Visualization Of Tumor Cell Apoptosis Induced By Crude Anticancer Drugs And Cell Surface Receptors Recognition

This dissertation includes two parts:(1) The molecular mechanisms of cell death induced by 3 crude anticancer drugs(taxol,Xiao-ai-ping(XAP) and Chan-su(CS)) were studied in human lung adenocarcinoma(ASTC-a-1) cells by fluorescence techniques;(2) Visualization of cell surface receptors recognition was performed by laser scanning confocal microscopy(LSCM) and atomic force microscopy(AFM). In the first part,the inhibitory effect and caspase-3 activation induced by taxol,XAP and CS on ASTC-a-1 cells were studied.Gene plasmid transfection,fluorescence emission spectra and fluorescence resonance energy transfer(FRET) were used to study the caspase-3 activation during the three anticancer drugs-induced ASTC-a-1 cell apoptosis.The main results are the following:(1) The cell activity of ASTC-a-1 cell was inhibited by Taxol with resultant resulting cell swelling,cytoplasmatic vacuolization and cell death,which,however,did not involve caspase-3.A novel nonapoptotic PCD resembling the paraptosis in ASTC-a-1 cells may be induced by Taxol.(2) The cell activity of ASTC-a-1 cell was inhibited by XAP in a dose-dependent manner and cell death can be induced by XAP;the SCAT3 inside living cells was cleaved completely 72 h after the treatment of XAP,implying that a great deal of pro-caspase-3 was activated by XAP;caspase-3 was not activated significantly by XAP within 24 h.(3) The cell activity of ASTC-a-1 cell was inhibited by CS in a dose-dependent manner and cell death can be induced by CS;SCAT3 inside living cells were cleaved significantly by caspase-3 in a time-dependent manner, and these results were verified by the acceptor photobleaching experiments of SCAT3 too,implying that caspase-3 was activated by CS and cell apoptosis was induced.In the second part,a method involving the conjugation of wheat germ agglutinin (WGA) with fluorescent quantum dots(QDs) simultaneously acting as fluorescent and topographic probes(WGA-QD probe) prior to cell surface staining is developed for LSCM and AFM.This method provided at least two advantages:(1) An amplified fluorescence of WGA-QD aggregates,strongly resistant to photobleaching,ensures repeated or real-time observations of the probe-labeled cells or cellular membrane surface by LSCM.(2) The enlarged size of WGA-QD probe makes it possible for labeled WGA to be distinguished from other membrane proteins by AFM.This method would be very useful for the integrated system combining fluorescence/confocal microscope and AFM.The WGA-binding site distributive patterns on the cell surface were compared by confocal microscopy and especially AFM between human erythrocytes and human MCF-7 breast cancer cells.The main results are the following:(1) We utilized AFM to image the WGA-binding sites on cell surfaces using WGA-QD complexes as topographical probes and directly "saw" the in situ distributions of WGA-binding sites on various types of cells,.We found that the WGA-binding sites on the cell surface distributed more in the donut rings of the concave erythrocytes and dispersed throughout the whole cancer cell surface with large dense areas mainly in the connecting zones between the center and periphery of MCF-7 cancer cells.(2) Using this WGA-QD probe,we also developed a method to rapidly detect the WGA-induced rigidity/stiffness alternation of the whole cells:the values of several parameters evaluated the rigidity of whole individual cells were rapidly obtained by utilizing the software equipped with the AFM to measure the topography of whole individual cells in bulk stained by WGA-QD probe,which is efficient and has the potential of being developed to a useful tool in clinical diagnosis.