Cloning, Expression, Antitumor Activity And Mechanisms Analysis Of ZS-TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily. As a potential anti-tumor agent, TRAIL has drawn a great deal of attention. The reason for this is that TRAIL selectively induces apoptosis in many cancer cell lines, but with minimal cytotoxicity toward normal cells. Domestic and international clinical phase I studies showed a high efficiency of a combinatorial TRAIL-based therapy without dose limiting toxicities and immunogenicity. Accordingly clinical phase II studies have begun recently. However, its low biological activity obstructs its extensive application. The addition of a trimerization domain has been used to enhance TNF-ligand stability and biological activity. Therefore, in this study, a human origin, trimeric coiled-coil domain (ZS) was fused with TRAIL for creating more active proteins.The results were shown as follows:1. Cloning, expression and purification of TRAIL and ZS-TRAIL. The human TRAIL gene (encoding residues 114-281) and ZS-TRAIL gene was cloned by RT-PCR and overlap-extension PCR, respectively. Then, the genes were cloned into plasmid pET-28a and expressed in E. coli BL21(DE3). TRAIL and ZS-TRAIL were purified by Ni-affinity chromatography and ion-exchange chromatography combinational. Purity of the prepared TRAIL and ZS-TRAIL, determined by HPLC, was 96.5% and 99%, respectively. Western blot analysis showed specific reaction of the purified recombinant TRAIL and ZS-TRAIL with mouse anti-human TRAIL monoclonal antibody. Molecule weight of TRAIL and ZS-TRAIL, determined by ESI-MS was matched well with the theoretic molecule weight. CD analysis showed the secondary and tertiary structure of TRAIL and ZS-TRAIL were ordered.2. The cytotoxicity of TRAIL and ZS-TRAIL in vitro. The cytotoxicity of TRAIL and ZS-TRAIL in cancer cells (SPC-A1, HCT-8, BEL-7402 and HL-60) and normal cells (LO2) was tested by SRB in vitro. Both TRAIL and ZS-TRAIL demonstrated the cytotoxicity by dose-dependent relationship in SPC-A1, HCT-8, BEL-7402 and HL-60 cells, but no cytotoxicity in normal LO2 cells. ZS-TRAIL was with much more cytotoxicity to cancer cells than TRAIL.3. The cytotoxicity of TRAIL and ZS-TRAIL in vivo. Nude mice xenograft tumor models of human colon cancer HCT-8 cells and lung cancer SPC-A1 cells were established to evaluate the antitumor activity of TRAIL and ZS-TRAIL in vivo. It exhibited that ZS-TRAIL was more active than TRAIL in the two models. High-dose(80mg/kg) of ZS-TRAIL and TRAIL had no hepatoxicity to the nude mice.4. Analysis the reason why ZS-TRAIL is more active than TRAIL in vitro and in vivo. Oligomerization state analysis showed that both ZS-TRAIL and TRAIL appeared as homologous trimers. Stability analysis showed that ZS-TRAIL was more stable than TRAIL under physiological condition (37℃), especially when the solution containing 1% Human Serum Albumin. Tm of TRAIL was higher than ZS-TRAIL, that means TRAIL was more thermal stable than ZS-TRAIL. Results of pharmacokinetic studies proved that the half-lives of TRAIL and ZS-TRAIL were almost the same. Accordingly, we speculated that the enhanced antitumor activity of ZS-TRAIL was related to its stability under physiological condition, and had nothing to do with the proportion of homologous trimers, the half-life and thermal stability. Further studies explored that the enhanced zinc-binding ability of ZS-TRAIL was the real reason for its improved antitumor activity.5. Optimization the culture conditions of ZS-TRAIL. The optimized culture conditions were determined by response surface analysis (RSM). The final composition of the optimized conditions were as follows: yeast extract, 11.38 g/L; NaCl, 10 g/L; typtone, 10 g/L; kanamycin, 30μg/mL; IPTG, 0.1 mM; ZnSO₄, 0.458 mM; induction temperature, 29.2℃; induction time, 6h. The production of soluble ZS-TRAIL in E.coli. BL21(DE3) was improved from 158.6 mg/L to 561.3 mg/L after optimization. Furthermore, high-cell-density fermentation was performed. Production of soluble ZS-TRAIL concentration reached to 4.26g/L under the fermentation conditions.