction of the therapeutic rNDV vectors inserting TRAIL gene. GE: gene end; GS: gene start. (B) Comparison of viral growth kinetics. (C) Growth kinetics of recombinant Newcastle disease viruses in B16-F10 cells. (D) Expression of the TRAIL gene in tumor cell lines infected with rNDV-TRAIL. Experiments were repeated three times. One-way ANOVA revealed a significant effect. **P 0.01, vs. control.
Figure 2: Biological activity of TRAIL expressed by rNDV-TRAIL. (A-D) Tumor cytotoxicity of the TRAIL inhibited the tumor cells growth. All the values are the mean and SEM of triplicate samples. ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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TRAIL-delivered by NDV for Cancer Gene Therapy was detected by ELISA using anti-TRAIL-specific antibody. The result showed that all tumor cells infected with rNDVTRAIL expressed the desired gene products, while no foreign genes were detected in the tumor cells infected with rNDV (Figure 1D). We concluded that TRAIL gene was expressed in rNDV-TRAIL-infected tumor cells. Identification of Biological Activity of TRAIL Expression in rNDV-TRAIL-infected Tumor Cells After confirmation expression of TRAIL in rNDV-TRAILinfected tumor cells, biological activity of TRAIL expression in rNDV-TRAIL-infected tumor cells need to identified. To address the bioactivity of TRAIL expressed by rNDVTRAIL, in vitro, we first analyzed TRAIL-R2 expression on HepG2, B16-F10, A549 and CT-26 cells (Figure 2A). In order to identify biological activity of the TRAIL expressed by rNDV-TRAIL, we ruled out that replicating virus in the supernatant used UV inactivation to avoid virus interference. The TRAIL protein (TRAIL, 100 ng/ml) was then added directly into the culturing tumor cells as commercial TRAIL as control. As shown in Figure 2B-2E, the cytotoxicity of the TRAIL for tumor cells was efficient at the appointed time. The results exhibited that TRAIL expressed by rNDV could obviously inhibited HepG2, B16-F10, A549 and CT-26 cells growth after 72 hours post infection.
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Real-time PCR Analyzed TRAIL-induced Tumor Cells Apoptosis TRAIL induces apoptosis of tumor cells by binding to death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. These receptors possess an intracellular death domain, which triggers the activation of the caspase signaling cascade with or without the involvement of mitochondria after receptor-ligand association (21). Previous study has identified TRAIL induced apoptosis by both mitochondrial pathway and extrinsic apoptotic pathway (22). BAX, BCL-2 and caspase-9 are TRAIL-induced apoptosis gene of mitochondrial pathway and caspase-8 is TRAIL-induced apoptosis gene of extrinsic apoptotic pathway. Fas-L and caspase-3 are shared with both mitochondrial pathway and extrinsic apoptotic pathway (21). To confirm the mechanism of apoptosis induced by rNDV and TRAIL, we investigated the relationship between rNDV-TRAIL and apoptosis-related genes after studied the expression of TRAIL-R2 in tumor cells in experiment. Several expressing level of mRNA related to TRAIL-induced apoptosis were analyzed, including, BAX, BCL-2, Fas-L, caspase-8, caspase-3 and caspase-9. The mRNA from cDNA of detergent-lysed tumor cells was analyzed using a Real-Time PCR at different time. As shown in Figure 3A-3F, apoptosis-related genes were increased in tumor cells after infected with rNDV-TRAIL (MOI 5 0.5) compared to rNDV-infected group (MOI 5 0.5).
Figure 3: NDV inserted TRAIL enhanced induce-tumor apoptosis potency. (A-F) Regression analyzed the rNDV-TRAIL-induced tumor cell lines apoptosis. Expression of each gene was calculated relative to the expression of housekeeping gene, β-actin and the results are expressed as the n-fold difference relative to β-actin. All the values are the mean and SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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Figure 4: TRAIL enhanced NDV inhibition of tumor cells growth. (A-D) rNDV-TRAIL enhanced the anti-tumor potency in vitro. Student’s paired twotailed t-test revealed a significant effect. *P 0.05, **P 0.01, vs. control. (E) The rNDV-TRAIL enhanced the ability to induce apoptosis of tumor cells. X axis: cells labeled Annexin V-FITC. Y axis: cells labeled PI. All the values are the mean and SEM of triplicate samples. Student’s paired two-tailed t-test revealed a significant effect. *P 0.05, **P 0.01, vs. control.
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Anti-tumor Effects of rNDV were Enhanced though Inserting TRAIL Gene As anti-cancer drugs, the cytotoxic effects of agent for malignant tumor cells are important in cancer therapy. Therefore, we investigated the anti-tumor potential of rNDV-TRAIL in tumor cell lines with rNDV as control. The tumor cell lines HepG2, B16-F10, A549 and CT-26 were infected with rNDV-TRAIL or rNDV at an MOI of 0.01. Cytotoxicity was assessed by MTT assay potency at 24, 48, 72 and 96 hours post infection. The result in Figure 4A-4D suggested that the inserting TRAIL greatly enhanced the cytotoxic effects of rNDVL for tumor cells, which inhibited the growth of tumor cells in vitro compared with control. Also, apoptotic rates for B16-F10 and H22 cells treated with the rNDV and rNDVTRAIL were detected by apoptosis assay using flow cytometry. As shown in Figure 4E, TRAIL enhanced potency of rNDV to induce early (Q4) and late (Q2) stage apoptosis of B16-F10 and HepG2 cells. Xenograph Mice Treated by rNDV-TRAIL Inhibited Tumor Growth In Vivo and Prolonged the Survival Time of Animals After confirmation of the success for TRAIL gene expression and biological activity in vitro, we examined the anti-cancer efficacy of rNDV-TRAIL in melanoma model. None of the animals exhibited significant weight loss over the study period (data not shown) during treatment. The most common side effect was development of localized swelling at the injection site, which subsided over the next few days after the last inoculation. We randomly selected 8/16 mice in each group to minor the tumor size. As shown in Figure 5A, the tumor size in the virus-treated groups was significantly smaller than that in the PBS-treated group. Furthermore, the tumor size of rNDV- TRAIL-treated mice was smaller than rNDV-treated mice, which is statistically significant (Student’s paired two-tailed t-test revealed a significant effect, **P 0.01) compared with rNDV-treated group. As shown in Figure 5B, over the next 120 days, remaining 6/8 animals in the rNDV group and 2/8 animals in the rNDV-TRAIL group in footpad melanoma developed significant tumors and needed to be sacrificed. The remaining animals in each group either completely cleared or had persistent pigmented nodules that did not change in size. The survival rate results suggested that rNDV-TRAIL will be an ideal drug for melanoma therapy, which enhances oncolytic ability by combining oncolytic TRAIL gene. TRAIL Expressed in Tumors and rNDV-TRAIL Only Infected Tumor Cells not Normal Cells Also, we detected the expression of foreign genes in tumors by ELISA. As shown in Figure 6A, the productions of foreign
Figure 5: rNDV-TRAIL effectively suppressed tumor growth and prolongs animal survival in melanoma model. (A) Short-term tumor growth in B16-F10 melanoma-bearing mice treated with rNDV, rNDV-TRAIL with PBS as control (n 5 8 in each group). (B) Survival of the B16-F10 melanoma model animals in 120 days period after treatment with the recombinant viruses. The tumor-bearing mice were sacrificed when the tumor volume developed to a significant size (diameter 18 mm). All the values are the mean and SEM of triplicate samples. Student’s paired two-tailed t-test revealed a significant effect, *P 0.05, **P 0.01.
genes were only detected in the tumors of rNDV-infected mice and were not detectable in serum of mice (data not shown). We further confirmed that the recombinant NDV viruses selectively replicate in tumor cells. As shown in Figure 6B, enhanced green fluorescent protein expression could be detected in B16-F10 melanoma cells infected by rNDV-EGFP, but not in MCF-10A normal cells, suggesting that the recombinant NDV retained tumor-selectivity. After confirm the safety of rNDV-TRAIL for human cells, we detected rNDV-TRAIL in organization of mice including heart, liver, brain, spleen and tumors. No HA titer was occurred in organization of mice, but rNDV-TRAIL only replicated in tumor (data not shown). These phenomena further tested that lentogenic NDV is a safe antitumor drug for human use. In addition, we observed spleens of mice treated with rNDV-TRAIL were stimulated and enlarged (data not shown). The reason for this phenomenon may be due to tumor cell lysed by TRAIL, and inclusions stimulated the immune system in mice.
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Bai et al. mesogenic NDVs have been forbidden in many countries for its high pathogenicity for chicken. Notably, previous study has reported that mesogenic strain was 1555 times stronger than lentogenic strain in killing human normal cells (25). The lentogenic NDVs are seldom used in cancer therapy for they are considered to be non-lytic and thus less likely to have direct cytolytic effects (6). However, the innately targeted lentogenic NDV strains may have meaningful potential in treating cancers. And our previous study has confirmed the therapeutic effects of lentogenic NDV for cancers (26). Based on this, avirulent strains LoSota may be a better candidate in future clinical use. It was used as a vaccine for prevention of NDV worldwide. Thus, the safety of LaSota for humans and animals has been well clarified (25). TRAIL is a member of the TNF superfamily, interacts with its functional death receptors and induces apoptosis in a wide range of cancer cell types (10). Therefore, TRAIL has been considered as an attractive agent for cancer therapy. However, TRAIL appears to be unable to induce apoptosis in normal cells while the physiological role of the apoptosisinducing TRAIL in cancer therapy is not enough (27-29). Here we provide the first demonstration that TRAIL delivered by rNDV can selectively suppress growth of melanoma without side effect to melanoma-bearing mice. Furthermore, our data indicate that TRAIL is an efficient drug for cancer therapy, which is consistent with previous study (30).
Figure 6: TRAIL only expressed in tumors and rNDV only replicated in tumor cells. (A) Tumor homogenates was analyzed in melanoma models. (B) rNDV-TRAIL retains tumor-selective cytotoxicity. A total of 106 cells/well were placed in a six-well plate. Non-tumor cells MCF-10A and tumor cells B16-F10 were infected with rNDN-EGFP at an MOI of 0.1. The infected cells were examined with fluorescent microcopy at 96 hours post-infection. All the values are the mean and SEM of triplicate samples. One-way ANOVA revealed a significant effect. *P 0.05, **P 0.01, vs. control.
Discussion and Conclusion NDV is a highly contagious viral disease, which infects almost all species of domestic and wild birds, especially the mesogenic and velogenic strains. Currently, naturally occurring and recombinant NDV viruses have been shown to be effective oncolytic agents against human cancers (1, 2, 23, 24). The mesogenic NDVs and some lentogenic NDVs with the modified F protein as oncolytic agents are widely used in cancers virotherapy. But such highly infectious strains of NDV (mesogenic) are problematic in clinical use for the possible unintentional release of pathogen in the environment. Mesogenic strains of NDV may cause losses of poultry industries. Eggs production of chicken without maternal antibody will drop when they are infected with mesogenic NDV. In addition,
In this study, we proceed to investigate the anti-tumor potential of rNDV-TRAIL based on the antineoplastic potency of NDV and TRAIL. We also tested its curative effects in melanoma model. The results of this study demonstrated that rNDV and TRAIL were interacting partners in cancer therapy, which opened a new horizon for cancer therapy. Although expression of a function gene by rNDV is not somewhat surprising, we have to particularly stress the efficiency of rNDV-TRAIL in aggressive cancer suppression. Importantly, therapeutic effects in the melanoma tumor model revealed that TRAIL delivered by NDV significantly inhibited tumor growth and prolonged animals’ survival. Previous study reported that increasing expression of oncolytic protein was beneficial to inhibit tumor growth (31). However, high concentration of TRAIL had side effect to animals and human beings (32). Therefore, the appropriate concentration of TRAIL would be better in human cancer therapy. In this study, expression level of TRAIL delivered by rNDV was delivered and had no potential toxicity for cancer patients, suggesting that rNDVTRAIL will be an optimal agent for cancer therapy. In summary, rNDV-TRAIL is thought to exert its tumor suppressor and therapy role by regulating tumor apoptosis. This study provides significant evidences supporting the antineoplastic effects of rNDV-TRAIL and suggests that rNDVTRAIL has potency in treatment of melanoma. Based on the
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TRAIL-delivered by NDV for Cancer Gene Therapy oncolytic therapy of the TRAIL and anti-tumor properties of rLaSota, rNDV-TRAIL has potential to be used for clinical application in melanoma patients. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments I would like to thank support of Medical Research Council. This work is supported by National Natural Science Foundation of China (NSFC, Grant No. 31200121), Heilongjiang Funds for Young Scientists (QC2012C099) and Northeast Agriculture University Funds for Ph.D. (2012RCB43). References 1. Cassel, W. A., Murray, D. R. Treatment of stage II malignant melanoma patients with a Newcastle disease virus oncolysate. Natural Immunity and Cell Growth Regulation 7, 351-352 (1988). PMID: 3065642 2. Steiner, H. H., Bonsanto, M. M., Beckhove, P., Brysch, M., Geletneky, K., Ahmadi, R., Schuele-Freyer, R., Kremer, P., Ranaie, G., Matejic, D., Bauer, H., Kiessling, M., Kunze, S., Schirrmacher, V., HeroldMende, C. Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol 22, 4272-4281 (2004). DOI: 10.1200/JCO.2004.09.038 3. Bateman, A. R., Harrington, K. J., Kottke, T., Ahmed, A., Melcher, A. A., Gough, M. J., Linardakis, E., Riddle, D., Dietz, A., Lohse, C. M., Strome, S., Peterson, T., Simari, R., Vile, R. G. Viral fusogenic membrane glycoproteins kill solid tumor cells by nonapoptotic mechanisms that promote cross presentation of tumor antigens by dendritic cells. Cancer Res 62, 6566-6578 (2002). PMID: 12438252 4. Vigil, A., Park, M. S., Martinez, O., Chua, M. A., Xiao, S., Cros, J. F., Martinez-Sobrido, L., Woo, S. L., Garcia-Sastre, A. Use of reverse genetics to enhance the oncolytic properties of Newcastle disease virus. Cancer Research 67, 8285-8292 (2007). DOI: 10.1158/00085472.CAN-07-1025 5. Vigil, A., Martinez, O., Chua, M. A., Garcia-Sastre, A. Recombinant Newcastle disease virus as a vaccine vector for cancer therapy. Molecular Therapy: the Journal of the American Society of Gene Therapy 16, 1883-1890 (2008). DOI: 10.1038/mt.2008.181 6. Krishnamurthy, S., Takimoto, T., Scroggs, R. A., Portner, A. Differentially regulated interferon response determines the outcome of Newcastle disease virus infection in normal and tumor cell lines. Journal of Virology 80, 5145-5155 (2006). DOI: 10.1128/JVI.02618-05 7. Grosse-Wilde, A., Voloshanenko, O., Bailey, S. L., Longton, G. M., Schaefer, U., Csernok, A. I., Schutz, G., Greiner, E. F., Kemp, C. J., Walczak, H. TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development. J Clin Invest 118, 100-110 (2008). DOI: 10.1172/JCI33061 8. Zhang, X. D., Franco, A. V., Nguyen, T., Gray, C. P., Hersey, P. Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. J Immunol 164, 3961-3970 (2000). PMID: 10754286 9. Ivanov, V. N., Bhoumik, A., Ronai, Z. Death receptors and melanoma resistance to apoptosis. Oncogene 22, 152-161 (2003). DOI: 10.1038/ sj.onc.1206456 10. Zhang, X. D., Franco, A., Myers, K., Gray, C., Nguyen, T., Hersey, P. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. Cancer Research 59, 2747-2753 (1999). PMID: 10364001
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