title: Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis.
abstract: Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Cui
authors.forename: Zhuanhua
authors.initials: Z
authors.lastname: Wang
authors.forename: Yuying
authors.initials: Y
authors.lastname: Li
authors.forename: Chen
authors.initials: C
authors.lastname: Li
mesh_terms: D001692:Biological Transport; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D019612:Fagopyrum / Q000235:genetics / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D002966:Clathrin; D010940:Plant Proteins; D014361:Trypsin Inhibitors
keywords: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; Buckwheat; Cell penetration mechanism; DAPI; DMSO; Drug delivery; Endocytosis; FITC; HEPES; MDC; Noc; PAI-1; Tf; Trypsin inhibitor; dimethyl sulfoxide; fluorescein isothiocyanate; monodansylcadaverine; nocodazole; plasminogen activator inhibitor type-l; rBTI; recombinant buckwheat trypsin inhibitor; transferrin; uPA; urokinase-type plasminogen activator