Objective:To evaluate the effects of tetrandrine citrate, a novel tetrandrine salt with high water solubility, on the growth of imatinib (IM)-resistant chronic myeloid leukemia (CML) in vitro and in vivo, and reveal action molecular mechanisms. Methods:Cell viability in vitro was measured using methyl thiazolyl tetrazolium (MTT) assay. CML cell growth in vivo was assessed using a xenograft model in nude mice. Bcr-Abl and β-catenin protein levels were determined using Western blotting. Bcr-Abl messenger RNA (mRNA) was measured by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry (FCM) was used to determine cell cycle status. Results:Tetrandrine citrate inhibited the growth of IM-resistant K562 cells, primary leukemia cells, and primitive CD34 + leukemia cells, and their inhibition concentration that inhibited 50% of target cells (IC 50 ) ranged from 1.20 to 2.97 μg/ml. In contrast, tetrandrine citrate did not affect normal blood cells under the same conditions, and IC 50 values were about 10.12-13.11 μg/ml. Oral administration of tetrandrine citrate caused complete regression of IM-resistant K562 xeno-grafts in nude mice without overt toxicity. Western blot results revealed that treatment of IM-resistant K562 cells with tetrandrine citrate resulted in a significant decrease of both p210 Bcr-Abl and β-catenin proteins, but IM did not affect the Bcr-Abl protein levels. Proteasome inhibitor, MG132, did not prevent tetrandrine-mediated decrease of the p210 Bcr-Abl protein. RT-PCR results showed that tetrandrine treatment caused a decrease of Bcr-Abl mRNA. FCM analysis indicated that tetrandrine induced gap 1 (G 1 ) arrest in CML cells. Conclusions:Tetrandrine citrate is a novel orally active tetrandrine salt with potent anti-tumor activity against IM-resistant K562 cells and CML cells. Tetrandrine citrate-induced growth inhibition of leukemia cells may be involved in the depletion of p210 Bcr-Abl mRNA and β-catenin protein.
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 doseand 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.
