The Human Mda-7/IL-24, Its Expression And Bioactivities

The control of malignant tumor has been a challenged task for decades in medical practice.As we know, angiogenesis is one of the important factors in tumor growth. Solid tumors can express some moleculars to promote angiogenesis resulting in metastasis. It is very important to find out how the tumor growth and angiogenesis is inhibited so as to prevent tumor transfer and gene therapy does provide this possibility. The perfect molecule used in gene therapy should be able to inhibite tumor cell proliferation, induce tumor cell appoptosis and prevent angiogenesis, but has no side effect on normal cells. The overexpressed mda-7/IL-24 bears such characteristics and has therefore been used in phase I clinical study.Although mda-7/IL-24 can function through many different ways, its mechanism is not clear yet. There is no report update whether it can act on esophageal tumor and small cell lung cancer. We cloned its cDNA, overexpressed it and analyzed its role on esophagus tumor cell Eca-109, lung cancer H460 (large cell cancer)and small cell lung cancer NCI-H466 cell line. What we got is as follow:1. mda-7/IL-24 cDNA was cloned from PHA treated human peripheral blood lymphocytes with RT-PCR and confirmed by DNA sequencing. The recombinant protein with His6-tag was expressed in E coli and used as immunogen to prepare the polyclonal antibody against MDA-7/IL-24 protein. The antiserum could be used in Western-blot to analysis the overexpressed MDA-7/IL-24 in E coli, carcinoma cells and normal cells.2. The effect of tag in different position of target protein on the distribution of MDA-7/IL-24 was analyzed by transient transfecting recombinant plasmids constructed GFP-mda-7/IL-24 and mda-7/IL-24-GFP respectively into monkey embryo kidney Cos7 cell line. The MDA-7/IL-24 protein with GFP-tag at NH₄-terminal localized in cytoplasm and nucleus. On the other hand, the MDA-7/IL-24 protein with GFP-tag at COOH-terminal was found in the cytoplasm and around the nuclear membrane and accumulated near the nuclear membrane too. The result implied that the position of tag of recombinant protein could change the localization of MDA-7/IL-24 protein.3. The recombinant plasmid pcNDA3-mda-7/IL-24 was transfected transiently into the lung cancer H460 cell line. The MTT analysis showed that overexpressed mda-7/IL-24 could inhibit proliferation of H460 cells.4. The recombinant adenvirus Ad.mda-7/IL-24 and Ad were prepared according to the protocol of pAdEasy system and identified by PCR and Western-blot. Their titers were measured by TCID₅₀ method were 6.3 × 10¹⁰ pfu/ml and 2×10¹⁰ pfu/ml respectively. The Ad.mda-7/IL-24 could infect some tumor cell lines, such as lung cancer cell H460, small cell lung cancer cell NCI-H446, esophageal tumor cell Eca-109, uterine cervical carcinoma cell HeLa and normal human embryo lung filbroblast cell HEL. MTT results...