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 ① 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.②The 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.③ Western-blot was used to analyze the expression of Nf-KB and IKB.④ The level of AML 1-ETO were analyzed by RT-PCR and Western-blot.⑤ After being incubated with different concentrations of ZGDH-1(50,100,200,500,1000μg/L)and controls(negative control and DMSO control)for 48h,we used FCM to detect the changes of mitochondrial membrane protein(Apo 2.7),△ψm and ROS.⑥Leukemia 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.Results①ZGDH-1 inhabited the growth of Kasumi-1 in a concentration and time-dependent manner.②The 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.③ZGDH-1 could induce the expression of IkB and down regulate the expression of Nf-KB to inhibit the growth of the cells.④ AML 1-ETO fusion-protein was deeply degraded by ZGDH-1.⑤The changes of mitochondrial III membrane protein(Apo 2.7)and △ψm 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.⑥ 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.