The Study Of An Exopolysaccharide From Trichoderma Pseudokoningii Inducing Apoptosis In Leukemic Cells And Its Molecular Mechanisms

Objective: To study exopolysaccharide from Trichoderma pseudokoningii inducing leukemia cell apoptosis and apoptotic molecular mechanisms of K562 cells.Methods: 1.The effect of EPS on the proliferation of murine leukemia cells WEHI-3 andhuman leukemia cells K562 was evaluated by MTT assay.2.To observe the changes of nuclear morphology of WEHI-3 cells and K562 cells treated with EPS by Hoechst33258 fluorescent dye.3.The effect of EPS on K562 cells cycle arrest was evaluated by PI staining.4.The activity of caspase-3,caspase-9,caspase-4 and caspse-8 in K562 cells was detected by caspase assay kit after EPS intervention.5.The changes of calcium concentration in K562 cells after EPS treatment were detected using Fluo-3AM probe.6.RT-q PCR technology was used to detect the m RNA transcription of GRP78 gene and CHOP gene in K562 cells after EPS treatment.7.Western Blotting method was used to detect the expression of proteins relating withapoptosis in K562 cells after EPS treatment.Result:1.EPS inhibited WEHI-3 and K562 cells proliferation in concentration-dependent manner compared with control group.the rate of inhibition was 18.7%,19.27%,21.79%,22.03%,26.04% and 26.94% when WEHI-3 cellstreated with EPS at the dose of 0.0625,0.125,0.25,0.5,0.75 and 1 mg/m L for 72 h,the inhibition rate was 11.47%,12.51%,15.82%,25.42%,33.82% and 39.83% when K562 cellstreated with EPS at the dose of 0.0625,0.125,0.25,0.5,0.75 and 1 mg/m L for 72 h.2.The nucleus of WEHI-3 and K562 cells showed obvious morphological changes,whichappeared chromatin condensation after treatment with EPS for 48 h.3.The results showed that the proportion of S phase cells increased in concentrationdependently manner in K562 cells treated with EPS for 48 h.4.Compared with the control group,the activity of caspase-3,8,4,9 were significantly enhancedafter treatedcells with 0.25,0.5,1 mg/m L EPS for 48 h,especially caspase-4 and caspase-9.5.Compared with the control group,the concentration of itracellular calciumin K562 cellswas increased after exposed to EPS for 48 h6.RT-q PCR results demonstrated that the level of GRP78 m RNA was down-regulated and the level of CHOP m RNA was up-regulated in K562 cells treated with EPScompared with control group7.Western blotting results showed that expression of Bcl-2 down-regulation,the expression of Bax and cyt-C protein up-regulation,the expression of x IAP protein was decreased relating to the mitochondrial pathway;The expression of FADD protein up-regulation relating to death receptor pathway;and the expression of P53 protein increased.The expression of CHOP protein increased,the expression of GRP78 protein decreased relating to endoplasmic reticulum pathway after K562 cells treated with EPS for 48 h.Conclusion: EPS can inhibit the proliferation of murine leukemia WEHI-3 cells and human chronic myeloid leukemia K562 cells in vitro.The experimental results showed that EPS can inhibit the proliferation of K562 cells by inducing apoptosis,and its mechanism is relating to the three classic pathways of apoptosis.