Studies On Antitumor Effects Of NS1A Recombination Protein From Avian Influenza Virus Combining With Baicalin

Lung cancer is one of the most severe diseases threating human's health and life. With the accelerating development of modern industry, the serious pollution of atmosphere and the increasing number of smokers, the incidence and death rate of lung cancer has been increasing globally. In the world, 600 thousand new patients contracted lung cancer annually. Its death rate is the highest of various cancers. At present, Lung cancer is removed primarily through surgery, radiotherapy or chemothrerapy and so on . Although traditional therapies can remove lung cancer, death rate, particularly in advanced lung cancers, remains high.As incidence of lung cancer has been increasing, the cure rate of lung cancer is still less than 15 percent. Now, drug cocktail is going to be a tendency of clinical treatment of cancers. The research is study on antitumor effects of NS1A recombination protein from H9N2 avian influenza virus combining with Baicalin on human tumor cell lines. We hope to achieve synergistic effects. All of these researches datas lay foundation to clinical treatment of cancer.To investigate the effects on human lung cancer cells SPC-A1 with NS1A from Avian influenza virus. The recombinant plasmid pVAX1-NS1A was transfected into human lung cancer cells SPC-A1 by lipotransamine 2000. To detect viability of SPC-A1, tetrazolium (MTT) method was employed. Results showed that, comparing with control group, viability of experimental group was decreased significantly at 72h. Its effects have time-dependency . Under light microscopy, morphology of SPC-A1 transfected with pVAX1-NS1A is round. With the time increasing, recombinant plasmid could lead to obviously morphological apoptotic changes of SPC-A1 ,such as nuclear shrinkage and agglutinated staining with Hoechst33258. FACS was used to detect apoptosis. Apoptosis rate is 43.17%. The proportion of cells at G0/G1 phase increased. It suggested NS1A inhibited the synthesis of DNA. Agarose gel electrophoresis results showed that SPC-A1 treated with recombinant plasmid pVAX1-NS1A for 72h had DNA ladder .The above results indicated apoptosis could be induced by NS1A from Avian influenza virus. The research datas showed that we should explore activity and function of NS1A recombination protein in vitro.The above results indicated NS1A can be used as a therapeutic agent. It can selectively inhibit cancer cells provided it could be transferred into tumor cells in great amount. It has been confirmed that there are many receptors of epidermic growth factor(EGF) existing on the surface of tumor cells. Bacillus pyocyaneus exotoxin II domain (P II) encodes a transmembrane protein. Therefore if the genes encoding EGF and P II could be integrated into the gene encoding NS1A, the ability of NS1A to promote the apoptosis of tumor cells would be enhanced greatly. So recombinant expression plasmid pET-20b-ETN was constructed. Then the positive recombinants were transformed into the host strain BL21 (DE3) , NS1A recombination protein expression was induced by IPTG. The specific protein expression band(about 45kDa) was detected by SDS-PAGE analyses. Expression level of recombination protein was amounting to 10.7% of the total bacterial protein detected by thin-layer scanning . Target protein mainly in solvable form. Purified recombination protein was used as antigen to immune rabbits .Titers of polyclonal antibody were detected by indirected ELISA.Although prokaryotic expression system has low cost, it can not have an effective post-translational modification of exogenous target protein.. Therefore, target protein can not fold at a correct conformation. To get a high activity of target protein, we employed Bac-to-Bac baculovirus expression systems . The ETN gene was cloned into a transfer vector pFastBac1. Recombinant bacmid was harvested according to the manufacturer's protocol. Recombinant bacmid was transfected into sf9. SDS-PAGE analysis showed that the highest expression level of recombination protein could be achieved at 72-96h after recombinant baculovirus infection.The indirect immunofluorescent assay(IFA)showed target protein could be expressed in sf9 cell furtherly. The recombinant protein was purified using Ni-NTA resin conveniently .The effects of recombination protein on lung cancer cells in vitro was assessed. The results showed that , comparing with control group, viability of SPC-A1 cells was decreased significantly in time and pose dependent manners . Under light microscopy, cell morphology became round. Double fluorescence staining assisted cell sorting was used to detect apoptosis rate.. The result was 23.17%. The proportion of cells at G0/G1 phase increased. Nucleate endonuclease actived agarose gel electrophoresis showed that SPC-A1 treated with recombination protein for 48h had DNA ladders. The above results indicated apoptosis induced by NS1A recombination protein may be resulted from the inhibiting the synthesis of DNATraditional Chinese medicine have distinct effect on cancer treatment.It attracts scientists all over the world, Due to extensive effect and low poison. Scutellaria baicalensis georgi comes from nature, it is safty and harmless. Long-term clinical practice indicate that it is effective to prevent many neoplastic diseases. Baicalin is one of the major effective ingredients. It belongs to glucuronide .Modern pharmacological researches indicate that it has been presented heat-cleaning and detoxicating anti-inflammatory and increasing immunity. It is has been suggested potent antitumor activity from different routes, such as inducing apoptosis and cell differentiation, and reversing drug resistance, etc. To investigate the apoptosis effects of Baicalin on human lung cancer cells SPC-A1,Baicalin was incubated with human lung cancer cells SPC-A1. The results showed that viability of SPC-A1 cells was decreased significantly in time- and dose-dependent manners , particularly at 48h. Compared with control group, SPC-A1 became round under light microscopy. FACS was used to detect apoptosis rate, 20.19%. cells at G0/G1 phase. Agarose gel electrophoresis results of SPC-A1 had DNA ladder morphology. The above results indicated Baicalin could induce SPC-A1 apoptosis.In this research, we aim at using drug cocktail to study tumor cells apoptosis. We can obtain new method of apoptosis. To investigate the antitumor effects from NS1A recombination protein combining with Baicalin on human lung cancer cells SPC-A1 .Inhibitory action of SPC-A1 cells was detected by tetrazolium (MTT) method. Result showed that synergistic effect enhances with the increasing of Baicalin concentration after combination of NS1A recombinant protein and Baicalin for 48h . Double staining FACS analysis show discrepancy between two groups is significant , Experimental group apoptosis rate is 55.79%. The decrease of mitochondrial transmembrane potential and activation of Caspase-3 is tested . The above results indicated combination of NS1A recombinant protein and Baicalin can inhibite the growth of SPC-A1 cells obviously and have synergistic effect. Our results were significant for further study on the cancer therapy.