Comparison Of Tumoricidal Effect Of Three NDV Strains On Throat Cancer Cells

Background and Objective:Newcastle disease virus (Newcastle disease virus, NDV) belongs to Avulavirus genera of the Paramyxoviridae family. It is highly pathogenic to birds, but in rare cases it can infect humans. A slight conjunctivitis or mild respiratory symptoms is the symptom of NDV infected [1]. The study showed that the replication efficiency of NDV in human tumor cell is 10 000-folds than in the normal cells, which allows people to take it as a potential anti-cancer biological agent [2]. At present, NDV anti-tumor therapy has become a complementary and alternative medical method (Complementary andalternative medicine, CAM) for the treatment of human cancer and has been carried out extensive research [3].In order to find NDV strains which could anti-throat cancer effective without deadly risk to normal cell, we chose human throat cancer Hep-2 cell line, human normal cell xenograft in athymic mice to evaluate the anti-tumor effcetiveness and safe of 3 NDV strains. Furthermore, we investigated the anti-tumor effect of NDV Lytic strain D817 and NDV Nonlytic strains 7793, La Sota therapy on xenotransplant of Hep-2 in athymic mice and explored the mechanism of this virotherapy.Materials and Methods:One NDV strain isolated in Hong Kong, one NDV strain isolated in Jiangxi Poyang Lake and one commercial veterinary vaccine La Sota(a lentogenic strain) were involved in this research.Those NDV were inoculated in chick embryo for virus amplification, 48 hours later the allantoic fluid contaning viruses were harvested and tested haemagglutination(HA) titer of virus, when HA titer≥1:1024 dilute the allantoic fluid to 1×108 PFU/ml, then stored at -80℃.African green monkey kidney cell line Vero-E6, human throat cancer cell line Hep-2 and human normal cell were grown according to ATCC recommendation, The cytotoxic effect of 3 NDV strains on human throat cancer cell line Hep-2 and human normal cell were measured by MTT staining in order to select NDV strains which have the potential to kill throat cancer cell efficiently and safely to normal cell as target strains. Amplification of the purified virus was done by passage through chick embryo. HA test and MTT staining were carried out to detect the cytotoxic effect of the purified virus on throat cancer cell and normal cell.The logarithmically growing Hep-2 cells were digested by 0.25% trypsin associated with 0.02% EDTA and then dilute/concentrate to 5×107cell/ml by RPMI-1640 medium. Hep-2 cells were implanted into athymic mice to generate xenograft by subcutaneous injection (0.2ml) of previous cell suspension, monitored the growth of xenograft and physique of mice daily. Once tumor reached at least 5mm in both long and wide dimensions, mice were randomized into treatment groups.Virus was removed from the allantoic fluid by aldehydated erythrocyte adsorption and diluted to 1×107PFU/ml by PBS then injected (0.1ml) to mice through intratumoral route. Tumor volume and body weight of mice were measured at regular time-intervals, then draw the tumor growth curve. After 6 weeks of first NDV infection mice were killed, the xenograft tumor was removed and weighed to calculate tumor growth inhibition rate. Furthermore, the spleen was also weighted to calculate spleen index (spleen weight/body weight) for evaluating NDV toxicity. One part of tumor tissue was placed in 10% formalin. After 24 hours they were taken out to make the dyeing of HE for observing pathological changes of tumor cells. At last the expression of apoptosis-related protein: Bax and Bcl-2 were analyzed through S-P IHC assay. Results:The three NDV strains selected in this study can multiple and kill Hep-2 cells in vitro, the killing effect is correlated with virus dosage and incubation time, but this effect has not be seen in the normal throat tissue cell (P<0.05). After addition of viruses into cell cultural media, we compared their effects on throat cancer Hep-2 cell line and the normal throat tissue cell by using MTT method, the three NDV strains anti-tumor cells effects are strong. When the virus dilution is 1×10-1, the mortality rates of NDV D817 strain, NDV 7793 strain and NDV La Sota strain were 96.65%, 94.95% and 95.35% after infected 60h; The killing effect in normal cells of these three NDV strains are relatively small (mortality rates after 60h≤25%), the differences has statistical significance. The killing effect showed a decrease of these three NDV strains after infected 72h. In the plaque experiment, the plaque of NDV D817 strain appears earliest, and the number of plaque is largest, the plaque of NDV 7793 strain appears latest, and the number of plaque is least, the plaque of NDV La Sota strain in middle.In order to accurately evaluate the killing effect on tumor of the three NDV strains, in the stageⅡexperiment, we study the killing effect of the three NDV strains on throat cancer Hep-2 cell line through the establishment of nude mice experimental model in vivo. The block tumor can be seen about 4d after inoculation in growth, length (L) and short diameter (W) can be up to 5mm after tumor inoculated 12d, nude tumor rate was 100%. In the course of treatment, tumor volume in early treatment of each group: PBS-negative control group, the average tumor volume was (183.5±15.2) mm3; NDV D817 group, the average tumor volume was (175.4±19.4) mm3; NDV 7793 group, the average tumor volume was (176.9±18.7) mm3; NDV La Sota group, the average tumor volume was (185.4±20.4) mm3; Cisplatin injection positive control group, the average tumor volume was (168.5±21.5) mm3.Among the three groups no significant difference. Tumor volume during the treatment of each group: PBS negative control group was fastest-growing, Cisplatin injection positive control group was slowest-growing, NDV groups were middle. Tumor volume at the end of the treatment of each group: NDV and Cisplatin injection positive control groups were significantly lower than PBS negative control group (P <0.01). After the treatment, stripping tumor tissue and weighing, the mortality rates of NDV D817 group, NDV 7793 group, NDV La Sota group and Cisplatin injection positive control group were 56.7%, 51.2%, 39.4% and 90.4%.In the toxicity experiment, the weight of PBS negative control group and NDV groups nude mice were slightly decreased than pre-treatment after treatment, the difference was not significant, body weight of after treatment / body weight of pre-treatment > 0.8;Positive control group of Cisplatin injection body weight after treatment compared to pre-treatment significantly decline (P<0.01), body weight of after treatment / body weight of pre-treatment <0.8, while the nude mice activity significantly decreased with the treatment. In the impact of organ, nude mice spleen weight and spleen index of NDV groups compare to PBS negative control group, there was no significant difference. Nude mice spleen weight and spleen index of positive control group of Cisplatin injection was significantly lower than PBS negative control group (P <0.01). Nude mice liver biopsy observations of NDV groups have not found clear pathological changes. In the experiments of pathological observations, NDV D817 group was found there was more died region in tumor tissue then PBS negative control group: smaller, rounder, and then out the near cell. In the High-power microscope, membrane processes, appear foam-like, membrane lysis, nucleolus disappear. Compared with positive control group of Cisplatin injection, cell fragmentation was not obviously. Flow cytometry analysis showed that tumor cell cycle at G0/G1 phase cells was reduced, S phase cells was reduced, while the G2/M phase cells was increased after treatment by NDV (P <0.01). Immunohistochemical detection showed that Bax, Bcl-2 protein of NDV 7793 and NDV La Sota groups expression brown cytoplasm deeper than PBS negative control group in nude mice subcutaneously transplanted tumor cells, Bax expression was significantly higher than PBS negative control group, the differences was statistically significant (P <0.01). Compare NDV 7793 and NDV La Sota groups to PBS negative control group, the Bcl-2 expression rates has no significant difference .Conclusions:1. 3 NDV strains in this research could replicate and kill throat cancer cell selectively, as potential anticancer agents they could be applied to cancer biotherapy.2. NDV strain 7793 has the capability to cause tumor regression of human throat cancer xenograft in athymic mice.3. The mechanism of anticancer effect maybe due to that NDV strains infection may upregulate the apoptosis and necrosis passes.