Roles of Phosphorylated p210bcr/abl and Hsp90 Protein in Apoptosis Induced by Berbamine in K562 Cells.

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic stem cell disorder characterized by accumulation of mature and immature granulocytes in peripheral blood and bone marrow due to uncontrolled growth and resistance to apoptosis. The dysregulated activity of the bcr/abl oncoprotein tyrosine kinase, which is encoded by the bcr-abl fusion gene generated from the chromosomal translocation of t(9; 22) and present in approximately 95% of CML patients, has been shown to be responsible for these malignant phenotypes. Numerous studies have demonstrated that only being phosphorylated that p210bcr/abl oncoprotein can promote cell proliferation and survival, and block apoptosis of tumor cells. Bcr/abl tyrosine kinase has chaperone association with heat shock protein 90 (Hsp90), which plays an essential role in stabilization of bcr/abl protein. Here we describe the activity of a natural small molecular compound, berbamine from plant Berberis amurensis that can selectively induce cell death of both Gleevecsensitive and -resistant Ph+ CML cells, but little is known about its exact mechanisms. We investigated the the expression levels of phosphorylated p210bcr/abl protein and Hsp90 protein in apoptosis induced by berbamine in K562 cells by means of various technologies, including cell culture system, flow cytomerty, western blot and immunoprecipitation (IP).

Methods: Human Ph+ CML leukemia K562 cells, which express endogenous p210bcr/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 apoptosis of leukemic cells; FCM and cytoperm/cytofix plus Caspase-3-McAb-PE were employed to measure leukemic cells with activated Caspase-3. Phosphorylation of p210bcr/abl protein in leukemic cells were assessed by a combination of immunoprecipitation (IP) with c-abl antibody and Western blot with p-Tyr(pY99)antibody. The protein levels of P210bcr/abl, Hsp90 and Hsp70 in leukemic cells were determined by Western blot with antibodies to c-abl, Hsp90 and Hsp70, respectively.

Results:

1. After treatment with berbamine at 8 μg/ml for 48 h, the percentages of leukemic cells expressing activated caspase-3 and apoptotic cells were 45.69% and 48.43%, respectively.

2. IP and WB results showed that berbamine at low concentration markedly inhibited phosphorylation of p210bcr/abl protein in leukemia cells, and the amount of phosphorylated p210bcr/abl in 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 P210bcr/abl down-regulated.

3. Berbamine also down-regulated chaperone Hsp90 protein, and the amount of Hsp90 protein in leukemia cells treated with berbamine at 8 μg/ml for 48 h accounted for 18.37% of that of untreated leukemia cells. Interestingly, berbamine at 8 μg/ml had no obvious effects on chaperone Hsp70 protein expression associated with the resistance of leukemia cells to apoptosis.

Conclusions:

1. Berbamine could induce caspase-3-mediated apoptosis of Ph+ leukemia cells through inhibiting phosphorylation of p210bcr/abl protein and down-regulating its chaperone Hsp90 protein.

2. Unlike Hsp90 inhibitor GA that could upregulate Hsp70, berbamine had no obvious effects on chaperone Hsp70 protein expression in leukemia cells, suggesting that berbamine may be a novel class of Hsp90 inhibitor, and further study is required.