| OBJECTIVEMyelodysplastic syndromes (MDS) represent a heterogenous group of clonal stem cell disorders with quality and quantity abnormity of blood cells and a high probability of evolving to acute leukemia. These patients are characterized by ineffective hematopoiesis and peripheral blood cytopenias. Now the therapy of MDS is still a tough question and a clinical hot spot. Recently, novel approaches are made by all kinds of new therapy schedule, new drugs and chemotherateutic drugs combined reasonably. Intensive induction chemotherapy in oder to reduce the malignant clone and reconstruct normal hematopoiesis is a classic therapy of MDS, especially high risk MDS. Topotecan (TPT), a semisynthetic water-soluble derivative of camptothecin, is a potent inhibitor of DNA topoisomerase I and has been most extensively studied in hematologic malignances. But the mechanisms of topotecan induce-apoptosis remain unknown. The aim of this paper is try to prove TPT can induce MUTZ-1 cells apoptosis and its new mechanism. This study will give us insight into further understanding of the role TPT may play in regulation of apoptosis in tumor cells. The data generate from this study will also provide valuableinformation for improve the clinical outcome of MDS and broaden the extent of clinical application of TPTMATERIALS AND METHODS1 In this study, using MTT assay we investigated the effect of TPT on MUTZ-1 growth. Cell apoptosis induced by TPT is proved by transmission electron microscope, DNA gel electrophoresis and flow cytometry (FCM). Cell cycle shift were observed by FCM.2 Semi-quantitative RT-PCR was used to evaluate the mRNA expression of survivin, XIAP, Bcl-2, Bax, cIAPl, cIAP2 and P21 in cell lines treated with TPT.3 Using the probe of JC-1 and DCFH-DA, detect the potential of mitochondrial membrane(MMP).RESULTS1.The results demonstated that TPT was capable of inhibit the MUTZ-1 cells growth by time- and dose-dependent manners: The result showed that following treatment with TPT(0.5 mol/L) for 24 h, the proliferation of MUTZ-1 cells was inhibited significantly, in comparison with the blank control group (P<0.01). IC50 of TPT was 5.011 mol/L, 1.297 mol/L and 0.483nmol/L at 24h, 48h and 72 h. 2. In vitro, TPT can induce MUTZ-1 cells apoptosis by dose-denpentant manner: The apoptosis morphology observed under a transmission electron microscope. The result displayed a series of typical morphological features of apoptosis were observed following treatment with TPT(l~10 mol/L) such as vacuolization of cell plasma, shrinkage of cell and nucleus, condensation of nuclear chromatin, marginated against the nuclear membranes, karyorrhexis and convolution of nuclear, and membrane-bound apoptotic bodies.The result of DNA ladder show that following treatment with 5 mol/L TPT for 24h, a ambiguous DNA ladder was observed; and10 mol/L TPT for 24h, a typical DNA ladder was observed.With the flow cytometry detecting the translocation of phosphatidyl serine(PS), After 24h culture with TPT (1, 5 10 mol/L), the apoptotic rates are individually 11.69 0.51% 34.07 1.73% 48.59 2.01%, comparing with the control group(3.47% 0.3 % ), it has apparently significant (F=31.642, P<0.01).3. TPT-induced change of cell cycle in the present of TPT. After MUTZ-1 cells were been treated with lu,mol/L TPT for 24h禄 the results showed that the percentage of MUTZ-1 cells in the G2/M phase of the cell cycle decreased, while in the S phase increased. The majority of the cells were arrested in S phase.4. After 24h culture with TPT (1, SN 10 u mol/L), TPT significantly down-regulate survivin, XIAP, cIAPl and cIAP2 mRNA expression (P<0.01), the down-regulation of survivin, XIAP, cIAPl and clAP2 mRNA was negative correlation to TPT-induced cells apoptotic rate (P<0.05) .5. After 24h culture with TPT ((0 1 5 10 u mol/L), with the increasing of dose of TPT, Bax and Bcl-2 have no change on mRNA expression (P>0.05).6. After 24h culture with TPT (0 1 5 10 mol/L), with the increasing of dose of TPT, mRNA expression of p21... |
PDF Full Text Request