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


CONCLUSIONS
(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.
Sun
Jian-rong
JR
Department of Hematology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
Zhang
Xiao-hong
XH
He
Zhi-wen
ZW
Gu
Ying
Y
Yu
Ying-zi
YZ
Fang
Yong-ming
YM
Lü
Qing-hua
QH
Dong
Qing-hua
QH
Xu
Rong-zhen
RZ
D000470:Alkaloids / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D044182:Benzylisoquinolines / Q000627:therapeutic use*; D053148:Caspase 3 / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism / Q000473:pathology*; D016374:Oncogene Proteins v-abl / Q000378:metabolism; D008517:Phytotherapy; D051025:bcl-Associated Death Protein / Q000378:metabolism
D000470:Alkaloids; D044182:Benzylisoquinolines; D018841:HSP90 Heat-Shock Proteins; D016374:Oncogene Proteins v-abl; D051025:bcl-Associated Death Protein; D053148:Caspase 3; C027870:berbamine