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jnci.oxfordjournals.org JNCI |Articles 1069
DOI: 10.1093/jnci/djq198 © The Author 2010. Published by Oxford University Press. All rights reserved.
Advance Access publication on May 26, 2010. For Permissions, please e-mail: journals.permissions@oxfordjournals.org.
The proteasomal degradation pathway rids cells of excess and mis-
folded proteins and regulates the cellular levels of proteins that are
responsible for processes, such as cell cycle progression, DNA
repair, and transcription [reviewed in (1)]. The proteasomal path-
way of protein degradation is initiated by the sequential enzymatic
activities of ubiquitin ligases E1, E2, and E3, which add chains
of ubiquitin molecules onto the lysine residues of proteins
to mark them for degradation [reviewed in (2,3)]. Ubiquitin-
tagged proteins are degraded by the 26S proteasome, a multimeric
enzyme complex consisting of a and b subunits located in the
nucleus and cytoplasm. Chemical and peptidyl inhibitors of the
proteasome prevent ubiquitin-mediated protein degradation. In
vitro and in vivo studies demonstrate that proteasome inhibitors
induce cell death in malignant cells and inhibit tumor growth in
mouse models of malignancy (4), thus supporting the development
of proteasome inhibitors as therapeutic agents for the treatment of
malignancies.
All chemical proteasome inhibitors currently approved or
under clinical evaluation, such as bortezomib and NPI-0052, bind
threonine residues in the active sites of the b subunits of the
20S proteasome (the core complex of the 26S proteasome in
eukaryotes, which degrades ubiquitinated target molecules in an
ARTICLE
Effect of Noncompetitive Proteasome Inhibition on Bortezomib
Resistance
Xiaoming Li, Tabitha E. Wood, Remco Sprangers, Gerrit Jansen, Niels E. Franke, Xinliang Mao, Xiaoming Wang, Yi Zhang,
Sue Ellen Verbrugge, Hans Adomat, Zhi Hua Li, Suzanne Trudel, Christine Chen, Tomasz L. Religa, Nazir Jamal, Hans Messner,
Jacqueline Cloos, David R. Rose, Ami Navon, Emma Guns, Robert A. Batey, Lewis E. Kay, Aaron D. Schimmer
Manuscript received June 29, 2008; revised April 27, 2010; accepted April 30, 2010.
Correspondence to: Aaron D. Schimmer, MD, PhD, 610 University Ave, Toronto, ON, Canada M5G 2M9 (e-mail: aaron.schimmer@utoronto.ca).
Background Bortezomib and the other proteasome inhibitors that are currently under clinical investigation bind to the cata-
lytic 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% [IC50] of AMC release in intact cells ranged from 0.57 to 5.03 µM), induced cell death in intact cells from
leukemia and myeloma cell lines (mean IC50 values for cell growth ranged from 0.94 to 3.85 µM), and preferen-
tially 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 b5 proteasome subunit (mean IC50 for cell death in the absence of bortezomib, wild-type
THP1: 3.7 µM, 95% confidence interval = 3.4 to 4.0 µM; THP1/BTZ500: 6.6 µM, 95% confidence interval = 5.9 to
7.5 µM). 5AHQ interacted with the a 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.
J Natl Cancer Inst 2010;102:1069–1082
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1070 Articles |JNCI Vol. 102, Issue 14 | July 21, 2010
ATP-dependent manner), thereby competitively inhibiting the
enzymatic activity of the proteasome (5–7). In clinical trials, the
proteasome inhibitor bortezomib has demonstrated clinical efficacy
in patients with new diagnosed and relapsed multiple myeloma and
mantle cell lymphoma (8,9). However, most patients who are
treated with this drug alone do not achieve complete remission,
and the majority of responders ultimately relapse (8,9). Several
mechanisms of resistance to bortezomib have been identified (10),
including mutation and overexpression of the b5 subunit of
the proteasome to which bortezomib binds (11). Molecules that
inhibit the proteasome through a mechanism distinct from that of
bortezomib could be useful for overcoming some forms of resis-
tance to this drug or used in combination with bortezomib to
improve clinical outcomes.
Previously, we demonstrated inhibition of the proteasome by
chloroquine and clioquinol, compounds that share a common
CONTEXT AND CAVEATS
Prior knowledge
Proteasome inhibitors that are currently approved or under clinical
investigation, including bortezomib, bind to the catalytic sites of
proteasomes and are competitive inhibitors. Proteasome inhibitors
that act through a noncompetitive mechanism might overcome
some forms of bortezomib resistance.
Study design
Screening of a 27-compound chemical library based on the quino-
line pharmacophore identified 5-amino-8-hydroxyquinoline (5AHQ)
as a proteasome inhibitor. The mechanism of inhibition was exam-
ined in isolated proteasomes. The effects of 5AHQ on proteasome
inhibition and on cell viability and apoptosis were tested in leuke-
mia and myeloma cell lines and in primary cells from myeloma and
leukemia patients. 5AHQ efficacy was evaluated in three leukemia
xenograft mouse models.
Contribution
5AHQ bound to the a subunits of the 20S proteasome at non-
catalytic sites. 5AHQ inhibited the proteasome and induced cell
death in leukemia and myeloma cell lines and preferentially
induced cell death in primary myeloma and leukemia cells com-
pared with normal hematopoietic cells. 5AHQ was equally cyto-
toxic to a bortezomib-resistant myeloma cell line and the parental
cell line from which it was derived. Orally administered 5AHQ
inhibited tumor growth in all three mouse models of leukemia
without overt toxicity.
Implications
5AHQ may represent a new strategy for proteasome inhibition in
cancer cells and a potential lead for a new class of therapeutic
agents.
Limitations
The possibilities that 5AHQ simultaneously binds to a and b sub-
units, that a 5AHQ metabolite is primarily responsible for protea-
some inhibition, and that 5AHQ or a metabolite also inhibits the
proteasome through indirect effects were not excluded. 5AHQ may
have additional targets beyond the proteasome, and inhibition of
these other targets may also contribute to its anticancer effects.
From the Editors
quinoline pharmacophore (12,13). Chloroquine binds the a sub-
units of the proteasome and inhibits the enzyme noncompetitively
(13). However, because supra-pharmacological concentrations of
chloroquine are required to observe these effects, we could not
investigate the effects of chloroquine on proteasomal function in
intact cells alone or in combination with bortezomib. Clioquinol is
more active than chloroquine, but its poor solubility in water
precludes detailed mechanistic studies. Therefore, we screened a
library of chemical compounds that were based on the quinoline
pharmacophore to identify proteasome inhibitors that may be
more amenable than chloroquine and clioquinol for in vitro and in
vivo studies. Here, we characterize 5-amino-8-hydroxyquinoline
(5AHQ), the most potent inhibitor identified in this screen in vitro
and in mouse xenograft models.
Materials and Methods
Cell Lines
Human multiple myeloma KMH11, KMS18, LP1, My5, and
UTMC2 cells (14), whose identities were confirmed by cytoge-
netic profiling, were grown in Iscove modified Dulbecco’s medium
(Ontario Cancer Institute, Toronto, ON, Canada). Human OCI-
AML2, NB4, KG1A (14), and K562 (American Type Culture
Collection, Manassas, VA) and murine MDAY-D2 (15) leukemia
cell lines, whose identities were verified by gene expression profiling,
were maintained in RPMI-1640 medium (Ontario Cancer Institute).
Human myelomonocytic THP1 cells and the bortezomib-resistant
derivative cell lines THP1/BTZ50, THP1/BTZ100, THP1/
BTZ200, and THP1/BTZ500 (11) were grown in RPMI-1640
medium supplemented with 20 mM HEPES and 2 mM glutamine;
THP1/BTZ50, THP1/BTZ100, THP1/BTZ200, and THP1/
BTZ500 were grown in the presence of 50, 100, 200, and 500 nM
bortezomib, respectively (Millennium Pharmaceutics, Cambridge,
MA). Bortezomib-resistant THP1 cells were maintained in
medium containing bortezomib for at least 3 days before their use
in an experiment. All media were supplemented with 10% fetal calf
serum, 100 µg/mL penicillin, and 100 U/mL streptomycin (all
from Hyclone, Logan, UT).
Primary Cells
Primary human acute myeloid leukemia (AML) and chronic
lymphocytic leukemia (CLL) cells were isolated by Ficoll (Sigma-
Aldrich, St Louis, MO) density gradient centrifugation from
peripheral blood samples obtained from AML (n = 4) and CLL
(n = 5) patients, respectively, attending the Princess Margaret Hospital
(Toronto, ON, Canada) for whom at least 80% of the mononu-
clear cells in their peripheral blood were malignant. Bone marrow
aspirates were obtained from patients with multiple myeloma (n =
3) attending the Princess Margaret Hospital. Primary normal he-
matopoietic cells were obtained from healthy volunteers (n = 4)
donating peripheral blood stem cells (PBSCs) for allotransplanta-
tion. Mononuclear cells were isolated by Ficoll density centri-
fugation. Primary cells were cultured at 37°C in Iscove modified
Dulbecco’s medium supplemented with 10% fetal calf serum,
1 mM l-glutamine, 100 µg/mL penicillin, and 100 U/mL strep-
tomycin. All patients and healthy volunteers provided written
informed consent. The collection and use of human tissue for this
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study were approved by the local ethics review board (University
Health Network, Toronto, ON, Canada).
Assessment of Proteasome Enzymatic Activity
To assess the effects of 5AHQ on the enzymatic activity of the
proteasome in vitro, cellular proteins were extracted from mye-
loma UTMC2, KMH11, and KMS18 cells and from leukemia
OCI-AML2, NB4, KG1A, MDAY-D2, and K562 cells with lysis
buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1% Triton
X-100, and 2 mM ATP). For each assay, 2 µg of protein was incu-
bated for 2 hours at 37°C with increasing concentrations of 5AHQ
(Sigma-Aldrich) diluted in assay buffer (50 mM Tris–HCl [pH 7.5]
and 150 mM NaCl). After incubation, the fluorogenic proteasome
substrate N-Succinyl-Leu-Leu-Val-Tyr-AMC (7-amino-4-
methylcoumarin) (Suc-LLVY-AMC; BIOMOL International,
Plymouth Meeting, PA) was added to each reaction at a final con-
centration of 40 µM and the amount of free AMC released was
measured with the use of a fluorescent spectrophotometric plate
reader at excitation and emission wavelengths of 380 and 460 nm,
respectively. Experiments were performed at least in duplicate and
repeated at least twice (n = 4–12 data points).
To assess the effects of 5AHQ on the enzymatic activity of the
proteasome in intact cells, leukemia OCI-AML2, NB4, KG1A,
and MDAY-D2 cells and myeloma UTMC2, KMH11, KMS18,
and MY5 cells were incubated with increasing concentrations of
5AHQ for 22 hours at 37°C. The cells were lysed in lysis buffer.
For each assay, Suc-LLVY-AMC was added to 2 µg of protein and
the generation of free AMC was measured over time with the
use of a fluorescent spectrophotometric plate reader as described
above. Experiments were performed at least in duplicate and
repeated at least twice.
To assess the effects of 5AHQ on the enzymatic activity of
purified proteasomes, we isolated proteasome complexes from
rabbit muscle and from Thermoplasma acidophilum as described
previously (13,16). Briefly, Escherichia coli BL21-CodonPlus(DE3)
cells (Stratagene, Wilmington, DE) expressing the T acidophilum
a and b proteasome subunits were grown in lysogeny broth (10 g
tryptone, 5 g yeast extract, and 10 g NaCl in 1 L water, pH 7.4).
Protein was purified on nickel–nitriloacetic acid (Ni-NTA) resin
(Qiagen, Mississauga, ON, Canada), followed by cleavage of an
amino-terminal hexa-His purification tag on the a subunit using
tobacco etch virus (TEV) protease that was purified from E coli
carrying a plasmid that overexpresses the His-tagged protease
(13) followed by gel filtration. The proteasome is spontaneously
formed in the E coli cells from the a and b subunits. The protea-
some consisting of a subunits and lacking the b subunits, also
called the half-proteasome, was produced by overexpressing the
a subunits that carried a TEV-cleavable His tag in E coli BL21-
CodonPlus(DE3) cells. Purification of the half-proteasome was
performed as above.
Methyl labeling of the proteasome subunits for nuclear mag-
netic resonance (NMR) analysis was achieved by growing E coli
BL21-CodonPlus(DE3) cells in D2O-based minimal medium
with 2H- and 12C-labeled glucose as the carbon source. One
hour before induction of protein expression with isopropyl b-d-1-
thiogalactopyranoside, E coli were treated with a-ketobutyric acid
(60 mg/L; one methyl group labeled with 13CH3, and the other was
labeled with 12CD3) and a-ketoisovaleric acid (one methyl group
labeled with 13CH3; 100 mg/L; both from Sigma-Aldrich). These
precursor molecules are metabolized by the E coli cells, resulting in
labeling of methyl groups with 13CH3 in the Ile, Leu, and Val
residues in an otherwise fully deuterated 12-carbon–labeled protein
(17). Proteins were purified by Ni-NTA affinity resin and size
exclusion chromatography.
Rabbit proteasomes were isolated from homogenates of the
psoas muscles from Zealand white male rabbits that were centri-
fuged (100 000g) and applied to a 100-mL DE52 column. Bound
protein was eluted, concentrated, dialyzed, and applied to a Mono
Q column (Amersham Pharmacia, Piscataway, NJ). Bound protein
was eluted, concentrated, and applied to a Superose 6 gel filtration
column (Amersham Pharmacia). Fractions were collected, and the
active fractions (those that released AMC from Suc-LLVY-AMC)
were pooled, dialyzed, and applied to a DEAE Affi-Gel Blue col-
umn (Sigma-Aldrich). Active fractions were then collected and
pooled for use in subsequent experiments.
Increasing concentrations of 5AHQ (0, 1.25, 2.5, 5, and 10 µM),
MG132 (0, 0.078, 0.156, 0.312, 0.625, and 1.25 µM; BIOMOL
International), and bortezomib (0, 2.5, 5, and 10 nM) were added
to isolated rabbit proteasomes in 50 mM HEPES (pH 7.5), 1 mM
dithiothreitol, and 0.018% sodium dodecyl sulfate (SDS) (18) and
to purified T acidophilum proteasomes in 50 mM Tris–HCl (pH 7.5)
and 150 mM NaCl (13). After 1 hour of incubation at 37°C, the
AMC-conjugated proteasomal substrate benzyloxycarbonyl-l-
leucyl-l-leucyl-l-glutamyl-methylcoumarylamide (Z-LLE-AMC;
BIOMOL International) was added at final concentrations of 500,
250, 125, 62.5, and 31.25 µM, and the rate of free AMC release was
measured over time with the use of a fluorescent spectrophoto-
metric plate reader as described above. The kinetics by which
5AHQ and MG132 inhibited purified proteasome was determined
using SigmaPlot software 11.0 and Enzyme Kinetics 1.3 (both
from Systat Software, San Jose, CA).
Identification of 5AHQ
We compiled a chemical library of 27 compounds (all from Sigma)
that were based on the quinoline pharmacophore and included
various halogenated and alkyl-substituted quinolines, hydroxyqui-
nolines, and quinoline hydrazones because we have previously
shown that quinoline compounds can inhibit the proteasome (13).
Aliquots of this library (final concentration of 3.9 µM) were added
to 2 µg of whole-cell extract from MDAY-D2 leukemia cell line in
reaction buffer (same as described above). After incubation for
2 hours at 37°C, the fluorogenic proteasome substrate Suc-LLVY-
AMC (final concentration 40 µM) was added to each reaction. The
amount of free AMC released was measured with the use of a fluo-
rescent spectrophotometric plate reader at excitation and emission
wavelengths of 380 and 460 nm, respectively, as described above.
NMR Assessment of Inhibitor Binding to the Proteasome
NMR experiments with the a7-a7 half-proteasome were performed
on a 600-MHz spectrometer (Varian, Palo Alto, CA) at 50°C using
samples that contained approximately 4 µM of proteasome (ap-
proximately 50 µM of a-monomer), 100% D2O, 25 mM potassium
phosphate (pH 6.8), 50 mM NaCl, 1 mM EDTA, 0.03% NaN3,
and 2 mM dithiothreitol, similar to that previously described (16,19).
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NMR data were processed and analyzed with the nmrPipe/
nmrDraw software package (20).
Cell Viability and Apoptosis Assays
The viability of leukemia and myeloma cells treated with 5AHQ
or bortezomib was assessed with the use of a CellTiter 96 AQueous
One Solution Cell Proliferation Assay (Promega, Madison,
WI), which is a form of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner
salt (MTS) assay, or with fluorescence-based AlamarBlue cell
viability reagent (Invitrogen, Carlsbad, CA) according to manufac-
turer’s instructions and as described previously (21,22) or by
trypan blue staining. Apoptosis was measured by staining cells
treated with 5AHQ with annexin V–fluorescein isothiocyanate and
propidium iodide (both from Biovision Research Products,
Mountain View, CA) and flow cytometry according to manufac-
turer’s instructions and as previously described (23). Experiments
were performed at least in duplicate and repeated at least twice
(n = 4–20 data points). Viable primary myeloma cells were identi-
fied by staining with phycoerythrin-conjugated mouse monoclonal
anti-CD138 antibody (20 µL/106 cells; Beckman Coulter, Brea,
CA). The percentage of myeloma cells that were CD138 positive
and annexin V negative after 5AHQ treatment compared with
untreated samples was quantified as a marker of cell viability as
previously described (24).
Mouse Xenograft Models
Mouse MDAY-D2 leukemia cells (5 × 105 cells per mouse) were
injected intraperitoneally into sublethally irradiated (3.5 Gy) 5- to
6-week-old male and female nonobese diabetic/severe combined
immunodeficient (NOD/SCID) mice (Ontario Cancer Institute).
Beginning the next day, the mice were treated by oral gavage with
5AHQ at 50 mg/kg body weight in 0.4% Tween 80 in phosphate-
buffered saline (vehicle) or vehicle control once per day for 8 days,
a duration of treatment that was based on previous experiments to
permit evaluation of differences in tumor size without causing
distress to the mouse (data not shown) (n = 10 mice per group [five
male and five female]). The number of mice per group was selected
based on the numbers that could be managed effectively during the
experiment. Mice were killed by CO2 inhalation, and the weight
and volume of the single intraperitoneal tumor that developed in
each mouse were measured.
Human leukemia K562 (3 × 106) or OCI-AML2 (2 × 106)
cells were injected subcutaneously into one flank of sublethally
irradiated 5- to 6-week-old male and female NOD/SCID mice.
When tumors were palpable (approximately 1 week after injec-
tion), the mice were treated with 5AHQ (50 mg/kg body
weight) in vehicle or vehicle control by oral gavage once daily
for 10 days (n = 10 mice per group for K562 [five male and five
female] and n = 9 per group for OCI-AML2 [four male and five
female]). The number of mice per group was selected based on
the numbers that could be managed effectively during the
experiment. The duration of treatment was based on previous
experiments to permit evaluation of differences in tumor
size without causing distress to the mouse (data not shown).
Mice were killed by CO2 inhalation, and their tumors were
weighed.
To assess the effect of 5AHQ on the enzymatic activity of the
proteasome in tumors, the leukemia xenograft tumors harvested
from mice bearing subcutaneous OCI-AML2 tumors from the
experiment described above were disaggregated to produce a
single-cell suspension in phosphate-buffered saline by grinding the
tumor with a 3-mL plunger of a syringe while the tumor was on
top of a 40-µm nylon cell strainer (BD Biosciences, San Jose, CA).
The resulting cells were lysed in lysis buffer, and the enzymatic
activity of the proteasome was determined as described above.
Mouse studies were carried out according to the regulations of
the Canadian Council on Animal Care and with the approval of the
local ethics review board.
Immunoblotting
Whole-cell lysates were prepared from untreated MY5 (myeloma)
and OCI-AML2 and K562 (leukemia) cells, as described previously
(21). Whole-cell lysates were also prepared from myeloma LP1 cells
treated for 24 hours with 2.5, 5, or 10 µM 5AHQ or buffer control.
Briefly, 1 × 106 cells were washed with phosphate-buffered saline
and resuspended in immunoblot lysis buffer (10 mM Tris [pH 7.4],
150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate,
and 5 mM EDTA) containing Complete Protease Inhibitor cocktail
tablets (Roche, Indianapolis, IN). Protein concentrations were
determined by the Bradford assay. Equal amounts of protein were
resolved on to SDS–polyacrylamide gels followed by transfer to
nitrocellulose membranes. Membranes were incubated with a
rabbit polyclonal anti-human ubiquitin antibody (1:1000 dilution;
Calbiochem, San Diego, CA), a rabbit polyclonal anti-human b
5 subunit antibody (1:1000 dilution; BIOMOL International), a
mouse monoclonal anti-human a 7 subunit antibody (1:1000 dilu-
tion; BIOMOL International), or a mouse monoclonal anti-b-actin
antibody (1:10 000 dilution; Sigma-Aldrich), followed by incubation
with horseradish peroxidase–conjugated goat anti-mouse or anti-
rabbit IgG (each at 1:3000 dilution; Amersham Bioscience UK,
Little Chalfont, UK). Antibody binding was detected by use of the
enhanced chemical luminescence method (Pierce, Rockford, IL).
Enzyme-Linked Immunosorbent Assay of NF-Kappa B
Activity
NF-Kappa B activity was measured with the use of a Trans-AM
NF-Kappa B p65 transcription factor assay kit (Active Motif,
Carlsbad, CA) according to the manufacturer’s instructions.
Briefly, 3.2 × 106 MDAY-D2 cells were treated with increasing
concentrations of 5AHQ for 24 hours. Cells were then treated
with tumor necrosis factor alpha (TNF-a; 10 nM) or buffer con-
trol (RPMI-1640) for 1 hour before harvesting. Nuclear extracts
were prepared by using a Nuclear Extract kit (Active Motif)
according to the manufacturer’s instructions. An enzyme-linked
immunosorbent assay with a chemiluminescence readout was
used to measure binding of the NF-Kappa B transcription factor
subunit p65 to a DNA consensus sequence (5′-GGGACTTTCC-3′)
cross-linked to 96-well plates. Experiments were performed in
triplicate and repeated three times (n = 9 data points).
Pharmacokinetic Studies
To partially characterize the pharmacokinetics of 5AHQ, suble-
thally irradiated NOD/SCID mice were injected subcutaneously
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in one flank with 1 × 106 MDAY-D2 cells. Seven days after tumor
cell injection, when palpable tumors had formed, mice were
treated once with 5AHQ (50 mg/kg body weight) by oral gavage,
and at increasing times after treatment, mice were killed by CO2
inhalation, and their blood and tumors were harvested, frozen on
dry ice, and stored at 270°C (n = 3 mice per time point; n = 27
mice total). Plasma was isolated from the whole blood and frozen
on dry ice and stored at 270°C. Plasma samples were thawed on
ice; 10 µL of 4 µg/mL 5-fluoro-2′-deoxyuridine (internal stan-
dard) and 10 µL of 2 M HCl and 100 mM sodium bisulfite were
added to a 25 µL sample of plasma, followed by addition of 75 µL
of acetonitrile. The samples were mixed by vortexing and centri-
fuged at 15 000g for 5 minutes. The supernatant was collected,
dried in a SpeedVac (Savant/Thermo, Waltham, MA), and
reconstituted in 50 µL of 5 mM HCl and 2 mM dithiothreitol.
Frozen tumor samples were weighed, two volume equivalents of
water were added, and the tumor was homogenized with the use
of a Powergen 125 homogenizer (Fischer, Ottawa, Canada). The
homogenates were stored at 270°C.
Levels of 5AHQ in the mouse plasma and tumor homogenates
were determined by liquid chromatography–mass spectrometry
analysis using an Acquity UltraPerformance Liquid Chromatography
System coupled to an eLambda PDA detector in line with a
Quattro Premier mass spectrometer (all from Waters, Milford,
MA). A BEH C18 column (2.1 × 100 mm, 1.7 µm; Waters) was
used for separations with the following elution gradient: 0.2
minutes 2% B, 4.2 minutes 40% B, and 4.4 minutes 100% B, where
A is 0.1% formic acid in water and B is methanol. A methanol flush
and re-equilibration following the gradient resulted in a final run
time of 8 minutes. The mass spectrometry instrument was oper-
ated in ES+ mode at unit resolution with 3.5 kV capillary voltage,
source and desolvation temperatures of 120°C and 350°C, respec-
tively, cone and desolvation N2 gas flows of 50 and 1000 L/hour,
respectively, and an Argon collision gas pressure of 5.1 mbar.
Calibration was carried out using 5AHQ standards that ranged
from 0.01 to 4 µg/mL, each containing 5-fluoro-2′-deoxyuridine
internal standards at 0.8 µg/mL. An additional series of standards
comprising the oxidized form of 5AHQ was also used to quantify
this metabolite. The m/z of 5AHQ was 161, and the m/z of the
oxidized form was 162. The ultraviolet–visible spectra produced a
spectral pattern similar to the mass spectrometry spectra. The m/z
of the amino-linked glucuronide of 5AHQ was predicted based on
a peak mass m/z of 337 (adduct mass of 176, glucuronic acid minus
H2O), a primary fragment m/z of 161, and a ultraviolet–visible
spectrum similar to that of 5AHQ. Retention times for glucuroni-
dated 5AHQ, 5AHQ, oxidized 5AHQ, and 5-fluoro-2′-deoxyuridine
were 1.9, 2, 2.6, and 3.2 minutes, respectively.
Phamacokinetic analysis was carried out using Phoenix
WinNonlin software version 1.0.0 (Pharsight, St Louis, MO).
Toxicology Studies
Studies to evaluate the toxicity of 5AHQ were performed by
ChemPartners (Shanghai, China). CD1 mice (n = 5 mice of each
sex per treatment group; ChemPartners) were treated with 5AHQ
at 0 (control), 100, 200, or 300 mg/kg body weight/d by oral ga-
vage for 14 days. An additional five mice of each sex were assigned
to the control and 300-mg/kg dose groups for a recovery during
which mice did not receive 5AHQ for a 1-week period after the
14-day treatment with 5AHQ. In the high-dose group, the dose
of 5AHQ was increased to 450 mg/kg body weight/d starting on
day 10 because of a lack of overt toxicity. Body weight, food con-
sumption, behavior, and appearance were measured over time.
Mice were weighed to record their body weight. Changes in the
weight of food in the cage were recorded as a measure of food
consumption. Mice were observed daily for physical abnormalities
and abnormal behavior. At the end of the treatment and recovery
periods, mice were killed by CO2 inhalation, their blood sent
for chemistry and hematology analysis, and their organs were
examined for signs of gross toxicity.
Statistical Analysis
Data are presented as mean values with 95% confidence intervals
(CIs) unless otherwise indicated. For in vivo studies, the Mann–
Whitney rank sum nonparametric method was used to test for
differences between treatment groups in the weight of the tumors.
The t test was used for comparisons of two groups in the in vitro
studies. All statistical tests were two-sided, and a P value less than
.05 was considered statistically significant.
The kinetics by which 5AHQ and MG132 inhibited protea-
somes purified from rabbit muscle were determined using
SigmaPlot 11.0 and Enzyme Kinetics 1.3 software (both from
Systat Software). Isobologram analysis to evaluate the combination
of 5AHQ and bortezomib on cell viability and the enzymatic ac-
tivity of the proteasome was performed with CalcuSyn software
(Biosoft, Ferguson, MO, and Cambridge, UK), in which a com-
bination index (CIN) less than 0.9 indicates synergism, a CIN
greater than 1.1 indicates antagonism, and a CIN of 0.9–1.1
indicates additivity (25).
Results
Effect of 5AHQ on the Proteasome
Novel inhibitors of the proteasome might be useful probes to
understand this enzymatic complex and leads for therapeutic
agents that can overcome resistance to existing proteasome inhib-
itors, such as bortezomib. We screened a chemical library of
compounds that were based on the quinoline pharmacophore and
included various halogenated and alkyl-substituted quinolines,
hydroxyquinolines, and quinoline hydrazones for compounds that
inhibited the enzyme activity of the proteasome when added to
protein extracts from malignant cells. The most potent chemical
inhibitor of the proteasome identified in this screen was 5AHQ
(Figure 1). When added to protein extracts derived from mouse
leukemia MDAY-D2 cells, 5AHQ inhibited the enzymatic activity
of the proteasome at low micromolar concentrations (0 vs 3.9 µM
5AHQ, mean relative residual proteasome activity = 100% vs
48.3%, difference = 51.7%, 95% CI = 50.2% to 53.2%; P < .001
[t test]). 5AHQ had similar inhibitory effects on proteasome
activity when it was added to protein extracts derived from a panel
of human leukemia and myeloma cell lines (Table 1) (mean IC50
for proteasome inhibition, defined as the concentration that caused
50% inhibition of AMC release, OCI-AML2: 4.2 µM, 95% CI =
3.61 to 4.79 µM; NB4: 2.4 µM, 95% CI = 2.22 to 2.58 µM; KG1A:
5.3 µM, 95% CI = 4.53 to 6.07 µM; MDAY-D2: 2.64 µM, 95%
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1074 Articles |JNCI Vol. 102, Issue 14 | July 21, 2010
CI = 2.17 to 3.11 µM; UTMC2: 2.54 µM, 95% CI = 2.34 to 2.74
µM; KMH11: 1.32 µM, 95% CI = 0.94 to 1.70 µM; KMS18: 0.57
µM, 95% CI = 0.56 to 0.58 µM).
Mechanism of 5AHQ Inhibition
To investigate the mechanism by which 5AHQ inhibits the protea-
some, we conducted detailed enzymatic studies using purified pro-
teasomes isolated from rabbit muscle. By Lineweaver–Burk plot
analysis, 5AHQ inhibited the rabbit proteasome with a mean Ki of
2.1 µM (95% CI = 2.08 to 2.33 µM). The pattern of inhibition fits
best to a noncompetitive model (sum of squares = 0.531, Akaike’s
information criterion (AICc) = 2121.679, Sy.x [SD of the vertical
distance of the point from the line] = 0.135) (Figure 2, A and
Supplementary Figure 1, A [available online]). Notably, a plot of
the enzyme activity vs the inhibitor concentration did not fit well to
competitive (sum of squares = 1.897, AICc = 280.927, Sy.x = 0.256)
or uncompetitive (sum of squares = 4.597, AICc = 252.609, Sy.x =
0.398) models of inhibition (Figure 2, A, inset, and data not shown,
respectively). 5AHQ also inhibited a recombinant proteasome from
the archaebacterium T acidophilum with noncompetitive kinetics
(Supplementary Figure 2, available online). However, 5AHQ was a
less potent inhibitor of the archaebacterial proteasome, with a mean
Ki of 161 µM (95% CI = 153 to 169.6 µM), which may reflect small
differences in the binding pocket for the inhibitor between the
archaebacterial and eukaryotic proteasomes (26,27). By contrast,
Table 1. IC50 values of 5-amino-8-hydroxyquinoline (5AHQ) in leukemia and myeloma cell lines*
Cell line†
Mean IC50, µM (95% CI)
Proteasome inhibition in cell extracts Proteasome inhibition in intact cells Cell viability
Leukemia
AML2 4.2 (3.61 to 4.79) 3.89 (3.78 to 4.00) 3.46 (3.13 to 3.79)
NB4 2.4 (2.22 to 2.58) 2.15 (1.82 to 2.48) 1.38 (1.34 to 1.42)
KG1A 5.3 (4.53 to 6.07) 5.03 (4.17 to 5.89) 3.85 (3.27 to 4.43)
MDAY-D2 (mouse) 2.64 (2.17 to 3.11) 0.57 (0.47 to 0.61) 1.96 (1.85 to 2.07)
Myeloma
UTMC2 2.54 (2.34 to 2.74) 2.62 (2.44 to 2.80) 2.29 (2.17 to 2.41)
KMH11 1.32 (0.94 to 1.70) 3.96 (3.89 to 4.03) 0.94 (0.9 to 0.98)
KMS18 0.57 (0.56 to 0.58) 2.23 (1.98 to 2.48) 1.31 (1.21 to 1.41)
* To measure the effects of 5AHQ on the proteasomal activity in cell extracts, cellular proteins were extracted from leukemia and myeloma cell lines and treated
with increasing concentrations of 5AHQ for 2 hours. To measure the effects of 5AHQ on the proteasomal activity in intact cells, leukemia and myeloma cell
lines were treated with increasing concentrations of 5AHQ for 22 hours and then cellular proteins were extracted. The fluorogenic substrate Suc-LLVY-AMC
(7-amino-4-methylcoumarin) was added to equal concentrations of proteins, and the rate of free AMC was measured over time. Data represent the mean values
and 95% CIs relative to untreated control cells. Experiments were performed in duplicate and repeated twice (n = 4). To measure the effects of 5AHQ on cell
viability, leukemia and myeloma cell lines were treated with increasing concentrations of 5AHQ for 72 hours. After treatment, cell viability was measured by
the AlamarBlue fluorescence assay. Data represent the mean values and 95% CIs relative to cells treated with buffer alone. Experiments were performed at
least in duplicate and repeated at least twice (n = 8–10). CI = confidence interval; IC50 = concentration that caused a 50% loss of proteasome activity or cell
viability compared with untreated control cells.
† Human unless otherwise indicated.
QHA5
)eniloniuqyxordyh-8-onima-5(
N
HO
HN 2
Figure 1. Chemical structure of 5-amino-8-hydroxyquinoline (5AHQ).
the proteasome inhibitor MG132, which binds the active site of the
proteasome, inhibited the rabbit proteasome competitively, as
described previously (13) (Figure 2, B and Supplemental Figure 1,
A [available online]). Thus, 5AHQ inhibits the proteasome through
a mechanism distinct from that of MG132.
Combined Effect of 5AHQ and Bortezomib on
Proteasome Activity
Given that 5AHQ inhibited the proteasome through a mechanism
distinct from competitive proteasome inhibitors such as MG132
[Figure 2; (12)] and bortezomib (7), we evaluated the effect of the
combination of 5AHQ and bortezomib on the proteasome using
isobologram analyses. 5AHQ acted synergistically with bortezomib
to produce greater proteasome inhibition than was observed either
agent alone, with CIN values of 0.46, 0.63, and 0.86 at the con-
centrations lethal to 10%, 25%, and 50%, respectively, of cells
(Figure 3, A and data not shown).
5AHQ Binding to the 20S Proteasome
Given that 5AHQ inhibits the proteasome through a unique
mechanism, we evaluated its interaction with this complex by
NMR. Our initial NMR studies to examine the interaction
between 5AHQ and the full T acidophilum proteasome comprising
a and b subunits were not successful because the elevated temper-
atures (65°C) and long recording times necessary to obtain high-
quality NMR spectra of this 670 kDa complex (12–16 hours at a
proteasome concentration of approximately 4 µM as was used here)
(16) led to degradation of 5AHQ (data not shown). By contrast,
NMR spectra of a smaller half-proteasome construct comprising a
pair of heptameric a-rings (a7-a7; molecular mass = 360 kDa) can be
recorded rapidly, and the spectra of the half-proteasome in complex
with 5AHQ are of high quality, even at only 50°C. Therefore, we
evaluated the interactions of 5AHQ with a7-a7.
The interaction of 5AHQ with a7-a7 produced clear spectral
changes localized to residues Ile159, Val113, Val87, Val82,
Leu112, Val89, Val134, Val24, and Leu136, all of which are
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jnci.oxfordjournals.org JNCI |Articles 1075
located inside the regulatory antechamber of the proteasome and
outside of the catalytic site in the proteolytic chamber (data not
shown and Figure 3, B). By contrast, MG132, which binds the
catalytic sites of the b subunits in the proteolytic chamber, pro-
duced shifts in the b rings of the full archaebacterial proteasome
(data not shown) as expected and as previously described (13).
Thus, 5AHQ interacts with the antechamber of the proteasome at
a site distinct from the active site where other proteasome inhibi-
tors such as MG132 and bortezomib bind. However, because these
NMR studies were conducted using a7-a7, we cannot exclude the
possibility that 5AHQ also interacts with sites on the b subunits.
Effect of 5AHQ on the Proteasome in Intact Cells
Given the ability of 5AHQ to inhibit the enzymatic activity of the
proteasome in cell extracts, we next assessed the effects of 5AHQ
on proteasome function in intact cells. OCI-AMl2, NB4, KG1A,
and MDAY-D2 leukemia and UTMC2, KMH11, and KMS18
myeloma cell lines were treated with increasing concentrations of
5AHQ for 22 hours. The cells were harvested and lysed, and the
chymotrypsin-like activity of the proteasome was measured by
monitoring the rate of cleavage of the fluorescent substrate Suc-
LLVY-AMC. 5AHQ inhibited the rate of Suc-LLVY-AMC
cleavage in the malignant cell lines with mean IC50s of less than 5
µM (mean IC50 for inhibition of the proteasome in intact cells,
OCI-AML2: 3.89 µM, 95% CI = 3.78 to 4.00 µM; NB4: 2.15 µM,
95% CI = 1.82 to 2.48 µM; KG1A: 5.03 µM, 95% CI = 4.17 to
5.89 µM; MDAY-D2: 0.57 µM, 95% CI = 0.47 to 0.61 µM;
UTMC2: 2.62 µM, 95% CI = 2.44 to 2.80 µM; KMH11: 3.96 µM,
95% CI = 3.89 to 4.03 µM; KMS18: 2.23 µM, 95% CI = 1.98 to
2.48 µM). Thus, 5AHQ inhibits the enzymatic activity of the pro-
teasome in intact tumor cells (Table 1). Of note, 5AHQ did not
alter levels of the proteasomal enzymes: We observed no change in
expression of the b5 proteasome subunit when 5AHQ was added
to intact cells at concentrations up to 10 µM for 24 hours (data not
shown). Likewise, 5AHQ did not alter the b5 levels when added to
cell extracts (data not shown).
To further assess the effects of 5AHQ on the function of the
proteasome in intact cells, human myeloma LP1 cells were treated
for 24 hours with increasing concentrations of 5AHQ. The cells
were harvested, lysates of total protein were prepared, and equal
amounts of protein were subjected to immunoblot analysis to
examine the abundance of ubiquitinated proteins in the cells. At
concentrations as low as 2.5 µM, 5AHQ increased the amount
of ubiquitinated protein compared with the amount in un-
treated samples, consistent with inhibition of the proteasome
(Figure 4, A).
Through its effects on the proteasome, bortezomib inhibits the
NF-Kappa B signaling pathway, which is important for cellular
proliferation (28). Therefore, we evaluated the effect on 5AHQ on
NF-Kappa B signaling in cells. MDAY-D2 cells were treated with
increasing concentrations of 5AHQ for 24 hours, followed by
treatment for 1 hour with TNF-a to stimulate NF-Kappa B sig-
naling or buffer control to evaluate basal NF-Kappa B signaling.
The cells were harvested, nuclear protein extracts were prepared,
and binding of the NF Kappa B p65 subunit to a consensus DNA-
binding sequence (which is required for signaling) was measured
by a chemiluminescent-based enzyme-linked immunosorbent
assay as an indicator of NF Kappa B signaling. 5AHQ inhibited
Figure 2. Effect of 5-amino-8-hydroxyquinoline (5AHQ) on isolated
proteasomes. Proteasomes isolated from rabbit muscle were treated
for 1 hour with 0 µM (black circle), 1.25 µM (open circle), 2.5 µM
(black triangle), or 5 µM (open triangle) 5AHQ (A) or 0 µM (black
circle), 0.078 µM (open circle), 0.156 µM (black triangle), 0.312 µM
(open triangle), 0.625 µM (black square), or 1.25 µM (open square)
MG132 (B). After incubation, the fluorogenic substrate Z-LLE-AMC
(7-amino-4-methylcoumarin) was added at increasing concentra-
tions (31.25, 62.5, 125, and 250 µM) and free AMC was measured
over time with the use of a florescence spectrophotometric plate
reader (excitation = 380 nm, emission = 460 nm). Data points repre-
sent the mean rate of AMC release from an experiment performed in
triplicate (n = 3); error bars correspond to 95% confidence intervals.
Lineweaver–Burk plots are presented as a fit to a noncompetitive
model of inhibition for 5AHQ (A) and a competitive model for MG132
(B). Insets: Lineweaver–Burk plots are presented demonstrating a
poor fit to a competitive model of inhibition for 5AHQ and a noncom-
petitive model for MG132.
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1076 Articles |JNCI Vol. 102, Issue 14 | July 21, 2010
both basal and TNF-a–stimulated NF-Kappa B activity at concen-
trations at which it inhibited the enzymatic activity of the protea-
some (mean basal NF-Kappa B activity: 0 vs 5 µM 5AHQ = 28 682.3
vs 7944.8 relative chemiluminescent units [RLU], difference =
20 737.5 RLU, 95% CI = 20 661.4 to 20 813.8 RLU, P < .0001;
mean TNF-a–stimulated NF-Kappa B activity: 0 vs 5 µM
5AHQ = 50 946.5 vs 8176.2 RLU, difference = 42 770.3 RLU,
95% CI = 40 171.1 to 45 369.4 RLU; P < .001) (Figure 4, B).
Induction of Cell Death by 5AHQ in Malignant Cells and
Normal Cells
Inhibition of the proteasome induces cell death in malignant cells
(4). Therefore, we assessed the effects of 5AHQ on cell viability in
malignant cell lines and in primary malignant cells isolated from
patients with leukemia and myeloma. Human (OCI-AML2, NB4,
and KG1A) and mouse (MDAY-D2) leukemia cells and human
myeloma UTCMC2, KMH111, and KMS18 cells were treated with
increasing concentrations of 5AHQ for 72 hours, and cell viability
was measured by the AlamarBlue assay. Concentrations of 5AHQ
required to inhibit 50% inhibition of growth (IC50s) were in the low
Figure 3. Effect of the combination of 5-amino-8-hydroxyquinoline
(5AHQ) and bortezomib on proteasome activity. A) Proteasome inhibi-
tion assay. Proteasomes isolated from rabbit muscle were treated for
1 hour with the indicated concentrations of bortezomib (gray bars),
5AHQ (white bars), or the combination of bortezomib and 5AHQ (black
bars). After incubation, the release of free 7-amino-4-methylcoumarin
(AMC) from the fluorogenic substrate Z-LLE-AMC was measured using
a florescence spectrophotometric plate reader (excitation = 380 nm,
emission = 460 nm). Data represent mean proteasomal activity as
defined by the amount of free AMC in treated cells divided by the
amount of free AMC untreated control cells multiplied by 100.
Experiments were performed at least in duplicate and repeated three
times (n = 6); error bars represent 95% confidence intervals. All P values
are from two-sided t tests. B) Molecular model. Inside view of the
a-rings of the Thermoplasma acidophilum half-proteasome high-
lighting residues (darkened region) that are affected in nuclear mag-
netic resonance spectra upon addition of 5AHQ.
Figure 4. Effect of 5-amino-8-hydroxyquinoline (5AHQ) on proteasome
activity when added to intact cells. A) Immunoblot analysis of ubiquit-
inated proteins. Multiple myeloma LP1 cells were treated with in-
creasing concentrations of 5AHQ for 24 hours, then harvested, total
protein was isolated, and equal concentrations of protein were sub-
jected to immunoblot analysis with anti-ubiquitin and anti-b-actin anti-
bodies to assess the abundance of ubiquitinated proteins and b-actin
expression, respectively. B) NF-Kappa B activity. Mouse leukemia
MDAY-D2 cells were treated with increasing concentrations of 5AHQ for
24 hours followed by treatment for 1 hour with 10 nM tumor necrosis
factor alpha (black bars) or buffer control (gray bars). The cells were
harvested, nuclear proteins were extracted, and NF-Kappa B activity
was measured based on the ability of the p65 subunit to bind its DNA
consensus sequence using an enzyme-linked immunosorbent assay as
described in the “Materials and Methods.” Data represent the mean
values of NF-Kappa B activity in relative chemiluminescent units (RLU)
from three experiments performed in triplicate (n = 9); error bars
correspond to 95% confidence intervals. P values are from two-sided
t tests.
micromolar range (mean IC50 for inhibition of cell growth, OCI-
AML2: 3.46 µM, 95% CI = 3.13 to 3.79 µM; NB4: 1.38 µM, 95%
CI = 1.34 to 1.42 µM; KG1A: 3.85 µM, 95% CI = 3.27 to 4.43 µM;
MDAY-D2: 1.96 µM, 95% CI = 1.85 to 2.07 µM; UTMC2: 2.29
µM, 95% CI = 2.17 to 2.41 µM; KMH11: 0.94 µM, 95% CI = 0.9 to
0.98 µM; KMS18: 1.31 µM, 95% CI = 1.21 to 1.41 µM) (Table 1).
Cell death was confirmed by annexin V staining (data not shown).
We also tested the effects of 5AHQ on the viability of primary
malignant and normal hematopoietic cells isolated from patient
samples. Primary AML, CLL, multiple myeloma, and normal
PBSC were treated with increasing concentrations of 5AHQ for
48 hours. After incubation, cell viability was measured by MTS
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(AML and normal PBSC), AlamarBlue (CLL), and annexin V
staining (myeloma). 5AHQ induced cell death in primary AML,
CLL, and myeloma cells with IC50s in the low micromolar range
(Figure 5, A and B). By contrast, 5AHQ was not cytotoxic to normal
PBSC at concentrations up to 62.5 µM (62.5 µM 5AHQ: mean rela-
tive percentage of viable PBSC vs AML cells = 85.1% vs 30.6%,
difference = 54.5%, 95% CI = 31.8% to 76.9%, P < .001; 62.5
µM 5AHQ: mean relative percentage of viable PBSC vs CLL cells =
85.1% vs 13.2%, difference = 71.9%, 95% CI = 52.4% to 91.3%,
P < .001; 31.25 µM 5AHQ: mean relative percentage of viable
PBSC vs AML cells = 92.5% vs 30%, difference = 62.5%, 95% CI =
32.7% to 92.3%, P < .001; 31.25 µM 5AHQ: mean relative percentage
of viable PBSC vs CLL cells = 92.5% vs 10.5%, difference = 82%,
95% CI = 57% to 107%, P < .001) (Figure 5, A and B).
Combined Effect of 5AHQ and Bortezomib on
Cell Viability
The combination of 5AHQ and bortezomib synergistically inhib-
ited the proteasome (Figure 3, A). We therefore evaluated the cy-
totoxicity of these two inhibitors in OCI-AML2 cells treated for
48 hours with increasing concentrations of the two compounds.
5AHQ acted synergistically with bortezomib to induce cell death
at levels greater than those observed with either compound alone,
with CIN values of 0.31, 0.48, and 0.74 at the concentration of
5AHQ and bortezomib lethal to 10%, 25%, and 50%, respec-
tively, of cells (Figure 5, C and data not shown).
Effect of 5AHQ on Cell Viability in Bortezomib-Resistant
Cells
The efficacy of bortezomib as an anticancer drug for patients with
hematologic malignancies is hampered by the emergence of drug
resistance, which may be due, in part, to overexpression and muta-
tion of the b5 proteasome subunit to which bortezomib binds (11)
or to overexpression of multidrug-resistant pump proteins (29).
Given that 5AHQ inhibited the proteasome through a mechanism
distinct from that of bortezomib, we evaluated the cytotoxictiy of
5AHQ in human leukemia THP1 cells that were selected for their
resistance to increasing concentrations of bortezomib and that
overexpress a mutated b5 proteasome subunit (11). We treated
THP1/BTZ50, THP1/BTZ100, THP1/BTZ100, and THP1/
BTZ500 cells with increasing concentrations of 5AHQ (in the
absence of bortezomib) or bortezomib for 72 hours and then mea-
sured cell viability by use of a trypan blue assay. THP1/BTZ50,
THP1/BTZ100, THP1/BTZ100, and THP1/BTZ500 cells,
which are 45-, 79-, 129-, and 237-fold more resistant to borte-
zomib, respectively, compared with wild-type THP1 cells, had
essentially the same sensitivity to 5AHQ-induced cell death as
THP1 cells (10) (mean IC50 for 5AHQ in the absence of bortezomib:
THP1/WT: 3.7 µM [95% CI = 3.4 to 4.0 µM]; THP1/BTZ50:
6.6 µM [95% CI = 6.2 to 7.0 µM]; THP1/BTZ100: 6.3 µM
[95% CI = 5.9 to 6.7 µM]; THP1/BTZ200: 6.4 µM [95% CI = 5.7
to 7.1 µM]; THP1/BTZ500: 6.6 µM [95% CI = 5.9 to 7.5 µM]).
Similar results were obtained when the cells were treated with
5AHQ in presence of bortezomib (Table 2).
To further test the ability of 5AHQ to induce cell death in cells
resistant to bortezomib, we examined the effect of 5AHQ cell via-
bility and proteasome activity in K562 cells, which are naturally
resistant to bortezomib. Compared with MY5 myeloma and OCI-
AML2 leukemia cells, K562 leukemia cells overexpress the b5
proteasome subunit (Figure 6, A). Compared with MY5 and
OCI-AML2 cells, K562 cells were more resistant to bortezomib-
mediated inhibition of proteasomal enzymatic activity (Figure 6, B)
0
52
05
57
001
+831DC
-831DC
amoleyM
elpmastneitap
% Viable cells
321
AC
B
#
*
5.263.13
PBSC
AML
CLL
02
04
06
08
001
021
6.518.79.30.20.10.0
0
02
04
06
08
001
021
5.263.136.518.79.30.20.10.0
% Viable cells
(QHA5 µ)M
01
02
03
04
05
06
07
08
100.<
100.<
100.<
100.<
)Mn(ZTB
404202
0
01
02
03
04
05
06
07
08
P100.<
P100.<
P100.<
P100.<
5AHQ (µM)
)Mn(ZTB
404202
2525012.512.50
% Dead cells
Figure 5. Cytotoxicity of 5-amino-8-hydroxyquinoline (5AHQ), alone
and in combination with bortezomib, in malignant cells vs normal
hematopoietic cells. A) Growth and viability of primary cells treated
with 5AHQ. Primary acute myeloid leukemia (AML) blasts (open trian-
gles) (n = 4), chronic lymphocytic leukemia (CLL) (open diamonds) (n = 5),
or normal peripheral blood stem cells (PBSCs) (black squares) (n = 4)
were obtained from the peripheral blood of consenting patients
with AML and CLL or donors of PBSC for allotransplantation, respec-
tively. Mononuclear cells were isolated by Ficoll separation and
treated for 48 hours with increasing concentrations of 5AHQ. Cell
viability was measured by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt)
MTS (AML and PBSC) or AlamarBlue fluorescence (CLL) assay. Data
represent the mean percentage of viable cells in treated vs untreated
controls tested in triplicate (n = 12–15). Error bars correspond to 95%
confidence intervals. #P < .001 for PBSC vs AML at 62.5 µM 5AHQ
(two-sided t test); *P < .001 for PBSC vs CLL at 62.5 µM 5AHQ
(two-sided t test). B) Viability of primary myeloma cells treated with
5AHQ. Bone marrow samples were obtained from three patients with
multiple myeloma. Mononuclear cells were isolated by Ficoll separa-
tion and treated for 48 hours with 5 µM 5AHQ or buffer control. Viability
of the myeloma (CD138 positive, black bars) and normal hematopoietic
(CD138 negative, gray bars) cells was measured by costaining with
phycoerythrin-labeled anti-CD138 and fluorescein isothiocyanate–
labeled annexin V and flow cytometry. Data represent the percentages
of viable cells in three individual patient samples. C) Cell death with the
combination of 5AHQ and bortezomib (BTZ). OCI-AML2 cells were
treated for 48 hours with the indicated concentrations of 5AHQ (white
bars), bortezomib (gray bars), or both agents combined (black bars).
Cell viability was measured by annexin V and propidium iodide stain-
ing. Data represent mean percentage of dead cells in treated sample
relative to untreated control cells. Experiments were performed in
duplicate and repeated twice (n = 4); error bars correspond to 95% con-
fidence intervals. P values are from two-sided t tests.
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1078 Articles |JNCI Vol. 102, Issue 14 | July 21, 2010
and bortezomib-induced cell death (Figure 6, C). However, K562
cells remained fully sensitive to proteasome inhibition and cell
death by 5AHQ, consistent with a mechanism of proteasome inhi-
bition distinct from bortezomib (Figure 6). We also examined the
effect of 5AHQ on cell viability in leukemia CEM cells, which are
resistant to a variety of chemotherapeutic drugs (30–33) including
bortezomib due to overexpression of the multidrug-resistant
pumps P glycoprotein, breast cancer resistance protein, or multi-
drug resistance protein 1. These multidrug-resistant cells also
remained equally sensitive to 5AHQ-induced cell death compared
with CEM wild-type cells (data not shown). Thus, 5AHQ can
overcome at least some forms of bortezomib resistance and does
not appear to be a substrate for common multidrug resistance
drug efflux transporters that can confer a multidrug-resistant
phenotype.
Effect of 5AHQ on Tumor Growth in Xenograft Models
We next evaluated the effects of oral administration of 5AHQ on
tumor growth in three mouse models of leukemia. Sublethally
irradiated NOD/SCID mice were injected subcutaneously with
human leukemia OCI-AML2 or K562 cells or intraperitoneally
with murine leukemia MDAY-D2 cells. The day after injecting the
MDAY-D2 cells or approximately 1 week after K562 and OCI-
AML2 cell injection, when the subcutaneous tumors were pal-
pable, the mice were treated once per day for 8 days (MDAY-D2)
or 10 days (K562 and OCI-AML2) with 5AHQ (50 mg/kg body
weight) or buffer control by oral gavage (MDAY-D2 and K562:
n = 10 mice per group; OCI-AML2: n = 9 mice per group). The mice
were monitored daily for changes in behavior and body weight,
and at the end of the treatment period, the tumors were excised
Table 2. Growth inhibitory effects of 5-amino-8-hydroxyquinoline
(5AHQ) against human leukemia cells with acquired resistance to
bortezomib*
Cell line Mean IC50, µM (95% CI)
Resistance factor†
5AHQ Bortezomib‡
THP1/WT 3.7 (3.4 to 4.0) 1 1
THP1/BTZ50
2bortezomib 6.6 (6.2 to 7.0) 1.8 45
+bortezomib 5.9 (5.5 to 6.3) 1.6
THP1/BTZ100
2bortezomib 6.3 (5.9 to 6.7) 1.7 79
+bortezomib 6.2 (5.2 to 5.8) 1.7
THP1/BTZ200
2bortezomib 6.4 (5.7 to 7.1) 1.7 129
+bortezomib 6.2 (5.8 to 6.6) 1.7
THP1/BTZ500
2bortezomib 6.6 (5.9 to 7.5) 1.8 237
+bortezomib 5.3 (5.5 to 6.1) 1.4
* Human leukemia THP1/BTZ50, THP1/BTZ100, THP1/BTZ200, and THP1/
BTZ500 cells were grown in the absence or presence of 50, 100, 200, or
500 nM bortezomib, respectively, for at least 3 days and then were treated
with increasing concentrations of 5AHQ or bortezomib for 72 hours. After
incubation, cell viability was assessed by trypan blue staining. Data represent
the mean values and 95% CIs for three separate experiments performed in
triplicate (n = 9). CI = confidence interval; IC50 = concentration that caused a
50% loss of cell viability compared with untreated control cells.
† IC50 resistant cell line/IC50 parental cell line (THP1/WT).
‡ Data from Oerlemans et al. 2008 (11).
Figure 6. Cytotoxicity of 5-amino-8-hydroxyquinoline (5AHQ) in borte-
zomib-resistant cell lines. A) Immunoblot analysis of b5 and a7 subunit
expression. The abundance of the b5 subunit and a7 subunit of the
proteasome and b-actin expression was assessed by immunoblotting
with anti-b5 subunit, anti-a7 subunit and anti-b-actin antibodies, re-
spectively, in protein lysates from My5, OCI-AM2, and K562 cells. B)
Proteasome inhibition. Cellular proteins were extracted from My5, OCI-
AM2, and K562 cells and treated with increasing concentrations of
5AHQ (black bars) or bortezomib (gray bars) for 2 hours. After incubation,
the fluorogenic substrate Suc-LLVY-AMC (7-amino-4-methylcoumarin)
was added and the rate of free AMC was measured over time as
described in “Materials and Methods.” Data represent the mean IC50s,
where IC50 represents the concentration of drug required to inhibit 50%
of the proteasomal activity of buffer-treated cells. Experiments were
performed in duplicate and repeated twice (n = 4); error bars corre-
spond to 95% confidence intervals. C) Cell growth. My5, OCI-AM2, and
K562 were treated for 72 hours with increasing concentrations of 5AHQ
(black bars) or bortezomib (gray bars). After treatment, cell viability was
measured by the AlamarBlue fluorescence assay. Data represent mean
IC50s (error bars correspond to 95% confidence intervals), where the IC50
represents the concentration of drug required to reduce cellular growth
by 50% of buffer-treated cells. Experiments were performed in dupli-
cate and repeated two to 10 times (n = 4–20).
and weighed. In all three mouse models, compared with control,
orally administered 5AHQ suppressed tumor growth without
causing weight loss or signs of organ toxicity (OCI-AML2 model:
median tumor weight [interquartile range {IQR}], 5AHQ vs
control = 95.7 mg [61.4–163.5 mg] vs 247.2 mg [189.4–296.2 mg],
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jnci.oxfordjournals.org JNCI |Articles 1079
P = .002; K562 model: median tumor weight [IQR], 5AHQ vs
control = 105 mg [40.8–178.4 mg] vs 209.5 mg [134.9–267 mg],
P = .01; MDAY-D2 model: median tumor weight [IQR], 5AHQ vs
control = 819.9 mg [551.7–1111.5 mg] vs 1163.1 mg [1024.9–
1281.3 mg], P = .001) (Figure 7, A–C).
We examined the effects of 5AHQ on proteasome activity in
the OCI-AML2 xenograft model by adding the fluorogenic pro-
teasome substrate Suc-LLVY-AMC to protein lysates made from
the excised OCI-AML2 tumor xenografts and measuring the gen-
eration of free AMC. Proteasome activity (expressed as relative
fluorescence units [RFU] of free AMC) was lower in tumor lysates
from mice treated with 5AHQ than in tumor lysates from mice
treated with buffer control (mean proteasome activity, 5AHQ vs
control: 1221.16 vs 1921.58 RFU, difference = 700.42 RFU, 95%
CI = 191.08 to 1209.76 RFU; P = .008 [t test]) (Figure 7, D).
To partially characterize the pharmacokinetics of 5AHQ,
NOD/SCID mice bearing subcutaneous MDAY-D2 tumors were
treated 1 week after tumor cell injection with a single dose of
5AHQ (50 mg/kg body weight) by oral gavage. At increasing times
after treatment, mice (n = 3 per time point) were killed by CO2
inhalation and their plasma and tumors were collected and sub-
jected to mass spectrometry to measure levels of 5AHQ and con-
version products and metabolites of 5AHQ. 5AHQ was detected
in the plasma at 15 minutes after dosing with a median peak con-
centration (Cmax) of 2.3 µg/mL (IQR = 1.4–6.75 µg/mL) (equivalent
to 14.3 µM). Also detected in the plasma at 15 minutes after dosing
were the oxidized form of 5AHQ (dihydroxyquinoline) with a
median Cmax of 0.23 µg/mL (IQR = 0.14–0.39 µg/mL) (equivalent
to 1.4 µM) and glucuronidated 5AHQ with a median Cmax of 18.6
µg/mL (IQR = 13.2–32.0 µg/mL) (equivalent to 115 µM). At
30 minutes after dosing, 5AHQ was detected in the tumors with
a median Cmax of 0.39 µg/mL (IQR = 0.27–1.78 µg/mL) (equivalent
to 2.4 µM). Also detected in the tumors at 30 minutes after dosing
were dihydroxyquinoline, with a median Cmax of 0.03 µg/mL (IQR =
0.0–0.04 µg/mL) (equivalent to 0.18 µM), and glucuronidated
5AHQ, with a median Cmax of 0.54 µg/mL (IQR = 0.29–1.5 µg/mL)
(equivalent to 3.3 µM).
To better understand the potential toxicity of 5AHQ, mice
(n = 5 of each sex) were treated with 5AHQ at 0 (control), 100,
200, or 300 mg/kg body weight/d by oral gavage daily for 14 days.
An additional five mice of each sex were assigned to the control
and highest dose groups for a 1-week recovery during which mice
received no 5AHQ for 7 days after the 14-day treatment period. In
the highest dose group, the dose of 5AHQ was increased to 450/kg
body weight/d starting on day 10 because no overt toxic effects
were observed. At the completion of the treatment and recovery
periods, mice were killed, their blood sent for chemistry and hema-
tology analysis, and their organs were observed for gross signs of
toxic effects. The only detectable toxic effect was an increase in the
plasma level of aspartate aminotransferase (AST), a marker of liver
inflammation, in male mice that were treated with the highest dose
of 5AHQ compared with control (Table 3), which was reversed
during the 1-week recovery period (mean AST level on day 14 of
treatment, 0 vs 300 (increased to 450 on day 10) mg/kg/d 5AHQ =
39.6 vs 140.4 U/L, difference = 100.8 U/L, 95% CI = 188.6 to
13.04 U/L, P < .001; mean AST level after 1-week recovery, 0 vs
300 (increased to 450 on day 10) mg/kg/d 5AHQ: 35.2 vs 39.1
U/L, difference = 3.9 U/L, 95% CI = 23.1 to 10.5 U/L, P = .79).
We also detected a non-statistically significant increase in total
bilirubin in male mice treated for 14 days with 300 mg/kg/d
(increased to 450 mg/kg/d on day 10) 5AHQ compared with
control that was also reversed during the 1-week recovery period
Figure 7. Effect of 5-amino-8-hydroxyquinoline
(5AHQ) on tumor growth in mouse models of
leukemia. (A–C) Scatter plot analyses of final
tumor weights. Sublethally irradiated nonobese
diabetic/severe combined immunodeficient (NOD/
SCID) mice were injected subcutaneously with
K562 (A) or OCI-AML2 (B) leukemia cells or intra-
peritoneally with MDAY-D2 (C) leukemia cells.
Starting the day after tumor injection (MDAY-D2)
or approximately 7 days after injection when the
tumors were palpable (K562 and OCI-AML2), mice
were treated daily for 8 (MDAY-D2) or 10 (OCI-
AML2 and K562) days by oral gavage with buffer
or 50 mg 5AHQ/kg body weight dissolved in buffer
(0.4% Tween 80 in phosphate-buffered saline)
(n = 10 mice per treatment group for K562 and
MDAY-D2 and n = 9 per treatment group mice for
OCI-AML2). The mice were killed, the subcuta-
neous or intraperitoneal tumors were excised,
and the weights of the tumors were measured.
Horizontal bars indicate the median values.
Asterisks indicate statistically significant dif-
ferences for 5AHQ vs control (K562: P = .01;
OCI-AML2: P = .002; MDAY-D2: P = .001) by the
Mann–Whitney nonparametric test (two-sided).
D) Proteasome activity in OCI-AMl2 xenograft
tumors. OCI-AML2 leukemia xenograft tumors were excised from the
mice treated with 5AHQ (n = 9) or buffer control (n = 9) (described
above), and protein extracts were made. To equal concentrations of
protein, the proteasome fluorogenic substrate, Suc-LLVY-AMC (7-amino-
4-methylcoumarin), was added and the generation of free AMC was
measured over time with a fluorescence spectrophotometric plate reader.
Data represent mean free AMC fluorescence in relative fluorescence units
(RFU), and error bars correspond to 95% confidence intervals (n = 9 tumors
per treatment group) from the one experiment. Asterisk indicates statisti-
cally significant difference for control vs 5AHQ (P = .008, two-sided t test).
0
005
0001
0051
0002
0052
QAH5lortnoC
*
lortnoC QHA5
0
05
001
051
002
052
003
053
Tumor weight (mg)
0
005
0001
0051
QHA5lortnoC
Tumor weight (mg)
*
*
Tumor weight (mg)
0
001
002
003
QHA5lortnoC
Proteasome activity
AMC flourescence
( RFU)
*
AB
CD
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1080 Articles |JNCI Vol. 102, Issue 14 | July 21, 2010
(mean total bilirubin level on day 14, 0 vs 300 mg/kg/d 5AHQ =
2.0 vs 3.3 mmol/L, difference = 1.3 mmol/L, 95% CI = 20.3 to
3.01 mmol/L, P = 0.09). No changes in AST or bilirubin were
observed in the female mice (data not shown). No differences in
body weight, food consumption, hematology, or renal function
were observed between mice that received buffer control and mice
in any of the 5AHQ treatment groups. The only abnormality
noted on necropsy was dark yellow discoloration of the liver in one
male mouse that was treated with the highest dose of 5AHQ.
Thus, 5AHQ was well tolerated in mice at doses up to sixfold
higher than the dose required for antitumor effects.
Discussion
In this study, we identified 5AHQ, a quinoline-based compound,
and showed that it inhibited the proteasome noncompetitively.
Consistent with a mechanism of action distinct from bortezomib,
5AHQ was cytotoxic to cells that are resistant to bortezomib.
Finally, 5AHQ induced cell death in primary myeloma and AML
cells preferentially over normal hematopoietic cells. Moreover, it
inhibited tumor growth in mouse models of leukemia.
Proteasome inhibitors improve the clinical outcome of patients
with multiple myeloma and mantle cell lymphoma and are cur-
rently being evaluated for the treatment of other malignancies,
including leukemia (8,9,34). However, bortezomib and all of the
other chemical proteasome inhibitors currently under clinical eval-
uation block the proteasome complex competitively by binding the
active sites of the enzymes (5–7). Here, we describe the activity of
5AHQ, a proteasome inhibitor that is unique in that it inhibits the
enzyme complex in a noncompetitive fashion. NMR studies estab-
lished that 5AHQ binds the a subunits of the antechamber of the
20S proteasome, a novel binding site that is distinct from the
binding site of bortezomib.
5AHQ induced cell death in leukemia and myeloma cell lines at
the same concentrations at which it inhibited the proteasome
and blocked NF-Kappa B signaling. These results suggest that the
cytotoxicity of 5AHQ is related to its effects on the proteasome.
Because 5AHQ and bortezomib inhibited the proteasome through
distinct mechanisms, we evaluated the effect of these agents in
combination in tumor cells. The combination of 5AHQ and bort-
ezomib inhibited the proteasome and induced cell death in a syn-
ergistic fashion. Thus, combining competitive and noncompetitive
proteasome inhibitors could be a novel strategy to increase
response rates to proteasome inhibition in patients with leukemia
or myeloma. Alternatively, if the side effect profiles of these two
agents in patients are distinct, using them in combination could
permit the use of lower doses of bortezomib, which would reduce
the incidence and severity of bortezomib’s toxic effects. Although
our toxicology studies suggest that 5AHQ was well tolerated in
mice, further investigation of the toxicology of 5AHQ in addi-
tional species is necessary before advancing this compound into
clinical trials. In addition, toxicology studies with the combination
of 5AHQ and bortezomib would be useful to determine whether
the combination produces excessive or unexpected toxic effects.
Several mechanisms of bortezomib resistance have been identi-
fied, including increased levels of heat-shock protein 27 (35) and
increased expression of multidrug resistance pumps (29). Previously,
Oerlemans et al. (11) demonstrated that an acquired mutation in,
or overexpression of, the b5 subunit of the proteasome can also
confer resistance to bortezomib. Our finding that 5AHQ retained
full activity in cell lines that carried mutated b5 subunits or that
overexpressed b5 subunits is consistent with 5AHQ binding a site
on the proteasome distinct from that of bortezomib. Moreover,
5AHQ retained activity in cells that overexpressed multidrug-
resistant pumps. Therefore, 5AHQ overcomes some forms of
bortezomib resistance. These findings support the development of
5AHQ or an analogue for the treatment of some patients with
myeloma who relapse after bortezomib treatment. However, it is
unknown how frequently mutations or overexpression of the b5
subunit occur in patients with bortezomib-resistant disease.
To our knowledge, the mechanism by which 5AHQ noncom-
petitively inhibits the proteasome is not known. However, one
possibility is that 5AHQ binding outside of the active site produces
a conformational change that prevents substrates from entering
the proteolytic chamber of the proteasome complex. In 2003,
Gaczynska et al. (36) showed that a 39-amino acid peptide named
Table 3. Toxicology of 5-amino-8-hydroxyquinoline (5AHQ) in male mice*
Treatment group ALT, U/L AST, U/L ALP, U/L TBil, µmol/L BUN, mmol/L Cr (µmol/L)
Control 23.2 (18.5 to 27.9) 39.6 (31.8 to 47.4) 139.3 (103.3 to 175.3) 2.0 (0.84 to 3.09) 7.8 (5.85 to 9.67) 9.8 (7.68 to 11.9)
5AHQ 36.6 (19.9 to 53.3) 140.4 (35.0 to 245.8) 79.3 (44.6 to 114) 3.3 (1.7 to 5.0) 5.3 (2.3 to 8.4) 6.3 (3.4 to 9.2)
Difference† 13.4 (21.0 to 27.8) 100.8 (13.0 to 188.6) 260 (2101.6 to 218.49) 1.3 (20.3 to 3.0) 22.4 (25.4 to 0.6) 1.5 (24.3 to 1.4)
P‡ .06 <.001 .01 .09 .10 .2
Control recovery 25.7 (21.9 to 29.45) 35.25 (27.4 to 43.1) 147.1 (107.5 to 186.7) 2.4 (2.1 to 2.8) 8.0 (7.2 to 8.7) 7.8 (7.2 to 8.5)
5AHQ recovery 23.4 (14.6 to 32.3) 39.1 (30.0 to 48.3) 150.0 (117 to 182.6) 0.7 (0.1 to 1.3) 9.6 (7.0 to 12.1) 11.7 (5.5 to 17.9)
Difference§ 1.9 (27.3 to 3.5) 3.7 (23.1 to 10.5) 7.5 (223.2 to 38.2) 21.8 (22.3 to 21.4) 1.9 (0.2 to 3.5) 4.1 (0.5 to 7.6)
P‡ .48 .79 .87 <.001 .03 .03
* CD1 male mice (n = 5 mice per group) were treated with 5AHQ at 300 mg/kg body weight/d or buffer control by oral gavage daily for 14 days. An additional five
mice were assigned to the control group and to the highest dose group for a 1-week recovery period during which the mice received no drug for 1 week after
the 14-day treatment. In the high-dose group, the dose of 5AHQ was increased to 450 mg/kg body weight/d starting on day 10 because no overt toxic effects
were observed. At the completion of the experiment, mice were killed and their blood was sent for chemistry analysis. Data represent mean values and
95% confidence intervals. ALT = alanine transaminase; ALP = alkaline phosphatase; AST = aspartate aminotransferase; BUN = blood urea nitrogen;
Cr = creatinine; TBil = total bilirubin.
† 5AHQ vs control.
‡ 5AHQ recovery vs control recovery.
§ Two-sided t test.
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jnci.oxfordjournals.org JNCI |Articles 1081
PR39 binds the 20S proteasome and inhibits the enzyme noncom-
petitively. This peptide was postulated to bind to a site distal from
the catalytic region and function via an allosteric mechanism of
action whereby binding leads to changes in proteasome structure.
However, characterization of the binding sites of PR39 on the
proteasome has not yet, to our knowledge, been reported. More
recently, we demonstrated that chloroquine also inhibits the pro-
teasome noncompetitively by binding a region of the a subunits in
the T acidophilum proteasome that is similar but does not overlap
with the one that 5AHQ binds (13). However, chloroquine is a
much weaker proteasome inhibitor than 5AHQ, and the concen-
trations of chloroquine that are required to inhibit the proteasome
in intact mammalian cells are not pharmacologically achievable
in animals or humans. Detailed crystal structures of the 5AHQ–
proteasome interaction are needed to better discern the mecha-
nism by which 5AHQ inhibits the proteasome. Furthermore,
having a defined interaction site may lead to the development of
more potent proteasome inhibitors, some of which could be leads
for new therapeutic agents.
We recognized a number of limitations of this study. First,
the experimental limitations discussed above prevented us from
excluding the possibility that 5AHQ simultaneously binds to a
and b subunits. However, the kinetic assays support a mecha-
nism of inhibition that differs from that of bortezomib. Second,
although results of the NMR studies suggest that 5AHQ has a
direct effect on proteasomal function, we cannot exclude the
possibility that a 5AHQ metabolite is primarily responsible for
proteasome inhibition. In addition, we cannot fully exclude the
possibility that 5AHQ or a metabolite also inhibits the proteasome
through indirect effects. Finally, we cannot exclude the possi-
bility that 5AHQ has additional targets beyond the proteasome
and that inhibition of these other targets may also contribute to
its anticancer effects.
In summary, we have found that 5AHQ is a novel noncom-
petitive inhibitor of the proteasome that synergizes with and
overcomes resistance to the competitive proteasome inhibitor
bortezomib. As such, 5AHQ may represent a new strategy for in-
hibition of the proteasome and a potential lead for a new class of
therapeutic agents.
Supplementary Data
Supplementary data can be found at http://www.jnci.oxfordjournals
.org/.
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Funding
The Princess Margaret Hospital Foundation; Ontario Institute for Cancer
Research through the Ministry of Research and Innovation; Leukemia and
Lymphoma Society.
Notes
L. E. Kay is a Canada Research Chair in Biochemistry. A. D. Schimmer is a
Leukemia and Lymphoma Scholar in Clinical Research. S. Trudel has received
research funding from OrthoBiotech (which markets bortezomib in Canada).
X. Li, T. E. Wood, R. Sprangers, X. Mao, X. Wang, H. Adomat, Y. Zhang, S.
E. Verbrugge, Z. H. Li, T. L. Religa, and N. E. Franke designed research,
analyzed data, performed research, and edited the article. G. Jansen, S. Trudel,
H. Messner, J. Cloos, D. R. Rose, R. A. Batey, E. Guns, and L. E. Kay analyzed
data and supervised research. C. Chen, N. Jamal, and A. Navon provided critical
reagents. A. D. Schimmer designed and supervised the research, analyzed data,
and wrote the article. All authors reviewed and edited the article. The study
sponsors did not have a role in the study, writing the manuscript, or the decision
to submit the manuscript for publication.
Present address: Department of Biology, University of Waterloo, Waterloo,
ON, Canada (D. R. Rose).
We thank Dr Doug Kuntz for helpful advice and discussion and Dr Tony
Panzarella for statistical advice.
Affiliations of authors: Ontario Cancer Institute, Princess Margaret
Hospital, Toronto, ON, Canada (XL, TEW, XM, XW, YZ, ZHL, ST, CC, NJ,
HM, DRR, ADS); Department of Chemistry (TEW, YZ, RAB, RS, TLR,
LEK), Department of Medical Genetics (RS, TLR, LEK), Department of
Biochemistry (RS, TLR, LEK), Department of Medicine (ST, CC, HM,
ADS), and Department of Medical Biophysics (ST, HM, DRR, ADS),
University of Toronto, Toronto, ON, Canada; Department of Pediatric
Oncology and Department of Rheumatology, VU University Medical
Centre, Amsterdam, the Netherlands (GJ, NEF, SEV, JC); Prostate
Centre at Vancouver General Hospital, Vancouver, BC, Canada (HA, EG);
Department of Biological Investigation, Weizmann Institute of Science,
Rehovot, Israel (AN); Department of Urologic Sciences, University of
British Columbia, Vancouver, BC, Canada (EG).
at University of Adelaide on January 28, 2015http://jnci.oxfordjournals.org/Downloaded from
