| BackgroundLeukemia is a common hematological malignant disease, but its machnism is remain uncertain. Currently, Chemotherapy is one of major approaches to treat it. However, the serious side effects of chemotherapy and MDR often lead to the failure of this treatment. Thus, it is important to find out novel and more efficient antitumor drugs to improve the cure rate. Berbamine is a kind of bis-benzylisoquinoline alkaloids in Chinese herbs. A number of studies showed that berbamine and its derivatives belong to calmodulin antagonist which could influence many functions of mammalian cells by binding with calmodulin. It has been used to deal with leukopenia for many years . Recently, some studies demonstrated that it could greatly inhibit the proliferationof a variety of tumor cell lines in vitro and reverse the raultidrug resistance of some leukemia cell lines. In this study, we tried to determine effects of berbamine on growth of NB4 leukemia cell line and explore its possible mechanisms.Materials and MethodsBerbamine compound, which was provided by the Fu Shun Traditional Chinese Medicine Factory, dissolved to the concentration of 1mg/ml with 0. 9% sodium chloride and diluted to desired concentrations with culture medium. Various working concentrations of berbamine were used in this studies ( 2 g/ml, 4 g/ml, 8 g/ml, 12 g/ml and 16 g/ml). NB4 leukemia cell line, which is a human acute promyelocyte leukemia cell line, was provided by ZheJiang University Cancer Research Institution. MTT assay was used to examine the effect of berbamine on growth of NB4 cell line. Morphological analysis and DNA agarose electrophoresis were used to detect the characteristic changes of apoptosis in NB4 cells, and apoptosis rate was measured by flow cytometry. The PML/RARa mRNA was determined by nested -PCR, and the Survivin mRNA was tested by RT-PCR. The expression of Caspase 3 protein in NB4 cell line was evaluated by flow cytometry.ResultsOur study results showed that berbamine inhibited the NB4 cells' growth significantly after treated with berbamine at different concentrations for different time points. When the concentration ofberbamine was at 8 g/ml , the inhibition rate of NB4 cells at 24 hours was 69. 8% , and at 48 hours and 72 hours, the inhibition rate werw 82. 5% and 82.8% respectively. When NB4 cell line were treated with berbamine after 48 hours, the inhibition rate increased from 70.6% at 4 g/ml to 82. 5% at 8 g/ml . The value of IC50 were 7. 241 g/ml at 24 hours, 3. 860 u g/ml at 48 hours and 2. 276 g/ml at 72 hours. In contrast, berbamine displayed no significant inhibition for normal bone marrow cells, and the value of IC50 was 1648. 342 u g/ml at 24 hours . Morphology analysis showed the characteristic of apoptosis, and the DNA agarose electro-phoresis showed a typical DNA ladder. The apoptosis rate increased from 2. 83% to 58. 44% after treated with berbamine at 12 g/ml for 48 hours. The PML/RARa mRNA wasn' t changed obviously .however, Survivin mRNA was decreased dramatically. The protein expression of Caspase3 increased obviously from 2. 06% to 70. 89% after treated with berbamine at a concentration of 12 g/ml for 48 hours.ConclusionsBerbamine could inhibit the growth of NB4 1reukemia cell line. And induction of apoptosis in NB4 cell line is one of mechanisms for its suppressing the growth of NB4 leukemia cell line. Berbamine might have no effect on the expression of PML/RAR in NB4 cell line at mRNA level. Inhibition of Survivin mRNA and upregalation of Caspase 3 protein might be one of mechanisms for this apoptosis. |
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