小檗胺对人慢性粒细胞白血病细胞发生凋亡的诱导机制研究  

The mechanism of apoptosis of chronic myeloid leukemia cells induced by the novel p210 bcr/abl inhibitor berbamine

作　　者：孙建荣[1] 张晓红[1] 何智文[2] 古莹[1] 虞瑛姿[1] 方永明[2] 吕庆华[2] 董庆华[2] 徐荣臻[1] 

机构地区：[1]浙江大学医学院附属第二医院血液科,杭州310009 [2]浙江大学肿瘤研究所

出　　处：《中华医学杂志》2006年第32期2246-2251,共6页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30270572);浙江省自然科学基金资助项目(Y204113)

摘　　要：目的研究新型p210 bcr／aN抑制剂小檗胺诱导人慢性粒细胞白血病细胞凋亡分子的机制。方法培养表达内源性p210 bcr／abl蛋白的Ph+人慢性粒细胞白血病细胞系K562,用小檗胺按指定时间和剂量干预细胞。应用膜联蛋白荧光素(Annexin-V-Fluos)／碘化丙啶(propidium iodide, PI)试剂盒和流式细胞术定量分析凋亡细胞百分比;用cytoperm／cytofix和天冬氨酸特异的半胱氨酸蛋白水解酶-3-McAb-PE定量检测含活化天冬氨酸特异的半胱氨酸蛋白水解酶-3(Caspase-3)细胞百分比;以免疫共沉淀技术(c-abl抗体)和Western印迹[p-Tyr(pY99)抗体]定量分析p2m bcr／abl蛋白磷酸化;p210 bcr／abl蛋白总量直接用Western印迹(c-abl抗体)检测;H8p90和Hsp70等分子伴侣蛋白水平的变化用Western印迹(Hsp90和Hsp70抗体)。结果48 h IC50浓度小檗胺(8μg／ml)作用48 h后,45．69％K562白血病细胞表达活化的Caspase-3凋亡分子和48．43％白血病细胞发生凋亡。免疫印迹和免疫共沉淀结果显示,低剂量小檗胺可明显抑制白血病细胞内p210 bcr／abl磷酸化:8μg／ml浓度小檗胺处理6 h后,白血病细胞磷酸化p210 bcr／abl蛋白含量仅为对照组的8．41％,而p210 bcr／abl蛋白总量并无变化。小檗胺还能直接下调p210 bcr／abl分子伴侣Hsp90蛋白水平:白血病细胞经8μg/ml浓度小檗胺处理24 h时的Hsp90水平只有对照组的18．37％,而且对能诱导白血病细胞产生凋亡抵抗的Hsp70蛋白水平影响不明显。结论(1)小檗胺是一种新型p210 bcr／abl蛋白磷酸化抑制剂,能通过抑制p210 bcr／abl蛋白磷酸化和诱导Caspase-3通路介导的Ph+白血病细胞发生凋亡; (2)与已知Hsp90抑制剂格尔德霉素(GA)不同,小檗胺能直接下调Hsp90蛋白水平,而对与肿瘤细胞凋亡抵抗有关的Hsp70蛋白表达影响不大,这提示小檗胺可能还是一种新型蛋白分子伴侣Hsp90抑制剂,值得进一步研究。Objective To investigate the mechanism of apoptosis of chronic myeloid leukemia （CML） cells induced by the novel p210 bcr/abl inhibitor berbamine. Methods Human Ph ＋ CML leukemia K562 cells, which express endogenous p210 bcr/abl protein, were cultured in RPMI 1640 and treated with berbamine as indicated time and dose. Flow cytometry （ FCM ） and Annexin-V-Fluos/PI staining kit were used to evaluate the apoptosis of leukemic cells ; FCM and cytoperm/cytofix plus Caspase-3-McAb- PE were employed to measure the leukemic cells with activated Caspase-3. Phosphorylation of p210 bcr/abl protein in the leukemic cells were assessed by a combination of immunoprecipitation （IP） with c-abl antibody and Western blotting with p-Tyr（pY99） antibody. The protein levels of p210 bcr/abl, Hsp90 and Hsp70 in the leukemic cells were determined by Western blotting with antibodies to c-abl, Hsp90, and Hsp70 respectively. Results After treatment with berbamine at 8μg,/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45. 69% and 48.43% respectively. IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210 ber/abl protein in the leukemia cells, and the amount of phosphorylated p210 bcr/abl in the leukemia cells exposured to berbamine at 8 μg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of p210 ber/abl down-regulated. Significantly, berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in the leukemia cells treated with berbamine at 8 μg/ml for 48 h accounted for 18. 37% of that of the untreated leukemia cells. Berbamine at 8μg/ml had no obvious effect on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis. Conclusion （1） Berbamine induces caspase-3-mediated apoptosis of Ph ＋ leukemia cells through inhibiting phosphorylation of p210 ber/abl protein and down-regulating its chaperone Hsp90 protei

关 键 词：小檗胺 P210 bcr/abl蛋白 热休克蛋白90 白血病 髓样 慢性 脱噬作用 

分 类 号：R733.72[医药卫生—肿瘤]