拓扑异构酶Ⅰ抑制剂对K562细胞的杀伤与诱导凋亡作用

Killing and Proapoptotic Effects of Topoisomerase ⅠInhibitor on K562 Cells

背景与目的:近年发现,拓扑异构酶Ⅰ抑制剂对加速期或急变期的慢性髓细胞白血病有较好疗效。为了深入理解拓扑异构酶Ⅰ抑制剂这一新的药理作用,本研究采用具有慢性髓细胞白血病特征性异常染色体犤t(9;22)犦的K562细胞株为实验对象,进一步探讨拓扑异构酶Ⅰ抑制剂拓扑替康(topotecan)对靶细胞的杀伤与诱导凋亡活性。方法:采用MTT法测定拓扑替康对K562细胞的杀伤作用;通过形态学与AnnexinVFITC染色,研究拓扑替康对靶细胞的促凋亡活性;采用caspase-8特异性抑制剂IETD-fmk,分析拓扑替康介导的细胞杀伤或凋亡和caspase活化的关系。结果:经0.15μmol/L拓扑替康处理至12、24、48及72h时,K562细胞的存活率与对照相比,逐渐降至(92±36)%犤P>0.05vs(94±27)%犦、(68±21)%犤P<0.05vs(119±13)%犦、(54±15)%犤P<0.05vs(132±31)%犦及(21±10)%犤P<0.01vs(114±19)%犦;同时,靶细胞出现磷脂酰丝氨酸外翻、细胞固缩、染色质边集、核碎裂,最终解离为大量凋亡小体;经caspase-8抑制剂与拓扑替康联合处理至24、48h时,K562细胞的存活率依然维持在(95±29)%与(87±11)%,后者显著高于单用拓扑替康者犤P<0.05vs(54±15)%犦,且无明确的凋亡小体形成。

Background and Objective:Recently,it has been demonstrated that topoisomerase Ⅰ(TopoⅠ) inhibitor is a useful alternative for treatment of the patients with chronic myelogenous leukemia(CML) in accelerated or blast crisis. In order to get insight into pharmalogic activity of TopoⅠinhibitor,this study was designed to investigate the killing and proapoptotic activity of TopoⅠinhibitor(Topotecan,TPT)using K562 cell line,which carry abnormal chromosome of characteristics of CML.Methods:The cytotoxic effect of TPT on K562 cells was determined using MTT assay;TPT induced apoptosis in K562 cells was identified by morphological analysis and Annexin V FITC staining;correlation between TPT mediated killing or apoptosis and caspase 8 activation was investigated using caspase 8 inhibitor IETD fmk. Results:After incubation of K562 cells with 0 15 μmol/L TPT for 12,24,48,and 72 h,the survival rates,compared with control,progressively reduced to (92±36)%[P >0 05 vs (94±27)%], (68±21)%[P< 0 05 vs (119±13)%], (54±15)%[P< 0 05 vs (132±31)%], and (21±10)%[P< 0 01 vs (114±19)%]. Targeted cells meanwhile demonstrated phosphatidylserine externalization,cell shrinkage,chromatin margination,karyorrehxis and eventually disintegrate into numerous apoptotic bodies. After exposure to TPT in combination with IETD fmk for 24 h and 48 h,K562 cells were still survival at viability of (95±29)%and (87±11)%, the latter was significantly higher than that of to TPT only [P< 0 05 vs (54±15)%]. Conclusion:Topoisomerase Ⅰinhibitor TPT possess killing activity and was induce apoptosis to K562 cells, which could be dependent on activation of caspase 8.