Berbamine inhibits proliferation and induces apoptosis of KU812 cells by increasing Smad3 activity*

Objective: The cytotoxic effect of berbamine on chronic myeloid leukemia (CML) cell line KU812 was evaluated, and the mechanisms of its action were explored. Methods: The effect of berbamine on the KU812 cell growth was determined by methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry was used to profile cell cycle alteration upon berbamine treatment. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to determine the transcripts of transforming growth factor-β (TGF-β) receptors (TβRs), Smad3, c-Myc, cyclin D1, p21Cip1(p21), and p27Kip1(p27). Changes in the protein levels of total Smad3, phosphorylated Smad3, the downstream targets of Smad3, and specific apoptosis-related factors were evaluated by Western blotting. Results: Berbamine inhibited KU812 cell proliferation in a dose- and time-dependent manner, and the half maximal inhibitory concentration (IC50) values for treatments of 24, 48, and 72 h were 5.83, 3.43, and 0.75 μg/ml, respectively. Berbamine induced G1 arrest as well as apoptosis in KU812 cells. Transcriptions of Smad3 and p21 were up-regulated, while those of TβRI, TβRII, c-Myc, cyclin D1 and p27 were not changed significantly. The protein levels of both total Smad3 and phosphorylated Smad3 were both up-regulated after berbamine treatment, together with decreased c-Myc and cyclin D1 and increased p21. Meanwhile, the levels of the anti-apoptotic proteins, such as Bcl-2 and Bcl-xL, were decreased, whereas pro-apoptotic Bax was increased. Conclusions: Berbamine suppresses KU812 cell proliferation through induction of cell cycle arrest in G1 and apoptosis. It activates Smad3 without additional stimulation of TGF-β, and alters the levels of the Smad3 downstream targets, including c-Myc, cyclin D1 and p21. Our findings suggest that berbamine is a promising drug in the treatment of advanced stage patients with CML