title: Tetrandrine citrate eliminates imatinib-resistant chronic myeloid leukemia cells in vitro and in vivo by inhibiting Bcr-Abl/β-catenin axis.
abstract: 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 xenografts 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.
authors.affiliation: Department of Hematology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China.
authors.forename: Xiao-hua
authors.initials: XH
authors.lastname: Xu
authors.forename: Yi-chao
authors.initials: YC
authors.lastname: Gan
authors.forename: Gen-bo
authors.initials: GB
authors.lastname: Xu
authors.forename: Ting
authors.initials: T
authors.lastname: Chen
authors.forename: Hong
authors.initials: H
authors.lastname: Zhou
authors.forename: Jin-fen
authors.initials: JF
authors.lastname: Tang
authors.forename: Ying
authors.initials: Y
authors.lastname: Gu
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.forename: Ying-ying
authors.initials: YY
authors.lastname: Xie
authors.forename: Xiao-ying
authors.initials: XY
authors.lastname: Zhao
authors.forename: Rong-zhen
authors.initials: RZ
authors.lastname: Xu
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D044182:Benzylisoquinolines / Q000737:chemistry / Q000494:pharmacology*; D048708:Cell Growth Processes / Q000187:drug effects; D002951:Citrates / Q000737:chemistry / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004347:Drug Interactions; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005260:Female; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints / Q000187:drug effects; D006801:Humans; D000068877:Imatinib Mesylate; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000473:pathology; D010879:Piperazines / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D011897:Random Allocation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023041:Xenograft Model Antitumor Assays; D051176:beta Catenin / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D001549:Benzamides; D044182:Benzylisoquinolines; D002951:Citrates; D010879:Piperazines; D011743:Pyrimidines; D012333:RNA, Messenger; D051176:beta Catenin; C009438:tetrandrine; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl