title: Berbamine inhibits proliferation and induces apoptosis of KU812 cells by increasing Smad3 activity.
abstract: 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, p21(Cip1)(p21), and p27(Kip1)(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.
authors: Department of Hematology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
authors: Yun
authors: Y
authors: Liang
authors: Xi
authors: X
authors: Qiu
authors: Rong-zhen
authors: RZ
authors: Xu
authors: Xiao-ying
authors: XY
authors: Zhao
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D044182:Benzylisoquinolines / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D019938:Cyclin D1 / Q000032:analysis; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000032:analysis; D016193:G1 Phase / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000473:pathology; D018125:Receptors, Transforming Growth Factor beta / Q000502:physiology; D051900:Smad3 Protein / Q000378:metabolism*
chemical_list: D044182:Benzylisoquinolines; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018125:Receptors, Transforming Growth Factor beta; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D019938:Cyclin D1; C027870:berbamine