Construction, Expression, Purification Of Human IL-24and IL-24-CN And The Effects On Human Malignant Melanoma Cell

In recent years, with the increasing of environmental deterioration, the aging population and bad habits of people, the number of cancer incidence and the deaths due to cancer were increasing. Metastasis is of the utmost clinical relevance, as it is responsible for more than90%of cancer-associated mortality. The traditional methods to treat cancer generally have toxic side-effects on the human body and are not always effective. Therefore, a cost-effective anticancer drug urgently needed. Interleukin(IL)-24is a novel cytokine, belongs to IL-10family, with the active that selectively inducing cancer cells apoptosis and inhibiting cancer cell proliferation in vivo and in vitro, but having little effect on normal cells. Therefore, IL-24has become a key target in the field of anti-tumour drug research for the great potential for application. Contortrostatin (CN) is a novel homodimeric disintegrin isolated from the venom of Agkistrodon contortrix contortrix snake and displays two RGD motifs (one on each chain) in the conformational flexibility of C-terminal. The interaction of CN with integrins on tumor cells and angiogenic vascular endothelial cells, which modulated by the RGD motifs, can significantly inhibit tumor cells metastasis and angiogenesis. CN is the only venom toxin has yielded superb potential for the treatment of metastatic cancer as demonstrated by in vitro and in vivo experimental studies. In this paper, we studied the recombinant expression of IL-24and IL-24-CN and their activities in cancer cells.In order to achieve the non-fusion expression of IL-24(nrIL-24) in E. coli, we optimised the secondary structure of the translation initiation region (TIR). The Gibbs free energy (â–³G) of the region was decreased from-22.0to-9.07kcal/mol, potentially promoting a loose secondary structure formation and improving the translation initiation efficiency. The IL-24mutant gene was cloned into pET-32a(+) and expressed in E. coli. The results indicated that the expression of nrIL-24was increased to26%of the total cellular protein from barely expression. The nrIL-24was refolded after denaturation of the inclusion bodies. Finally, we obtained nrIL-24with the purity of93%by ion exchange chromatography.In order to enhance the anti-tumor activity of IL-24, we constructed the IL-24-CN fusion expression plasmid pET32a-IL24-CN, and transformed into E.coli Rosetta-gami B(DE3) pLysS. The results showed that the fusion protein was expressed as soluble form and comprised up to39.3%of the total cellular supernatant protein. We have established the purification process for obtained the high purity IL-24-CN.The inhibitory effects of nrIL-24and IL-24-CN on the growth and metastasis of tumor in vitro were studied.(1) MTT assay showed nrIL-24and IL-24-CN suppresses cancer cells growth in a concentration-dependent manner. To further determine whether this growth inhibition produced by the induction of apoptosis, FCM was carried out. The results revealed that the apoptotic rate of A375cells inducing by nrIL-24was16.8%, lower than the IL-24-CN of26.8%. These supported that the anti-tumor activity of IL-24-CN is higher than the nrIL-24.(2) We analyzed the effects of recombinant protein onA375cells migration using wound healing experiments. The migration distance was measured by photo shop after photograph. The mobility of A375cells treatment with PBS was33.07%, nrIL-24was26.59%and IL-24-CN was16.82%, respectively. These suggested that nrIL-24and IL-24-CN can inhibit A375cells migration and IL-24-CN with a higher activity than nrIL-24.(3) Transwell experiments verified the above results. The nrIL-24slightly inhibits the migration and invasion in A375cells with the inhibitionsof24.68%and13.56%respectively. And IL-24-CN has a more robust activity, the inhibiting rates were51.95%and45.76%respectively.(4) Cell adhesion assay described that IL-24-CN has a more vigorous anti-tumor activity compared with nrIL-24due to the higher affinity of IL-24-CN to tumor cells. The results supported that CN can improve the targeting of IL-24to the tumor cells surface.In a summary, the present study achieved the efficient preparation of recombinant IL-24by the non-fusion expression strategy. The purified nrIL-24can significantly induces apoptosis and inhibits proliferation of A375cells. In addition, nrIL-24can slightly blocks migration and invasion of A375cells. This study also constructed the soluble IL-24-CN expression system and established a purfication process of the fusion protein. IL-24-CN has a higher anti-cancer activity than nrIL-24and can obviously inhibits A375cells migration and invasion. These results suggested that the fusion espression strategy of IL-24and CN may increase its anti-cancer activity by a synergistic effect. The study laid the foundation for IL-24further research and cancer therapy.