Research On Trail In Combination With Lidamycin, Its Mechanism And The Expression Of Trail

1. Studies on the synergistic effect and its possible mechanisms of lidamycin in combination with TRAIL in NSCLCBACKGROUND&OBJECTIVE:To investigate the effect and its possible mechanisms of lidamycine (LDM) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human non-small cell lung cancer (NSCLC) cells. METHODS:MTT assay was used to determine the growth inhibition of LDM, TRAIL and the two on H460 cells. Apoptosis was examined by Annexin V-FITC/PI staining and DNA-specific dye Hoechst33342 staining. The level of TRAIL receptor and apoptosis-associated protein expression was detected by Western blot analysis. RESULTS:The combination of LDM and TRAIL exerted more potent than LDM or TRAIL alone in decreasing the survival and inducing apoptosis of H460 cells (non-small cell lung cancer cells). There existed synergistic effect between LDM and TRAIL in inhibiting the proliferation of tumor cells because CDI value was less than 1 by MTT assay. The IC50 value of LDM and TRAIL for H460 cells was 4.60x10-10 mol/L and 915 ng/mL separately. The IC50 value of LDM reduced to 3.06×10-11 mol/L when the H460 cells were treated with different concentrations of LDM combined with TRAIL at the concentration of 50 ng/mL and 1.61×10-11 mol/L when combined with TRAIL at the concentration of 100 ng/mL. After the cells were treated with TRAIL (50 ng/mL), LDM (1×10-10 mol/L) and the combination, the apoptotic percentage was 62.3%,26.1% and 74.7%, respectively. Furthermore, the induction of the cleavage of PARP and the activation of caspase-3 and caspase-8 by the combination were more effective than LDM or TRAIL alone. At last, the level of death receptor 5 (DR5) expressions was increased in a dose-dependent fashion and time-related pattern. CONCLUSION:LDM inhibits the growth of H460 cells in vitro. DR5 induction contributes to enhancement of TRAIL-induced apoptosis by lidamycin in human non-small lung cancer cells. 2. The construction, expression and purification of TRAIL and the analysis of its biological activityBACKGROUND&OBJECTIVE:The Study of first section have confirmed that there is synergistic effect on inhibiting the proliferation of NSCLC between lidamycin and TRAIL. Animal experiment will be used to examine the synergy in vivo. Therefore the construction, expression and purification of TRAIL is necessary in order to obtain a great deal TRAIL protein. This part was to isolate TRAIL gene from human placenta, to construct a recombinant expression vector contaning TRAIL gene which was transformed into E coli. and expressed, and at last to analyze the biological activity of recombinant TRAIL protein. METHODS:TRAIL gene was obtained from human placenta by two-step RT-PCR and sTRAIL sequence inserted with His-Tag encoding sequence at 3'terminal was acquired via PCR amplification. The recombinant gene verified by enzyme-digestion and sequenced was inserted into pET30a(+) expression vector and then it was transformed into E.coli BL21(DE3) starTM. The expression of TRAIL protein was induced by 0.2 mmol/L IPTG and analyzed by SDS-PAGE. Protein in the inclusion bodies was denatured after cell lysis and renatured after purification via Ni2+ affinity chromatography. The protein was identified using Western Blot. The binding activity between the product and its receptor was assessed by ELISA. RESULT:The vector containing TRAIL gene was successfully constructed and the encoded proteins were expressed in the inclusion bodies of E.coli. Fusion proteins were purified by Ni2+ affinity chromatography and the production of active TRAIL was 3 mg per liter fermentation broth. The result of Western blot showed that the purified protein contained His-Tag. Strong binding activity was found between the recombinant protein and H460 cells treated by lidamycin. CONCLUSION:TRAIL gene and sTRAIL protein with His-Tag in the C-terminal were acquired. The TRAIL protein was isolated and purified from the inclusion bodies. ELISA assay showed high binding activity between TRAIL and H460 cells treated by LDM.