Abstract
Myelodysplastic syndromes (MDS) represent a heterogenous group of clonal stem cell disorders with qualitative and quantitative abnormalities of blood cells and a high probability of evolving to acute leukemia. Intensive induction chemotherapy in order 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 extensively studied in hematologic malignances. However, little is known about how TPT acts against neoplastic cells. The aim of this study is to evaluate apoptotic effect of TPT on the MDS cell line MUTZ-1 and its associated changes in the expression of inhibitors of apoptosis protein (IAPs). The effect of TPT on MUTZ-1 growth was determined by using MTT assay. Characteristics associated with apoptosis induced by TPT were evaluated by transmission electron microscope, DNA gel electrophoresis and flow cytometry (FCM). Cell cycle shift were observed by FCM. Semi-quantitative RT-PCR was used to evaluate the mRNA expression of members of IAP gene family, including survivin, XIAP, Bcl-2, Bax, cIAP1 andcIAP2. The potential of mitochondrial membrane potential (MMP) was determined by using JC-1 probe. The results demonstrated that TPT significantly inhibited MUTZ-1 cell growth in a time- and dose-dependent manner with IC50 of 5.011 mmol/L, 1.297mmol/L and 0.483mmol/L at 24h, 48h and 72h respectively. Morphological features associated with TPT-induced apoptosis observed by transmission electron microscopy included cytoplasmic and nuclear shrinkage, karyorrhexis, nuclear convolution, chromatin condensation and margination, cytoplasmic vacuolization, and membrane-bound apoptotic bodies. An ambiguous DNA ladder was observed following treatment with 5mmol/L TPT for 24h, and a typical DNA ladder was observed with 10mmol/L TPT for 24h. The apoptotic rates were 11.69±0.51%, 34.07±1.73%, and 48.59±2.01%, respectively, after 24h culture with TPT as 1, 5, 10 μmol/L, significantly higher than that of the control (3.47%±0.3%; F=31.642, P<0.01). The percentage of MUTZ-1 cells in G2/M phase of the cell cycle decreased while in S and G0/G1 phase increased after treatment with 1mmol/L TPT for 24h,. The majority of the cells were arrested in S phase. After 24h culture with TPT at1, 5, and 10μmol/L, the mRNA levels of survivin, XIAP, cIAP1 and cIAP2 were decreased (P<0.01). This down-regulation was negatively correlated with TPT-induced apoptotic rates(P<0.05). There was no significant change in the Bax and Bcl-2 mRNA levels after TPT treatment (P>0.05). After 24h culture with 1μmol/L TPT, the MMP of TPT treated cells decreased significantly(P<0.01). Together, we conclude TPT can inhibit the growth and induce apoptosis of MUTZ-1 cells in a time- and dose- dependent manner. TPT can also induce the cell cycle changes, with the majoritoy of cells being arrested in S phase. The TPT-induced apoptosis in MUTZ-1 cells is associated with down-regulation of suvivin, XIAP, cIAP1and cIAP2 mRNA expresison. As well, MMP may be play a important role in the apoptotic process of MUTZ-1 cells induced by TPT.
Inducible expression of Oct-3/4 reveals synergy with Klf4 in targeting Cyclin A2 to enhance proliferation during early reprogramming
