Ionophores are hydrophobic organic molecules that disrupt cellular transmembrane potential by permeabilizing membranes to specific ions. Gramicidin A is a channel-forming ionophore that forms a hydrophilic membrane pore that permits the rapid passage of monovalent cations. Previously, we found that gramicidin A 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 gramicidin A inhibits HIF in RCC cells. We found that gramicidin A 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 gramicidin A 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, gramicidin A reduced the growth of human RCC xenograft tumors without causing significant toxicity in mice. Gramicidin A–treated tumors also displayed physiologic and molecular features consistent with the inhibition of HIF-dependent angiogenesis. Taken together, these results demonstrate a new role for gramicidin A as a potent inhibitor of HIF that reduces tumor growth and angiogenesis in VHL-expressing RCC. Mol Cancer Ther; 13(4); 788–99. ©2014 AACR.

Kidney cancer is a relatively rare but deadly disease that is among the top 10 causes of cancer-related deaths in men in the United States (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 RCC (ccRCC; 70%), papillary RCC (pRCC; 10%–15%), chromophobe (5%), and collecting duct (<1%), is associated with unique morphologic and genetic characteristics (3).

RCC characteristically exhibits molecular and biochemical features associated with chronic responses to low oxygen (hypoxia; ref. 4). Adaptation to hypoxia is mediated by an O2-sensitive transcription factor known as hypoxia-inducible factor (HIF; ref. 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; ref. 4). Hydroxylation of HIF-α creates a binding interface for the von Hippel–Lindau tumor suppressor protein (VHL) that 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 endothelial cells (e.g., sunitinib, sorafenib, bevacizumab, etc.) are now routinely used for patients with ccRCC, 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 about their utility for non-ccRCC subtypes as these patients were excluded from clinical trials (8).

Another antiangiogenesis therapeutic strategy is to target HIF directly, and several points of regulation have been exploited to develop novel HIF-inhibiting agents. These drugs include (i) mTOR inhibitors (rapamycin, temsirolimus, and everolimus) that interfere with the translation of HIF-α subunit transcripts; (ii) histone deacetylase inhibitors, HSP90 inhibitors, and nonsteroidal anti-inflammatory drugs that enhance HIF-α subunit protein degradation; (iii) anthracyclines (doxorubicin and daunorubicin) and DNA-intercalating agents (echinomycin) that interfere with the binding of HIF to DNA; and (iv) 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 radiosensitization, 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).

In contrast with the mobile carriers, use of channel formers as antitumor agents has not been widely evaluated. Gramicidin A is a prototypical channel-forming ionophore that forms a 4-Å pore that can accommodate water, protons, and monovalent cations. Channel formation results in Na+ influx, K+ efflux, osmotic swelling, and cell lysis in biologic systems (18, 19) and confers gramicidin A with potent antibiotic activity against gram-positive bacteria, fungi, and protozoa (20, 21). We have previously demonstrated that gramicidin A is toxic to RCC cells and induces metabolic dysfunction and cellular energy depletion (22). In this study, we investigated whether treatment with gramicidin A specifically affects HIF in RCC cells. We found that gramicidin A 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 gramicidin A 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 gramicidin A 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 (i) specifically inhibit HIF-dependent hypoxia responses, and (ii) specifically upregulate a tumor suppressor (VHL). Our results reveal an additional “targeted” role for gramicidin A as a potent inhibitor of HIF and suggest its utility as an antiangiogenic therapeutic agent for RCC tumors that express wild-type VHL.

Cell culture

Human ccRCC (A498, 786-O, SN12C, Caki-1, UMRC6, and UMRC6 + VHL), pRCC (ACHN), and embryonic kidney (HEK293T) cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 2 mmol/L 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) with a regulated environment of 1% O2, 5% CO2, and 94% N2 at 37°C. Of note, 786-O, Caki-1, and HEK293T cells were purchased from the American Type Culture Collection 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 before use.

Reagents

The following chemicals were purchased from Sigma-Aldrich; gramicidin A, monensin, valinomycin, calcimycin (A23187), MG132, and cobalt chloride.

Antibodies

We purchased primary antibodies specific for HIF-1α (BD Biosciences), HIF-2α, CA-IX (carbonic anhydrase IX; Novus Biologicals), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), α-tubulin, HA (Cell Signaling Technology), GLUT-1 (glucose transporter 1; Alpha Diagnostic International), β-actin (Sigma-Aldrich), and VHL (EMD Chemicals). Horseradish peroxidase (HRP)–conjugated secondary antibodies were purchased from Cell Signaling Technology.

Plasmids and transfections

Plasmids pGL2-HRE-luciferase (Addgene plasmid 26731; ref. 25), pcDNA3-ODD-luciferase (Addgene plasmid 18965; ref. 26), pcDNA3-HA-HIF1α (Addgene plasmid 18949; ref. 27), and pcDNA3-HA-HIF1α-P402A/P564A (Addgene plasmid 18955; ref. 28) were purchased from Addgene. Plasmid pcDNA3 vector was purchased from Life Technologies and plasmid phRL-renilla was purchased from Promega Corporation. Transient transfections were accomplished using lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Transfection of Caki-1 cells with td-Tomato-N1 (Clonetech) was accomplished by electroporation with a Nucleofector II (Lonza) using Kit V according to the manufacturer's instructions. Cells were examined using a Leica DMI microscope (Leica Microsystems) and single cells expressing red fluorescent protein were picked after 2 weeks of selection with 800 μg/mL G418 (Geneticin) to establish stable cell lines. These cells were used for in vivo studies.

Immunoblot analysis

Cell lysates were prepared in a buffer containing 95 mmol/L NaCl, 25 mmol/L Tris pH 7.4, 0.5 mmol/L EDTA, and 2% SDS. Tumor lysates were prepared by mincing tumor samples and then lysing in a buffer containing 150 mmol/L NaCl, 20 mmol/L Tris pH 7.4, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 1% IGEPAL (octylphenoxypolyethoxyethanol), 1 mmol/L β-glycerol phosphate, 1 mmol/L Na3VO4, 2.5 mmol/L Na4P2O7, 50 mmol/L NaF, and 12 mmol/L deoxycholate. Lysates were sonicated, centrifuged, and the protein concentrations of the supernatants were determined using the detergent-compatible protein assay (Bio-Rad). Equal amounts of protein were then resolved by SDS–PAGE and transferred to nitrocellulose. The membranes were blocked in 5% nonfat milk in TBS 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% nonfat milk/TBS-T at room temperature for 1 hour. The protein bands were visualized using Amersham ECL Prime (GE Healthcare). Images were acquired using Photoshop (Adobe Systems Inc.) and relative quantification was performed using ImageJ (NIH, Bethesda, MD).

Quantitative real-time PCR

Total RNA was extracted using TRizol reagent (Life Technologies) and reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad) as per the respective manufacturer's instructions. The cDNA was amplified via real-time PCR (RT-PCR) using the SYBR Green PCR Master Mix (Applied Biosystems). 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′; and GAPDH 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). Variation in cDNA loading was normalized to GAPDH expression, which remained constant at the 24 hours incubation periods used, and relative expression was determined using the ΔΔCt method of relative quantification. Graphs represent the average relative quantification value with error bars (SE of the relative quantification value) from one representative of three independent experiments. Graphs were generated using the GraphPad Prism Software (GraphPad Software).

Luciferase activity assay

HEK293T cells were cotransfected with 100-ng phRL-renilla and 2 μg of pGL2-HRE-luciferase or 1 μg of pcDNA3-ODD-luciferase using lipofectamine 2000 and incubated for 24 hours before drug treatment. Following drug treatment, the Dual-Luciferase Reporter Assay (Promega Corporation) was performed according to the manufacturer's instructions. Briefly, lysates were prepared using the provided buffer and then diluted 1:10, then 2 μL of diluted sample lysate was added in triplicate to a white-walled 96-well plate, mixed with 100 μL of firefly luciferase assay substrate, and luminescence was immediately recorded using a VictorX4 plate reader (PerkinElmer). Then 100 μL 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.

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- to 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 (gramicidin A) groups of 8 mice each with an average initial tumor volume of approximately 85 mm3 in each group. Gramicidin A (0.22 mg/kg body weight) was diluted in a 1:1 solution of ethanol and saline, and mice were dosed thrice weekly with 50 μL of either vehicle or gramicidin A 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 |${\rm (length} \times {\rm width}^{\rm 2} {\rm)/2}$|⁠, 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) using the following settings: excitation (EX), 550 nm; emission (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 samples of vehicle and gramicidin A–treated tumors were prepared using routine procedures. Of note, 5-μm sections were floated onto charged slides and dried for 1 hour at 65°C. Following deparaffinization in xylene and graded alcohols, tissues underwent heat-induced epitope retrieval using the Decloaking Chamber and Reveal Decloaking Buffer (Biocare Medical) according to the recommended manufacturer's protocols. The VHL polyclonal antibody (#PA5-17477; Thermo Fisher Scientific) and polyclonal CD31 (#LS-B1932; LifeSpan Biosciences) were diluted in 1% BSA (Sigma-Aldrich) and applied overnight at 4°C. Slides were incubated with PromARK anti-rabbit HRP polymer (BioCare Medical) and stained with diaminobenzidine using the Betazoid DAB Kit (Biocare Medical) according to the recommended manufacturer's protocols. Nuclei were stained with hematoxylin (EMD Millipore) and Bluing Reagent (Thermo Fisher Scientific), cleared, and mounted for microscopic analysis.

Statistical analysis

Quantitative RT-PCR (qRT-PCR) results were analyzed using one-way ANOVA followed by the Dunnett multiple comparison test. All other analyses were performed using the two-tailed unpaired Student t test.

Gramicidin A reduces HIF-1α and HIF-2α protein expression

Because constitutive activation HIF is central to RCC pathogenesis, we investigated whether gramicidin A affects the expression of HIF in RCC cells. Using Caki-1, SN12C, and ACHN cell lines, we found that treatment with gramicidin A for 24 hours provoked a dose-dependent decrease in the expression of both HIF-1α and HIF-2α protein in these cell lines (Fig. 1A, left). Because HIF-α subunits are stabilized by hypoxia (1% O2), we next assessed whether gramicidin A reduces HIF-α expression in hypoxia. Exposure to 1% O2 dramatically increased HIF-1α and HIF-2α as expected, but treatment with gramicidin A prevented this increase in a dose-dependent manner (Fig. 1A, right). Strikingly, 1 μmol/L gramicidin A 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 gramicidin A significantly altered transcript expression for only HIF-2α in SN12C cells (P = 0.01 by one-way ANOVA; Fig. 1B), suggesting that gramicidin A primarily affects only HIF protein levels. Finally, we assessed whether mobile-carrier ionophores also reduce hypoxic HIF protein expression. We compared equal doses (0.5 μmol/L) of gramicidin A with monensin (Na+-selective), valinomycin (K+-selective), and calcimycin (Ca2+-selective) in hypoxic SN12C cells. We observed that monensin slightly reduced HIF-1α and HIF-2α at 72 hours, valinomycin moderately reduced both proteins from 24 to 72 hours, and calcimycin had no effect on either protein (Fig. 1C). Only gramicidin A elicited a profound decrease in both isoforms that persisted from 24 to 72 hours (Fig. 1C). These data reveal that only gramicidin A is a potent inhibitor of HIF-1α and HIF-2α protein expression.

Figure 1.

Gramicidin A (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 24 hours and protein expression was measured by immunoblot analysis. 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, mean ± SE of three independent experiments. *, P < 0.05. C, hypoxic SN12C cells were treated with vehicle or 0.5 μmol/L of the indicated ionophore for the indicated time points and protein expression was measured by immunoblot analysis.

Figure 1.

Gramicidin A (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 24 hours and protein expression was measured by immunoblot analysis. 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, mean ± SE of three independent experiments. *, P < 0.05. C, hypoxic SN12C cells were treated with vehicle or 0.5 μmol/L of the indicated ionophore for the indicated time points and protein expression was measured by immunoblot analysis.

Close modal

Gramicidin A reduces HIF transcriptional activity and target gene expression

Next, we analyzed the effect of gramicidin A upon the transcriptional activity of HIF. We used 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 gramicidin A 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, GLUT-1, and GAPDH were all decreased by gramicidin A 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 gramicidin A attenuates hypoxia responses by preventing the transcriptional activation of HIF-responsive genes.

Figure 2.

GA blocks HIF activity and reduces HIF target expression. A, HEK293T cells were cotransfected with HRE–luciferase and renilla–luciferase plasmids and treated with vehicle or GA in the absence or presence of hypoxia for 24 hours 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 48 hours and protein expression was measured by immunoblot analysis.

Figure 2.

GA blocks HIF activity and reduces HIF target expression. A, HEK293T cells were cotransfected with HRE–luciferase and renilla–luciferase plasmids and treated with vehicle or GA in the absence or presence of hypoxia for 24 hours 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 48 hours and protein expression was measured by immunoblot analysis.

Close modal

Gramicidin A destabilizes HIF through proline hydroxylation

O2-dependent downregulation of HIF-α depends upon the proteasome to degrade ubiquitylated HIF. To elucidate whether gramicidin A uses this mechanism, we first measured HIF expression in HEK293T cells treated with increasing doses of gramicidin A in the absence or presence of the well-known proteasomal inhibitor MG-132 (10 μmol/L; ref. 29). Treatment with gramicidin A failed to reduce HIF-1α and HIF-2α protein expression in cells treated with MG-132 (Fig. 3A, left) indicating that gramicidin A 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 (1 mmol/L) stabilized HIF-1α and HIF-2α as expected but completely blocked destabilization of these proteins by gramicidin A (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 gramicidin A–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 gramicidin A 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; ref. 27) or ODD-mutant HIF-1α (HA-HIF1α-P402A/P564A; ref. 28). Treatment of these cells revealed that wild-type HIF-1α but not ODD-mutant HIF-1α was reduced by gramicidin A (Fig. 3C). Altogether, these results demonstrate that gramicidin A uses the O2-dependent regulatory mechanism to destabilize HIF protein via PHD-dependent hydroxylation of its ODD.

Figure 3.

GA destabilizes HIF protein through proline hydroxylation. A, HEK293T cells were treated with vehicle or GA in the absence or presence of 10 μmol/L MG-132 (left) or 1 mmol/L CoCl2 (right) and protein expression was measured by immunoblot analysis. B, HEK293T cells were cotransfected with ODD–luciferase and renilla–luciferase plasmids and treated with vehicle or GA for 24 hours before luciferase activity was measured. *, P < 0.05. NS, not significant. C, HEK293T cells were transfected with empty vector (pcDNA3), HA-HIF-1α-wt, or HA-HIF-1α-mut and treated with vehicle or 1 μmol/L GA for 24 hours before protein expression was measured by immunoblot analysis.

Figure 3.

GA destabilizes HIF protein through proline hydroxylation. A, HEK293T cells were treated with vehicle or GA in the absence or presence of 10 μmol/L MG-132 (left) or 1 mmol/L CoCl2 (right) and protein expression was measured by immunoblot analysis. B, HEK293T cells were cotransfected with ODD–luciferase and renilla–luciferase plasmids and treated with vehicle or GA for 24 hours before luciferase activity was measured. *, P < 0.05. NS, not significant. C, HEK293T cells were transfected with empty vector (pcDNA3), HA-HIF-1α-wt, or HA-HIF-1α-mut and treated with vehicle or 1 μmol/L GA for 24 hours before protein expression was measured by immunoblot analysis.

Close modal

Gramicidin A 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 gramicidin A destabilizes HIF through proline hydroxylation and proteasomal degradation. To ascertain whether VHL is involved in gramicidin A–mediated degradation of HIF, we used the naturally VHL-deficient ccRCC cell line UMRC6 and found that gramicidin A 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, right). HIF-1α expression was undetectable in this cell line. These data demonstrate that VHL is essential for gramicidin A–mediated destabilization of HIF.

Figure 4.

GA upregulates VHL to destabilize HIF. A, cells were treated with vehicle or GA for 24 hours and HIF protein expression was measured by immunoblot analysis. B, cells were treated with vehicle or GA for 24 hours and VHL protein expression was measured by immunoblot analysis. C, cells were treated with vehicle or GA for 24 hours and relative transcript expression was measured by qRT-PCR. *, P < 0.05.

Figure 4.

GA upregulates VHL to destabilize HIF. A, cells were treated with vehicle or GA for 24 hours and HIF protein expression was measured by immunoblot analysis. B, cells were treated with vehicle or GA for 24 hours and VHL protein expression was measured by immunoblot analysis. C, cells were treated with vehicle or GA for 24 hours and relative transcript expression was measured by qRT-PCR. *, P < 0.05.

Close modal

Although hypoxia reduces proline hydroxylation of HIF, it does not completely abolish it (34). Because gramicidin A treatment reduced HIF expression even in hypoxic conditions (Fig. 1) and uses the O2-dependent degradation mechanism (Fig. 3), we speculated that gramicidin A enhances a component of this pathway to accelerate HIF destabilization. We investigated this possibility and observed that treatment with gramicidin A 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 gramicidin A inhibits HIF by enhancing VHL expression.

Gramicidin A 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 gramicidin A may reduce tumor growth in vivo by disrupting tumor angiogenesis. We previously found that gramicidin A reduced the in vivo growth SN12C tumor xenografts in mice (22). To evaluate the antiangiogenic efficacy of gramicidin A, 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 approximately 85 mm3, the mice were randomized into two groups (each n = 8) and administered 50 μL of either vehicle solution or gramicidin A (0.22 mg/kg) by intratumoral injection thrice weekly for 26 days. As shown in Fig. 5A and B, the control tumors were noticeably larger than the gramicidin A–treated tumors. We found that the average mass of the gramicidin A–treated tumors was 52% less than that of the control tumors (Fig. 5C; P = 0.017 by t test). Analysis of tumor growth revealed that the tumors of the gramicidin A group essentially failed to grow once treatment with gramicidin A 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 gramicidin A 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 gramicidin A inhibits the growth of VHL-expressing RCC tumors.

Figure 5.

GA reduces the growth of Caki-1 tumor xenografts. A, mice were euthanized and tumor fluorescence from three representative tumors from each group were visualized. B, tumors were excised and five 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, mean ± SE of 8 mice in each group. *, P < 0.05.

Figure 5.

GA reduces the growth of Caki-1 tumor xenografts. A, mice were euthanized and tumor fluorescence from three representative tumors from each group were visualized. B, tumors were excised and five 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, mean ± SE of 8 mice in each group. *, P < 0.05.

Close modal

To confirm that reduced tumor growth was due to a blockade of tumor angiogenesis, we histologically examined the tumor tissue. Gramicidin A–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 gramicidin A–treated tumors (vehicle = 7.13 ± 0.18 vs. gramicidin A = 3.04 ± 0.54; P = 0.0004; Fig. 6A and B). In agreement with these data, immunoblot analysis revealed that HIF-2α and GAPDH protein expression was also substantially reduced in the gramicidin A–treated tumors (Fig. 6C). HIF-1α was not detectable by immunoblot analysis 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 gramicidin A inhibits tumor growth through the suppression of HIF-dependent angiogenesis.

Figure 6.

GA reduces tumor microvasculature and HIF expression in vivo. A, immunohistochemical staining of representative sections from the control and GA-treated Caki-1 tumors. Magnification, ×20. Arrows, CD31+ microvessels. B, quantification of CD31+ microvessels from 10 random fields of each tumor at ×40 magnification. Graph, mean ± SD of four tumors from each group. *, P < 0.05. C, immunoblot analysis of HIF-2α and GAPDH expression from the Caki-1 tumors.

Figure 6.

GA reduces tumor microvasculature and HIF expression in vivo. A, immunohistochemical staining of representative sections from the control and GA-treated Caki-1 tumors. Magnification, ×20. Arrows, CD31+ microvessels. B, quantification of CD31+ microvessels from 10 random fields of each tumor at ×40 magnification. Graph, mean ± SD of four tumors from each group. *, P < 0.05. C, immunoblot analysis of HIF-2α and GAPDH expression from the Caki-1 tumors.

Close modal

Here, we report for the first time that gramicidin A is a novel inhibitor of tumor angiogenesis. We have demonstrated that treatment with gramicidin A 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 gramicidin A has therapeutic potential as an angiogenesis inhibitor for VHL-positive RCCs and possibly for other cancers that express VHL.

Gramicidin A–mediated destabilization of HIF-α subunits required both proline hydroxylation and VHL expression indicating that gramicidin A used the O2;-dependent degradation mechanism to target HIF. Strikingly, gramicidin A 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 gramicidin A 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 gramicidin A 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 gramicidin A increases VHL expression remains to be elucidated. We previously reported that treatment of RCC cells with gramicidin A 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 gramicidin A–treated cells indicating that either the translation of VHL transcripts or the stability of VHL protein is increased by gramicidin A. 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, and cullin 2; ref. 36). Our results clearly show that VHL was active in mediating the degradation of HIF in gramicidin A–treated cells, so it is possible that gramicidin A 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 gramicidin A–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 and colleagues 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 gramicidin A failed to reduce HIF-1α and HIF-2α expression in VHL-deficient cells implying that gramicidin A may not be effective as an antiangiogenic therapy for patients with ccRCC with functional inactivation of VHL. However, constitutive activation of HIF is also a characteristic of certain nonclear cell RCC subtypes (45–48) even though VHL mutation is exceedingly rare in these tumors (49). Because VHL is functional in these subtypes, gramicidin A is likely to have therapeutic utility in this traditionally underserved patient population (8). Furthermore, gramicidin A 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 gramicidin A by either intravenous or intraperitoneal injection was lethal to mice. However, we found that repeated intratumoral injection of gramicidin A 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 (noninjected) tumors by enhancing the processing of tumor-associated antigens expressed in cell debris from the injected tumor (51). Nevertheless, systemic administration of gramicidin A may be possible in the near future. Chemical modification of gramicidin A has been shown to change the characteristics of the peptide enough to increase microbial targeting and/or decrease nonspecific toxicity (18, 19, 52, 53), and encapsulation of gramicidin A within a targeted drug carriers such as nanoparticles may be a plausible method to safely deliver gramicidin A to only the tumor (54). Whether these approaches can be effectively applied to the use of gramicidin A as a novel cancer therapy is an essential area of future investigation.

J.M. David and A.K. Rajasekaran have ownership interest in a patent. No potential conflicts of interest were disclosed by the other authors.

Conception and design: J.M. David, A.K. Rajasekaran

Development of methodology: J.M. David, A.K. Rajasekaran

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.M. David, T.A. Owens, L.J. Inge, A.K. Rajasekaran

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J.M. David, T.A. Owens, L.J. Inge, A.K. Rajasekaran

Writing, review, and/or revision of the manuscript: J.M. David, T.A. Owens, L.J. Inge, R.M. Bremner, A.K. Rajasekaran

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.A. Owens, A.K. Rajasekaran

Study supervision: A.K. Rajasekaran

The authors thank 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). The authors also thank Dr. Sonali P. Barwe and Vinu Krishnan for technical assistance in conducting the in vivo work.

This work was supported by NIH grants P20GM103464, R01 DK56216, and Nemours Foundation (to A.K. Rajasekaran), Heart and Lung Research Initiative, St. Joseph's Foundation (to R.M. Bremner and L.J. Inge).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Siegel
R
,
Naishadham
D
,
Jemal
A
. Cancer statistics, 2012
.
CA Cancer J Clin
2012
;
62
:
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
:
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
:
133
55
.
4.
Haase
VH
. 
Renal cancer: oxygen meets metabolism
.
Exp Cell Res
2012
;
318
:
1057
67
.
5.
Shen
C
,
Kaelin
WG
 Jr
. 
The VHL/HIF axis in clear cell renal carcinoma
.
Semin Cancer Biol
2013
;
23
:
18
25
.
6.
Semenza
GL
. 
Defining the role of hypoxiacite-inducible factor 1 in cancer biology and therapeutics
.
Oncogene
2010
;
29
:
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
:
585
96
.
8.
Singer
EA
,
Bratslavsky
G
,
Linehan
WM
,
Srinivasan
R
. 
Targeted therapies for nonclear renal cell carcinoma
.
Target Oncol
2010
;
5
:
119
29
.
9.
Melillo
G
. 
Targeting hypoxia cell signaling for cancer therapy
.
Cancer Metastasis Rev
2007
;
26
:
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
:
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
:
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
:
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 Opin Drug Discov
2009
;
4
:
109
46
.
14.
Kart
A
,
Bilgili
A
. 
Ionophore antibiotics: toxicity, mode of action, and neurotoxic aspect of carboxylic ionophores
.
J Anim Vet Adv
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
:
3175
85
.
17.
Huczynski
A
. 
Polyether ionophores-promising bioactive molecules for cancer therapy
.
Bioorg Med Chem Lett
2012
;
22
:
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
:
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
:
225
32
.
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
271
5
.
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
:
982
9
.
35.
Folkman
J
. 
Angiogenesis: an organizing principle for drug discovery?
Nat Rev Drug Discov
2007
;
6
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
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
:
853
60
.
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
:
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
:
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
:
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
:
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
:
2199
210
.