Anti-leukemia Effects Of Perifosine And The Mechanism Of Resistance In CML Cells

Section1:Perifosine reduces cell viability and induces apoptosis in AML and CML cell linesAim:To study and compare cytotoxic effects and apoptosis induced by Akt inhibitor perifosine on CML and AML cells lines.Methods:Viability of cell growth was measured by MTT assay. Annexin V-FITC/propidium iodide and Hoechst staining were used for detecting apoptosis. The expression of caspase-3, caspase-9, PARP and associatied proteins in AML and CML cell lines were examined by Western blotting.Results:(1)24h IC50of perifosine on CML cell lines K562, K562/G and AML cell lines Kasumi-1、HL-60was121.74,311.3,11.08,11.14μmol/L, respectively. And48h IC50was56.28、141.75、4.24、3.62μmol/L. It suggested that perifosine is more sensitive to AML cell lines than CML cell lines.(2) After treatment with10μmol/L perifosine for 24h, the apoptosis ratio in K562、K562/G、Kasumi-1and HL-60cells was8.23%,7.37%、26.72%、25.80%, respectively, which indicated that perifosine could effectively induce apoptosis in AML cell lines,but not in CML cell lines.(3)Treated with10ol/L perifosine for24h, apoptotic body appeared in Kasumi-1and HL-60cells, but not in K562and K562/G cells, which confirmed that perifosine could induce apoptosis in AML cell lines but not CML cell lines.(4)As measured by western-blot, caspase-3, caspase-9and PARP were cleaved and activated in AML cell lines but not in CML cell lines.(5) In AML cell lines treated with perifosine for24h, the expression of AKT, p-AKT were down-regulated and the expression of JNK, p-JNK were up-regulated.Section2:The mechanism of resistance to perifosine in CML cell lines and the role of autophagy in itAim:Based on the former research, we further studied the potential mechanism of resistance to perifosine in CML cell lines, and the role of autophagy in it.Methods:Autophagy was evaluated using electron microscopy, acridine orange staining method and the examination of GFP-LC3under a flurescence microscopy. The expression of autophagy related genes were examined by Western blotting. Viability of cells growth was assessed by a MTT assay.Results:(1) Perifosine could induce autophagy in a dose-dependent manner in CML cell lines.(2) The autophagy accompanied with upregulation of Atg5is Beclin-1independent.(3) Inhibition of autophagy using chloroquine elicited a significant suppression of cell growth and apoptosis in CML cells. Conclusion:Perifosine induces apoptosis, and inhibits Akt activation in AML cells, but not in CML cells. Treatment of CML cells with perifosine induces cytoprotective Beclinl-independent autophagy, with the upregulation of ATG5. Inhibiton of such a perifosine-mediated autophagic response led to accelerated apoptotic cell death. These findings may contribute to the resistance partly.