title: Potent antitumor effect elicited by RGD-mda-7, an mda-7/IL-24 mutant, via targeting the integrin receptor of tumor cells.
abstract: The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, an mda-7/IL-24 mutant, RGD-mda-7, which includes the cell adhesive sequence 164Arg-165Gly-166Asp (RGD motif), was constructed and evaluated for bioactivity. RGD peptide binds to integrins α(V)β(3) and α(V)β(5), which are selectively expressed in tumor neovasculature and in the surface of some tumor cells. The wtmda-7/IL-24 and RGD-mda-7 were expressed in Escherichia coli and then purified and renatured. The immunostimulatory activity of RGD-mda-7 was assayed by stimulating peripheral blood mononuclear cells. The results suggested that the abilities of RGD-mda-7 to induce IL-6, TNF-α, and IFN-γ production were higher than wtmda-7/IL-24. Tumor targeting of RGD-mda-7 was assayed using cell adhesion experiments. The antitumor effect of the purified RGD-mda-7 on cell proliferation in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, cell apoptosis by staining with fluorescent probes of FITC-annexin V and DAPI, and caspase-3 expression and activity. The in vitro results showed that RGD-mda-7 inhibited the proliferation of multiple tumor cell lines (Hela, ACHN, HepG2, and A549). Staining with fluorescent probes of FITC-annexin V and DAPI indicated that RGD-mda-7 could induce apoptosis more effectively in four tumor cell lines than wtmda-7/IL-24, but has no effect on normal cell line NHLF. Western blotting showed that treatment of tumor cells with RGD-mda-7 could activate apoptotic pathway by cleavage of caspase-3 as same as wtmda-7/IL-24. Further, RGD-mda-7 group showed a higher cleaved level of caspase-3, but not in NHLF cells. These results demonstrate that RGD-MDA-7 possesses more potent antitumor effects than wtmda-7/IL-24 and therefore merits further investigation in preclinical and clinical studies.
authors: Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, China.
authors: Bao-Fu
authors: BF
authors: Zhang
authors: Jun-Jie
authors: JJ
authors: Liu
authors: Dong-Sheng
authors: DS
authors: Pei
authors: Zhi-Xia
authors: ZX
authors: Yang
authors: Jie-Hui
authors: JH
authors: Di
authors: Fei-Fei
authors: FF
authors: Chen
authors: Hui-Zhong
authors: HZ
authors: Li
authors: Wei
authors: W
authors: Xu
authors: Yong-Ping
authors: YP
authors: Wu
authors: Jun-Nian
authors: JN
authors: Zheng
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D048708:Cell Growth Processes; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D007371:Interferon-gamma / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D007378:Interleukins / Q000235:genetics / Q000494:pharmacology*; D008545:Melanoma / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D058990:Molecular Targeted Therapy; D009842:Oligopeptides / Q000235:genetics / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis; D000099180:Interleukin-24
chemical_list: D016023:Integrins; D007371:Interferon-gamma; D015850:Interleukin-6; D007378:Interleukins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D000099180:Interleukin-24; C047981:arginyl-glycyl-aspartic acid