Effect Of Decitabine In Combination With Imatinib Mesylate In K562 Cells

ObjectiveAlthough tyrosine kinase inhibitor imatinib mesylate (imatinib), as the first line treatment for chronic myelogenous leukemia (CML), is generally highly effective and well tolerated, resistance to imatinib is increasingly recognized as a clinical problem, especially in patients with accelerated phase (AP) or blastic phase (BP). Thus, identification of alternative strategies to effectively kill CML cells through a mechanism different from the inhibition of tyrosine kinase activity would have important therapeutic implications in CML. DNA methylation plays an important role in development and progression of cancer. In CML, DNA methylation increases in concert with disease progression. Previously studies showed that decitabine, a hypomethylating agent, has single agent clinical activity in CML, including imatinib resistant cases. Expression of BIM in CML cells is regulated by promoter hypermethylation. And the expression of BIM is required for imatinib induced apoptosis and inhibition of proliferation in CML. Down regulation of BIM is found in 1/3 CML patients who show less effective to imatinib. We hypothesized that expression of BIM is up regulatied by promoter hypermethylation induced by decitabin (DAC) and strengthen effective of imatinib. This study evaluated DAC alone or combined with imatinib capable of killing K562 cells and influencing the expression of BCR/ABL mRNA and Bim protein.MethodsWe culture the K562 cells, group randomly:control group, IM group(0.1 mol/L,0.2 mol/L), DAC group(1 mol/L,4 mol/L), combination groups(IM 0.1 mol/L+DAC 1 mol/L,IM 0.1 mol/L+ DAC 4 mol/L,IM 0.2 mol/L+ DAC 1 mol/L及IM 0.2 mol/L+DAC 4 mol/L), investigate the inhibitory rate of drug intervention, early apoptosis, cell cycle after 24h,48h,72h, and BCR / ABL mRNA ,Bim protein expression after 48h:(1) The cell proliferating activity and the concentration was assessed by tetrazolium blue (MTT) method;(2) Cell cycle and apoptotic rate was examied by flow cytometry (FCM) ;(3) The BCR / ABL mRNA expression was detected by RT PCR ;(4) The Bim protein expression was detected by Western blot. Results(1) The role of single agent or combination on the inhibition rate of K562 cellsBoth DAC and IM caused time and concentration dependent induction of cell death. DAC and IM in combination produced a greater inhibition of growth against K562 cells, DAC 4 mol / L inconbination with IM 0.2 mol / L for 48h and 72h caused the combined effects of inhibition rate 56.7%, 90.7%, respectively.The main effect and interaction between two drugs was statistically significant (P <0.001) after 24h,48h,72h.(2) The role of single agent or combination on K562 cell cycle and early apoptosisCycle arrest and early apoptosis were obviously in 48h. After 48h, cells in the control group S phase accounted for 53.323%, and G1 phase cells accounted for 46.629 percent. With the increase of drug concentration, S phase cells gradually decreased, G1 phase cells increased gradually. IM 0.2 mol / L combined with DAC 4 mol / L for 48h the role of S phase cells decreased 27.641%, G1 phase cells gradually increased to 68.863%, accounting for 28.7% of apoptotic cells. Treatment with IM0.2 mol / L for 48h caused 6.7% early apoptotic cells, IM0.2 mol / L combined with DAC4 mol / L showed 8.4% in early apoptosis.(3) The BCR / ABLmRNA expression level before and after treatmentBefore treatment the BCR / ABL mRNA expression was 0.828±0.052, the BCR / ABL mRNA expression was 0.493±0.048 after treated by IM 0.2 mol / L for 48h, the expression was 0.763±0.035 after treated by 48h DAC4 mol / L, IM0 .2Μmol / L combinated with DAC4 mol / L for the same time showed the expression was 0.336±0.084. The difference was statistically significant (P <0.05).But no statistically significant interaction between two drugs(P =0.154).(4) Bim expression changes before and after treatmentBim protein expression was 0.374±0.015 before treatment, the expression of Bim was 0.536±0.019 after treated with IM 0.2 mol / L for 48h, the expression was 0.649±0.011 after treated with DAC4 mol / L for 48h, IM0 .2Μmol / L combinated with DAC4 mol / L for the same time showed the expression was 0.884±0.008. The difference was statistically significant (P <0.05). The interaction between two drugs was statistically significant (P <0.001).Conclusion(1) DAC caused time and concentration dependent induction of cell death in K562 cells;(2) DAC indused cell death involved in inhibition of proliferation of K562, cell cycle arrest at G1 phase, reduced the proportion of S phase cells; reduced BCR / ABLmRNA and increased expression of Bim; (3) IM can increase the expression of Bim;(4) DAC conbinated with IM can inhibit the proliferation of K562, and the mechanism involved in reducing BCR / ABL mRNA and increased expression of Bim protein;(5) DAC conbinated with IM was an option of patients with CML AP or BP and patients who is resistant or less sensitive to IM.