Anti-Tumor Effect Of Newcastle Disease Virus 7793 And D817 Strins Against Nude Mice Xenografts Of Human Colon Carcinoma

Background and objectiveNewcastle disease virus (NDV) belongs to Avulavirus genera of the Paramyxoviridae family, which are enveloped viruses containing single-stranded negative-sense RNA. NDV are pathogenic poultry viruses, only in rare cases of human infection, and for the self-limiting disease. NDV therefore are considered non-toxic to humans, has been used as a Complementary and alternative medicine (CAM) and researched extensively.The situation of NDV inhibited the transfer of advanced gastric cancer has becoming a cause for concern in the1850's. It was reported that NDV could infect various human tumor cells, multiplying and replicating independently and selectively kill them, which was contributed to the treatment of cancer patients. The former study demonstrated that different NDV strains have different potentials and preferences in killing tumor cells. In recent years Studies from abroad figured out that 73-T,PV 701 and MTH-68/H strains have entered the stageⅡ,Ⅲclinical trials, and some experimental results showed good prospects in clinical application. But, in China, NDV research is less, mainly concentrated in a nonlytic NDV vaccine strain La Sota. Based on enriched source of the virus, our research group selected two stronger, better anti-tumor effect of the natural strains of NDV. And this test is the preliminary study on the anti-tumor effect of these two NDV strains against human colon carcinoma in vitro and in vivo.Materials and Methods1. Purification of virusTwo NDV strains (DK/HK/817/1980 and WDK/JX/7793/2004, kindly offered by Professor Y. Guan, HongKong University) were inoculated in SPF level egg, incubated in 35℃for 48 hours for virus amplification. Viruses were purified by plaque assay. Purified virus were amplified in SPF level egg again and the allantoic fluids containing viruses were harvested after testing haemagglutination (HA) titre of virus, and then stored at -80℃.2. In vitroWe selected human colon cancer cell lines( LoVo, Ls174t) and one strain of normal colon cell line as subjects. Plaque assay were carried out to detect the infectious effect of the purified virus. The cytotoxic effect of NDV strains on the three colon cancer cell lines were performed by using Lactate dehydrogenase (LDH) assay, The HA test was conducted on the purified virus to determine the HA titre.3. In vivoLoVo cells were implanted into athymic mice to generate xenograft by subcutaneous injection (0.2ml) of cell suspension. Once tumor reached at least 5mm in both long and wide dimensions, mice were randomized into treatment groups. It is PBS group, 5-FU group; NDV 7793 high, medium and low dose group and NDV D817 high, medium and low dose group. Each group has multi-point and bit injection inside and around the tumor. It is injected (0.1ml) once a week for a total of three weeks. After stopping injection, mice were continued to observe 4 weeks. From the first day of injection, the growth of xenograft and physique of mice were monitored daily. It is the conventional measurements of long and wide dimensions every three days. After 7 weeks of first injection, mice were killed and continued the following tests:1.1 Tumor growth curves were mapped, and the tumors were removed and weighed to calculate tumor growth inhibition rate. One part of tumor tissues were place in 10% formalin. After 24 hours they were taken out to make the dyeing of HE for observing pathological changes of tumor cells.1.2 The changes of mice body weight were calculated. The appearance and pathological changes of the livers were observing the degree of damage.1.3 Cell apoptosis morphology was observed by TEM. Cancer cell apoptosis rate and necrosis rate were detected by FCM. Cyt-C, Bax, and Bcl-2 were also detected.1.4 NDV live virus were detected by HA test in mice tumor and other normal tissues.Results1. In vitro1.1 In plaque assay, the time of plaque occurring of NDV 7793 is later than NDV D817. Diameters and PFU were smaller than that of NDV D817. So the toxicity of NDV 7793 was less than NDV D817.1.2 NDV D817, NDV 7793 were more sensitive on LoVo. The anti-tumor of NDV 7793 on two human colon cancer cells was stronger than NDV D817. And the best effects of anti-tumor of different NDV strains were relevant with the best time and the optimal virus concentration.1.3 NDV D817, NDV 7793 were visible in the growth of tumor cells to copy. Two NDV strains have a stronger capable of replication in in LoVo cell line. And the capable of replication of NDV 7793 was stronger than NDV D817.1.4 NDV D817 strain has a feature of membrane fusion, and in line with the lytic NDV strain, but NDV 7793 has not. This result was consistent with the previous extrapolate of NDV 7793 is nonlytic strain and NDV D817 is lytic strain.1.5 NDV D817, NDV 7793 can not reproduction at the normal of colon cells, and has no significant killing effect to them.2. In vivo2.1 It can be observed that the tumor growth of mice of 5-FU group, NDV 7793 high and medium dose group , NDV D817 medium dose group were slower than the other groups. The tumor inhibition rate of 5-FU group, NDV 7793 high and medium dose group , NDV D817 medium dose group is 37.14% ( P<0.01), 50.14%( P<0.01), 37.86%( P<0.01), and 47.98% ( P<0.01) respectively. In addition, NDV 7793 showed a better dose-effect relationship.2.2 After stopped injection, it can be observed that the tumor volume of the 5-FU group growth than the above three groups.2.3 After the experiment, the body weight of NDV 7793 high dose group was higher than PBS group, the other groups has a weight decline to a varying degree ( P<0.01).2.4 The appearance of liver of tumor-bearing mice in each group was normal. It was not found metastasis in pathological sections, but has a certain degree of injury. The side effects of 5-FU are most serious, and 1/3 tumor-bearing mice in this group can be observed liver tissue steatosis.2.5 5-FU caused the death of cancer cells is mainly necrosis. But the high dose NDV 7793 and medium dose NDV D817 can significantly improve the rate of apoptosis in cancer cells ( P<0.01). 2.6 NDV 7793 of high dose and NDV D817 of medium dose can enhanced the permeability of mitochondrial membrane significantly, and improved Cyt-C in cytoplasm.2.7 NDV 7793 high dose group and NDV D817 medium dose group cancer cells expressed the Bax protein in the strongest; and Bcl-2 protein expression in each group has no significant difference.2.8 NDV 7793 and NDV D817 can be confined to proliferated in the tumor tissue, accorded with the bio-security criterion.Conclusions1. Further verified in this experiment for NDV 7793 strain is a attenuated virulent strain. It belongs to nonlytic strain. While NDV D817 strain is high virulent strain which belongs to lytic strain.2. NDV 7793 strain and NDV D817 strain possesses powerful anti-tumor potency.3. Apart from the selectively replicate in cancer cells, the mechanism of anti-tumor of NDV 7793 strain and NDV D817 strain may be related to the promotion of Cyt-C, increase of Bax to enhance cancer cell apoptosis.4. In respect to the safity and effectiveness for biological treatment, NDV 7793 strain is a more appropriate stain in anti-tumor therapy, accounting for its potent effect on tumor cells wihout harming human normal cells, also for its attenuated virulent feacture.