Soluble Human MDA-7/IL-24: Characterization of the Molecular Form(s) Inhibiting Tumor Growth and Stimulating Monocytes

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Abstract
Interleukin-24 (IL-24), also known as melanoma differentiation-associated gene-7 (mda-7), is a member of the IL-10 family that exhibits both tumor suppressor and proinflammatory properties. We describe the purification of this novel dual-function tumor suppressor/cytokine from the supernatant of IL-24 gene-transfected HEK 293 cells and define the biochemical and functional properties of the soluble human IL-24 protein. Size exclusion chromatography demonstrates that an IL-24 macromolecular complex fractionates in a broad peak with a median of 110 kDa and comprises several IL-24 isoforms, identified by immunoblotting with anti-IL-24 polyclonal antibody after reducing SDS-PAGE analysis. IL-24 was found to associate with two serum components, albumin and C1q. Cation exchange purification results in the isolation of at least two N-linked glycosylated IL-24 dimers covalently associated via intermolecular disulfide bonds. These molecularly defined N-glycosylated IL-24 dimers elicited dose-dependent secretion of tumor necrosis factor-α (TNF-α) and IL-6 from human monocytes, as well as cytotoxicity to human melanoma cell lines. Thus, we demonstrated that the secreted, glycosylated, dimeric, human IL-24 is immunomodulatory to monocytes and exhibits tumor cell growth inhibition.