Antileukemia Effect And Mechanism Of A Novel CA-4 Analogue-MZ3

Aims:Evaluate the antileukemia effect of MZ3,a novel CA-4 analogue, in vivo and in vitro,and explore the antileukemia mechanisms.Methods:MTT method was used to evaluate the inhibition effect of MZ3 on human leukemia cells(NB4,Molt-4,HL60,K562,HL60R,K562R),IC₅₀ values were calculated;SCID mice model of human leukemia engrafts was used to evaluate the antileukemia effect of MZ3 in vivo,survival curves were drawn and mediam survival time(MST)were calculated.DAPI stain was used to observe apoptotic bodies in K562 cells;DNA fragmentation test and flow cytometry were employed to detect the apoptosis in HL60 and K562 cells.JC1 stain and flow cytometry were used to test the change of mitochondrial membrane potential(ΔΨm)in HL60 cells;Carboxy-DCFDA stain and flow cytometry were used to test the content of ROS in HL60 cells. The cell cycle distribution of K562 cells was measured using PI stain and flow cytometry;the cellular microtubule networks was visualized by immunofluorescence.All the proteins expression and activation in HL60 and K562 cells were measured by western blotting.Results:1.MZ3 inhibited proliferation of six selected human leukemia cell lines, including MDR-positive cell lines,with IC50s＜8μM.The IC₅₀values of MZ3 on NB4,Molt-4,HL60,K562,HL60R and K562R are 1.2±0.2μM,4.2±0.4μM,2.7±0.2μM,3.4±0.1μM,3.5±0.5μM and 8.0±0.3μM.2.Human leukemia SCID mice in control group were all dead in 20 days after enxograft,MST is 15 days;in CTX group and MZ3 group,the first death happened at 25^thand 26^thday,MST were 26.5 and 33.5 days.CTX (10.0mg/kg)and MZ3(10.0mg/kg)both prolonged the MST of leukemia SCID mice more,and the MZ3's effect was more potent.3.DAPI stain showed that 16.0μM MZ3 24 h-treatment caused apoptotic bodies in K562 cells.DNA fragmentation test demonstrated that HL60 cells treated with 4.0μM,8.0μM and 16.0μM MZ3 for 24 h showed DNA ladder; K562 cells after treated with 8.0μM and 16.0μM MZ3 for 24 h showed DNA ladder.4.Flow cytometry showed that after 0 h,6 h,12 h,24 h,48h and 72h treated with 8.0μM MZ3,0.9%,2.5%,14.2%,30.5%,65.5%and 85.4% HL60 were induced apoptosis in a time-depenent manner;after treatment with 16.0μM MZ3 for 0 h,6 h,12 h,24 h,2.5%,2.8%,5.8%and 31.3%K562 cells were induced apoptosis in a time-depenent manner;and after treatment with 0.0μM,4.0μM,8.0μM and 16.0μM MZ3 for 24 h,4.4%,35.0%,36.6% and 31.3%K562 cells were induced apoptosis.5.Western blotting showed that 12 h and 24 h treatment with 8.0μM MZ3 induced the Procaspase-3 cleavage and PARP hydrolyzation in HL60 cells; 24 h treatment of 4.0μM,8.0μM and 16.0μM MZ3 induced the Procaspase-3 cleavage and PARP hydrolyzation in K562 cells.6.JC1 stain showed that 0 h,8 h,16 h and 24 h treatment of 8.0μM MZ3 causedΔΨm decrease in 2.0%,3.0%,18.9%and 48.4%HL60 cells in a time-dependent manner.7.Carboxy-DCFDA stain showed that ROS increased to peak at 2 h after 8.0μM MZ3 treatment in HL60 cells,and after that,decreased to normal level.8.NAC(200.0μM),an inhibitor to ROS,can not inhibit the cytotoxicity of MZ3.9.Western blotting showed that in HL60 cells 6 h,12 h and 24 h treatment of 8.0μM MZ3 increased Bax in a time-dependent manner and decreased Bcl-2 in a time-dependent manner,and the ratio of Bax/Bcl2 were 2.2,15.7 and 28.5 seperately.6 h,12 h and 24 h treatment of 8.0μM MZ3 decreased P-ERK1/2,and increased P-p38 and P-JNK.These data indicated that MZ3 could affect MAPK and Bcl-2 protein family in HL60 cells,which can activate mitochondra-apoptosis and cause apoptosis.10.MZ3 can induce G2/M phase arrest in K562 cells in both dose- and time- dependent manner.0 h,6 h,12 h and 24 h treatment of 16.0μM MZ3 can arrest 6.1%,11.1%,31.1%and 38.1%K562 at G2/M phase;24 h treatment of 0.0μM,4.0μM,8.0μM and 16.0μM MZ3 can arrest 10.8%, 25.7%,28.2%and 38.1%K562 cells at G2/M phase.11.24 h treatment of 16.0μM MZ3 can cause disturbance of the celluar microtubulue network and decrease of microtubule in cell plasma.These results indicate that MZ3 can arrest cells at G2/M phase through disrupting microtubule polymerization.12.24 h treatment of 0.0μM,4.0μM,8.0μM and 16.0μM MZ3 can increase cyclin B1 and MPM-2,decrease cdc2c and dc25C and activate Wee1.These results suggested that MZ3 can arrest cells at G2/M phase through affect cell cycle regulating protein.Conclusion:MZ3 exhibits good antileukemia effect both in vivo and in vitro.MZ3 can induce apoptosis in HL60 cells and K562 cells,the possible mechanisms are:1)affect MAPK and Bcl-2 protein family,and eventually activate the mitochondria-apoptosis pathway;2)affect key G2/M phase regulatory protein and cellular microtubule polymerization,which lead the cell arrested in G2/M phase.