Acriflavine inhibits HIF-1 dimerization, tumor growth,
KangAe Leea,b, Huafeng Zhanga,c, David Z. Qiana,c, Sergio Reya,b, Jun O. Liuc,d, and Gregg L. Semenzaa,b,c,e,1
aVascular Program, Institute for Cell Engineering,bMcKusick–Nathans Institute of Genetic Medicine, Departments ofcOncology,dPharmacology,
andePediatrics, Medicine, Radiation Oncology, and Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Contributed by Gregg L. Semenza, August 18, 2009 (sent for review July 17, 2009)
HIF-1 is a heterodimeric transcription factor that mediates adaptive
responses to hypoxia and plays critical roles in cancer progression.
Using a cell-based screening assay we have identified acriflavine as
a drug that binds directly to HIF-1? and HIF-2? and inhibits HIF-1
dimerization and transcriptional activity. Pretreatment of mice
bearing prostate cancer xenografts with acriflavine prevented
tumor growth and treatment of mice bearing established tumors
resulted in growth arrest. Acriflavine treatment inhibited intratu-
moral expression of angiogenic cytokines, mobilization of angio-
genic cells into peripheral blood, and tumor vascularization. These
results provide proof of principle that small molecules can inhibit
dimerization of HIF-1 and have potent inhibitory effects on tumor
growth and vascularization.
cancer ? chemotherapy ? hypoxia ? xenograft
invasion, metastasis, and patient mortality (2). The adaptation of
cancer cells to hypoxia is critical for their survival. Hypoxia-
inducible factor 1 (HIF-1) activates transcription of genes encoding
proteins that mediate major adaptive responses to hypoxia (2). For
example, HIF-1 activates the expression of vascular endothelial
growth factor (VEGF), a key regulator of angiogenesis, as well as
glucose transporters (e.g., GLUT1) and glycolytic enzymes (e.g.,
hexokinase [HK] 1 and 2), which are required for high levels of
glucose uptake and metabolism (2). The fact that HIF-1 regulates
the expression of multiple gene products involved in tumor metab-
olism and vascularization suggests that a greater anticancer effect
may be achieved by inhibition of HIF-1, as compared to a down-
stream gene product, such as VEGF.
HIF-1? subunits, which belong to the family of basic helix–loop–
(PAS) domain (3). In contrast to the constitutively expressed
HIF-1? subunit, high levels of HIF-1? are induced in response to
hypoxia. HIF-1? is constantly synthesized and, in well-oxygenated
cells, is hydroxylated on proline residue 402 and/or 564, which is
required for binding of the von Hippel-Lindau protein, the recog-
nition subunit of an E3 ubiquitin ligase that targets HIF-1? for
proteasomal degradation (4). Asparagine 803 is also hydroxylated,
which inhibits recruitment of the coactivator proteins p300 and
CREB binding protein (CBP) to the transcriptional activation
O2for their catalytic activity. Under hypoxic conditions, hydroxy-
lation decreases, HIF-1? accumulates and dimerizes with HIF-1?
to form a functional transcription factor capable of DNA binding
at hypoxia response elements (HREs) and transcriptional activa-
tion. HIF-2? is another bHLH-PAS protein that is O2-regulated,
dimerizes with HIF-1?, and binds to HREs (4).
Increased HIF-1? or HIF-2? levels are found in human lung,
colon, breast, and prostate carcinomas, and are associated with
disease progression and increased patient mortality (2). A large
body of experimental data indicates that disruption of HIF-1
signaling inhibits tumor growth in mouse models (2). A growing
olid tumors frequently contain hypoxic regions because they
have high rates of cell proliferation and form aberrant blood
number of drugs have been identified that (i) inhibit HIF-1 activity
binding, or transactivation of target genes; and (ii) have anticancer
activity in preclinical studies (5). However, no drugs have been
identified that bind directly to HIF-1.
Dimerization of HIF-1? with HIF-1? or HIF-2?, which is
required for HIF-1 DNA binding and transcriptional activity, is
mediated by bHLH and PAS domains located in the amino-
terminal half of each subunit (3, 6). A small molecule that targets
HIF-1 dimerization might function as a selective HIF-1 inhibitor,
but current pharmacological dogma holds that small molecules are
unlikely to disrupt large dimerization interfaces, such as the com-
bined HLH and PAS domains, which span over 200 amino acids
(aa). In this study, we identified an inhibitor of HIF-1 dimerization,
which decreased HIF-1 transcriptional activity and showed anti-
cancer efficacy in vivo that was due at least in part to its antian-
Screening for Inhibitors of HIF-1 Dimerization. We developed a
cell-based dimerization assay based on complementation of split
and HIF-1? bHLH-PAS domains provided the mechanism for
complementation (Fig. 1A). Interaction of HIF-1?12–396and HIF-
1?11–510causes the N- and C-terminal halves of Rluc to be closely
approximated, thereby reconstituting RLuc activity. Vectors en-
coding NRLuc-HIF-1?12–396and HIF-1?11–510-CRLuc fusion pro-
teins were cotransfected with control reporter pGL2-promoter, in
which firefly luciferase (Fluc) coding sequences are downstream of
an SV40 promoter. Rluc activity was detected in cells expressing
NRLuc-HIF-1?12–396and HIF-1?11–510-CRLuc, but no Rluc activity
was observed when either NRLuc-HIF-1?12–396or HIF-1?11–510-
CRLuc was expressed alone (Fig. 1B).
To validate the specificity of the split Rluc assay, we coex-
pressed NRLuc-HIF-1?11–510and found that it competes with
HIF-1?11–510-CRLuc for binding to NRLuc-HIF-1?12–396, and
thereby blocks reconstitution of Rluc activity (SI Appendix, Fig.
S1A). We also coexpressed a double-mutant form of HIF-1?
(HIF-1?DM) that contains Pro-to-Ala substitutions at residues
402 and 564, thereby preventing hydroxylation and rendering
HIF-1? stable under nonhypoxic conditions. HIF-1?DM
competed with NRLuc-HIF-1?12–396for dimerization with HIF-
1?11–510-CRLuc (SI Appendix, Fig. S1B). The finding that in-
creasing concentrations of NRLuc-HIF-1?11–510 or HIF-1?DM
led to decreasing Rluc activity confirmed that Rluc activity was
dependent on NRLuc-HIF-1?12–396::HIF-1?11–510-CRLuc inter-
action and indicated that the assay was suitable for identifying
inhibitors of HIF-1 dimerization.
Author contributions: K.L., D.Z.Q., and G.L.S. designed research; K.L., H.Z., D.Z.Q., and S.R.
performed research; J.O.L. contributed new reagents/analytic tools; K.L., H.Z., D.Z.Q., S.R.,
and G.L.S. analyzed data; and K.L. and G.L.S. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
October 20, 2009 ?
vol. 106 ?
We previously screened a collection of 3,120 drugs that are
approved by the FDA or have entered phase II clinical trials and
identified 336 drugs that inhibited hypoxia-induced transcription
of HIF-1-dependent reporter plasmid p2.1 by ?50% at a con-
centration of 10 ?M (8). The top 200 hits were subjected to
secondary screening using the split Rluc assay. The most potent
inhibitor of HIF-1 dimerization was acriflavine (ACF), which
inhibited Rluc activity by 94% at a concentration of 5 ?M (Fig.
1C). ACF inhibited Rluc in a dose-dependent manner with an
IC50of approximately 1 ?M.
Acriflavine Inhibits the Dimerization of HIF-1? (or HIF-2?) with HIF-1?.
To confirm that ACF inhibits dimerization of HIF-1? and HIF-1?,
we performed coimmunoprecipitation (co-IP) assays. HEK293
cells were treated with ACF or vehicle and exposed to 20 or 1% O2
for 24 h. IP with anti-HIF-1? antibodies (Ab) showed that ACF
decreased interaction between endogenous HIF-1? and HIF-1? in
hypoxic cells (Fig. 2A). We also incubated a purified GST-HIF-
1?11–510 fusion protein with lysates from cells transfected with
Flag-HIF-1?DM(Fig. 2B). Flag-HIF-1?DMwas pulled down with
decreased when cells were treated with ACF. ACF also decreased
the interaction between HIF-2? and endogenous HIF-1? (SI
Appendix, Fig. S2A) or purified GST-HIF-1?11–510(SI Appendix,
An in vitro binding assay was also performed using purified
GST-HIF-1?11–510and His-HIF-1?12–395(or His-HIF-2?3–351) pro-
teins. His-HIF-1?12–395(Fig. 2C) or His-HIF-2?3–351(SI Appendix,
Fig. S2C) was pulled down by GST-HIF-1?11–510, but not by GST
alone, and these interactions were inhibited by ACF. The ability of
ACF to disrupt HIF-1 (Fig. 2D) or HIF-2 (SI Appendix, Fig. S2D)
dimerization was dependent on the concentration of ACF. The
observed IC50was approximately 1 ?M and 5 ?M led to almost
Acriflavine Binds Directly to HIF-1? and HIF-2?. To determine
whether ACF binds to HIF-1?, purified GST-HIF-1?11–510 was
preincubated with ACF and the complex was captured by gluta-
thione beads, washed 1–3 times to eliminate unbound ACF, and
then incubated with purified His-HIF-1?12–395. Preincubation of
GST-HIF-1?11–510with ACF did not inhibit dimerization of GST-
was washed extensively (Fig. 3A; ?2 and ?3 wash). Moreover,
His-HIF-1?12–395 levels captured by GST-HIF-1?11–510 did not
decrease with increasing concentration of ACF during preincuba-
tion (Fig. 3B). These results indicate that HIF-1? is not a target of
ACF. Next, purified His-HIF-1?12–395was preincubated with ACF,
captured by Ni-NTA-agarose beads, washed, and incubated with
GST-HIF-1?11–510. Preincubation of His-HIF-1?12–395with ACF
decreased binding of GST-HIF-1?11–510even after extensive wash-
ing (Fig. 3C). This effect was dependent on the ACF concentration
during preincubation (Fig. 3D). These results indicate that ACF
binds to HIF-1? and disrupts its interaction with HIF-1?.
To confirm these conclusions, we determined the direct binding
affinity of ACF to HIF-1? or HIF-2? by following the innate
fluorescence of ACF at ?ex ? 463 nm and ?em ? 490 nm (SI
Appendix, Fig. S3). Purified GST-HIF-1?3–350or GST-HIF-2?3–351
was incubated with 0–250 ?M ACF and captured by glutathione
agarose beads. The protein-drug complex was washed 3 times and
analyzed for fluorescence intensity (FI; Fig. 3E). The FI of GST-
HIF-1?3–350or GST-HIF-2?3–351increased in a dose dependent
manner as ACF concentration increased, whereas neither GST nor
GST-HIF-1?11–510showed increased FI even at high ACF concen-
trations. A significant difference between HIF-? subunits and
FI was maximal at 100 ?M.
Acriflavine Specifically Binds to PAS-B Subdomain of HIF-1? and
HIF-2?. We tested purified proteins that contained different do-
A and B). ACF increased the FI of all GST fusion proteins that
contained the PAS-B domain, whereas no difference in FI was
observed after ACF vs. vehicle treatment of all fusion proteins that
lacked PAS-B. Maximal FI was captured by GST-HIF-1?235–350,
which contained only PAS-B, whereas FI was not captured by
GST-HIF-1?90–155, which contained only PAS-A. Similar results
were also observed for HIF-2? (Fig. 4B and SI Appendix, Fig. S4 C
and D). For both HIF-1? and HIF-2?, a significant difference in
captured FI between fusion proteins that included vs. excluded
PAS-B was first observed at a concentration of 1 ?M ACF (SI
Appendix, Fig. S4 B and D Inset). These results indicate that ACF
interacts specifically with the PAS-B subdomain of HIF-1? or
The aryl hydrocarbon receptor (AHR) is a bHLH-PAS tran-
of ACF to GST-fusion proteins containing AHR residues 1–410
(bHLH-PAS) or 281–410 (PAS-B) was not significantly different
from GST alone (SI Appendix, Fig. S5), indicating that ACF does
not bind to AHR. To test whether ACF blocks heterodimerization
N-terminal and C-terminal portions of Rluc were attached to HIF-1?12–396and
HIF-1?11–510, respectively. (B) The ratio of Renilla/firefly luciferase activity
(Rluc/Fluc) was determined using cells co-transfected with pGL2-promoter,
which encodes Fluc, and NRluc-HIF-1?12–396(NR-1?) and/or HIF-1?11–510-CRluc
(1?-CR). Each value was normalized to the results for empty vector (EV). Data
represent mean ? SEM (n ? 6).**, P ? 0.01 (Student’s t test) vs. EV. C, HEK293
cells were cotransfected with pGL2-promoter, NR-1?, and 1?-CR vectors and
treated with acriflavine (ACF) for 24 h. The Rluc/Fluc ratio in cell lysates was
determined and normalized to the results for NR-1?. Mean ? SEM are shown
(n ? 6).*, P ? 0.05;**, P ? 0.01 vs. 0 ?M ACF (Student’s t test).
Split Rluc system for identifying inhibitors of HIF-1 dimerization. (A)
Lee et al.PNAS ?
October 20, 2009 ?
vol. 106 ?
no. 42 ?
by inducing HIF-1? homodimerization, purified GST-HIF-1?3–350
or GST-HIF-1?11–510 was incubated with lysates obtained from
HEK293 cells exposed to 1% O2in the presence of ACF or vehicle.
interaction between HIF-1? and GST-HIF-1?11–510, whereas no
HIF-1? was captured by GST-HIF-1?2–350in the presence of ACF
(SI Appendix, Fig. S6A). His-HIF-1?12–395also failed to capture
GST-HIF-1?3–350in the presence of ACF (SI Appendix, Fig. S6 B
and C). Thus, ACF does not induce homodimerization of HIF-1?.
Acriflavine Inhibits HIF-1 DNA Binding and Transcriptional Activity.To
investigate the functional consequences of disrupting HIF-1 dimer-
ization, we first determined the effect of ACF on the binding of
endogenous HIF-1 to DNA in living cells. Chromatin-bound
HIF-1? was immunoprecipitated from HEK293 cells after expo-
sure to 20% or 1% O2in the presence of 0–10 ?M ACF. HIF-1
binding sites from the VEGF and PDK1 genes were specifically
amplified by PCR from chromatin that was immunoprecipitated
from cells exposed to 1% O2but not from chromatin that was
immunoprecipitated from cells exposed to 20% O2, demonstrating
hypoxia-induced binding of HIF-1 in the absence of drug (Fig. 5A).
Treatment of hypoxic cells with ACF inhibited binding of HIF-1?
to DNA in a dose dependent manner. ACF also blocked binding of
Fig. S7). We next determined the effect of ACF on HIF-1 tran-
scriptional activity. HEK293 cells were cotransfected with HIF-1-
dependent Fluc reporter p2.1 and pSV-Renilla, and exposed to
20% or 1% O2. ACF significantly inhibited the hypoxic induction
of Fluc activity in a dose dependent manner with an IC50 of
approximately 1 ?M and complete inhibition at 5 ?M (Fig. 5B).
Hypoxic induction of VEGF and GLUT1 mRNA was also blocked
Acriflavine Does Not Affect HIF-1?:Hsp90 Interaction or MYC-medi-
ated Transcription. HIF-1? interacts with the chaperone Hsp90 and
disruption of HIF-1?:Hsp90 association leads to proteasomal deg-
radation of HIF-1? (9). Similar levels of Hsp90 were coimmuno-
precipitated with HIF-1? from lysates of hypoxic HEK293 cells
treated with ACF or vehicle, indicating that ACF does not disrupt
We also tested whether ACF affects MYC-mediated transcription
using human P493 cells with tetracycline-repressible MYC activity
(10). Expression of fibrillarin (FBL) and apurinic/apyrimidinic
exonuclease (APEX) mRNAs, which are products of MYC target
not affect FBL or APEX mRNA levels, whereas ACF specifically
decreased hypoxia-induced GLUT1 mRNA expression.
Effect of ACF on HIF-1?:HIF-1? interaction was deter-
mined using coimmunoprecipitation (co-IP). HEK293
or 1% O2for 24 h. IP of whole cell lysates (WCL) was
products were assayed by immunoblot (IB) to detect
HIF-1?, HIF-1?, and ?-actin. (B) GST-HIF-1?11–510 was
vector encoding Flag-HIF-1?DMin the presence or ab-
sence of ACF. Proteins were pulled down with glutathi-
one-Sepharose-4B beads and subjected to IB assays us-
ing anti-FLAG and anti-GST Ab. An aliquot of WCL was
GST in the presence of ACF or vehicle overnight at 4 °C.
Binding was determined by GST pull down and IB using
anti-His and anti-GST Ab. An aliquot of His-fusion pro-
tein was analyzed directly by IB assay (Input). (D) Dose-
dependent effects of ACF on HIF-1 dimerization in vitro
Inhibition of HIF-1 dimerization by ACF. (A)
preincubated with vehicle (-) or ACF (?) and the complex was captured by
glutathione beads, subjected to one to three washes, incubated with purified
His-HIF-1?12–395, washed again, and proteins bound to glutathione were ana-
lyzed by IB using anti-His and anti-GST Ab. An aliquot of His-HIF-1?12–395was
analyzed directly by IB assay (Input). (B) Effect of preincubating GST-HIF-1?11–510
with ACF was determined as described above. (C) His-HIF-1?12–395was preincu-
bated with ACF, captured by Ni-NTA agarose, subjected to one to three washes,
analyzed by IB using anti-His and anti-GST Ab. Aliquots of GST and GST-HIF-
1?11–510were analyzed directly by IB assay (Input). (D) His-HIF-1?12–395was pre-
incubated with ACF and then incubated with GST-HIF-1?11–510and analyzed as
described above. (E) GST fusion proteins were incubated with 0–250 ?M ACF,
(FI; mean ? SEM; n ? 6). FI analysis at low ACF concentrations is shown (Inset).
www.pnas.org?cgi?doi?10.1073?pnas.0909353106Lee et al.
Acriflavine Inhibits Tumor Xenograft Growth. We examined whether
inhibition of HIF-1 dimerization by ACF affects the growth of
human cancer xenografts. ACF did not affect HIF-1? or HIF-2?
mRNA or protein levels in PC-3 human prostate cancer cells and
Hep3B-c1 cells (SI Appendix, Figs. S9 and S10). ACF also did not
affect the levels of HIF-1?, c-Myc, Hsp90, or ?-Actin protein in
nonspecific effects of ACF on transcription or translation at con-
centrations that block HIF-1 activity. In contrast, HRE-dependent
transcription (SI Appendix, Fig. S9B) and the expression of VEGF,
stromal-derived factor 1 (SDF1), stem cell factor (SCF), and
GLUT1 mRNAs, which are all encoded by HIF-1 target genes (SI
Appendix, Fig. S9C), were significantly inhibited by treatment of
PC-3 cells with ACF. ACF had no effect on PC-3, P493, or
Hep3B-c1 cell cycling, proliferation, or survival at concentrations
that block HIF-1 activity (SI Appendix, Fig. S11).
To investigate the effect of ACF on tumor growth, vehicle or
ACF was administered by daily i.p. injection to severe combined
immune deficiency (SCID) mice that received s.c. PC-3 cell xeno-
grafts. Treatment was initiated 3 days before implantation and
by day 12 and grew to approximately 500 mm3by day 32, whereas
mice treated with ACF did not show any significant tumor growth
even after one month (SI Appendix, Fig. S12). Daily administration
of ACF did not cause weight loss (SI Appendix, Fig. S13A),
suggesting ACF has no major systemic toxicity.
When treatment was delayed until 14 days after s.c. implantation
100 mm3, ACF inhibited tumor growth after 7 days of administra-
without any effect on body weight (SI Appendix, Fig. S13B).
Expression of VEGF, SDF1, SCF, GLUT1, HK1, and HK2
mRNAs was decreased in tumors after treatment with ACF (Fig.
6B), whereas HIF-1? protein was highly expressed in tumors from
mice treated with vehicle or ACF (SI Appendix, Fig. S14).
Next, Hep3B-c1 hepatoma cells stably transfected with the
HIF-1-regulated FLuc reporter gene p2.1 were implanted in SCID
mice. Before initiation of treatment (day 25), tumor volume (?100
mm3) was similar across groups (Fig. 6C). Analysis of HIF-1-
dependent FLuc activity by whole-body Xenogen imaging revealed
bioluminescence at the tumor site that was similar in both groups
(Fig. 6D Upper). Mice were then treated with ACF or vehicle by
daily i.p. injection and Xenogen imaging was repeated 4 h after
treatment on day 28. The bioluminescence in tumors before and
after treatment with vehicle was similar. In contrast, FLuc activity
was markedly decreased after treatment with ACF for 4 days (Fig.
proteins were generated containing indicated HIF-1? residues (Top), incu-
bated with ACF or vehicle, captured with glutathione beads, washed, and
analyzed for fluorescence intensity (FI) (Bottom). Mean ? SEM are plotted
(n ? 6)***, P ? 0.001 vs. GST (two-way ANOVA with Bonferroni correction).
(B) GST-HIF-2? proteins were generated containing indicated HIF-2? residues
(Top). ACF binding was determined by FI (Bottom). Mean ? SEM is shown
(n ? 3).***, P ? 0.001 vs. GST (two-way ANOVA with Bonferroni correction).
ACF binds to the PAS-B domain of HIF-1? and HIF-2?. (A) GST-HIF-1?
isolated from an aliquot of lysate before IP. The remaining lysate was divided
and incubated with anti-HIF-1? Ab or rabbit IgG for IP. PCR was performed
using immunoprecipitates as template to amplify sequences from the VEGF
and PDK1 promoters, which contain known HIF-1 binding sites. PCR products
were analyzed by gel electrophoresis and ethidium bromide staining. M, size
marker. (B) Cells were cotransfected with reporter plasmid p2.1, in which
transcription of Fluc sequences was driven by an HRE upstream of an SV40
promoter, and reporter plasmid pSV-Renilla, in which Rluc sequences were
with ACF and exposed to 20% (white bar) or 1% (black bar) O2for 24 h. Cells
were lysed and Fluc/Rluc ratio was determined (mean ? SEM; n ? 6).*, P ?
exposed to 20% (white bar) or 1% (black bar) O2for 24 h in the presence of
vehicle (Con) or 5 ?M ACF. Total RNA was isolated for determination of VEGF
and GLUT1 mRNA levels. HIF-1 target gene mRNA levels relative to 18S
rRNA were calculated as 2??(?Ct)where ?Ct ? Cttarget? Ct18Sand ?(?Ct) ?
?Ctcontrol ? ?Cttreatment where control ? vehicle?treated cells at 20% O2.
of cells cultured at 20% or 1% O2in the presence of 0–10 ?M ACF for 20 h.
ACF inhibits HIF-1 DNA-binding and transcriptional activity. (A)
Lee et al.PNAS ?
October 20, 2009 ?
vol. 106 ?
no. 42 ?
6D, Lower), indicating that ACF inhibited HIF-1 activity in tumors,
which preceded the significant inhibition of tumor growth that was
first observed on day 36 (Fig. 6C). ACF administration for 15 days
did not cause any loss of body weight (SI Appendix, Fig. S13C).
Acriflavine Inhibits Angiogenic Cell Mobilization and Tumor Vascular-
ization. VEGF, SCF, and SDF1 induce the mobilization from bone
marrow and other sites into peripheral blood of circulating angio-
including endothelial progenitor, mesenchymal stem, myeloid, and
other cell types that home to tumors and stimulate angiogenesis.
HIF-1 plays a critical role in these processes (11). To determine the
effect of ACF on CACs, SCID mice bearing PC-3 xenografts of
approximately 50 mm3were treated with ACF for 9 days (SI
Appendix, Fig. S15). Four hours after the last dose, blood was
collected for flow cytometric analysis of CACs, as defined by the
and either VEGFR2 or CXCR4 (Fig. 7A). The number of
VEGFR2?/CD117?, VEGFR2?/CD34?, and CXCR4?/ScaI?
CACs was increased approximately 5-fold in tumor-bearing mice
compared with mice without tumors and no significant differences
were observed between untreated and vehicle-treated tumor-
bearing mice. In contrast, ACF treatment significantly decreased
the number of CACs in tumor-bearing mice to levels observed in
mice without tumors. ACF inhibited the expression of mRNAs
encoding VEGF, SDF1, and SCF (which are the ligands bound by
VEGFR2, CXCR4, and CD117, respectively) in PC-3 xenografts
(Fig. 6B). SDF-1 protein levels were increased in blood from
tumor-bearing vs. nontumor-bearing mice and ACF treatment for
9 days reduced SDF-1 in the blood of tumor-bearing mice to levels
similar to those in nontumor bearing mice (Fig. 7B). These effects
of ACF were associated with a significant reduction in tumor
vascularization (Fig. 7C).
of the critical role that it plays in cancer progression (1, 2, 5).
desirable as a defined and selective mechanism of action, although
conventional wisdom holds that the large interfaces involved in
protein dimerization are difficult to disrupt by small molecules. We
used a cell-based split-Rluc assay to identify ACF as drug that
inhibits HIF-1 dimerization (Figs. 1–3) by binding to the PAS-B
DNA-binding and transcriptional activity (Figs. 5 and 6), leading to
inhibition of tumor growth, CAC mobilization, and tumor vascu-
larization (Figs. 6 and 7).
PAS domains of HIF-1?, HIF-2?, HIF-1?, and other bHLH-
PAS proteins are organized into PAS-A and PAS-B subdomains,
which contribute to dimerization by providing, in addition to the
bHLH domain, secondary interaction surfaces that increase the
specificity of dimerization. NMR spectrometry revealed that hy-
in the HIF-2? PAS-B subdomain mediate heterodimerization with
HIF-1? (12). The HIF-2? PAS-B crystal structure was shown to
contain an internal cavity, which accommodated a small molecule
that partially disrupted the heterodimerization of isolated HIF-2?
and HIF-1? PAS subdomains in vitro but was not reported to have
any effect on heterodimerization of the full length proteins or on
HIF-1 transcriptional activity (13). We show that ACF binds to the
PAS-B subdomain of HIF-1? or HIF-2?, thereby blocking het-
were treated by daily i.p. injection of vehicle or ACF for 14 days. (A) Tumor volume (mean ? SEM; n ? 4) is shown.*, P ? 0.05;**, P ? 0.01 vs. vehicle (two-way
ANOVA with Bonferroni correction). (B) Mice were euthanized on day 28 (4 h after last dose) and tumors were collected. mRNA levels relative to 18S rRNA in
*, P ? 0.05;**, P ? 0.01 (two-way ANOVA with Bonferroni correction). (C-D) mice bearing Hep3B-c1 xenografts were treated with vehicle or ACF starting on
day 25. (D) HRE-driven FLuc activity was determined by Xenogen imaging before treatment (Upper) and 4 h after treatment on day 28 (Lower).
ACF inhibits tumor growth, HIF-1 target gene expression, and HIF-1 activity. (A and B) PC-3 xenografts were grown to approximately 100 mm3and mice
www.pnas.org?cgi?doi?10.1073?pnas.0909353106Lee et al.
transcription in cultured cells and tumor xenografts.
ACF has known trypanocidal, antibacterial, and antiviral activ-
ago (15). In the present study, we have demonstrated that ACF
administration inhibited the expression in prostate cancer xeno-
grafts of VEGF, SDF1, and SCF mRNA, which encode angiogenic
cytokines that are critical for tumor vascularization through mobi-
and CD117, respectively. ACF treatment decreased tumor-induced
CAC mobilization and vascularization, providing a mechanism for
tumor growth arrest. ACF inhibited HIF-1 at concentrations that
do not affect cell proliferation or survival ex vivo. However, the
drug concentrations achieved in vivo were not determined and
other mechanisms of action besides HIF-1 inhibition, such as
NF-?B inhibition (16), may contribute to the anticancer effects
ACF is a mixture of 3,6-diamino-10-methylacridinium chloride
(trypaflavin) and 3,6-diaminoacridine (proflavine) (SI Appendix,
Fig. S16). Our results provide proof-of-principle that small mole-
cule inhibitors of HIF-1 dimerization in vivo can be identified.
directly to both HIF-1? and HIF-2?. ACF has been administered
to patients for at least 5 months without major side effects (14),
suggesting that it may be a candidate for clinical trials. ACF can
also serve as the lead compound for development of drugs to
treat patients with cancer subtypes in which increased HIF-1? or
HIF-2? levels are associated with disease progression and patient
mortality (2, 5).
Materials and Methods
Detailed materials and methods are available online in SI Appendix.
Split Rluc System. DNAs encoding HIF-1? residues 12–396 and HIF-1? residues
the Rluc N-terminal region (residues 1–229) in pCMV-Nrluc or upstream of the
Rluc C-terminal region (residues 230–311) in pCMV-Crluc, respectively (7).
ng of HIF-1?11–510-CRluc, and 80 ng of pGL2-promoter, using Fugene-6 (Roche).
Quantitative Real Time-Reverse Transcription-PCR Assay. Primers (SI Appendix,
Table S2) were designed using Beacon Designer software (Bio-Rad).
Preparation of His-tagged and GST Fusion Proteins. HIF-1?12–395and HIF-2?3–351
were cloned into pET-28c (Novagen). HIF-1?, HIF-1?, and HIF-2? residues were
amplified using specific primers (SI Appendix, Table S4) and the PCR products
were inserted into pGEX-5X-1 (GE Healthcare).
Chromatin IP (ChIP) Assay. The ChIP Assay Kit (Upstate-Cell Signaling Solution)
was used with rabbit polyclonal anti-HIF-1? or HIF-2? Ab (Novus Biologicals) or
rabbit IgG. VEGF and PDK1 sequences were detected by PCR (SI Appendix,
ACKNOWLEDGMENTS. We are grateful to S. Gambhir and P. Ramasamy (Stan-
luciferase vectors and anti-HIF-2? Ab; G. Wang for assistance with assay devel-
opment; J. Kim for assistance with fluorometry; K. Miyake for assistance with
Armstrong Professor at Johns Hopkins University School of Medicine.
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