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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。结论:拓扑异构酶Ⅰ抑制剂拓扑替康对K562细胞具有较强的杀伤活性与诱导凋亡作用
BACKGROUND & OBJECTIVE: It has been found in recent years that topoisomerase I inhibitors have a good effect on chronic myeloid leukemia in accelerated or blastic phase. In order to further understand the new pharmacological effects of topoisomerase I inhibitors, we used K562 cell line with characteristic aberrant chromosome 犤t (9; 22) 慢 of chronic myeloid leukemia as the experimental object, Killing and induction of apoptotic activity of target Ⅰ by topotecan, an enzyme Ⅰ inhibitor. Methods: MTT assay was used to determine the killing effect of topotecan on K562 cells. Morphological and AnnexinVFITC staining was used to investigate the anti-apoptotic activity of topotecan on target cells. The caspase-8 specific inhibitor IETD-fmk was used to analyze the topological Ticiconb-mediated cytotoxicity or apoptosis and caspase activation. Results: After treated with 0.15μmol / L topotecan for 12, 24, 48 and 72h, the survival rate of K562 cells was decreased to (92 ± 36)% vs 0.05 vs 94 ± 27 % 犦, (68 ± 21)% 犤 P <0.05 vs (119 ± 13)% 犦, (54 ± 15)% 犤 P <0.05 vs (132 ± 31)% 犦 and (21 ± 10)% 犤 P < 0.01vs (114 ± 19)% 犦. At the same time, the target cells appeared phosphatidylserine valgus, cell shrinkage, chromatin margination, nuclear fragmentation and eventually dissociated into a large number of apoptotic bodies; the caspase-8 inhibitor The survival rate of K562 cells maintained at (95 ± 29)% and (87 ± 11)% when combined with topotecan treatment up to 24,48 h was significantly higher than that of topotecan alone (P <0.05 vs (54 ± 15)% 犦, and no clear formation of apoptotic bodies. CONCLUSION: Topotecan, a topoisomerase Ⅰ inhibitor, has strong killing activity and induces apoptosis on K562 cells