Induction Of G2/M Phase Arrest And Apoptosis By ZGDH-1 In Leukemia Cells

Objective Kasumi-1 cells were used as target cells to investigate the effect of ZGDH-1 on the proliferation and apoptosis of leukemia cells.The mechanism of Kasumi-1 arrested at G2/M phase was also deeply discussed.A theoretical foundation of using ZGDH-1 and other traditional Chinese medicinal materials was set up.Methods ? To verify the effect of ZGDH-1 on cell growth,Kasumi-1 cells were treated with increasing concentration of ZGDH-1 for 24h,48h and 72h.?The appearance of morphologic characteristic of Kasumi-1 cells was also detected by Wright and Hoechst 33258 staining.We also tested the changes of Caspase-3,cleaved Caspase-3(which is the active part of Caspase-3)and PARP(which is the substrate of Caspase-3)by Western-blot.? Western-blot was used to analyze the expression of Nf-KB and IKB.? The level of AML 1-ETO were analyzed by RT-PCR and Western-blot.? After being incubated with different concentrations of ZGDH-1(50,100,200,500,1000?g/L)and controls(negative control and DMSO control)for 48h,we used FCM to detect the changes of mitochondrial membrane protein(Apo 2.7),??m and ROS.?Leukemia cell cycles were analyzed by flow cytometry.The expressiona of cyclin,CDKs and CKIs in G2/M were analyzed after Kasumi-1 cells being treated by ZGDH-I with FCM and Western-blot method.The expression changes of these proteins were also analyzed after the inhibitor of CHK1,CHIR-124,was added to the drug.Results?ZGDH-1 inhabited the growth of Kasumi-1 in a concentration and time-dependent manner.?The positive result of AV/PI used FCM revealed the early stage of apoptosis after Kasumi-1 treated with ZGDH-1 for 24h.Caspase-3 decreased and cleaved Caspasse-3 greatly increased with dose manner,the cleaved fragments of PARP was also easily observed which suggested that Caspase-3 was actually activated after ZGDH-1 treated.?ZGDH-1 could induce the expression of IkB and down regulate the expression of Nf-KB to inhibit the growth of the cells.? AML 1-ETO fusion-protein was deeply degraded by ZGDH-1.?The changes of mitochondrial III membrane protein(Apo 2.7)and ??m reflects the integrity of mitochondrial membrane was destroied.And there was ROS accumulated in Kasumi-1 cells.The changes of Bcl-2,Bad and Bax by FCM and Western-blot showed that ZGDH-1 induces the apoptosis through mitochondrial pathway.? Kasumi-1 cells in G2/M were significantly accumulated with concentration dependent manner.The protein level of cdc2 and cyclinB1 were obviously decreased with concentration-dependent manner,which led to the obstruction of Kasumi-1 cells entering into mitosis.The level of cdc25c was also decreased,while phosphor-cdc25c was increased to inhibit the combination of cdc2 and cyclin B1.P53,was activated;while,p27,as CKIs,was up-regulated to induce G2/M arrest.When CHIR-124 added,the phenomenon of G2/M arrested was reversed.Conclusions ZGDH-1 inhabits the growth of Kasumi-1 cells by regulate the expression of Nf-KB and AML1-ETO.It induces apoptosis through mitochondrial pathway with ROS accumulated in Kasumi-1 cells.ZGDH-1 also influenced the cell cycles of leukemia cells.Definitly mechanisms and target on that ZGDH-1 inducing G2/M arrest of leukemia cells was clearly revealed.All these conclusions set up firm theoretical foundation of clinical use of ZGDH-1 with other traditional Chinese medicinal materials.