title: HIV-TAT-fused FHIT protein functions as a potential pro-apoptotic molecule in hepatocellular carcinoma cells.
abstract: Accumulating evidence has demonstrated that FHIT (fragile histidine triad) is a bona fide tumour suppressor gene in a large fraction of human tumours, including hepatocellular cancer. A virus-based delivery system has been developed to transfer the FHIT gene into many types of cancer cells to inhibit growth or even induce apoptosis. However, a protein-based replacement strategy for FHIT has not been performed in cancer cells. Here, we used HIV-TAT (transactivator of transcription)-derived peptide to transfer the purified FHIT protein into HCC (hepatocellular carcinoma) cells and determine the biological effect of this fusion protein in inducing apoptosis. Affinity chromatography was used to purify TAT peptide-fused human FHIT (TAT-FHIT) protein from BL21 Escherichia coli. Immunofluorescence staining and Western blot analysis were performed to identify the expression and internalization of TAT-FHIT in HCC cells compared with the purified FHIT protein. Our study showed that TAT-FHIT protein can translocate into cancer cells in 1 h after incubation at 37°C. Furthermore, the results of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, Annexin-V staining and Western blotting demonstrated that TAT-FHIT can robustly inhibit growth and induce apoptosis of HCC cells in vitro. In addition, a mechanistic study showed that both exogenous and intrinsic apoptotic pathways were involved in TAT-FHIT-mediated apoptosis and this effect could be attenuated partially by a mitochondrial protector TAT-BH4, indicating that mitochondrion plays a critical role in TAT-FHIT-mediated pro-apoptotic effect in cancer cells. Taken together, our study suggests that TAT-FHIT is a potential pro-apoptotic molecule in HCC cells and strengthen the hypothesis of its therapeutic application against HCC.
authors.affiliation: State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University, 710032 Xi'an, People's Republic of China.
authors.forename: Gui-Rong
authors.initials: GR
authors.lastname: Yu
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Qin
authors.forename: Ji-Peng
authors.initials: JP
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Hua
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Meng
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Bo
authors.initials: B
authors.lastname: Yan
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhang
authors.forename: Lin-Tao
authors.initials: LT
authors.lastname: Jia
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: An-Gang
authors.initials: AG
authors.lastname: Yang
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000096:biosynthesis / Q000494:pharmacology*; D004355:Drug Stability; D056945:Hep G2 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D010539:Permeability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis / Q000494:pharmacology*; D000099170:Bis(5'-adenosyl)triphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D053181:Caspase 8; D053453:Caspase 9; D057846:Cell-Penetrating Peptides; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D000099170:Bis(5'-adenosyl)triphosphatase; C505530:CASP8 protein, human; C506433:CASP9 protein, human