Tirchinella Spiralis Anti-tumor Protein Gene Cloning, Expression And The Research Of Inducing Apoptosis In H7402Cells

The investigation of World Health Organization shows that the number of globalpatients suffering from hepatocellular carcinoma drastically ascends over100million and died61million every year. In China, the morbidity and mortalitynumbers are340,000and110,000respectively. China has ranked top one global livercancer incidence and mortality. At present, operation has been regarded as the mosteffective treatment for earlycancer, but liver cancer usually were diagnosed inadvanced cancer, and accompanied with liver cirrhosis and other liver functionabnormalities, so the effectiveness of surgical resection has been restricted largely.Although, radiotherapy and chemotherapy powerfully inhibit the proliferation ofcancer cells, but their side effect block the therapies on the way. As technologyadvances, the development of anti-tumor drugs have extended to chemical drugs andtargeted drugs. Targeted drugs have become a study got pot in the field. Such assorafenib and other durgs target on tumor cell signal transduction, they have beenratified and used in liver cancer clinical therapy by USFDA. Therefore, intervene thecell signal transduction and induced liver cancer cell apoptosis by the biological drugwill greatly release the lack of anti-liver cancer drug status.Previousily, an anti-tumor gene named A200711from Trichinella spiralis hasbeen screened using human hepatocellular carcinoma cell line H7402and humanleukemia cell line K562for cell panning by our research group, The obtainedA200711gene was transfected into human hepatocellular carcinoma cells H7402andsignificantly inhibit tumor cells proliferation; upregulate the expression ofapoptosis-related genes caspase-3and caspase-9. Based on the above results, in thisstudy, the gene was prokaryotic expressed and purified. Our studies havedemonstrated the anti-tumor effect of Trichinella spiralis A200711proharyotic proteinboth in vitro and vivo, which pave the road for anti-tumor resrarch of Trichinellaspiralis. A200711gene was amplified from Trichinella spiralis cDNA libarary and cloned topET28a expression vector. The recombinant expression plasmid was expressed in E.coli BL21(DE3) and induced by IPTG; the expressed protein was purified andrefolded by Ni affinity chromatography column. Protein expression was tested bySDS-PAGE. BCA method was used to assaied protein concentration. Western blottinganalysis showed that the expressed protein was identified by positive sera of pig thatinfected with Trichinella spiralis for26days.Different concentrations of Trichinella spiralis A200711protein were co-culturedwith human hepatocellular carcinoma cell line H7402and human normal liver cellline H7702, respectively. CCK-8results showed Trichinella spiralis A200711roteininhibited the proliferation of human hepatocellular carcinoma cell line H7402significantly(p<0.05) and exist dose-dependent and time-effect relationship,Conversely, the relation is not detected in human normal liver cell line H7702(p>0.05).The result of Annexin V-FITC showed that Trichinella A200711protein inductedH7402cells apoptosis and exist a linear relationship. We also observed thatTrichinella spiralis A200711protein can encumber cell cycle of H7402cells by FCManalysis. Cell cycle was arrested at S phase and then a significant sub-G1apoptoticpeak was detected.Experiment in vivo was conduced on nude mice. Nude mice were grafted humanhepatocellular carcinoma cell line H7402. A200711protein was injected into tumortissue. Mice were injected with the same amount of PBS as negative control, and DDPas positive control. After21days’ treatment, tumor growth of experimental group wasinhibited. Contrast to DDP, A200711protein pose less effect on physiologicalconditions of nude mice. This study show that Trichinella A200711protein couldinhibit H7402cells proliferation, induce apoptosis of tumor cells in vivo and vitro, butit could not affect on the growth of human normal liver cells H7702. As comparedwith DDP, Trichinella spiralis A200711protein shielded tumor beared mice frommore side effects, All these results provides some basis dates for Trichinella A200711protein becoming an anti-tumor drug candidate in future.