The Expression And Purification Of Soluble TRAIL And The Preliminary Analysis Of TRAIL’s Effect On Three Colon Cancer Cells

Objective: 1. To express and purify TRAIL protein and to optimize induction condition. 2. To detect the effects of TRAIL to colorectal cancer cells and analyze the preliminary mechanismsMethods: 1. Expression and purification of TRAILThrough the large scale cultivation of E.coli containing p ET-d-TRAIL plasmid, TRAIL protein was induced by IPTG and purified by Ni affinity and ion-exchange chromatography. Through cultivation of Colorectal cancer cell line, The anti tumor activity of TRAIL was tested by the method of MTT. The effect of IPTG concentration and induction time spelled out on the most efficient way of TRAIL expression. In the process of affinity chromatography, two different elution methods were examined and different time of dialysis was used. The influence of these variation on the activity of TRAIL was determined.2. Preliminary analysis of the effect of TRAIL on the proliferation of colorectal cancer cell line HCT116、SW480 and SW620.Three colon cancer cells HCT116、SW480 and SW620 were treated with 0、100、200 and 400mg/ml TRAIL, the morphologic change was observed under the microscope; proliferation of Colorectal cancer cell was quantified by the method of MTT; expression levels of protein DR5、caspase-3、caspase-6、caspase-7、caspase-8 and XIAP were determined by western blot;caspase-8 plasmid was transfected into SW620, the sensitivity of SW620 cells in which caspase-8 was high expressed to TRAIL was further researched.Results: 1. The optimal induction conditions were determined: TRAIL expressing bacteria entered the logarithmic phase after culture 4 h, then bacteria was induced by 0.5mmol/L IPTG at 37℃ for 6 h, SDS-PAGE showed that TRAIL accounted for 20% of total protein.2. Successfully Purified soluble TRAIL protein whose purity was higher than 95%.3. In the iogarithmic phase colorectal cancer cells treated with 0、100、200 and 400mg/ml TRAIL cultured 12 h. Then MTT analysis showed that cell viability of HCT116 accounted for 65%、28%、6% of the control group. Cell viability of SW480 accounted for 85%、65%、42% of the control group. Cell viability of SW620 accounted for 95%、94%、91% of the control group.4. Western blot showed that the expression of caspase-3、caspase-6、caspase-7、caspase-8 in HCT116 SW480 and SW620 decreased gradually,expression of protein DR5 in HCT116、SW480 and SW620 increased gradually,the expression level of XIAP ordered from high to low was SW480、SW620 and HCT116.5. Treated with 100 mg/ml TRAIL,the viability of SW620 transfected with caspase-8 plasmid decreased 33% comparied with SW620 untransfected caspase-8 plasmid.Conclusion: 1. TRAIL can reduce proliferation of colorectal cancer HCT116、SW480 and SW620, the inhibitory effect was dependent on the concentration of TRAIL and time.2. Three colorectal cancer cell lines had varying tolerance. HCT116 was most sensitive, SW620 was most tolerance, SW480 was tolerance moderately. 3. The tolerance of SW620 to TRAIL might be relative to the low expression of Caspase-8.