title: Using adenovirus armed short hairpin RNA targeting transforming growth factor β1 inhibits melanoma growth and metastasis in an ex vivo animal model.
abstract: The transforming growth factor β (TGF-β) is the key molecule implicated in impaired immune function in human patients with malignant melanoma. TGF-β can promote tumor growth, invasion, and metastasis in advanced stages of melanoma. Blocking these tumor-promoting effects of TGF-β provides a potentially important therapeutic strategy for the treatment of melanoma. In this study, we used an adenovirus-based shRNA expression system and successfully constructed Ad/TGF-β1-RNA interference (RNAi) which mediated the RNAi for TGF-β1 gene silencing. We examined the effects of TGF-β1 protein knockdown by RNAi on the growth and metastasis of melanoma in C57BL/6 mice induced by the B16F0 cell line. The TGF-β1 hairpin oligonucleotide was cloned into adenoviral vector. The resulting recombinant adenoviruses infected murine melanoma cell line, B16F0, and designated as B16F0/TGF-β1-RNAi cells. The blank adenoviral vector also infected B16F0 cells and designed as B16F0/vector-control cells served as a control. TGF-β1 expression was reduced in B16F0/TGF-β1-RNAi cells compared with B16F0 cells and B16F0/vector-control cells. Three million wild-type B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells were injected subcutaneously into the right flanks of adult female syngeneic mice C57BL/6. The tumor sizes were 756.09 (65.35), 798.48 (78.77), and 203.55 (24.56) mm at the 14th day in the mice receiving B16F0 cells, B16F0/vector-control cells, and B16F0/TGFβ1-RNAi cells, respectively. The P value was less than 0.01 by 1-way analysis of variance. TGF-β1 knockdown in B16F0 cells enhanced the infiltration of CD4 and CD8 T cells in the tumor regions. C57BL/6 mice were evaluated for pulmonary metastasis after tail vein injection of 2 million B16F0 cells, B16F0/vector-control cells, and B16F0/TGF-β1-RNAi cells. The pulmonary metastasis also reduced significantly on days 14 day and 21 in mice injected with B16F0/TGF-β1-RNAi tumors. The blood vessel density of the tumors markedly reduced in B16F0/TGF-β1-RNAi tumors. Our results showed that Ad/TGF-β1-RNAi could induce silencing of the TGF-β1 gene effectively. Silencing of TGF-β1 expression in B16F0 cells by RNAi technology can inhibit the growth and metastasis of this tumor after being transplanted to C57BL/6 mice. This kind of adenoviral vector based on RNAi might be a promising vector for cancer therapy.
authors.affiliation: From the *General Education Center, Tzu Chi College of Technology; †Division of Plastic Surgery, Department of Surgery, Buddhist Tzu Chi General Hospital; and ‡Department of Surgery, Tzu Chi University, Hualien, Taiwan.
authors.forename: Kuo-Feng
authors.initials: KF
authors.lastname: Tai
authors.forename: Chien-Hsing
authors.initials: CH
authors.lastname: Wang
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005260:Female; D020868:Gene Silencing; D005822:Genetic Vectors; D007150:Immunohistochemistry; D008175:Lung Neoplasms / Q000556:secondary; D008545:Melanoma / Q000276:immunology* / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D034741:RNA, Small Interfering / Q000187:drug effects / Q000276:immunology* / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012878:Skin Neoplasms / Q000276:immunology* / Q000473:pathology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1