Screening Of The Effects Of Leukemia Cells Differentiation Induced By Retinoic Acid Derivatives And Effects Of 4-amino-2-trifluoromethyl-phenyl Retinate On Differentiation And Its Influence On Cell Cycle

Objective:To investigate the proliferation inhibition and differentiation induction activities of 10 retionoic acid derivatives in K562 and NB4 cells ,screening of the derivatives with pharmacological activity ,and investigate their pharmacodynamics and mechanism of inducing differentiation .Methods: 1,Cell proliferation was assessed by MTT assay before and after the treatment with retionoic acid derivatives in vitro.Morphologic changes were observed after Wright's staining using inverted phase contrast microscope.Cell differentiation indexs were analyzed by NBT reduction test.Cell cycle and the expression of the maturation specific cell surface markers CD_11b, CD₁₃ were determined by FCM analysis.2,After screening of the derivatives with pharmacological activity,the changes of growth curve induced by different concentrations of derivatives were analysed by cell counting. Cell differentiation indexs were analyzed by NBT reduction test.Cell cycle,the expression of the maturation specific cell surface markers CD₄₅,CD₇₁,CD₁₁₇ of K562 cells and CD11b,CD₁₃,CD₁₄ of NB4 cells were detected by FCM.The expression of cell cycle-related factors such as Cyclin D1,Cyclin E,PCNA,CDK2,CDK4,CDK6,P21^cip1,P27^kip1,P57^kip2 mRNA was measured by RT-PCR.Western blotting was performed to detect the expression of protein cyclin D1, CDK4 protein, PML / RARαand RARα.Results:1.The growth of K562 and NB4 cells was inhibited in a dose-dependent manner after the treatment with retinoic acid derivatives for 3 days.The changes of differentiation and maturation of leukemic cells induced by 10 retinoic acid derivatives at the concentration of 10-5 mol/L were observed through immersion objective.NBT reduction test indicated that the retinoic acid derivatives could induce differentiation of leukemic cells and increase the positive cell ratio.The expression level of the maturation specific cell surface marker CD11b was increased while the expression level of CD13 was decreased.The proportion of cells in G0/G1 phase was increased.Cell cycle progression was blocked in the G1 phase.2. Through the above analysis and comparison of experimental results, found that 4 -amino-2-trifluoromethyl-phenyl ester of retinoic acid (4-amino-2-trifluoromethyl-phenyl retinate, ATPR) has powerful activity in inbibiting proliferation and inducing differentiation . After the treatment with ATPR,the growth of leukemic cells was inhibited in a dose-dependent and time-dependent manner.The NBT positive cell ratio,the expression level of the maturation specific cell surface marker CD11b,CD14 of NB4 cells were increased while the expression level of CD13 was decreased,which was similar to ATRA.The longer, the greater the change.The proportion of cells in G0/G1 phase increased while S phase cells decreased. Cell cycle progression was blocked in the G1 phase. The expression level of Cyclin D1,Cyclin E,CDK2,CDK4,CDK6,mRNA decreased after 3 days, while PCNA mRNA decreased after 7 days treatment with ATPR and the expression level of P57 kip2 mRNA was significantly increased.Conclusion:1. Above of the all,the retinoic acid derivatives retinoid 2a-03, 4a-02 and 5a-02 have powerful activity in inhibiting proliferation and inducing cell differentiation., suggesting that it is significant to develop a better retinoic acid-type drug with amino trifluoromethyl phenyl retinate as the starting point.2. ATPR (4a-02) has powerful activity in inbibiting proliferation and inducing differentiation through increasing the expression level of P57 kip2 mRNA to inhibit Cyclin-CDK kinase which plays a role in cell cycle arrest.