The Transmembrane Transduction Mechanism Of A Peptide With High Affinity To Human Breast Cancer And Membrane Protein Structure Investigation

Objective:Detection cells affinity of PI and MDA-MB-231breast cancer cells; To investigate transmembrane transduction mechanism of human breast cancer cell line MDA-MB-231uptake a peptide (PI); co-immunoprecipitation analysis of MDA-MB-231breast cancer cells and Calu-1lung cancer cell membrane structure differences; For the future Continue to study the specificity of PI transduction mechanism of transmembrane experimental basis.Methods:(1) To investigate whether the inhibitors of cytomembrane passageway amiloride, methyl-β-cyclodextrin, chloropromazine have cytotoxicity effect on MDA-MB-231cells; PI were syntheticed and labeled FITC green fluorescence in its N-terminus. To investigate whether the transmembrane transduction mechanism of PI is associated with the macropinocytosis, caveolin-mediated, clathrin-mediated endocytosis, the distribution of FITC-PI in MDA-MB-231cells were detected using inverted fluorescence microscope and flow cytometry after pre-incubation with inhibitors of endocytosis, amiloride, methyl-β-cyclodextrin, chloropromazine.(2)To investigate whether the transmembrane transduction mechanism of PI is associated with the same membrane protein of the two cell lines (MDA-MB-231cell, Calu-1cell), the membrane proteins were detected by co-immunoprecipitation.Results:The inhibitors of cytomembrane passageway amiloride, methyl-β-cyclodextrin, chloropromazine have no cytotoxicity effect on MDA-MB-231cells, The inhibitors amiloride, methyl-β-cyclodextrin suppressed FITC-PI distribution. Co-immunoprecipitation analysis showed that membrane protein profiles of the two cell lines had three same protein straps.Conclusion:The transduction of PI is mediated by the macropinocytosis, caveolin-mediated endocytosis. The same proteins of the two cell lines may be also involved in this process.This study provides experimental evidence for further investigation of the transmembrane transduction mechanism.