Analysis Of Cinobufacini Induced Apoptosis Of MDA-231Breast Tumor Cell

Cinobufacini is a traditional Chinese anti-tumor drug and widely used in clinic experiences. But little is known about its effect on the cells. In this study, we used atmic force microscope (AFM), converted fluorescence microscope,CCK-8cell proliferation experiment, laser scanning confocal microscope (LSCM), flow cytometry (FCM) and transmission electron microscope (TEM) to detect the different tumor cells (stomach cancer SGC-7901cell, breast cancer MCF-7cell and MDA-MB-231cell) apoptosis after treated by cinobufacini and further discussed the MDA-MB-231cell mechanism induced by cinobufacini. The results are as follows:1. After the cells treated by different concentrations of cinobufacini (0、6.25、12.5、25、37.5and50μg·mL-1), we used AFM to detect the changes of the cell surface at the nanoscale level. The results told us that the cell-surface ultrastructure and the cell morphology were changed a lot. After the MDA-MB-231cells were treated by cinobufacini, the data showed cinobufacini could inhibit the MDA-MB-231cell growth effectively in dose-dependent and time-dependent manners. After the cells being treated with50μg/mL cinobufacini for48hours, the early apoptosis percentage (20.45+1.46%) is much higher than the normal group (7.73+1.21%). The cell cycle data indicated that cinobufacini caused a cell cycle arrest at S-phase. The cell membrane potential and the concentration of calcium ion were increased significantly after treated with cinobufacini of different concentrations. The ROS levels were significantly higher than control group and the mitochondrial membrane potential levels were lower than control groups significantly. ROS can make the mitochondrial membrane channel hole (MPTP) open, at the same time, ROS can make the calcium ions released from the endoplasmic reticulum, and then it caused cell membrane potential to depolarize, and calcium ion passage opened and calcium ions poured into the cells. At last calcium ion as the second messenger of the cell mediated the cell apoptosis.2. What’s more, cinobufacini can affect the disruption of cytoskeleton. The cells after treated with50μg·mL-1cinobufacini, the expression of F-actin has decreased to60%. It indicated that the cell membrane structure and cytoskeleton networks were destroyed and the cell tails were narrowed after the cell being treated with cinobufacini, and the nuclei changed markedly. The expressions of CD44were decreased to50%. At the molecular level, we proved cinobufacini can inhibit the cell migration.3. Bcl-2is the main target molecule in the mechanism of apoptosis. When the DNA was damaged, the bcl-2can stop the signal arrived at the target molecules. The results showed that after the MDA-MB-231cells were treated by cinobufacini, the expressions of bcl-2were decreased from58.32%to44.84%.In conclusion, this research indicated that cinobufacini could inhibit cellular proliferation significantly. It could induce cell cycle arrest at S phase and induce apoptosis effectively in MDA-MB-231cells, so it provided us to further understand the mechanism of cinobufacini. Mean while, the structure of the MDA-MB-231cell changes were detected by AFM at the nanometer scale, so AFM will be a potential sensitive tool for detecting cancer cells. The present study is to provide valuable new insights to understand the mechanism of cinobufacini in anti-tumor process.