Gramicidin a Induces Metabolic Dysfunction and Energy Depletion Leading to Cell Death in Renal Cell Carcinoma Cells

Published OnlineFirst September 4, 2013; DOI: 10.1158/1535-7163.MCT-13-0445

Molecular Cancer Small Molecule Therapeutics Therapeutics

Gramicidin A Induces Metabolic Dysfunction and Energy Depletion Leading to Cell Death in Renal Cell Carcinoma Cells

Justin M. David1,2, Tori A. Owens2, Sonali P. Barwe2, and Ayyappan K. Rajasekaran2

Abstract Ionophores are lipid-soluble organic molecules that disrupt cellular transmembrane potential by rendering biologic membranes permeable to speciﬁc ions. They include mobile-carriers that complex with metal cations and channel-formers that insert into the membrane to form hydrophilic pores. Although mobile-carriers possess anticancer properties, investigations on channel-formers are limited. Here, we used the channel- forming ionophore gramicidin A to study its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional treatments. We found that gramicidin A reduced the in vitro viability of several RCC cell lines at submicromolar concentrations < m (all IC50 1.0 mol/L). Gramicidin A exhibited similar toxicity in RCC cells regardless of histologic subtype or the expression of either the von Hippel-Lindau tumor suppressor gene or its downstream target, hypoxia- inducible factor-1a. Gramicidin A decreased cell viability equal to or greater than the mobile-carrier monensin depending on the cell line. Mechanistic examination revealed that gramicidin A blocks ATP generation by inhibiting oxidative phosphorylation and glycolysis, leading to cellular energy depletion and nonapoptotic cell death. Finally, gramicidin A effectively reduced the growth of RCC tumor xenografts in vivo. These results show a novel application of gramicidin A as a potential therapeutic agent for RCC therapy. Mol Cancer Ther; 12(11); 2296–307. 2013 AACR.

Introduction resistance, sensitize cells to chemo-/radiotherapy, and Ionophores are highly hydrophobic molecules that even interfere with oncogenic signaling (10). permeabilize membranes to various cations. They differ Gramicidin A is the simplest and best-characterized channel-forming ionophore. Produced by the bacterial based on cation specificity and mechanism of transport. Bacillus brevis Mobile-carriers physically associate with individual species and discovered in 1939 by Dr. Rene cations to form a complex that diffuses across lipid Dubos (11, 12), gramicidin A was the very first antibiotic bilayers, whereas channel-formers incorporate into the tested in a clinical setting (13). It is a linear 15-residue peptide of alternating L- and D-amino acids (Fig. 1A) and membrane to form hydrophilic transmembrane nano- b pores that permit rapid diffusion of cations through the adopts a -helix conformation within the lipid bilayer in membrane. Several ionophores are naturally produced which two gramicidin A monomers dimerize end-to-end ˚ and exhibit potent antibiotic activity (1). Research over the to form a functional nanopore of 4 A that spans the past decade has described a role for mobile-carrier iono- membrane (Fig. 1B; ref. 14). The gramicidin A channel is wide enough to permit the diffusion of water and inor- phores as anticancer therapeutics; monensin induces cell- þ þ cycle arrest and apoptosis in various cancer cell lines (2–7) ganic monovalent cations, resulting in Na influx, K and acts as a radiosensitizing agent (8), and salinomycin efflux, osmotic swelling, and cell lysis (14, 15). This confers preferentially targets breast cancer stem cells (CSC) and gramicidin A with potent antibiotic activity against bac- reduces in vivo growth and metastasis formation (9). Many teria, fungi, and protozoa (11, 12, 16, 17). However, groups have now reported selective targeting of CSCs by whether gramicidin A also possesses anticancer proper- salinomycin in a wide range of additional malignancies ties akin to the mobile-carrier ionophores has not been (10). Moreover, salinomycin can overcome multiple-drug established. Kidney cancer is a devastating disease that is among the top 10 causes of deaths related to cancer in men in the Authors' Affiliations: 1Department of Biological Sciences, University of United States (18). The majority (80%–85%) of kidney Delaware, Newark; and 2Nemours Center for Childhood Cancer Research, Alfred I. duPont Hospital for Children, Wilmington, Delaware cancers are classified as various histologic subtypes of renal cell carcinoma (RCC; ref. 19). RCC is characteristi- Corresponding Author: Ayyappan K. Rajasekaran, Nemours Center for Childhood Cancer Research, A.I. duPont Hospital for Children, 1701 Rock- cally resistant to both chemo- and radiotherapy, and the 5- land Road, Wilmington, DE 19803. Phone: 302-651-6593; Fax; 302-651- year disease-specific survival rate for invasive RCC is only 4827; E-mail: [email protected] 10% (19, 20). RCC development and progression is pri- doi: 10.1158/1535-7163.MCT-13-0445 marily due to chronic activation of the cellular response 2013 American Association for Cancer Research. to low oxygen (hypoxia; refs. 21, 22). Hypoxia-inducible

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Figure 1. Gramicidin A structure and function. A, chemical structure of the gramicidin A peptide. B, dimerization of two gramicidin A monomers produces the functional transmembrane channel. factor (HIF) is the master transcriptional regulator of ed by Dr. Michael I. Lerman (National Cancer Institute, hypoxia responses, and its expression is regulated by Bethesda, MD) in 2000 (25). All cell lines obtained from cellular oxygen levels. In normoxic conditions, HIF is investigators have been authenticated before use. hydroxylated and then bound by the von Hippel-Lindau (VHL) tumor suppressor protein that promotes its poly- Reagents ubiquitylation and subsequent degradation (23). Howev- Gramicidin A, monensin, ouabain, and rhodamine 123 er, HIF becomes stabilized in hypoxia as low oxygen were purchased from Sigma-Aldrich. prevents protein hydroxylation. Constitutive (i.e., oxy- Plasmids and transfections gen-insensitive) activation of the HIF transcriptional pro- pcDNA3-HA-HIF-1a (Addgene plasmid 18949; Wil- gram occurs in RCC via the functional inactivation of VHL liam Kaelin, Howard Hughes Medical Insititute, Dana- in the clear cell RCC subtype, or through various VHL- Farber Cancer Institute and Brigham and Women’s Hos- independent means in the other RCC subtypes (19). pital, Harvard Medical School, Boston, MA; ref. 26), Here, we have conducted the first systematic study of pcDNA3-HA-HIF-1a-P402A/P564A (Addgene plasmid the cytotoxic effects of gramicidin A in RCC cell lines. We 18955; William Kaelin; ref. 27) were purchased from report that disruption of cationic homeostasis by grami- Addgene. pcDNA3 vector was purchased from Life Tech- cidin A is toxic to RCC cells regardless of histologic nologies. Transfections were accomplished using Lipo- subtype or HIF expression/activity. We found that gram- fectamine 2000 (Life Technologies) according to the man- icidin A impaired cellular metabolism by disrupting both ufacturer’s instructions. oxidative phosphorylation and glycolysis, leading to profound energy depletion and subsequent nonapoptotic cell Antibodies in vivo death. Gramicidin A also reduced the growth of We purchased primary antibodies specific for HIF-1a RCC tumor xenografts without causing significant toxic- (BD Biosciences), HIF-2a (Novus Biologicals), hemagglu- ity in mice. These findings identify gramicidin A as a new tinin (HA)-tag, glyceraldehyde-3-phosphate dehydroge- potential anticancer therapeutic for RCC. nase (GAPDH), cleaved-PARP, a-tubulin, AMP-activated Materials and Methods protein kinase (AMPK), phospho-AMPK (T172), acetyl- coA carboxylase (ACC), phospho-ACC (S79; Cell Signal- Cell culture ing Technology), and b-actin (Sigma-Aldrich). Horserad- Human clear cell RCC (A498, 786-O, Caki-1, SN12C, ish peroxidase (HRP)–conjugated secondary antibodies þ UMRC6, and UMRC6 VHL), papillary RCC (ACHN), were purchased from Cell Signaling Technology. embryonic kidney (HEK293T), and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco’s Mod- Cell viability assay ified Eagle Medium (DMEM) supplemented with 10% FBS, The CellTiter-Blue cell viability assay was conducted 2 mmol/L L-glutamine, 25 U/mL penicillin, and 25 mg/mL according to the manufacturer’s instructions (Promega streptomycin. 786-O, Caki-1, HEK293T, and MDCK cells Corporation). Cells were seeded in triplicate into 96-well were purchased from the American Type Culture Collec- plates and allowed to attach overnight. Cells were then tion in 1995. A498, SN12C, and ACHN cells were kindly treated with vehicle (ethanol) or drug and incubated for 72 provided by Dr. Charles L. Sawyers (Memorial Sloan- hours. Next, 20 mL of CellTiter-Blue reagent was added to Kettering Cancer Center, New York City, NY) in 2005 each well and incubated for additional 4 hours. Fluores- (24). UMRC6 and UMRC6þVHL cells were kindly provid- cence was then read at 560 nm using a VictorX4 plate reader

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(PerkinElmer, Inc.). Readings were corrected for back- obtained using a VictorX4 plate reader (PerkinElmer, ground fluorescence using the fluorescence value from Inc.). Readings were corrected for background absorbance the media only and relative cell viability was normal- using the absorbance value from the media only. Percent- ized to vehicle-treated samples. Data represent the age cell death was calculated using the equation: %LDH ¼ þ mean SE of three independent experiments. The data released (A490 from medium)/(A490 from plate A490 obtained were further analyzed using Prism nonlinear from medium) 100, and was normalized to vehicle- regression software (GraphPad Software, Inc.) for treated samples. Data represent the mean SE of three curve fitting and determination of absolute IC50 values. independent experiments.

Immunoblot analysis DNA fragmentation assay Cell lysates were prepared in a buffer containing 95 Cells were seeded into 100-mm dishes, incubated over- mmol/L NaCl, 25 mmol/L Tris pH 7.4, 0.5 mmol/L night, and then treated with vehicle or drug for 48 hours. EDTA, and 2% SDS. Lysates were sonicated, centrifuged, Following the treatment, the cells were removed by scrap- and the protein concentrations of the supernatants were ing the dishes, washed in 1 PBS, and lysed in lysis buffer determined using the detergent compatible (DC) protein (100 mmol/L Tris–HCl pH 7.5, 10 mmol/L EDTA, 0.06% assay (Bio-Rad). Equal amounts of protein were then SDS). DNA was precipitated overnight at 4C by adding resolved by SDS-PAGE and transferred to nitrocellulose. NaCl to a final concentration of 1 mmol/L. The lysate was The membranes were blocked in 5% nonfat milk in TBS then thawed, centrifuged to spin down the high-molecu- with 0.1% Tween 20 (TBS-T) and then incubated overnight lar weight DNA, and the supernatant was subjected to at 4C with primary antibodies diluted in 5% bovine phenol/chloroform extraction. The low-molecular weight serum albumin (BSA)/TBS-T. The following day, the DNA contained in the aqueous phase was precipitated membranes were washed and incubated with HRP-con- with 100% ethanol, pelleted, washed with 70% ethanol, jugated secondary antibodies diluted in 5% nonfat milk/ and resuspended in 20 mL TE buffer (10 mmol/L Tris– TBS-T at room temperature for 1 hour. The protein bands HCl, 1 mmol/L EDTA, pH 7.4) containing RNase A at 20 were visualized using Amersham ECL Prime (GE Health- mg/mL for 30 minutes at room temperature. Samples were care). Following visualization, immunoblots of phosphor- electrophoresed on a 1.2% agarose gel and ethidium ylated proteins were stripped from the nitrocellulose bromide staining of the samples were visualized under membrane by washing with buffer containing 50 UV light and photographed using a Geliance 600 Imaging mmol/L Tris pH 8.0, 2% SDS, and 0.7% 2-mercaptoetha- System (PerkinElmer, Inc.). nol at 55C, and then immunoblotted for unphosphory- lated total proteins as described earlier. Images were Cellular ATP measurement generated using Photoshop (Adobe Systems, Inc.) and The CellTiter-Glo luminescent viability assay was used quantified using ImageJ (NIH, Bethesda, MD). to quantify cellular ATP and was conducted according to the manufacturer’s instructions (Promega). Briefly, cells Phase-contrast microscopy were seeded in triplicate into white-walled 96-well plates Phase-contrast images were acquired using a Leica DM and treated with thymidine (1 mmol/L) overnight to IL microscope equipped with a DCF 420 C camera and block cell proliferation and maintain a constant cell num- Leica Application Suite Software (Leica Microsystems ber. Cells were then treated with vehicle (ethanol) or drug GmbH). Images were processed using Photoshop (Adobe in culture media containing 1 mmol/L thymidine and Systems, Inc.). incubated for 24 hours. Plates were then equilibrated to room temperature for 30 minutes and then 100 mLof Cell death assay CellTiter-Glo reagent was added to each well. Plate con- The Cytotox96 nonradioactive assay was conducted tents were mixed for 2 minutes using an orbital shaker and according to the manufacturer’s instructions (Promega). then incubated an additional 10 minutes at room temper- Briefly, cells were seeded in triplicate into 12-well plates in ature before luminescence was recorded using a VictorX4 phenol-free DMEM supplemented with 0.1% to 0.5% FBS plate reader (PerkinElmer, Inc.). Luminescence values and allowed to attach overnight. Cells were then treated were corrected for background luminescence using the with vehicle (ethanol) or gramicidin A for 72 hours. reading from the media only, and corrected values were Conditioned medium was collected to assay for lactate normalized to vehicle-treated samples to calculate the dehydrogenase (LDH) released from the dead cells (A490 relative ATP levels. Data represent the mean SE of three from medium). An equivalent volume of medium was independent experiments. replaced in the plate and incubated at 80C for 60 minutes to lyse cells. This medium was then thawed and Redox activity assay collected to be assayed for LDH released from the viable Cells were seeded in triplicate into 96-well plates and cells. Samples were clarified by centrifugation and 50 mL treated with thymidine (1 mmol/L) overnight to block cell aliquots were added to 50 mL LDH substrate in a 96-well proliferation and maintain a constant cell number. Cells plate. The reaction was terminated after 30 minutes using were then treated with vehicle (ethanol) or drug in culture 50 mL stop solution and absorbance values at 490 nm were media containing 1 mmol/L thymidine and incubated for

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24 hours. Cellular redox activity was measured using the Biosciences) solution. SN12C tumors were allowed to CellTiter-Blue assay as described earlier. grow for 1 week before randomization into control (vehicle solution only) and drug (gramicidin A) groups of 5 Mitochondrial transmembrane potential assay mice each with an average initial tumor volume of approx- Cells were seeded into 60-mm dishes and treated with imately 100 mm3, as determined by caliper measurement thymidine (1 mmol/L) overnight to block cell prolifera- using the formula [(length width2)/2] where length was tion and maintain a constant cell number. Cells were then the longest of the two measurements. Gramicidin A (0.11 treated with vehicle (ethanol) or drug in culture media mg/kg body weight) was diluted in a 1:1 solution of containing 1 mmol/L thymidine and incubated for 24 ethanol and saline (0.9% NaCl), and mice were dosed hours. Cells were then washed twice with 1 PBS and twice weekly with 50 mL of either vehicle or gramicidin A then incubated with 10 mg/mL rhodamine 123 in 1 PBS solutions by intratumoral injection. Mouse body masses m supplemented with 100 mol/L CaCl2, 1 mmol/L MgCl2, were recorded before each injection. Upon completion of 0.2% BSA, and drug (gramicidin A or monensin) for 30 the study, mice were euthanized, the tumors were surgi- minutes at 37C in the dark. After incubation, the cells cally excised, and masses were recorded. were washed twice with 1 PBS and then lysed in 200 mL of 1 PBS supplemented with 0.02% SDS for 15 minutes. Statistical analysis Lysates were collected by scraping, added to the wells of a Dose-response viability curves were conducted by 96-well plate, and fluorescence was read at 535 nm using a ANOVA, all other analyses were conducted using VictorX4 plate reader (PerkinElmer, Inc.). Fluorescence unpaired two-tailed Student t test. values were corrected for background fluorescence using the measurement from the media only, and corrected Results values were normalized to vehicle-treated samples to Treatment with gramicidin A reduces the viability of calculate the relative fluorescence. Data represent the RCC cells in vitro mean SE of three independent experiments. The viability of several different cancer cell lines has been shown to be sensitive to treatment with mobile- Glycolysis assay carrier ionophores. However, the effect of channel-form- The Glycolysis Cell-Based Assay Kit was used to quan- ing ionophores upon RCC cell viability is not known. We tify extracellular L-lactate and was conducted according to measured the in vitro viability of four ccRCC cell lines the manufacturer’s instructions (Cayman Chemical Com- (A498, 786-O, Caki-1, and SN12C) and one papillary RCC pany). Cells were seeded in duplicate into 96-well plates, cell line (ACHN; ref. 28) treated with gramicidin A, and allowed to attach for 4 hours, and then treated with found that it caused a dose-dependent decrease in cell thymidine (1 mmol/L) overnight to block cell prolifera- viability (Fig. 2A). These cell lines responded similarly to tion and maintain a constant cell number. Cells were then gramicidin A as the absolute IC50 values for all cell lines treated with vehicle (ethanol) or drug in phenol-free were less than1 mmol/L (Table 1). This result shows that DMEM supplemented with 0.5% FBS and 1 mmol/L the viability of RCC cells is sensitive to treatment with thymidine. The cells were divided into two treatment gramicidin A. groups of either 0 to 12 or 12 to 24 hours. For the 12- to 24-hour samples, the media was removed after the initial Neither VHL nor HIF-1a expression significantly 12 hours of incubation and replaced with fresh media to alters cellular sensitivity to gramicidin A incubate for an additional 12 hours. Following incubation, Our analysis of cell viability revealed that the IC50 10 mL of conditioned media was mixed with the kit values of the VHL-deficient ccRCC cell lines (A498, 786- reactants in a 96-well plate and absorbance values at O) were higher than those of the VHL-expressing ccRCC 490 nm were obtained using a VictorX4 plate reader cell lines (Caki-1 and SN12C; Table 1). Given the signif- (PerkinElmer, Inc.). Readings were corrected for back- icance of VHL in renal cancer pathology, we sought to ground absorbance using the absorbance value from the ascertain whether its expression affects cellular sensitivity culture medium only and the corrected values were to gramicidin A in vitro. We first compared the viability of applied to a standard curve to calculate extracellular gramicidin A–treated isogenic UMRC6 cells (VHL-defi- lactate levels. Data represent the mean SE of three cient) with that of VHL-reconstituted UMRC6þVHL cells. independent experiments. We found that these cell lines responded very similarly to gramicidin A (Fig. 2B) yet the dose–response curves just Tumor growth assay barely achieved statistical significance (P ¼ 0.0403 by two- Animal experiments were carried out according to the way ANOVA). Further analysis revealed that although þ NIH guidelines and approved by the Nemours Institu- the UMRC6 VHL cells had a slightly higher IC50 value tional Animal Care and Use Committee and Institutional than the parental UMRC6 cells (Table 1), this difference Biosafety Committee. Female hairless Nu/J mice (Charles was not statistically significantly different (P ¼ 0.190 by t River Laboratories) that were 6- to 8-week old were test). In addition, because VHL deficiency stabilizes HIF, injected subcutaneously with a suspension of SN12C cells we also examined whether overexpression of HIF-1a (1.0 106) in a 50% growth factor–reduced Matrigel (BD affects the cellular response to gramicidin A. HEK293T

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Figure 2. Gramicidin A (GA) reduces the viability of RCC cell lines. A, RCC cells lines were treated with increasing doses of gramicidin A for 72 hours and cell viability was measured. B, stable isogenic UMRC6 and UMRC6þVHL cells were treated with increasing doses of gramicidin A for 72 hours and cell viability was measured. C, immunoblot of transfected HEK293T cells used in D to conﬁrm transgene expression. D, HEK293T cells were transiently transfected with the indicated plasmids and treated with increasing doses of gramicidin A for 72 hours and cell viability was measured.

cells were transiently transfected with either empty vector Gramicidin A affects cell viability comparable to (pcDNA3), HA-tagged wild-type HIF-1a (HA-HIF- monensin 1a; 26), or HA-tagged constitutively active mutant HIF- Next, we sought to compare the viability of RCC cells 1a (HA-HIF-1a-mut) in which prolines 402 and 564 had treated with either gramicidin A or the mobile-carrier been mutated to alanines to abrogate O2-dependent ionophore monensin. We selected monensin for this com- downregulation by VHL (Fig. 2C; ref. 27). No differences parison because (i) monensin has been shown to inhibit in the dose-response viability curves of these transfected the growth of RCC cells (2) and (ii) both monensin and þ þ cell lines were found in response to treatment with gram- gramicidin A exhibit selectivity for Na and K ions (29). icidin A (Fig. 2D; P ¼ 0.988 by one-way ANOVA). Anal- Using both VHL-deficient (786-O) and VHL-expressing ysis of the IC50 values (Table 1) also failed to yield any (SN12C) cell lines, we found that gramicidin A and mon- statistically significant differences (P > 0.5 for all possible ensin caused a dose-dependent decrease in cell viability as t combinations by test). Altogether, our results imply that expected (Fig. 3A and B). Although the absolute IC50 value gramicidin A reduces the viability of RCC cells in vitro in a for gramicidin A–treated 786-O cells was slightly less manner that is independent of both VHL and HIF-1a than monensin (0.430 vs. 0.622 mmol/L), the difference expression. in the dose-response was not found to be statistically significant (P ¼ 0.528 by two-way ANOVA). Conversely, we found that the viability of SN12C cells was 13.9-fold more sensitive to treatment with gramicidin A than with Table 1. Calculated IC50 values for gramicidin ¼ m A–treated cell lines monensin (IC50 0.104 vs. 1.443 mol/L), which was highly statistically significant (P < 0.0001 by two-way ANOVA). These data suggest that channel-forming iono- m Cell line IC50, mol/L phores reduce the viability of RCC cells comparable to or A498 0.420 greater than mobile-carrier ionophores in a cell-type– 786-O 0.430 specific manner. Caki-1 0.228 SN12C 0.104 Gramicidin A induces nonapoptotic cell death in RCC ACHN 0.783 cells UMRC6 0.253 Several investigations have reported that mobile-car- UMRC6þVHL 0.425 rier ionophores (i.e., monensin and salinomycin) induce HEK293TþpcDNA3 0.057 apoptotic cell death in cancer cells. Microscopic analysis HEK293TþHA-HIF-1a 0.058 of monensin-treated 786-O cells confirmed the presence HEK293TþHA-HIF-1a-mut 0.067 of typical apoptotic morphologic features such as cell rounding, membrane blebbing, and vacuolization (Fig. 4A,

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Figure 3. Gramicidin A (GA) reduces viability comparable to monensin (MON). A and B, 786-O (A) and SN12C (B) cells were treated with increasing doses of either gramicidin A or monensin for 72 hours and cell viability was measured. Graphs depict mean SE of three independent experiments.

middle). However, gramicidin A–treated 786-O cells than in 786-O cells, which is consistent with the higher did not display these characteristics but instead fea- sensitivity of this cell line to gramicidin A (Fig. 5A tured prominent lamellipodia (arrows) and rounding and Table 1). Importantly, gramicidin A decreased of the nuclei (Fig. 4A, right). In addition, these cells cellular ATP levels to a greater extent than monensin showed floating cellular debris (data not shown). Sim- in SN12C cells at 1 mmol/L (gramicidin A ¼ 18% 2% ilar characteristics were also observed in SN12C cells vs. monensin ¼ 41% 14%) and at 10 mmol/L in both (data not shown). To further analyze the cytotoxicity of cell lines (786-O ¼ 45% 10% vs. 70% 2%; SN12C ¼ gramicidin A, we measured the cell death–associated 10% 1% vs. 24% 0.3%). These observations show release of the stable cytosolic enzyme LDH. Treatment that gramicidin A depletes cellular energy in RCC of 786-O, SN12C, and Caki-1 cells (data not shown) with cells. gramicidin A resulted in a dose-dependent increase in Cellular energy homeostasis is regulated by AMPK. extracellular LDH (Fig. 4B), confirming that gramicidin This kinase is activated by an elevation of the intracellular A does induce cell death in RCC cells. Next, we sought ratio of AMP–ATP, which occurs in stressful conditions to verify our observations in Fig. 4A that gramicidin A (31). We investigated the activation of the AMPK pathway seems to elicit a nonapoptotic form of cell death. A in gramicidin A–treated 786-O and SN12C cells as a common marker of apoptosis is the proteolytic cleavage marker of cellular energy stress. Immunoblot analysis of PARP. Immunoblot analysis detected PARP cleavage revealed that treatment with gramicidin A increased the in monensin-treated 786-O and SN12C cells but not in phosphorylation of AMPKa and its substrate ACC at both gramicidin A–treated cells (Fig. 4C). Another indicator 24 and 48 hours (Fig. 5B). AMPKa phosphorylation was of apoptosis is the degradation of cellular chromatin more pronounced in SN12C cells than 786-O cells at 24 into nucleosome-length fragments that appear as a hours, which is consistent with the previously observed DNA "ladder" when separated by electrophoresis. Con- greater sensitivity of this cell line to ATP depletion (Fig. sistent with Fig. 4C, we observed electrophoretic DNA 5A). This observed activation of the AMPK pathway laddering in only monensin-treated 786-O and SN12C confirms that gramicidin A induces cellular energy stress cells (Fig. 4D). Altogether, these data signify that gram- in RCC cells. icidin A induces a nonapoptotic form of cell death in Next, we investigated whether cellular energy deple- RCC cells. tion caused by gramicidin A is due to the disruption of þ þ Na and K homeostasis. The Na,K-ATPase pump is a Gramicidin A depletes cellular energy in RCC cells key regulator of intracellular ionic homeostasis that trans- þ þ Although gramicidin A and monensin enhance the ports 3Na out of the cell and 2K into the cell against þ þ permeability of cells to Na and K , we were surprised their respective electrochemical gradients (32) Inhibition to find that these drugs induced differing forms of cell of the Na,K-ATPase blocks the active transport of these þ þ death. A potential determinant of cell death mechanism cations and permits the influx of Na and efflux of K is cellular energy status; apoptosis is an ATP-depen- similar to gramicidin A (33). Using the Na,K-ATPase dent process of self-degradation, whereas necrosis is inhibitor ouabain (0.5 mmol/L), we measured cellular typically associated with rapid bioenergetic compro- ATP and observed that ouabain treatment mimicked mise and energy insufficiency (30). To evaluate wheth- gramicidin A and produced a marked reduction in cel- er gramicidin A depletes cellular energy, we measured lular ATP in both 786-O (6% 2%) and SN12C (30% the ATP content of 786-O and SN12C cells treated with 15%) cell lines (Fig. 5C). Furthermore, combination treat- increasing doses of either gramicidin A or monensin. ment with both gramicidin A and ouabain decreased As depicted in Fig. 5A, we observed that treatment cellular ATP levels more than either drug alone (Fig. þ with gramicidin A for 24 hours decreased cellular ATP 5C). These results indicate that disruption of Na and þ in both 786-O and SN12C cell lines. ATP reduction by K balance is involved in gramicidin A–induced energy gramicidin A was more pronounced in SN12C cells depletion in RCC cells.

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Figure 4. Gramicidin A (GA) induces nonapoptotic cell death. A, phase- contrast images of 786-O cells treated with vehicle (ethanol) or 10 mmol/L of either monensin (MON) or gramicidin A for 48 hours; scale bar, 100 mmol/L. B, RCC cell lines were treated with increasing doses of gramicidin A for 72 hours and cell death was measured by LDH release assay. Graph depicts mean SE of three independent experiments; , P < 0.05 by t test. C, 786-O and SN12C cells were treated with either gramicidin A or monensin for 48 hours and immunoblotted for the presence of the 89 kDa cleavage product of PARP. D, 786-O and SN12C cells were treated with either gramicidin A or monensin for 48 hours and low-molecular weight DNA was isolated and analyzed for electrophoretic laddering. Positive control, DNA from serum-starved MDCK cells.

Gramicidin A induces metabolic dysfunction in RCC and produced only minimal inhibition in 786-O cells cells (gramicidin A ¼ 36% 3% vs. monensin ¼ 75% 6% at The profound loss of cellular energy that resulted from 10 mmol/L). These observations indicate that gramici- the treatment with gramicidin A may indicate that ATP din A interferes with general cellular metabolism in production was impaired. Because cellular metabolism RCC cells. depends upon oxidation–reduction (redox) reactions, we Proliferating cells produce ATP almost exclusively first measured whether gramicidin A interferes with from glucose via oxidative phosphorylation and glycoly- redox activity in RCC cells. We used the redox-sensitive sis. Oxidative phosphorylation occurs within mitochon- dye resazurin (CellTiter-Blue), which is reduced into dria and depends upon the establishment and mainte- highly fluorescent resorufin in the presence of metabol- nance of a proton gradient between the inner and outer ically active cells. Treatment of 786-O and SN12C cells mitochondrial membranes to energetically couple elec- with gramicidin A for 24 hours resulted in a significant tron transport with ATP synthesis. Gramicidin A is well 30% to 60% reduction in the cellular redox activity at known to conduct protons and depolarize mitochondrial all doses (P < 0.05; Fig. 6A). In contrast, monensin failed transmembrane potential, thereby blocking ATP synthe- to significantly reduce the activity in SN12C cells sis (34). To determine whether gramicidin A interferes

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Figure 5. Gramicidin A (GA) depletes cellular energy. A, 786-O and SN12C cells were treated with increasing doses of either gramicidin A or monensin (MON) for 24 hours and cellular ATP was measured. B, 786-O and SN12C cells were treated with gramicidin A or monensin and AMPK pathway activation was assessed by immunoblot. C, cells were treated with either 5 mmol/L gramicidin A (786-O) or 0.5 mmol/L gramicidin A (SN12C), 0.5 mmol/L ouabain (oua, both cell lines), or combination for 24 hours and cellular ATP was measured. Graphs depict mean SE of three independent experiments; , P < 0.05; , P < 0.005 by t test.

with mitochondrial function, we measured the uptake of 786-O cells showed a significant increase (þ92% 40.3%) the cell-permeable dye rhodamine 123 (Rh123), which in lactate production (Fig. 6C), whereas no change was accumulates within polarized mitochondria (35). As observed in SN12C cells (Fig. 6D). To further probe this shown in Fig. 6B, treatment with gramicidin A for 24 effect, we divided the 24-hour incubation into two 12-hour hours resulted in a 40% to 60% reduction in Rh123 uptake periods and found that gramicidin A induced an initial in 786-O and SN12C cells. In contrast, monensin did not increase in lactate levels in both cell lines during the first 0- significantly alter the Rh123 uptake in either cell line. This to 12-hour period (786-O ¼ 236% 17.7%; SN12C ¼ 136% result indicates that gramicidin A depolarizes mitochon- 14.8%; Fig. 6C and D). However, this elevation was not drial membrane potential and decreases oxidative phos- sustained as lactate levels fell significantly during the phorylation-dependent ATP synthesis. subsequent 12- to 24-hour period (786-O ¼ 83% Next, we sought to determine whether gramicidin A 13.4%; SN12C ¼ 23% 3.3%; Fig. 6C and D). The increased impairs glycolysis in RCC cells by measuring the produc- accumulation of lactate at 12 hours suggests that glycol- tion of extracellular L-lactate, the end product of glycolysis was initially stimulated but later inhibited by gram- ysis. Surprisingly, at 24 hours, the gramicidin A–treated icidin A.

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Figure 6. Gramicidin A (GA) impairs cellular metabolism. A, 786-O and SN12C cells were treated with gramicidin A or monensin (MON) for 24 hours and cellular redox activity was measured. B, 786-O and SN12C cells were treated with gramicidin A or monensin for 24 hours and mitochondrial transmembrane potential was measured. Graphs depict mean SE of three independent experiments. C and D, 786-O (C) and SN12C (D) cells were treated with 5 mmol/L or 1.0 mmol/L gramicidin A, respectively, for the indicated time periods and the production of extracellular lactate was measured. Graph depicts mean SD of one representative of three independent experiments. , P < 0.05; , P < 0.005 by t test; NS, not signiﬁcant.

Gramicidin A inhibits the growth of RCC tumor and administered 50 mL of either vehicle solution (50% xenografts ethanol in saline) or gramicidin A (0.11 mg/kg) by intra- To evaluate the in vivo antitumor efﬁcacy of gramicidin tumoral injection twice weekly for 14 days. As shown A, we engrafted human SN12C RCC cells by subcutane- in Fig. 7A, at necropsy, the average tumor mass was ous injection into female Nu/J athymic nude mice. When reduced by approximately 40% with gramicidin A treat- the tumors reached an average size of approximately 100 ment (230 24 mg vs. 140 20 mg; P ¼ 0.0228 by t test). mm3, the mice were randomized into two groups (n ¼ 5) Importantly, intratumoral injection of gramicidin A did

Figure 7. Gramicidin A (GA) reduces the growth of SN12C tumor xenografts. A, xenograft tumors were excised from euthanized mice and the mass of each tumor was measured. B, measurement of the average body masses of the mice. Graphs depict the mean SE of n ¼ 5 in each group.

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Cytotoxicity of Gramicidin A in Renal Cell Carcinoma

not elicit significant toxicity as no changes in the activity Cellular energy depletion can occur if the plasma mem- levels or the average body masses of the mice were brane is damaged and ATP leaks out of the cell (38). We observed (Fig. 7B). investigated this possibility but failed to detect ATP in the culture supernatant from gramicidin A–treated cells (data Discussion not shown). Enhanced ATP consumption can also con- In this study, we provide the first evidence that treat- tribute to the depletion of energy. The Na,K-ATPase is a ment with the channel-forming ionophore gramicidin A is very energy-intensive enzyme that uses more than 50% of toxic to RCC cells in vitro and in vivo. This finding is the cellular energy in the kidney (39) and it is stimulated þ þ significant because RCC is a highly therapy-resistant by the imbalance of Na and K provoked by gramicidin malignancy. Mechanistically, we showed that gramicidin A (29). However, we found that the inhibition of Na,K- A impairs oxidative phosphorylation and glycolysis lead- ATPase with ouabain failed to rescue gramicidin A– ing to cellular ATP depletion and nonapoptotic cell death. induced ATP depletion but actually enhanced it. We þ þ Disruption of Na and K homeostasis by gramicidin A is finally assessed whether gramicidin A blocks ATP gen- likely involved in this depletion of energy. Our findings eration as the sheer multitude of ATP-consuming pro- suggest that gramicidin A may have therapeutic potential cesses makes cells vulnerable to energy depletion once for the treatment of RCC and possibly other solid tumors. ATP synthesis is halted. We found that gramicidin A Constitutive activation of HIF plays a prominent role in reduced cellular redox activity, disrupted mitochondrial RCC pathophysiology. Using a panel of various VHL- transmembrane potential, and decreased extracellular lac- positive and -negative RCC cell lines, we found that tate production, indicating that gramicidin A impairs glu- gramicidin A reduced the in vitro viability of all of these cose catabolism. The inhibition of glycolysis likely accounts cell lines similarly. We also used stably and transiently for the bulk of energy depletion by gramicidin A, because transfected cell lines to manipulate VHL and HIF-1a RCC cells are known to have a distinct bioenergetic expression, respectively, and failed to find any significant organization in which ATP production is shifted heavily differences in the response of these cells to gramicidin A. in favor of glycolysis (23, 40). However, we cannot at this Furthermore, we observed that gramicidin A reduced time rule out a loss of energy from other sources (amino cellular energy and metabolism similarly in VHL-defi- acids, fatty acids, etc.) as contributing to gramicidin cient (786-O) and VHL-expressing (SN12C) cells. These A–induced energy stress. results show that histologic subtype, VHL status, and HIF- Although gramicidin A disrupts oxidative phosphory- 1a expression/activity are not major factors that deter- lation directly through energetic uncoupling (34), the mine the cellular response to gramicidin A. However, precise mechanism whereby gramicidin A initially stimu- given the prominent role of VHL/HIF in regulating tumor lates and then subsequently inhibits glycolysis is currently angiogenesis, it is possible that the in vivo response of RCC not understood. Glycolysis is likely stimulated to com- cells to gramicidin A will differ based on these factors. pensate for the loss of energy production by oxidative Experiments to test this possibility are currently under- phosphorylation. AMPK is known to increase glycolysis way in our laboratory. to replenish cellular energy in stressed cells (31). It is Monensin has been reported to induce cell-cycle arrest possible that the observed activation of AMPK might be and apoptosis in RCC cells (2) and we also observed involved in the stimulation of glycolysis. In addition, þ morphologic and molecular features consistent with apo- glycolysis depends upon the reduction of NAD to ptotic cell death in monensin-treated RCC cells. However, NADH, and we observed that gramicidin A produced a gramicidin A seems to induce a different cell death mech- marked decrease in cellular redox activity. It is possible anism. Despite obvious cytotoxicity, we did not observe that gramicidin A reduces glycolysis by preventing þ apoptotic features (membrane blebbing, PARP cleavage, NAD /NADH cycling or, alternatively, by activating þ and DNA laddering) in gramicidin A–treated cells. In enzymes that catalyze NAD -consuming ADP-ribosyla- addition, marked cellular energy depletion and inhibition tion reactions such as PARP (which remained intact in of glycolysis are both features of necrotic cell death (30), gramicidin A–treated cells). and both of these were observed in gramicidin A–treated Toxicity is an essential factor to consider in clinical drug cells. Furthermore, treatment of RCC cells with necrosta- development, yet most of the chemotherapeutics used tin-1, an inhibitor of necroptosis (36), failed to rescue clinically have substantial nonspecific toxicity toward gramicidin A–induced cell death (data not shown). Col- normal cells. Gramicidin A is known to cause hemolysis lectively, our observations signify that gramicidin A and is toxic to the liver, kidney, meninges, and olfactory induces a necrotic form of cell death in RCC cells. Necrosis apparatus (14, 41). We tested the toxicity of gramicidin A is viewed negatively because it provokes a robust inflam- using nontumorigenic kidney cell lines from various matory response, yet necrotic inflammation may also species [LLC-PK1 (porcine), HK-2 (human), and MDCK attract host leukocytes, thereby enhancing tumor antigen (canine)] and observed IC50 values that were comparable presentation and antitumor immunity (30, 37). Thus, to those of the RCC cell lines (data not shown). However, gramicidin A–induced necrotic cell death might also we have shown here that gramicidin A was both safe and facilitate antitumor immune responses in RCC tumors effective via intratumoral injection in mice. Intratumoral and must be addressed by future studies. administration improves the therapeutic index of drugs

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by increasing the tumor-to-organ ratio, and advances in Analysis and interpretation of data (e.g., statistical analysis, biostatis- tics, computational analysis): J.M. David, T.A. Owens, S.P. Barwe, A.K. the use of X-ray–computed tomography have made this Rajasekaran route of administration possible for even metastatic Writing, review, and/or revision of the manuscript: J.M. David, T.A. Owens, A.K. Rajasekaran and/or inoperable tumors (42). Alternatively, mutation Administrative, technical, or material support (reporting or organizing and/or chemical modification of the gramicidin A peptide data, constructing datases): A.K. Rajasekaran may be used to decrease the nonspecific toxicity (14), or Study supervision: A.K. Rajasekaran encapsulation of gramicidin A within an amphiphilic Acknowledgments drug carrier may also be used to simultaneously increase The authors thank Dr. Andrew Napper for providing the CellTiter-Glo solubility, decrease toxicity, and improve tumor targeting reagent and Dr. Landon Inge for providing the p-ACC and total ACC (43). Careful evaluation of these options will allow gram- antibodies. icidin A to reach its full therapeutic potential. Grant Support This study was supported by NIH grant, R01 DK56216, (A.K. Rajase- Disclosure of Potential Conflicts of Interest karan), P20GM103464 (Thomas Shaffer), and a grant by Nemours Foun- No potential conflicts of interest were disclosed. dation (A.K. Rajasekaran). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Authors' Contributions advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Conception and design: J.M. David, A.K. Rajasekaran this fact. Development of methodology: J.M. David Acquisition of data (provided animals, acquired and managed patients, Received June 3, 2013; revised August 9, 2013; accepted August 28, 2013; provided facilities, etc.): J.M. David, T.A. Owens, S.P. Barwe published OnlineFirst September 4, 2013.

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www.aacrjournals.org Mol Cancer Ther; 12(11) November 2013 2307

Gramicidin A Induces Metabolic Dysfunction and Energy Depletion Leading to Cell Death in Renal Cell Carcinoma Cells

Justin M. David, Tori A. Owens, Sonali P. Barwe, et al.

Mol Cancer Ther 2013;12:2296-2307. Published OnlineFirst September 4, 2013.

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