Post on 28-Jun-2020
1
Gramicidin A Blocks Tumor Growth and Angiogenesis Through Inhibition of Hypoxia-Inducible Factor in Renal Cell Carcinoma
Justin M. David,1,2* Tori A. Owens,2 Landon J. Inge,3 Ross M. Bremner,3 and Ayyappan K. Rajasekaran2
1Department of Biological Sciences, University of Delaware, Newark, DE 19716, 2Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, Wilmington, DE 19803, 3Center for Thoracic Disease and Transplantation, Heart and Lung Institute, St. Joseph's
Hospital and Medical Center, Phoenix, AZ 85711
* Current address: Justin M. David
Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20878
Corresponding Author:
Ayyappan K. Rajasekaran, Ph.D Nemours Center for Childhood Cancer Research
Alfred I. duPont Hospital for Children 1701 Rockland Road, Wilmington, DE 19803
Telephone: (610) 246-5705 Fax: (610) 793-1320 Email: arakaran687@gmail.com Running Title: Gramicidin A Inhibits Hypoxia-Inducible Factor Keywords: ionophore, hypoxia, renal cell carcinoma, angiogenesis, HIF, VHL Abbreviations: CA-IX (carbonic anhydrase IX), CAL (calcimycin), GA (gramicidin A), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), GLUT-1 (glucose transporter 1), HIF (hypoxia-inducible factor), HRE (hypoxia-response element), MON (monensin), ODD (oxygen-dependent degradation domain), PHD (prolyl-4-hydroxylase domain-containing protein), RCC (renal cell carcinoma), ccRCC (clear cell renal cell carcinoma), pRCC (papillary RCC), SAL (salinomycin), VAL (valinomycin), VHL (von Hippel-Lindau) Financial Support: NIH grants P20GM103464, R01 DK56216 and Nemours Foundation (A.K. Rajasekaran), Heart and Lung Research Initiative, St. Joseph’s Foundation (R.M. Bremner, L.J. Inge). Conflicts of Interest: Authors A.K. Rajasekaran and J.M. David have filed a patent. Word Count: abstract = 198, manuscript = 4,393 References: 54 Figures: 6
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
2
Abstract:
Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane
potential by permeabilizing membranes to specific ions. Gramicidin A (GA) is a channel-
forming ionophore that forms a hydrophilic membrane pore which permits the rapid passage of
monovalent cations. Previously, we found that GA induces cellular energy stress and cell death
in renal cell carcinoma (RCC) cell lines. RCC is a therapy-resistant cancer that is characterized
by constitutive activation of the transcription factor hypoxia-inducible factor (HIF). Here, we
demonstrate that GA inhibits HIF in RCC cells. We found that GA destabilized HIF-1α and
HIF-2α proteins in both normoxic and hypoxic conditions, which in turn diminished HIF
transcriptional activity and the expression of various hypoxia-response genes. Mechanistic
examination revealed that GA accelerates O2-dependent downregulation of HIF by upregulating
the expression of the von Hippel-Lindau (VHL) tumor suppressor protein, which targets
hydroxylated HIF for proteasomal degradation. Furthermore, GA reduced the growth of human
RCC xenograft tumors without causing significant toxicity in mice. GA-treated tumors also
displayed physiological and molecular features consistent with the inhibition of HIF-dependent
angiogenesis. Taken together, these results demonstrate a new role for GA as a potent inhibitor
of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
3
Introduction:
Kidney cancer is a relatively rare but deadly disease that is among the top ten causes of
cancer-related deaths in men in the USA (1). Most kidney tumors are classified as renal cell
carcinomas (RCC) and are highly therapy-resistant (2-4). RCC is actually a histologically
heterogeneous group of several distinct tumor subtypes that originate from the epithelial cells of
the renal tubule. Each subtype, including clear cell (ccRCC, 70%), papillary (pRCC, 10-15%),
chromophobe (5%), and collecting duct (<1%), is associated with unique morphological and
genetic characteristics (3).
RCC characteristically exhibits molecular and biochemical features associated with
chronic responses to low oxygen (hypoxia) (4). Adaptation to hypoxia is mediated by an O2-
sensitive transcription factor known as hypoxia-inducible factor (HIF) (4), and accumulated
genetic, clinical, and experimental evidence suggests that constitutive (i.e. O2-independent)
activation of HIF plays a causal role in the development and progression of RCC (4, 5). In
normoxic conditions, the α-subunit of HIF (HIF-α) is rapidly hydroxylated at specific proline
residues within the oxygen-dependent degradation domain (ODD) by prolyl-4-hydroxylase
domain-containing protein 2 (PHD2) (4). Hydroxylation of HIF-α creates a binding interface for
the von Hippel-Lindau tumor suppressor protein (VHL) which serves as the substrate recognition
component of an E3 ubiquitin ligase complex that promotes the polyubiquitylation and
subsequent proteasomal degradation of HIF-α (4). Conversely, reduced O2 in hypoxic conditions
prevents the hydroxylation/degradation of HIF-α. Stabilized HIF-α dimerizes with its β-subunit
(HIF-β) and activates various target genes that collectively govern a wide array of processes
relevant to cancer development and progression, most notably angiogenesis and metabolism (6).
Targeted therapies that block the action of proangiogenic growth factors and their receptors on
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
4
endothelial cells (e.g. sunitinib, sorafenib, bevacizumab, etc.) are now routinely used for ccRCC
patients, and have succeeded in increasing progression-free survival and quality of life.
However, these agents typically fail to achieve durable remission in most cases (7), and little is
known as to their utility for non-ccRCC subtypes as these patients were excluded from clinical
trials (8).
Another anti-angiogenesis therapeutic strategy is to target HIF directly, and several points
of regulation have been exploited to develop novel HIF-inhibiting agents. These drugs include
1) mTOR inhibitors (rapamycin, temsirolimus, and everolimus) that interfere with the translation
of HIF-α subunit transcripts; 2) histone deacetylase (HDAC) inhibitors, heat shock protein 90
(HSP90) inhibitors, and nonsteroidal anti-inflammatory drugs (NSAIDs) that enhance HIF-α
subunit protein degradation; 3) anthracyclines (doxorubicin, daunorubicin) and DNA
intercalating agents (echinomycin) that interfere with the binding of HIF to DNA; and 4)
dimerization inhibitors that block the binding of HIF-α subunits with HIF-β (6, 9-12). All of
these agents are in various stages of preclinical development, clinical trials, or clinical use.
Ionophores are lipophilic molecules that render membranes permeable to specific cations
and are classified as mobile-carriers and channel-formers. These drugs are potent antibiotics and
are used in veterinary medicine and as feed additives for agriculture (13, 14). Mobile-carrier
ionophores are known to exhibit broad-spectrum anticancer abilities and are capable of
overcoming drug resistance, improving chemo- and radio-sensitization, and inhibiting oncogenic
signaling (13, 15, 16). Accumulated research has now demonstrated that ionophores are not
simply nonspecific cytotoxic chemicals, but are also capable of working at multiple levels to
selectively disrupt cancer cell growth and survival (17).
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
5
In contrast to the mobile-carriers, use of channel-formers as antitumor agents has not
been widely evaluated. Gramicidin A (GA) is a prototypical channel-forming ionophore that
forms a 4Å pore that can accommodate water, protons, and monovalent cations. Channel
formation results in Na+ influx, K+ efflux, osmotic swelling, and cell lysis in biological systems
(18, 19) and confers GA with potent antibiotic activity against Gram-positive bacteria, fungi, and
protozoa (20, 21). We have previously demonstrated that GA is toxic to RCC cells and induces
metabolic dysfunction and cellular energy depletion (22). In this study, we investigated whether
treatment with GA specifically affects HIF in RCC cells. We found that GA destabilizes HIF-1α
and HIF-2α in both normoxia and hypoxia leading to reduced HIF transcriptional activity and
loss of target gene expression. We determined that GA accelerates the O2-dependent degradation
of HIF-α subunits via upregulation of the VHL tumor suppressor protein. Furthermore, we show
that in vivo administration of GA reduces the growth and angiogenesis of VHL-expressing RCC
cell line tumor xenografts without significant toxicity in mice. To our knowledge, this is the first
time that an ionophore has been reported to 1) specifically inhibit HIF-dependent hypoxia
responses, and 2) specifically upregulate a tumor suppressor (VHL). Our results reveal an
additional "targeted" role for GA as a potent inhibitor of HIF and suggest its utility as an anti-
angiogenic therapeutic agent for RCC tumors that express wild-type VHL.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
6
Materials and Methods
Cell culture
Human clear cell RCC (A498, 786-O, SN12C, Caki-1, UMRC6, and UMRC6+VHL),
papillary RCC (ACHN) and embryonic kidney (HEK293T) cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2mM L-
glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. For hypoxia experiments we
cultured the cells in a HERAcell 150 tri-gas cell incubator (Thermo Fisher Scientific, Waltham,
MA) with a regulated environment of 1% O2, 5% CO2, and 94% N2 at 37°C. 786-O, Caki-1, and
HEK293T cells were purchased from the American Type Culture Collection (Manassas, VA) in
1995. A498, SN12C and ACHN cells were kindly provided by Dr. Charles L. Sawyers
(Memorial Sloan-Kettering Cancer Center, New York City, NY) in 2005 (23). UMRC6 and
UMRC6+VHL cells were kindly provided by Dr. Michael I. Lerman (National Cancer Institute,
Bethesda, MD) in 2000 (24). All cell lines obtained from investigators have been authenticated
prior to use.
Reagents
The following chemicals were purchased from Sigma-Aldrich (St. Louis, MO);
gramicidin A, monensin, valinomycin, calcimycin (A23187), MG132, and cobalt chloride.
Antibodies
We purchased primary antibodies specific for HIF-1α (BD Biosciences, San Jose, CA),
HIF-2α, CA-IX (Novus Biologicals, Littleton, CO), GAPDH, α-tubulin, HA (Cell Signaling,
Danvers, MA), GLUT-1 (Alpha Diagnostic International, San Antonio, TX), β-actin (Sigma-
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
7
Aldrich, St. Louis, MO), and VHL (EMD Chemicals, Gibbstown, NJ). Horseradish peroxidase
(HRP)-conjugated secondary antibodies were purchased from Cell Signaling (Danvers, MA).
Plasmids and Transfections
Plasmids pGL2-HRE-luciferase (Addgene plasmid 26731, Navdeep S. Chandel) (25),
pcDNA3-ODD-luciferase (Addgene plasmid 18965, William G. Kaelin) (26), pcDNA3-HA-
HIF1α (Addgene plasmid 18949, William G. Kaelin) (27), and pcDNA3-HA-HIF1α-
P402A/P564A (Addgene plasmid 18955, William G. Kaelin) (28) were purchased from Addgene
(Cambridge, MA). Plasmid pcDNA3 vector was purchased from Life Technologies (Grand
Island, NY) and plasmid phRL-renilla was purchased from Promega Corporation (Madison, WI).
Transient transfections were accomplished using Lipofectamine 2000 (Life Technologies, Grand
Island, NY) according to manufacturer's instructions. Transfection of Caki-1 cells with td-
Tomato-N1 (Clonetech, Mountain View, CA) was accomplished by electroporation with a
Nucleofector II (Lonza, Walkersville, MD) using Kit V according to the manufacturer's
instructions. Cells were examined using a Leica DMI microscope (Leica Microsystems,
Bannockburn, IL) and single cells expressing red fluorescent protein were picked after 2 weeks
of selection with 800μg/mL G418 (Geneticin) to establish stable cell lines. These cells were
employed for in vivo studies.
Immunoblot Analysis
Cell lysates were prepared in a buffer containing 95 mM NaCl, 25 mM Tris pH 7.4, 0.5
mM EDTA, and 2% SDS. Tumor lysates were prepared by mincing tumor samples and then
lysing in a buffer containing 150mM NaCl, 20mM Tris pH 7.4, 1mM EDTA, 1mM EGTA, 1%
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
8
Triton X-100, 1% IGEPAL (octylphenoxypolyethoxyethanol), 1mM β-glycerol phosphate, 1mM
Na3VO4, 2.5mM Na4P2O7, 50mM NaF, and 12mM deoxycholate. Lysates were sonicated,
centrifuged, and the protein concentrations of the supernatants were determined using the DC
protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were then resolved by SDS-
PAGE and transferred to nitrocellulose. The membranes were blocked in 5% non-fat milk in
tris-buffered saline with 0.1% Tween 20 (TBS-T) and then incubated overnight at 4°C with
primary antibodies diluted in 5% bovine serum albumin (BSA)/TBS-T. The following day, the
membranes were washed and incubated with HRP-conjugated secondary antibodies diluted in
5% non-fat milk/TBS-T at room temperature for 1hr. The protein bands were visualized using
Amersham ECL Prime (GE Healthcare, Piscataway, NJ). Images were acquired using
Photoshop (Adobe Systems Inc., San Jose, CA) and relative quantification was performed using
ImageJ (NIH, Bethesda, MD).
Quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Life Technologies, Grand Island, NY)
and reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules,
CA) as per the respective manufacturer's instructions. The cDNA was amplified via real-time
polymerase chain reaction (RT-PCR) using the SYBR Green PCR Master Mix (Applied
Biosystems, Warrington, UK). The following primers used to measure specific target genes:
HIF-1α forward, 5'-CCACAGGACAGTACAGGATG-3', reverse 5'-
TCAAGTCGTGCTGAATAATACC-3'; HIF-2α forward, 5'-GTCTCTCCACCCCATGTCTC-3',
reverse 5'-GGTTCTTCATCCGTTTCCAC-3'; VHL forward, 5'-
ATGGCTCAACTTCGACGGC-3', reverse 5'-CAAGAAGCCCATCGTGTGTC-3'; GAPDH
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
9
forward, 5'-GCTGTCCAACCACATCTCCTC-3', reverse 5'-TGGGGCCGAAGATCCTGTT -3'.
Samples were assayed in a 384 well format in triplicate using a 7900HT Fast Real-Time PCR
system (Applied Biosystems, Foster City, CA). Variation in cDNA loading was normalized to
GAPDH expression, which remained constant at the 24hr incubation periods used, and relative
expression was determined using the ΔΔCt method of relative quantification (RQ). Graphs
represent the average RQ value with error bars (standard error of the RQ value) from one
representative of three independent experiments. Graphs were generated using the GraphPad
Prism Software (GraphPad Software, La Jolla, CA).
Luciferase Activity Assay
HEK293T cells were cotransfected with 100ng phRL-renilla and 2μg of pGL2-HRE-
luciferase or 1μg of pcDNA3-ODD-luciferase using Lipofectamine 2000 and incubated for 24hr
before drug treatment. Following drug treatment, the Dual-Luciferase Reporter Assay (Promega
Corporation, Madison, WI) was performed according to the manufacturer's instructions. Briefly,
lysates were prepared using the provided buffer and then diluted 1:10, then 2μL of diluted
sample lysate was added in triplicate to a white-walled 96-well plate, mixed with 100μL of
firefly luciferase assay substrate, and luminescence was immediately recorded using a VictorX4
plate reader (Perkin-Elmer, Waltham, MA). Then 100μL of renilla luciferase substrate was
added to each well and luminescence was immediately recorded using the plate reader. Values
were corrected for background luminescence using the reading from the media only, and the
corrected values were first normalized to renilla luciferase (internal control) and then to the
vehicle-treated samples to calculate the relative luciferase activity. Data represent the mean±SD
of one representative of three independent experiments.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
10
Tumor Growth Experiment
Animal experiments were performed according to the NIH guidelines and approved by
the Nemours Institutional Animal Care and Use Committee. Female hairless 6-8 week old Nu/J
mice were injected subcutaneously with a suspension of Caki-1-td-Tomato cells (1.5 × 106) in a
50% growth factor-reduced matrigel solution. Caki-1 tumors were allowed to grow for 1 week
before randomization into control (vehicle solution only) and drug (GA) groups of 8 mice each
with an average initial tumor volume of ~85mm3 in each group. GA (0.22mg/kg body weight)
was diluted in a 1:1 solution of ethanol and saline, and mice were dosed thrice weekly with 50μL
of either vehicle or GA solutions by intratumoral injection. Mouse body masses and tumor sizes
were recorded before each injection. Tumor size was measured using calipers and tumor volume
was estimated using the formula (length × width2) / 2 where length was the longer of the
measurements. Upon completion of the study, mice were euthanized and the tumors were
imaged, harvested, and prepared for immunohistochemical and immunoblot analysis.
Fluorescence signals from Caki-1 xenografts were acquired at the end of the study using the
Kodak In Vivo Multispectral FX PRO imaging system (Carestream, Woodbridge, CT) using the
following settings: Ex. 550 nm, Em. 600 nm, no binning, f/stop 2.8, focal plane 13.1 mm, field-
of-view 119.1 mm.
Immunohistochemistry
Formalin fixed paraffin embedded (FFPE) samples of vehicle and GA treated tumors were
prepared using routine procedures. 5μM sections were floated onto charged slides and dried for
one hour at 65°C. Following deparaffinization in xylene and graded alcohols, tissues underwent
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
11
heat-induced epitope retrieval using the Decloaking Chamber and Reveal Decloaking Buffer
(Biocare Medical, Concord, CA, USA) according to recommended manufacturer protocols. The
VHL polyclonal antibody (#PA5-17477, Thermo Fisher, Rockford, IL, USA) and polyclonal
CD31 (#LS-B1932, LifeSpan Biosciences, Seattle, WA) were diluted in 1% BSA (Sigma-
Aldrich) and applied overnight at 4°C. Slides were incubated with PromARK anti-Rabbit
horseradish peroxidase polymer (BioCare Medical) and stained with diaminobenzidine using the
Betazoid DAB kit (Biocare Medical) according to recommended manufacturer protocols.
Nuclei were stained with hematoxylin (EMD Millipore, Billerica, MA, USA) and Bluing
Reagent (Thermo Fisher Scientific, Waltham, MA, USA), cleared, and mounted for microscopic
analysis.
Statistics
qRT-PCR results were analyzed using one-way ANOVA followed by Dunnett's Multiple
Comparison Test. All other analyses were performed using two-tailed unpaired Student's T-test.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
12
Results:
GA reduces HIF-1α and HIF-2α protein expression:
Because constitutive activation HIF is central to RCC pathogenesis, we investigated
whether GA affects the expression of HIF in RCC cells. Using Caki-1, SN12C, and ACHN cell
lines, we found that treatment with GA for 24hr provoked a dose-dependent decrease in the
expression of both HIF-1α and HIF-2α protein in these cell lines (Fig. 1A, left). Since HIF-α
subunits are stabilized by hypoxia (1%O2), we next assessed whether GA reduces HIF-α
expression in hypoxia. Exposure to 1%O2 dramatically increased HIF-1α and HIF-2α as
expected, but treatment with GA prevented this increase in a dose-dependent manner (Fig. 1A,
right). Strikingly, 1μM GA was sufficient to reduce the hypoxic expression of both isoforms
below even their normoxic level (Fig. 1A, lane 8) with the exception of HIF-1α in ACHN cells.
Concomitant analysis of HIF mRNA expression revealed that GA significantly altered transcript
expression for only HIF-2α in SN12C cells (P = 0.01 by one-way ANOVA) (Fig. 1B) suggesting
that GA primarily affects only HIF protein levels. Finally, we assessed whether mobile-carrier
ionophores also reduce hypoxic HIF protein expression. We compared equal doses (0.5μM) of
GA with monensin (MON, Na+-selective), valinomycin (VAL, K+-selective), and calcimycin
(CAL, Ca2+-selective) in hypoxic SN12C cells. We observed that MON slightly reduced HIF-1α
and HIF-2α at 72hr, VAL moderately reduced both proteins from 24-72hr, and CAL had no
effect on either protein (Fig. 1C). Only GA elicited a profound decrease in both isoforms that
persisted from 24-72hr (Fig. 1C). These data reveal that only GA is a potent inhibitor of HIF-1α
and HIF-2α protein expression.
GA reduces HIF transcriptional activity and target gene expression:
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
13
Next we analyzed the effect of GA upon the transcriptional activity of HIF. We utilized
HEK293T cells transfected with a HIF-responsive luciferase reporter plasmid that contains three
hypoxia-response elements (HRE) from the PGK1 (phosphoglycerate kinase 1) gene upstream of
firefly luciferase (25). HIF-dependent luciferase activity was significantly stimulated by
hypoxia, but treatment with GA diminished this activity to nearly zero (Fig. 2A). Next we
measured the expression of various HIF targets in RCC cells. We found that hypoxic expression
of CA-IX (carbonic anhydrase 9), GLUT-1 (glucose transporter 1), and GAPDH (glyceraldehyde
3-phosphate dehydrogenase) were all decreased by GA in a dose-dependent manner in SN12C
cells (Fig. 2B left). Similar results were obtained using Caki-1 and ACHN cells (with the
exception of GAPDH in Caki-1 cells) (Fig 2B, right). Collectively, these results demonstrate
that GA attenuates hypoxia responses by preventing the transcriptional activation of HIF-
responsive genes.
GA destabilizes HIF through proline hydroxylation:
O2-dependent downregulation of HIF-α depends upon the proteasome to degrade
ubiquitylated HIF. In order to elucidate whether GA employs this mechanism, we first measured
HIF expression in HEK293T cells treated with increasing doses of GA in the absence or
presence of the well-known proteasomal inhibitor MG-132 (10μM) (29). Treatment with GA
failed to reduce HIF-1α and HIF-2α protein expression in cells treated with MG-132 (Fig. 3A,
left) indicating that GA destabilizes HIF by enhancing its degradation by the proteasome. This
regulatory mechanism also requires the hydroxylation of conserved proline residues located
within the ODD of HIF by PHD enzymes (30). Inhibition of PHD activity using the hypoxia
mimetic CoCl2 (1mM) stabilized HIF-1α and HIF-2α as expected but completely blocked
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
14
destabilization of these proteins by GA (Fig. 3A, right). Similar results were also observed using
CoCl2-treated Caki-1, SN12C, and ACHN cells (not shown). We then examined whether the
ODD of HIF is involved in the GA-mediated inhibition of HIF activity. Using HEK293T cells
transfected with a luciferase reporter plasmid that contains the ODD of HIF-1α fused in frame to
firefly luciferase (26) we determined that treatment with GA significantly reduced ODD-
luciferase activity (P < 0.05 by T-test, Fig. 3B). In a related experiment, we transfected
HEK293T cells with either HA-tagged wild-type HIF-1α (HA-HIF-1α) (27) or ODD-mutant
HIF-1α (HA-HIF1α-P402A/P564A) (28). Treatment of these cells revealed that wild-type HIF-
1α but not ODD-mutant HIF-1α was reduced by GA (Fig 3C). Altogether, these results
demonstrate that GA employs the O2-dependent regulatory mechanism to destabilize HIF protein
via PHD-dependent hydroxylation of its ODD.
GA upregulates VHL protein expression:
Mutational inactivation of VHL occurs extensively in sporadic ccRCC (up to 80%), and a
remaining proportion of tumors (<10%) silence the VHL gene through DNA methylation (31,
32). Loss of VHL cripples the ability of the cell to degrade HIF in normoxia yielding chronic
activation of the HIF transcriptional program (33). In our aforementioned experiments we used
VHL-expressing RCC cells to establish that GA destabilizes HIF through proline hydroxylation
and proteasomal degradation. In order to ascertain whether VHL is involved in GA-mediated
degradation of HIF we used the naturally VHL-deficient ccRCC cell line UMRC6 and found that
GA failed to reduce HIF-1α or HIF-2α expression (Fig. 4A, left). In contrast, treatment of VHL-
reconstituted UMRC6+VHL cells did yield a reduction in HIF-2α protein expression (Fig. 4A,
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
15
right). HIF-1α expression was undetectable in this cell line. These data demonstrate that VHL is
essential for GA-mediated destabilization of HIF.
Although hypoxia reduces proline hydroxylation of HIF, it does not completely abolish it
(34). Since GA treatment reduced HIF expression even in hypoxic conditions (Fig. 1) and
utilizes the O2-dependent degradation mechanism (Fig. 3), we speculated that GA enhances a
component of this pathway to accelerate HIF destabilization. We investigated this possibility
and observed that treatment with GA dramatically increased the expression of VHL protein in a
dose-dependent manner in HEK293T cells as well as Caki-1, SN12C, and ACHN RCC cells
(Fig. 4B). This increase was not reflected at the mRNA level as transcript expression was
significantly elevated in only SN12C cells (P < 0.001 by one-way ANOVA) (Fig. 4C). These
results demonstrate that GA inhibits HIF by enhancing VHL expression.
GA inhibits the growth and angiogenesis of VHL-expressing RCC tumor xenografts:
Tumor growth and development beyond a microscopic mass depends on the recruitment
of new blood vessels (35). Our in vitro data suggested that GA may reduce tumor growth in vivo
by disrupting tumor angiogenesis. We previously found that GA reduced the in vivo growth
SN12C tumor xenografts in mice (22). In order to evaluate the anti-angiogenic efficacy of GA,
we performed a similar experiment in which we engrafted human Caki-1 RCC cells that express
the red fluorescent protein td-Tomato and can be visualized in vivo. Once the average tumor
volume reached ~85mm3, the mice were randomized into two groups (each n = 8) and
administered 50μL of either vehicle solution or GA (0.22mg/kg) by intratumoral injection thrice
weekly for 26 days. As shown in Fig. 5A and B, the control tumors were noticeably larger than
the GA-treated tumors. We found that the average mass of the GA-treated tumors was 52% less
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
16
than that of the control tumors (Fig. 5C, P = 0.017 by T-test). Analysis of tumor growth
revealed that the tumors of the GA group essentially failed to grow once treatment with GA was
initiated (Fig. 5D). The difference in mean tumor volume achieved significance at day 5 and
continued throughout the duration of the study (P < 0.05). Significantly, the increased dose,
frequency, and duration of GA treatment did not elicit significant toxicity as no changes in
average body mass (Fig. 5E) or activity levels were observed in the mice. Taken together, these
data demonstrate that GA inhibits the growth of VHL-expressing RCC tumors.
In order to confirm that reduced tumor growth was due to a blockade of tumor
angiogenesis, we histologically examined the tumor tissue. GA-treated tumors recapitulated our
in vitro findings as we observed an overall increase in VHL immunostaining (Fig. 6A) and a
57% reduction in the average number of CD31 positive microvessels in the GA-treated tumors
(Veh = 7.13±0.18 vs. GA = 3.04±0.54, P = 0.0004) (Fig. 6A, B). In agreement with these data,
immunoblot analysis revealed that HIF-2α and GAPDH protein expression was also substantially
reduced in the GA-treated tumors (Fig. 6C). HIF-1α was not detectable by immunoblot but this
result was not surprising as it has been reported that RCC growth in vivo is driven by HIF-2α but
repressed by HIF-1α (5). Taken together, these results are consistent with our in vitro data and
indicate that GA inhibits tumor growth through the suppression of HIF-dependent angiogenesis.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
17
Discussion:
Here we report for the first time that GA is a novel inhibitor of tumor angiogenesis. We
have demonstrated that treatment with GA enhances VHL expression which destabilizes HIF-1α
and HIF-2α protein to suppress the transcription of various HIF targets. Loss of the HIF
transcriptional program leads to reduced tumor angiogenesis which curtails tumor growth in
vivo. These novel findings suggest that GA has therapeutic potential as an angiogenesis inhibitor
for VHL-positive RCCs and possibly for other cancers that express VHL.
GA-mediated destabilization of HIF-α subunits required both proline hydroxylation and
VHL expression indicating that GA utilized the O2-dependent degradation mechanism to target
HIF. Strikingly, GA reduced HIF expression even in hypoxic conditions. Although hypoxia
(1%O2) limits PHD-mediated hydroxylation by depleting molecular oxygen, it does not
completely abolish it (34). We determined that GA increases the expression of VHL protein to
accelerate O2-dependent degradation of HIF. Because upregulation of VHL was sufficient to
compensate for the inhibitory effects of hypoxia, we suggest that VHL levels are another
important limiting factor in the regulation of HIF in hypoxic conditions. However, whether GA
also increases PHD expression and/or activity is an additional possibility that remains for further
investigation.
To our knowledge, this is the first time that an ionophore has been shown to specifically
upregulate a tumor suppressor protein, yet precisely how GA increases VHL expression remains
to be elucidated. We previously reported that treatment of RCC cells with GA activates the
AMPK pathway (22), but whether AMPK-mediated stress responses are directly related to VHL
upregulation is not known. Our results show that VHL protein, but not mRNA, increases in GA-
treated cells indicating that either the translation of VHL transcripts or the stability of VHL
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
18
protein is increased by GA. VHL is known to be targeted for degradation by the ubiquitin-
proteasome pathway, and VHL is stabilized by association with ubiquitin ligase components
(elongin B, elongin C, RBX1, cullin 2) (36). Our results clearly show that VHL was active in
mediating the degradation of HIF in GA-treated cells, so it is possible that GA enhances complex
formation to stabilize and upregulate VHL protein. In addition, signaling by Src was recently
identified as a therapeutic target in RCC (37), and phosphorylation of tyrosine 185 by Src
destabilizes VHL (38). Several other proteins are also known to specifically target and
destabilize VHL, including E2-EPF ubiquitin carrier protein (39), casein kinase 2 (40), and
transglutaminase 2 (41). Whether inhibition of any of these proteins is involved in the GA-
mediated increase in VHL expression remains to be investigated.
The plausibility of VHL overexpression as a therapeutic strategy has been demonstrated
in various reports; Sun et al. first showed that VHL gene delivery using liposomes in vivo
reduced HIF-1α and VEGF expression, reduced tumor angiogenesis, and induced the regression
of murine thymic lymphoma tumor xenografts (42) and rat glioma tumor xenografts (43). More
recently, VHL overexpression by adenovirus infection was found to synergize with doxorubicin
to suppress the growth of murine hepatocellular carcinoma xenografts (44), and a novel small
molecule inhibitor of HIF-1α was shown to reduce the growth and vascularization of human
colorectal carcinoma tumor xenografts via VHL overexpression (34). These studies demonstrate
the effectiveness of enhancing VHL expression to block tumor growth as well as combining
VHL overexpression with other treatments to augment therapeutic efficacy.
Constitutive activation of HIF is regarded as a hallmark of RCC pathology. This is most
prominent in ccRCC in which the overwhelming majority of tumors feature inactivating
mutation of the VHL gene (31, 32). We observed that GA failed to reduce HIF-1α and HIF-2α
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
19
expression in VHL-deficient cells implying that GA may not be effective as an anti-angiogenic
therapy for ccRCC patients with functional inactivation of VHL. However, constitutive
activation of HIF is also a characteristic of certain non-clear cell RCC subtypes (45-48) even
though VHL mutation is exceedingly rare in these tumors (49). Since VHL is functional in these
subtypes, GA is likely to have therapeutic utility in this traditionally underserved patient
population (8). Furthermore, GA may also prove effective in other cancers as upregulation of
HIF occurs in the majority of solid tumors and generally correlates with poor survival (6).
Toxicity is an essential factor in clinical drug development. Our preliminary
investigations confirmed that systemic administration of GA by either intravenous or
intraperitoneal injection was lethal to mice. However, we found that repeated intratumoral
injection of GA blocked tumor growth without causing significant toxicity. Intratumoral
administration is by nature localized, and it improves the therapeutic index of drugs by
increasing the tumor-to-organ ratio which greatly reduces systemic toxicity (50). Although
systemic administration is commonly regarded as essential for the treatment of invasive cancer,
intratumoral injection is now a feasible approach for certain inoperable and/or metastatic tumor
sites through the use of X-ray computed tomography. Furthermore, intratumoral administration
can actually enhance immune responses against disseminated (non-injected) tumors by
enhancing the processing of tumor-associated antigens expressed in cell debris from the injected
tumor (51). Nevertheless, systemic administration of GA may be possible in the near future.
Chemical modification of GA has been shown to change the characteristics of the peptide
enough to increase microbial targeting and/or decrease non-specific toxicity (18, 19, 52, 53), and
encapsulation of GA within a targeted drug carriers such as nanoparticles may be a plausible
method to safely deliver GA to only the tumor (54). Whether these approaches can be
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
20
effectively applied to the use of GA as a novel cancer therapy is an essential area of future
investigation.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
21
Acknowledgements:
We acknowledge Dr. Navdeep S. Chandel for producing pGL2-HRE-luciferase and Dr.
William G. Kaelin for producing pcDNA3-ODD-luciferase, pcDNA3-HA-HIF-1α and pcDNA3-
HA-HIF-1α-P402A/P564A (see materials and methods section). We thank Dr. Sonali P. Barwe
and Vinu Krishnan for technical assistance in conducting the in vivo work.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
22
References: 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62(1):10-29. 2. Gupta K, Miller JD, Li JZ, Russell MW, Charbonneau C. Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature review. Cancer Treat Rev 2008;34(3):193-205. 3. Baldewijns MM, van Vlodrop IJ, Schouten LJ, Soetekouw PM, de Bruine AP, van Engeland M. Genetics and epigenetics of renal cell cancer. Biochim Biophys Acta 2008;1785(2):133-55. 4. Haase VH. Renal cancer: Oxygen meets metabolism. Exp Cell Res 2012;318(9):1057-67. 5. Shen C, Kaelin WG, Jr. The VHL/HIF axis in clear cell renal carcinoma. Semin Cancer Biol 2013;23(1):18-25. 6. Semenza GL. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 2010;29(5):625-34. 7. Kirchner H, Strumberg D, Bahl A, Overkamp F. Patient-based strategy for systemic treatment of metastatic renal cell carcinoma. Expert Rev Anticancer Ther 2010;10(4):585-96. 8. Singer EA, Bratslavsky G, Linehan WM, Srinivasan R. Targeted therapies for non-clear renal cell carcinoma. Target Oncol 2010;5(2):119-29. 9. Melillo G. Targeting hypoxia cell signaling for cancer therapy. Cancer Metastasis Rev 2007;26(2):341-52. 10. Lee K, Zhang H, Qian DZ, Rey S, Liu JO, Semenza GL. Acriflavine inhibits HIF-1 dimerization, tumor growth, and vascularization. Proc Natl Acad Sci U S A 2009;106(42):17910-5. 11. Miranda E, Nordgren IK, Male AL, Lawrence CE, Hoakwie F, Cuda F, et al. A cyclic peptide inhibitor of HIF-1 heterodimerization that inhibits hypoxia signaling in cancer cells. J Am Chem Soc 2013;135(28):10418-25. 12. Scheuermann TH, Li Q, Ma HW, Key J, Zhang L, Chen R, et al. Allosteric inhibition of hypoxia inducible factor-2 with small molecules. Nat Chem Biol 2013;9(4):271-6. 13. Kevin II DA, Meujo DAF, Hamann MT. Polyether ionophores: broad-spectrum and promising biologically active molecules for the control of drug-resistant bacteria and parasites. Expert Opinion on Drug Discovery 2009;4:109-46. 14. Kart A, Bilgili A. Ionophore Antibiotics: Toxicity, Mode of Action and Neurotoxic Aspect of Carboxylic Ionophores. Journal of Animal and Veterinary Advances 2008;7:748-51. 15. Naujokat C, Steinhart R. Salinomycin as a drug for targeting human cancer stem cells. J Biomed Biotechnol 2012;2012:950658. 16. Ketola K, Vainio P, Fey V, Kallioniemi O, Iljin K. Monensin is a potent inducer of oxidative stress and inhibitor of androgen signaling leading to apoptosis in prostate cancer cells. Mol Cancer Ther 2010;9(12):3175-85. 17. Huczynski A. Polyether ionophores-promising bioactive molecules for cancer therapy. Bioorg Med Chem Lett 2012;22(23):7002-10. 18. Wang F, Qin L, Pace CJ, Wong P, Malonis R, Gao J. Solubilized gramicidin A as potential systemic antibiotics. Chembiochem 2012;13(1):51-5. 19. Otten-Kuipers MA, Beumer TL, Kronenburg NA, Roelofsen B, Op den Kamp JA. Effects of gramicidin and tryptophan-N-formylated gramicidin on the sodium and potassium content of human erythrocytes. Mol Membr Biol 1996;13(4):225-32.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
23
20. Bourinbaiar AS, Coleman CF. The effect of gramicidin, a topical contraceptive and antimicrobial agent with anti-HIV activity, against herpes simplex viruses type 1 and 2 in vitro. Arch Virol 1997;142(11):2225-35. 21. Moll GN, van den Eertwegh V, Tournois H, Roelofsen B, Op den Kamp JA, van Deenen LL. Growth inhibition of Plasmodium falciparum in in vitro cultures by selective action of tryptophan-N-formylated gramicidin incorporated in lipid vesicles. Biochim Biophys Acta 1991;1062(2):206-10. 22. David JM, Owens TA, Barwe SP, Rajasekaran AK. Gramicidin A induces metabolic dysfunction and energy depletion leading to cell death in renal cell carcinoma cells. Mol Cancer Ther 2013;12(11):2296-307. 23. Thomas GV, Tran C, Mellinghoff IK, Welsbie DS, Chan E, Fueger B, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med 2006;12(1):122-7. 24. Gorospe M, Egan JM, Zbar B, Lerman M, Geil L, Kuzmin I, et al. Protective function of von Hippel-Lindau protein against impaired protein processing in renal carcinoma cells. Mol Cell Biol 1999;19(2):1289-300. 25. Emerling BM, Weinberg F, Liu JL, Mak TW, Chandel NS. PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a). Proc Natl Acad Sci U S A 2008;105(7):2622-7. 26. Safran M, Kim WY, O'Connell F, Flippin L, Gunzler V, Horner JW, et al. Mouse model for noninvasive imaging of HIF prolyl hydroxylase activity: assessment of an oral agent that stimulates erythropoietin production. Proc Natl Acad Sci U S A 2006;103(1):105-10. 27. Kondo K, Klco J, Nakamura E, Lechpammer M, Kaelin WG, Jr. Inhibition of HIF is necessary for tumor suppression by the von Hippel-Lindau protein. Cancer Cell 2002;1(3):237-46. 28. Yan Q, Bartz S, Mao M, Li L, Kaelin WG, Jr. The hypoxia-inducible factor 2alpha N-terminal and C-terminal transactivation domains cooperate to promote renal tumorigenesis in vivo. Mol Cell Biol 2007;27(6):2092-102. 29. Tsubuki S, Kawasaki H, Saito Y, Miyashita N, Inomata M, Kawashima S. Purification and characterization of a Z-Leu-Leu-Leu-MCA degrading protease expected to regulate neurite formation: a novel catalytic activity in proteasome. Biochem Biophys Res Commun 1993;196(3):1195-201. 30. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001;107(1):43-54. 31. Herman JG, Latif F, Weng Y, Lerman MI, Zbar B, Liu S, et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 1994;91(21):9700-4. 32. Nickerson ML, Jaeger E, Shi Y, Durocher JA, Mahurkar S, Zaridze D, et al. Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors. Clin Cancer Res 2008;14(15):4726-34. 33. Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 1999;399(6733):271-5.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
24
34. Lee K, Kang JE, Park SK, Jin Y, Chung KS, Kim HM, et al. LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1alpha via upregulation of VHL in a colon cancer cell line. Biochem Pharmacol 2010;80(7):982-9. 35. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 2007;6(4):273-86. 36. Kamura T, Brower CS, Conaway RC, Conaway JW. A molecular basis for stabilization of the von Hippel-Lindau (VHL) tumor suppressor protein by components of the VHL ubiquitin ligase. J Biol Chem 2002;277(33):30388-93. 37. Suwaki N, Vanhecke E, Atkins KM, Graf M, Swabey K, Huang P, et al. A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma. Sci Transl Med 2011;3(85):85ra47. 38. Chou MT, Anthony J, Bjorge JD, Fujita DJ. The von Hippel-Lindau Tumor Suppressor Protein Is Destabilized by Src: Implications for Tumor Angiogenesis and Progression. Genes Cancer 2010;1(3):225-38. 39. Jung CR, Hwang KS, Yoo J, Cho WK, Kim JM, Kim WH, et al. E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis. Nat Med 2006;12(7):809-16. 40. Ampofo E, Kietzmann T, Zimmer A, Jakupovic M, Montenarh M, Gotz C. Phosphorylation of the von Hippel-Lindau protein (VHL) by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription. Int J Biochem Cell Biol 2010;42(10):1729-35. 41. Kim DS, Choi YB, Han BG, Park SY, Jeon Y, Kim DH, et al. Cancer cells promote survival through depletion of the von Hippel-Lindau tumor suppressor by protein crosslinking. Oncogene 2011;30(48):4780-90. 42. Sun X, Kanwar JR, Leung E, Vale M, Krissansen GW. Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha. Gene Ther 2003;10(25):2081-9. 43. Sun X, Liu M, Wei Y, Liu F, Zhi X, Xu R, et al. Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1alpha eradicates gliomas. Cancer Gene Ther 2006;13(4):428-35. 44. Wang J, Ma Y, Jiang H, Zhu H, Liu L, Sun B, et al. Overexpression of von Hippel-Lindau protein synergizes with doxorubicin to suppress hepatocellular carcinoma in mice. J Hepatol 2011;55(2):359-68. 45. Baldewijns MM, van Vlodrop IJ, Vermeulen PB, Soetekouw PM, van Engeland M, de Bruine AP. VHL and HIF signalling in renal cell carcinogenesis. J Pathol 2010;221(2):125-38. 46. Kim CM, Vocke C, Torres-Cabala C, Yang Y, Schmidt L, Walther M, et al. Expression of hypoxia inducible factor-1alpha and 2alpha in genetically distinct early renal cortical tumors. J Urol 2006;175(5):1908-14. 47. Preston RS, Philp A, Claessens T, Gijezen L, Dydensborg AB, Dunlop EA, et al. Absence of the Birt-Hogg-Dube gene product is associated with increased hypoxia-inducible factor transcriptional activity and a loss of metabolic flexibility. Oncogene 2011;30(10):1159-73. 48. Roos FC, Evans AJ, Brenner W, Wondergem B, Klomp J, Heir P, et al. Deregulation of E2-EPF ubiquitin carrier protein in papillary renal cell carcinoma. Am J Pathol 2011;178(2):853-60.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
25
49. van Houwelingen KP, van Dijk BA, Hulsbergen-van de Kaa CA, Schouten LJ, Gorissen HJ, Schalken JA, et al. Prevalence of von Hippel-Lindau gene mutations in sporadic renal cell carcinoma: results from The Netherlands cohort study. BMC Cancer 2005;5:57. 50. Lammers T, Peschke P, Kuhnlein R, Subr V, Ulbrich K, Huber P, et al. Effect of intratumoral injection on the biodistribution and the therapeutic potential of HPMA copolymer-based drug delivery systems. Neoplasia 2006;8(10):788-95. 51. Goldberg EP, Hadba AR, Almond BA, Marotta JS. Intratumoral cancer chemotherapy and immunotherapy: opportunities for nonsystemic preoperative drug delivery. J Pharm Pharmacol 2002;54(2):159-80. 52. Sorochkina AI, Plotnikov EY, Rokitskaya TI, Kovalchuk SI, Kotova EA, Sychev SV, et al. N-terminally glutamate-substituted analogue of gramicidin A as protonophore and selective mitochondrial uncoupler. PLoS One 2012;7(7):e41919. 53. Lewis JC, Dimick KP, Feustel IC, Fevold HL, Olcott HS, Fraenkel-Conrat H. Modification of Gramicidin through Reaction with Formaldehyde. Science 1945;102(2646):274-5. 54. Krishnan V, Xu X, Barwe SP, Yang X, Czymmek K, Waldman SA, et al. Dexamethasone-loaded block copolymer nanoparticles induce leukemia cell death and enhance therapeutic efficacy: a novel application in pediatric nanomedicine. Mol Pharm 2013;10(6):2199-210.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
26
Figure Legend:
Figure 1: GA decreases HIF protein expression in RCC cells. (A) RCC cells were treated with
vehicle or GA in normoxic (21% O2) or hypoxic (1% O2) conditions for 24hr and protein
expression was measured by immunoblot. (B) RCC cells were treated with vehicle or
GA and relative transcript expression of HIF-1α (top) and HIF-2α (bottom) was measured
by qRT-PCR. Graphs depict mean±SE of three independent experiments. *, P < 0.05.
(C) Hypoxic SN12C cells were treated with vehicle or 0.5μM of the indicated ionophore
for the indicated time points and protein expression was measured by immunoblot.
Figure 2: GA blocks HIF activity and reduces HIF target expression. (A) HEK293T cells were
co-transfected with HRE-luciferase and renilla-luciferase plasmids and treated with
vehicle or GA in the absence or presence of hypoxia for 24hr before luciferase activity
was measured. *, P < 0.005, **, P < 0.00005 by T-test. (B) RCC cells were treated with
vehicle or GA in the absence or presence of hypoxia for 48hr and protein expression was
measured by immunoblot.
Figure 3: GA destabilizes HIF protein through proline hydroxylation. (A) HEK293T cells were
treated with vehicle or GA in the absence or presence of 10μM MG-132 (left) or 1mM
CoCl2 (right) and protein expression was measured by immunoblot. (B) HEK293T cells
were co-transfected with ODD-luciferase and renilla-luciferase plasmids and treated with
vehicle or GA for 24hr before luciferase activity was measured. *, P < 0.05. NS, not
significant. (C) HEK293T cells were transfected with empty vector (pcDNA3), HA-HIF-
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
27
1α-wt, or HA-HIF-1α-mut and treated with vehicle or 1μM GA for 24hr before protein
expression was measured by immunoblot.
Figure 4: GA upregulates VHL to destabilize HIF. (A) Cells were treated with vehicle or GA
for 24hr and HIF protein expression was measured by immunoblot. (B) Cells were
treated with vehicle or GA for 24hr and VHL protein expression was measured by
immunoblot. (C) Cells were treated with vehicle or GA for 24hr and relative transcript
expression was measured by qRT-PCR. *, P < 0.05.
Figure 5: GA reduces the growth of Caki-1 tumor xenografts. (A) Mice were euthanized and
tumor fluorescence from 3 representative tumors from each group were visualized. (B)
Tumors were excised and 5 representative tumors from each group were photographed.
Scale = cm. (C) Measured masses of the excised tumors. (D) Caliper measurements of
tumor growth. (E) Measurement of the body masses of the mice. Graphs depict
mean±SE of 8 mice in each group. *, P < 0.05.
Figure 6: GA reduces tumor microvasculature and HIF expression in vivo. (A) IHC staining of
representative sections from the control and GA-treated Caki-1 tumors. Magnification =
20X. Arrows indicate CD31+ microvessels. (B) Quantification of CD31+ microvessels
from ten random fields of each tumor at 40X magnification. Graph depicts mean±SD of
4 tumors from each group. *, P < 0.05. (C) Immunoblot analysis of HIF-2α and GAPDH
expression from the Caki-1 tumors.
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891
Published OnlineFirst February 3, 2014.Mol Cancer Ther Justin M. David, Tori A. Owens, Landon J. Inge, et al. Inhibition of Hypoxia-Inducible Factor in Renal Cell CarcinomaGramicidin A Blocks Tumor Growth and Angiogenesis Through
Updated version
10.1158/1535-7163.MCT-13-0891doi:
Access the most recent version of this article at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://mct.aacrjournals.org/content/early/2014/02/01/1535-7163.MCT-13-0891To request permission to re-use all or part of this article, use this link
on July 15, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 3, 2014; DOI: 10.1158/1535-7163.MCT-13-0891