Studies On The Effects Of Berbamine As A Calmodulin Antagonist On Human Leukemia K562 Cells And Its Mechanisms

BackgroundLeukemia is a common hematological malignant disease andfinding out some new antitumor drugs is the important way to improve the cure rate. Berbamine is one kind of bis-benzylisoquinoline alkaloids that was extracted from Chinese herbs. Recently, berbamine and its derivatives were determined to be calmodulin antagonist which could influence many functions of living cell by binding with calmodulin. It could inhibit the proliferation of different kinds of cell lines obviously in vitro and reverse the multidrug resistance of some leukemia cell lines partially. In this study we tried to elucidate the anti-proliferation effects of berbamine on leukemia cells and its mechanism.Materials and methodsBerbamine compound was dissolved to the concentration of Img/ml with 0.9% sodium chloride and diluted to desired concentrations with culture medium. The major working solution concentration of berbamine was from 4u,g/ml to 12|ag/ml. The human acute erythroleukemia cell line K562 cells, its adriamycin resistance cell line K562/adr cells and human acute granulocytic leukemia cell line HL-60 cells were provided by ZheJiang University Cancer Research Institution.MTT was used to examine the effect of berbamine on cell growth of K562 cells and K562/adr cells and HL-60 cells. It was also measured in primary leukemia cells obtained from AML patients and normal bone marrow cells. Morphological observation was used to detect the characteristic changes of apoptosis in K562 cells while apoptosis rate was measured by flow cytometry assay. The expression of apoptosis related genes bcl-2 and bax were determined by RT-PCR and that of bcr/abl was evaluated by nested-PCR treated with berbamine at 8jag/ml. At last, Caspase 3, the most important enzyme during the process of apoptosis, was determined by flow cytometry assay in K562 cells treated with berbamine at 12|ag/ml.ResultsBerbamine could inhibit the cell proliferation significantly in both K562 cells and HL-60 cells. And its IC50 was 5.23u,g/ml and 5.02^g/ml, respectively. In contrast, it had slight effect on normal bone marrow cells and its ICso value was 15.6 times higher than that ofK562 cells. The K562/adr cells and primary leukemia cells were also sensitive to berbamine and its ICso value was 10.97 u.g/ml and 6.8lug/ml, respectively. Berbamine was detected to induce apoptosis of K562 cells quickly by morphology and flow cytometry assay. K562 cells treated with 4fig/ml berbamine for 6 hours showed morphological characteristic changes of apoptosis, such as cell shrinkage and apoptotic bodies. The apoptosis rate of K562 was concertration-dependent measured by FCM. The apoptosis rate of K562 cells treated with 4|xg/ml berbamine increased from 4.0% to 25.3% cultured for 24 and 72 hours, respectively. And it increased from 17.5% to 70.7% treated with 8|ag/ml berbamine. K562 cells didn't express bcl-2 and expressed bax. No change of these genes expression had been detected treated with 8jag/ml berbamine until 72 hours later. Differently, the expression level (semiquantity value) of bcr/abl decreased quickly from 1.284-0.506 ,treated with 8ug/ml berbamine for 72 hours. The expression level of Caspase 3 after treatment with berbamine at 12ug/ml was determined to be increased significantly from 18.36 % to 38.25 % (p<0.001).ConclusionsOur results suggested that berbamine could significantly inhibit the proliferation of K562 cells, K562/adr cells, HL-60 cells as well as primary leukemia cells, but had no obvious inhibitory effect on growth of normal bone marrow cells. Apoptosis could be induced by berbamine in K562 and we suspected that it came out through decreasing the expression of bcr/abl and increasing that of Caspase 3in K562 cells. At the same time it didn't affect the expression of bcl-2 andbax .