Gramicidin A Blocks Tumor Growth and Angiogenesis Through ... · 1 Gramicidin A Blocks Tumor Growth and Angiogenesis Through Inhibition of Hypoxia-Inducible Factor in Renal Cell Carcinoma

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: [email protected] 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

http://mct.aacrjournals.org/

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.




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




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).




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.




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-




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%




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




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.




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




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.




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:




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




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,




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




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.




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




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




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




20

effectively applied to the use of GA as a novel cancer therapy is an essential area of future

investigation.




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.




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.




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.




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.




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.




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-




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.






















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

[email protected] 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



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-13-0891

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/early/2014/02/01/1535-7163.MCT-13-0891


Documents

Gramicidin A Blocks Tumor Growth and Angiogenesis Through ... · 1 Gramicidin A Blocks Tumor Growth and Angiogenesis Through Inhibition of Hypoxia-Inducible Factor in Renal Cell Carcinoma