Experimental Study On Anti-leukemic Effect Of Amifostine By Inhibiting Cell Cycle Process

Objective: The amifostine is the most commonly used pan-cell protective agentsin cancer radiotherapy and chemotherapy. Preliminary studies show that theamifosition protect normal cells, will not reduce the anti-tumor effect of radiotherapyand chemotherapy. With further research, amifosition also has many otherpharmacological effects, including hematopoietic cell growth factor-like role,inducing tumor cell apoptosis and inhibiting cell cycle progression andanti-angiogenic effect. At present, the impact of amifosition on leukemic cells is notclear. Therefore, based on bioinformatics prediction results, the purpose of this studyis to research the impact of amifosition on human leukemia K562cells proliferationand cell cycle, and the underlying molecular mechanisms.Methods:1.Bioinformatics prediction and analysis:Take Amifostine, Ethyl,WR-2721as key words, search-related gene and protein expression information inhuman gene expression database and proteomics database, screening differentiallyexpressed genes by chip database analysis methods. Then, cluster analysis byDAVID2.0software.2. K562cells were treated by0.35mg/ml,0.75mg/ml,1.5mg/mlconcentration of Amifostine for12h,24h and48h, observe cell morphology changesby an inverted microscope, to detect the proliferation of K562cells used CCK-8assay,cell cycle using propidium iodide (PI) staining flow cytometry, cell cycle-related geneexpression of Cyclin A2and Cyclin B1and Cyclin D1and Cyclin E1, CDK2, CDK4,CDK6and the mRNA expression level changes by fluorescent real-time quantitativePCR (real-time quantitive PCR, RT qPCR).Results:1. About5%of the genome-wide gene expression can be regulated byamifostine. There were139genes were known genes in the460differentiallyexpressed genes, of which77expressed raised62downregulated,13new expressedgenes, and5gene expression is completely inhibited. Cluster analysis showed that the139differentially expressed genes belong to11categories, mainly related to the cell cycle, hematopoietic and immune regulation and apoptosis. K562cells began togather into a group, larger and smaller, refraction loss, cytoplasm grainy obvioustreated by amifostine, And with the drug treatment time and concentration, the cellshrinkage, and nuclear fragmentation was significantly.3. K562cells proliferation canbe inhibited by the0.35mg/ml,0.75mg/ml,1.5mg/ml concentration of amifostine. Theinhibitory rates of K562cells respectively were14.4%,15.8%and28.6%by0.35mg/ml amifostine treated for12h,24h, and48h,31.1%、48.9%and66.7%by0.75mg/ml,83.7%、95.8%�'�99.2%by1.5mg/ml.The amifostine inhibited theproliferation of K562cells in a dose and time-dependent. The half inhibitoryconcentration (50%Inhibiting concentration, IC50) as0.64mg/ml after the K562cellwas treated for24h. Cell cycle analysis showed that the treated cells were increased inthe G1phase, compared with the control group, and this effect was dose and time–dependent.5.The RT-qPCR analysis showed that, compared with the control group,the K562cells treated by amifosition for24h had a decreased expression of themRNA of Cyclin A2, Cyclin B1and Cyclin D1(p <0.05), no significant effect on theexpression of Cyclin E1, and CDK2, CDK4and CDK6mRNA expression areincreased varying (p <0.05), Cyclin D1and CDK6mRNA expressed in aconcentration-dependent manner.Conclusion：The amifosition inhibit leukemia cell line K562proliferation. Theamifosition inhibit leukemia cell line K562proliferation, and part of this role is down-regulated the expression of cell cycle related protein,then the cells was arrested inG1/S phase.