Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China.
Food Chem. 2013 Dec 1;141(3):2625-33. doi: 10.1016/j.foodchem.2013.04.001. Epub 2013 Apr 11.
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.
重组荞麦胰蛋白酶抑制剂（rBTI）被研究用于评估其是否能够进入癌细胞，并确定其机制。异硫氰酸荧光素标记的荞麦胰蛋白酶抑制剂（FITC-BTI）以浓度依赖的方式进入 Hep G2 细胞。FITC-BTI 与标记的转铁蛋白（Tf）在点状结构中共定位，表明 rBTI 通过网格蛋白依赖的内吞作用进入 Hep G2 细胞。用不同的化学抑制剂孵育 Hep G2 细胞可消除弥散荧光，但点状荧光不受影响，因此表明膜电位在此过程中起关键作用。用网格蛋白重链的 RNAi 破坏网格蛋白介导的内吞作用，大大减少或完全消除了弥散和点状荧光，进一步支持了单一内吞途径的理论。与我们的工作假设一致，处于 M 期的 Hep G2 细胞没有显示任何囊泡或弥散的 FITC-BTI。我们从这些结果得出结论，rBTI 进入 Hep G2 细胞既需要内吞作用，也需要膜电位。
