| Purpose:Using cell culture technology in vitro, the apoptosis mechanism induced by norcantharidin (NCTD) in human breast cancer Bcap-37 cell lines was studied.Methods:In order to screen the optimum concentration and the best action time of NCTD, the inhibitory effects of NCTD on growth of the human breast cancer Bcap-37 cells for 24 h and 48 h were studied using MTT assay. The apoptosis changes and the DNA damage of Bcap-37 cells were analyzed with acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The morphological observation, DNA gel electrophoresis and Comet assay was analyzed, respectively. In addition, the laser scanning confocal microscope was employed to evaluate the effects of NCTD on mitochondrial membrane potential, generation of reactive oxygen species, and the expression of Hsp70, Caspase-3 proteins in Bcap-37 cells. Finally, the flow cytometry was used to research the relationship between cell apoptosis and the variation of cell cycle.Results:1.The results of MTT test indicated that NCTD could inhibit the growth of Bcap-37 cells and reduce the cell survival rate. In a certain range of NCTD concentration, the stronger the inhibitory effects on Bcap-37 cell growth is and the higher apoptotic rate, when the higher the concentration and the longer the acting time of drug. The optimum concentration of NCTD is about 10μg/ml. Stained with AO/EB, the morphological changes were detected by fluorescence microscope. The cells became round, membrane folded, nuclear fragmentation, etc, and the apoptotic bodies could be seen clearly.2.The results of flow cytometry analysis showed that the proprortion of G1 phase was significantly decreased, while the S and G2/M phase increased in the cell cycle, and the apoptosis rate rised also. DNA agarose gel electrophoresis and comet assay analysis demonstrated that a certain concentration of NCTD could cause the DNA damage and initiate trailing phenomenon of Bcap-37 cells, and the "ladder" bands which were composed of DNA fragment of about 200bp as the base could be seen.3.NCTD could obviously reduce mitochondrial transmembrane potentialΔΨm, enhance intracellular ROS contents, induce the generation of H2O2, and inhibit the activity of CAT enzyme at the same time. 4.Compared with the control group, the expression of Caspase-3 protein in the cells treated with NCTD for 24h could be up-regulated, and there is significant difference (p<0.01). However, the expression of heat shock protein Hsp70 was varied with the changes of drug concentration. It is matched with its dual regulatory function on tumor cells.Conclusion:1.NCTD could significantly inhibit the growth of Bcap-37 cells, and the toxic effect of NCTD was in a dose-time-dependent manner. With electrophoresis technology, the classic DNA "ladder" bands and Comet tailing phenomenon could be seen clearly.2.The Bcap-37 cells was prevented from entering the next cell cycle, which may be one of the NCTD-induced apotosis mechanisms. The results showed the phenomenon of cell cycle was blocked.3.The intracellular ROS levels in Bcap-37 cells was promoted and mitochondrial membrane potential was declined, which indicated that the way of mitochondrial oxidative damage could be one of the NCTD-induced apotosis mechanisms.4.NCTD caused Bcap-37 cells apotosis through regulating the expression of Caspase-3 and Hsp70 protein. The results demonstrated that NCTD could affect some gene's expression in tumor cells, change the physiological activity of enzyme, and play its anti-tumor effect.
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