title: Significant induction of apoptosis in renal cell carcinoma cells transfected             with cationic multilamellar liposomes containing the human interferon-β gene through             activation of the intracellular type 1 interferon signal pathway.
abstract: We previously reported that cationic multilamellar liposome containing the human interferon-β (huIFN-β) gene (IAB-1) demonstrated significant cytotoxic effect in the NC65 human renal cell carcinoma (RCC) cell line. In this study, we investigated the molecular mechanisms of IAB-1-induced apoptosis and cytotoxicity in RCC cells. Remarkable in vitro cytotoxic and apoptosis-inducing effects of IAB-1 against NC65 cells were observed by a colorimetric method and TUNEL staining, respectively. In contrast, treatment of NC65 cells with exogenously added huIFN-β protein induced low-level cytotoxicity without apoptosis. Neutralizing antibodies against huIFN-β significantly suppressed the cytotoxic effect of huIFN-β protein, but they were unable to block the effect of IAB-1. Cytotoxicity assays using transwell plates revealed that NC65 cells treated with IAB-1 did not secrete cytotoxic soluble factors other than IFN-β. Substantial enhancement of interferon-stimulated response element (ISRE) activity of NC65 cells by IAB-1 was demonstrated by promoter reporter assays. In addition, immunofluorescence using confocal microscopy revealed the intracellular expression of IFN-β and its receptor induced by IAB-1. The induction of c-Myc by IAB-1 was suggested by a cDNA macroarray and was confirmed by western blot analysis. These findings indicate that IAB-1 induces significant cytotoxicity and apoptosis in NC65 cells, possibly through enhanced ISRE activity, that is associated with increased intracellular localization of huIFN-β and IFN-receptor. Our data support the potential clinical application of IAB-1 gene therapy for RCC resistant to IFN.
authors.affiliation: Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kyoto 602-8566, Japan. ntakaha@koto.kpu-m.ac.jp
authors.forename: Natsuki
authors.initials: N
authors.lastname: Takaha
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakanishi
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kimura
authors.forename: Fumiya
authors.initials: F
authors.lastname: Hongo
authors.forename: Kazumi
authors.initials: K
authors.lastname: Kamoi
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kawauchi
authors.forename: Masaaki
authors.initials: M
authors.lastname: Mizuno
authors.forename: Jun
authors.initials: J
authors.lastname: Yoshida
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Wakabayashi
authors.forename: Tsuneharu
authors.initials: T
authors.lastname: Miki
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D003124:Colorimetry; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D006801:Humans; D020287:In Situ Nick-End Labeling; D050835:Interferon Regulatory Factors / Q000235:genetics / Q000378:metabolism*; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008081:Liposomes; D018613:Microscopy, Confocal; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D017471:Receptors, Interferon / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D015398:Signal Transduction; D014162:Transfection
chemical_list: D050835:Interferon Regulatory Factors; D008081:Liposomes; C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D017471:Receptors, Interferon; D016899:Interferon-beta