Sun Jian-rong, Zhang Xiao-hong, He Zhi-wen, Gu Ying, Yu Ying-zi, Fang Yong-ming, Lü Qing-hua, Dong Qing-hua, Xu Rong-zhen
Department of Hematology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
Zhonghua Yi Xue Za Zhi. 2006 Aug 29;86(32):2246-51.
To investigate the mechanism of apoptosis of chronic myeloid leukemia (CML) cells induced by the novel p210 bcr/abl inhibitor berbamine.
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.
After treatment with berbamine at 8 microg/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 bcr/abl protein in the leukemia cells, and the amount of phosphorylated p210 bcr/abl in the leukemia cells exposed to berbamine at 8 microg/ml for 6 h were only 8.41% of that of untreated leukemia cells without the protein levels of p210 bcr/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 microg/ml for 48 h accounted for 18.37% of that of the untreated leukemia cells. Berbamine at 8 microg/ml had no obvious effect on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis.
(1) Berbamine induces caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210 bcr/abl protein and down-regulating its chaperone Hsp90 protein. (2) Unlike Hsp90 inhibitor GA that upregulates Hsp70, berbamine has no obvious effect on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.
探讨新型p210 bcr/abl抑制剂小檗胺诱导慢性髓性白血病（CML）细胞凋亡的机制。
将表达内源性p210 bcr/abl蛋白的人Ph+ CML白血病K562细胞培养于RPMI 1640中，并按指定的时间和剂量用小檗胺处理。采用流式细胞术（FCM）和Annexin-V-Fluos/PI染色试剂盒评估白血病细胞的凋亡情况；运用FCM以及胞内固定/胞膜通透加Caspase-3单克隆抗体-PE检测活化Caspase-3的白血病细胞。通过用c-abl抗体进行免疫沉淀（IP）并结合用p-Tyr（pY99）抗体进行蛋白质印迹法来评估白血病细胞中p210 bcr/abl蛋白的磷酸化情况。分别用抗c-abl、Hsp90和Hsp70抗体通过蛋白质印迹法测定白血病细胞中p210 bcr/abl、Hsp90和Hsp70的蛋白水平。
用8μg/ml小檗胺处理48小时后，表达活化Caspase-3的白血病细胞百分比和凋亡细胞百分比分别为45.69%和48.43%。IP和WB结果显示，低浓度小檗胺可显著抑制白血病细胞中p210 bcr/abl蛋白的磷酸化，在8μg/ml小檗胺处理6小时的白血病细胞中，磷酸化p210 bcr/abl的量仅为未处理白血病细胞的8.41%，且p210 bcr/abl蛋白水平未下调。值得注意的是，小檗胺还下调了伴侣蛋白Hsp90的表达，在8μg/ml小檗胺处理48小时的白血病细胞中，Hsp90蛋白量占未处理白血病细胞的18.37%。8μg/ml小檗胺对与白血病细胞凋亡抗性相关的伴侣蛋白Hsp70的表达无明显影响。
（1）小檗胺通过抑制p210 bcr/abl蛋白的磷酸化并下调其伴侣蛋白Hsp90的表达，诱导Ph+白血病细胞发生Caspase-3介导的凋亡。（2）与上调Hsp70的Hsp90抑制剂GA不同，小檗胺对白血病细胞中伴侣蛋白Hsp70的表达无明显影响，提示小檗胺可能是一类新型的Hsp90抑制剂，有待进一步研究。
