Tetrandrine citrate eliminates imatinib-resistant chronic myeloid leukemia cells in vitro and in vivo by inhibiting Bcr-Abl/β-catenin axis

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： 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 mo- lecular 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 poly- merase 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 （ICso） ranged from 1.20 to 2.97 pg/ml. In contrast, tetrandrine citrate did not affect normal blood cells under the same conditions, and IC50 values were about 10.12-13.11 μg/ml. Oral administration of tetrandrine citrate caused complete regression of I M-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 p210Bcr-Ab1 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 p210scr-Ab1 protein. RT-PCR results showed that tetrandrine treatment caused a decrease of Bcr-Abl mRNA. FCM analysis indicated that tetrandrine induced gap 1 （G1） 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 p210scr-Abl mRNA and β-catenin protein.