Oncolytic Effect Of Newcastle Disease Virus On Small Cell Lung Cancer

Background and objective:At present malignant tumor has become a leading cause which influences people's living quality and threatens people's health severely.So the urgent affairs are studying the pathogenesis of tumor and developing effective anti-tumor approaches which can help the sufferers racked with cancer to improve their living quality and to prolong their life span.Recently researchers find there are some viruses have the capability to kill tumor cells.For the ability to kill cancer cells efficient and relatively safe to normal cells,Newcastle Disease Virus(NDV)attracts more and more attentions among these viral agents for cancer therapy.NDV belongs to Avulavirus genera of the Paramyxoviridae family.NDV could infect various human tumor cells,multiply and replicate independently and selectively kill them.Moreover,NDV is safe to most normal human cells.The former study demonstrated that different NDV strains have different potentials and preferences in killing tumor cells.Studies from abroad always focus on NDV lyric strains such as 73-T.The anti-tumor effects of those NDVs were evaluated in several pre-clinical studies.In China,nonlytic NDV strain LaSota was always chosen for the treatment of cancers.Until now,there isn't any document has been found about the isolation and incubation of tumor adapted NDV.In order to find NDV strains which could anti-lung cancer effective without deadly risk to normal cell,we chose human small cell lung cancer cell line NCI-H446,human embryonic lung fibroblast cell line HFL-1 and NCI-H446 xenograft in athymic mice to evaluate the anti-tumor effectiveness and security of 14 NDV strains.Furthermore,we investigated the anti-tumor effect of NDV lentogenic strain 7793 therapy on xenotransplant of NCI-H446 in athymic mice and explored the mechanism of this virotherapy.Materials and Methods:Twelve NDV strains 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 1×10⁸PFU/ml,then stored at -80℃.African green monkey kidney cell line Vero-E6,human small cell lung cancer cell line NCI-H446 and human embryonic lung fibroblast cell line HFL-1 were incubated according to ATCC recommendation.The cytotoxic effect of 14 NDV strains on human small cell lung cancer cell and human embryonic lung fibroblast were measured through MTT staining in order to select NDV strains which have the potential to kill lung cancer cell efficiently and safely to normal cell as target strains.Target strains were propagated in NCI-H446 cell line to adapt for lung cancer cell,and then triple-plaque purified in Vero-E6 cell line. Amplification of the purified virus was done by passage through chick embryo. HA test,TCID₅₀and MTT staining were carried out to detect the cytotoxic effect of the purified virus on lung cancer cell and normal cell.A multiplex RT-PCR assay was performed to differentiate the virulence of target strain primarily.The logarithmically growing NCI-H446 cells were digested by 0.25% trypsin associate with 0.02%EDTA and then dilute/concentrate to 5×10⁷ cell/ml by RPMI-1640 medium.NCI-H446 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 form 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 killed mice,the 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 place 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:1.14 NDV strains in this research showed more significant cytotoxic ability in cancer cell line NCI-H446 than normal cell line HFL-1 in most time (P＜0.01).2.Except NDV strain D713,NDV strains in this research made more obvious cytotoxic effect on NCI-H446 with increase of incubation time.3.48 hours after infection of D713 strain,the cell mortality rate of NCI-H446 was decreased with increase of virus concentration.4.The target strains:D417 strain and D922 strain of NDV had the capability of killing lung cancer cell efficiently while relative safe to normal cell,which were inoculated in cell lines for 48 hours,showed about 90%cell mortality rate of lung cancer cell and about 20%cell mortality rate of normal cell respectively. 5.Both D417 and D922 strains of NDV appeared to replicate much more efficiently in lung cancer cell than they did in normal cell.Their ability to replicate are 64- to 128-fold faster in lung cancer cell than in normal cell.6.In high virus dose(10⁶～10⁷ PFU/ml)there were no significant statistical differences between the lethality of lung cancer cell for NDV strain D417 and NDV strain D922 infection,however in low virus dose(10⁴～10⁵PFU/ml) the lethality of lung cancer cell after D417 infection was markedly higher than D922 in vitro.7.After selective passage in lung cancer cell NDV strains obtained more powerful oncolytic capability,while no significant changes in cytotoxic effect on normal cell.8.10 days after subcutaneous injection of tumor cells the xenograft model in athymic mice was successfully established.9.Both NDV 7793 and cisplatin injection showed inhibitory effect on tumor growth,the tumor growth inhibition rates were 34.1%and 50.7%.10.NDV 7793 therapy showed no significantly toxic or adverse effect,while obviously adverse effect was observed in cisplatin group.11.Pathological examination showed that there were more and bigger apoptosis/necrosis regions in tumors of NDV 7793 group than control group.12.The result of S-P IHC assay showed that expression of Bax protein in NDV 7793 group was markedly higher than control(P＜0.01).Compared the expression of Bcl-2 in NDV 7793 group and control group,the difference was not significant.Conclusions:1.14 NDV strains in this research could replicate and kill small cell lung cancer cell selectively,as potential anticancer agents they could be applied to cancer biotherapy.NDV strain D417 and NDV strain D922 possess more powerful anticancer potency among 14 NDV strains.2.NDV strains DK/HK/417/1978 and DK/HK/922/1980 belong to NDV velogenic strain.3.NDV strain WDK/JX/7793/2004 has the capability to cause tumor regression of human small cell lung cancer xenograft in athymic mice.4.The mechanism of anticancer effect maybe due to that NDV strain WDK/JX/7793/2004 infection may upregulate the expression of Bax protein.