title: Effect of noncompetitive proteasome inhibition on bortezomib resistance.
abstract: BACKGROUND
  Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the catalytic sites of proteasomes and are competitive inhibitors. We hypothesized that proteasome inhibitors that act through a noncompetitive mechanism might overcome some forms of bortezomib resistance.
  
  
  METHODS
  5-amino-8-hydroxyquinoline (5AHQ) was identified through a screen of a 27-compound chemical library based on the quinoline pharmacophore to identify proteasome inhibitors. Inhibition of proteasome activity by 5AHQ was tested by measuring 7-amino-4-methylcoumarin (AMC) release from the proteasome substrate Suc-LLVY-AMC in intact human and mouse leukemia and myeloma cells and in tumor cell protein extracts. Cytotoxicity was assessed in 5AHQ-treated cell lines and primary cells from myeloma and leukemia patients using AlamarBlue fluorescence and MTS assays, trypan blue staining, and annexin V staining. 5AHQ-proteasome interaction was assessed by nuclear magnetic resonance. 5AHQ efficacy was evaluated in three leukemia xenograft mouse models (9-10 mice per group per model). All statistical tests were two-sided.
  
  
  RESULTS
  5AHQ inhibited the proteasome when added to cell extracts and intact cells (the mean concentration inhibiting 50% [IC(50)] of AMC release in intact cells ranged from 0.57 to 5.03 microM), induced cell death in intact cells from leukemia and myeloma cell lines (mean IC(50) values for cell growth ranged from 0.94 to 3.85 microM), and preferentially induced cell death in primary myeloma and leukemia cells compared with normal hematopoietic cells. 5AHQ was equally cytotoxic to human myelomonocytic THP1 cells and to THP1/BTZ500 cells, which are 237-fold more resistant to bortezomib than wild-type THP1 cells because of their overexpression and mutation of the bortezomib-binding beta5 proteasome subunit (mean IC(50) for cell death in the absence of bortezomib, wild-type THP1: 3.7 microM, 95% confidence interval = 3.4 to 4.0 microM; THP1/BTZ500: 6.6 microM, 95% confidence interval = 5.9 to 7.5 microM). 5AHQ interacted with the alpha subunits of the 20S proteasome at noncatalytic sites. Orally administered 5AHQ inhibited tumor growth in all three mouse models of leukemia without overt toxicity (eg, OCI-AML2 model, median tumor weight [interquartile range], 5AHQ vs control: 95.7 mg [61.4-163.5 mg] vs 247.2 mg [189.4-296.2 mg], P = .002).
  
  
  CONCLUSIONS
  5AHQ is a noncompetitive proteasome inhibitor that is cytotoxic to myeloma and leukemia cells in vitro and inhibits xenograft tumor growth in vivo. 5AHQ can overcome some forms of bortezomib resistance in vitro.
authors.affiliation: Ontario Cancer Institute, Princess Margaret Hospital, Toronto, ON, Canada.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Li
authors.forename: Tabitha E
authors.initials: TE
authors.lastname: Wood
authors.forename: Remco
authors.initials: R
authors.lastname: Sprangers
authors.forename: Gerrit
authors.initials: G
authors.lastname: Jansen
authors.forename: Niels E
authors.initials: NE
authors.lastname: Franke
authors.forename: Xinliang
authors.initials: X
authors.lastname: Mao
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sue Ellen
authors.initials: SE
authors.lastname: Verbrugge
authors.forename: Hans
authors.initials: H
authors.lastname: Adomat
authors.forename: Zhi Hua
authors.initials: ZH
authors.lastname: Li
authors.forename: Suzanne
authors.initials: S
authors.lastname: Trudel
authors.forename: Christine
authors.initials: C
authors.lastname: Chen
authors.forename: Tomasz L
authors.initials: TL
authors.lastname: Religa
authors.forename: Nazir
authors.initials: N
authors.lastname: Jamal
authors.forename: Hans
authors.initials: H
authors.lastname: Messner
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cloos
authors.forename: David R
authors.initials: DR
authors.lastname: Rose
authors.forename: Ami
authors.initials: A
authors.lastname: Navon
authors.forename: Emma
authors.initials: E
authors.lastname: Guns
authors.forename: Robert A
authors.initials: RA
authors.lastname: Batey
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.forename: Aaron D
authors.initials: AD
authors.lastname: Schimmer
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001897:Boronic Acids / Q000493:pharmacokinetics / Q000494:pharmacology*; D000069286:Bortezomib; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004195:Disease Models, Animal; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006912:Hydroxyquinolines / Q000493:pharmacokinetics / Q000494:pharmacology*; D015151:Immunoblotting; D020128:Inhibitory Concentration 50; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000201:enzymology*; D016328:NF-kappa B / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000493:pharmacokinetics / Q000494:pharmacology*; D014183:Transplantation, Heterologous
chemical_list: C528550:5-amino-8-hydroxyquinoline; D000970:Antineoplastic Agents; D001897:Boronic Acids; D006912:Hydroxyquinolines; D016328:NF-kappa B; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011719:Pyrazines; D000069286:Bortezomib