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Therapeutics, Targets, and Chemical Biology

Mitochondria-Targeted Drugs Synergize with2-Deoxyglucose to Trigger Breast Cancer Cell Death

GangCheng1, Jacek Zielonka1, Brian P. Dranka1, DonnaMcAllister1, A. CraigMackinnon Jr2, Joy Joseph1, andBalaraman Kalyanaraman1

AbstractCancer cells are long known to exhibit increased aerobic glycolysis, but glycolytic inhibition has not offered a

viable chemotherapeutic strategy in part because of the systemic toxicity of antiglycolytic agents. However, recentstudies suggest that a combined inhibition of glycolysis andmitochondrial functionmay help overcome this issue.In this study, we investigated the chemotherapeutic efficacies of mitochondria-targeted drugs (MTD) incombination with 2-deoxy-D-glucose (2-DG), a compound that inhibits glycolysis. Using the MTDs, termedMito-CP and Mito-Q, we evaluated relative cytotoxic effects and mitochondrial bioenergetic changes in vitro.Interestingly, both Mito-CP and Mito-Q synergized with 2-DG to decrease ATP levels in two cell lines. However,with time, the cellular bioenergetic function and clonogenic survival were largely restored in some cells. In axenograft model of human breast cancer, combined treatment of Mito-CP and 2-DG led to significant tumorregression in the absence of significant morphologic changes in kidney, liver, or heart. Collectively, our findingssuggest that dual targeting ofmitochondrial bioenergeticmetabolismwithMTDs and glycolytic inhibitors such as2-DG may offer a promising chemotherapeutic strategy. Cancer Res; 72(10); 2634–44. �2012 AACR.

IntroductionEmerging research in cancer chemotherapy is focused on

exploiting the biochemical differences between cancer cell andnormal cell metabolism (1, 2). One of the fundamental changesthat occurs inmostmalignant cancer cells is the shift in energymetabolism from oxidative phosphorylation to aerobic glycol-ysis to generate ATP (the Warburg effect; refs. 3–5). Severalagents that specifically inhibit glycolyticmetabolismhave beenused as effective anticancer agents in cellular systems and inanimal models (5, 6). However, this approach has yielded fewerpositive results in human patients (with the exception ofimatinib), most likely due to dose-limiting side effects (e.g.,neurotoxicity; ref. 7). One of the most frequently used anti-glycolytic agents is 2-deoxy-D-glucose (2-DG), which is phos-phorylated by hexokinase and subsequently inhibits ATPgenerated via the glycolytic pathway (7, 8). However, highconcentrations (�20 mmol/L) of 2-DG were typically usedto inhibit the glycolytic metabolism in cancer cells (9). 2-DGis undergoing clinical trials for treatment of glioma and its

efficacy is limited by the systemic toxicity (10). A recentstrategy to "hypersensitize" tumor cells involved the com-bined use of mitochondrial inhibitors (oligomycin and anti-mycin) or delocalized cationic compounds with 2-DG (11,12). Dual targeting of mitochondrial and glycolytic pathwayswas suggested as a promising chemotherapeutic strategy(13, 14).

Recent work has revealed that cancer-promoting oncogenesand hypoxia-inducible factor (HIF-1a) also induce a glycolyticshift (15, 16). Activation of oncogenic signaling pathwaysinvolving PI3K/Akt/mTOR, c-Myc, Src, and Ras leads toenhanced glucose uptake and high glycolytic activity mimick-ing theWarburg effect in cancer cells (17, 18). Thus, targeting ofboth mitochondrial bioenergetic function and the glycolysispathway is an attractive experimental chemotherapeutic strat-egy. Previously, investigators have used agents (e.g., rhoda-mine-123, rotenone, antimycin, or oligomycin) that inhibitmitochondrial oxidative phosphorylation in combination withglycolytic inhibitors to eradicate tumor cells (19). In contrast, asimilar treatment (rhodamine-123 and 2-DG) failed to elicitcytotoxic effects in normal epithelial cells (20). The rationalefor this approach is that compromised oxidative phosphory-lation in tumor cells leads to stimulation of glycolytic metab-olism for ATP generation. Dichloroacetate (DCA), a pyruvatemimetic that inhibits pyruvate dehydrogenase kinase, shiftscellular metabolism from glycolysis to OXPHOS (21, 22). How-ever, DCA was selectively more toxic in cells with defectivemitochondrial electron transport chain (22, 23). As indicatedearlier, mitochondrial targeting of drugs combined with 2-DGor other glycolytic inhibitors may be an extremely effectivestrategy to eliminate slow growing hypoxic tumor cells presentin most solid tumors (12).

Authors' Affiliations: 1Department of Biophysics and Free RadicalResearch Center, Medical College of Wisconsin, Milwaukee, WI; and2Department of Pathology, Medical College of Wisconsin, Milwaukee,Wisconsin

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Balaraman Kalyanaraman, Department of Bio-physics and Free Radical Research Center, Medical College of Wisconsin,8701WatertownPlankRoad,Milwaukee,WI 53226. Phone: 414-456-4000;Fax: 414-456-6512; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3928

�2012 American Association for Cancer Research.

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Published OnlineFirst March 19, 2012; DOI: 10.1158/0008-5472.CAN-11-3928

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The accumulation of mitochondria-targeted delocalizedlipophilic cations such as Mito-CP (Fig. 1) in tumor cells iscontrolled by several factors including the lipophilicity and thechain length of mitochondria-targeted drugs (MTD), the neg-ative mitochondrial membrane potential, and the levels of p-glycoprotein-mediated MDR protein that actively pumps cat-ionic drugs out of the tumor cells (24). Recent research showedthatMito-Q (coenzyme-Q conjugated to an alkyl triphenylpho-sphonium cation) and Mito-CP (a 5-membered nitroxide, CP,conjugated to a TPPþ) potently inhibited proliferation ofbreast cancer cells (MCF-7 andMDA-MB-231) or human coloncancer cells (HCT-116; refs. 25, 26). These MTDs did notsignificantly affect normal cell proliferation (25). In relatedstudies, we have shown that theMTDs effectivelymitigated thetoxic side effects (cardiotoxicity or nephrotoxicity) induced byconventional chemotherapeutics such as doxorubicin or cis-platin (27). These results led us to postulate the followinghypothesis: The combined use of MTDs (Mito-CP and Mito-Q)and 2-DGwill synergistically enhance cytotoxicity selectively incancer cells compared with either drug alone. In this study, weinvestigated the chemotherapeutic effects of 2-DG in combi-nation with Mito-CP, Mito-Q, and other appropriate controlcompounds. Results from this study indicate that targetingmitochondria with small molecular weight antioxidants con-jugated to an alkyl TPPþ and glycolytic inhibitors is a poten-tially promising and effective cancer treatment strategy.

Materials and MethodsChemicalsMito-CPandMito-Qweresynthesizedaspreviously published

(refs. 28, 29; see Fig. 1 for chemical structures). 2-DG, carboxy-proxyl (CP), andmethyl triphenylphosphonium(Me-TPPþ)werepurchased from Sigma-Aldrich. D-Luciferin sodium salt wasobtained fromCaliper Life Sciences, Inc. (1-Decyl) triphenylpho-sphonium bromide (Dec-TPPþ) was obtained from Alfa Aesar.

Cell cultureMCF-7, MCF-10A, and MDA-MB-231 cells were acquired in

the last 2 years from the American Type Culture Collection,

where they are regularly authenticated. Cells were stored inliquid nitrogen and used within 6 months after thawing. Celllines were grown at 37�C in 5% CO2. MCF-7 cells were main-tained in MEM-a (Invitrogen) containing 10% FBS, bovineinsulin (10 mg/mL), penicillin (100 U/mL), and streptomycin(100 mg/mL). MCF-10A cells were cultured in Dulbecco'sModified Eagle's Medium (DMEM)/F12 media (1:1; Invitro-gen) supplemented with 5% horse serum, bovine insulin(10 mg/mL), epidermal growth factor (20 ng/mL), choleratoxin (100 ng/mL), and hydrocortisone (0.5 mg/mL), penicillin(100U/mL), and streptomycin (100mg/mL).MDA-MB-231 cellswere cultured in DMEM, 10% FBS, penicillin (100 U/mL), andstreptomycin (100 mg/mL). The MDA-MB-231-luc cell linestably transfected with luciferase was cultured under the sameconditions as the MDA-MB-231 cells described above. Thesecells were a kind gift from Dr. Michael B. Dwinell (Departmentof Microbiology and Molecular Genetics, Medical Collegeof Wisconsin, Milwaukee, WI) and were recently described indetail (30). They are regularly assessed for standard growthcharacteristics and tumorigenicity in nude mice.

Extracellular flux assayThe bioenergetic function of MCF-7 and MCF-10A cells in

response toMito-CP or 2-DGwas determined using a SeahorseBioscience XF24 Extracellular Flux Analyzer (Seahorse Biosci-ence). MCF-7 or MCF-10A cells were seeded in specialized V7Seahorse tissue culture plates. One hour before the start of theexperiment, cells were washed and changed to unbufferedassay medium adjusted to pH 7.4, final volume 675 mL(MEM-a forMCF-7, DMEM/F12 forMCF-10A). After establish-ing the baseline oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR), Mito-CP (1 mmol/L) or 2-DG(5 mmol/L) were administered through an automated pneu-matic injection port of XF24. The changes in OCR and ECARwere monitored for 4 hours. The resulting effects on OCR andECAR are shown as a percentage of the baseline measurementfor each treatment.

To determine the mitochondrial and glycolytic function ofMCF-7 andMCF-10A cells in response toMito-CP,Mito-Q, and2-DG, we used the bioenergetic function assay previouslydescribed with several modifications (31, 32). After seedingand treatment as indicated, MCF-7 cells and MCF-10A cellswere washed with complete media and either assayed imme-diately or returned to a 37�C incubator for 36 or 60 hours. Thecells were then washed with unbuffered media as describedabove. Five baseline OCR and ECAR measurements were thentaken before injection of oligomycin (1 mg/mL) to inhibit ATPsynthase, FCCP (1–3 mmol/L) to uncouple the mitochondriaand yield maximal OCR, and antimycin A (10 mmol/L) toprevent mitochondrial oxygen consumption through inhibi-tion of complex III. From these measurements, indices ofmitochondrial function were determined as previouslydescribed (31, 32).

Intracellular ATP measurementsMCF-7, MCF-10A, and MDA-MB-231 cells seeded at 2� 104

per well in 96-well plates were treated continuously with 2-DGin the presence and absence of 1mmol/L of the indicatedMTDs

Figure 1. Structures of 2-DG, Mito-CP, Mito-Q, Dec-TPPþ, Carboxyproxyl (CP), Me-TPPþ.

Selective Targeting of Metabolism in Breast Cancer

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(Mito-CP or Mito-Q) or appropriate controls for 6 hours.Intracellular ATP levels were determined in cell lysates usinga luciferase-based assay per manufacturer's instructions (Sig-ma Aldrich). Results were normalized to the total protein levelin cell lysatemeasured in eachwell determined by the Bradfordmethod (Bio-Rad).

Cell death and clonogenic assaysMCF-7 and MCF-10A cells seeded at 2 � 104 per well in 96-

well plates were treatedwith 2-DG in the presence and absenceof 1 mmol/L MTDs for 6 or 24 hours, and dead cells weremonitored by stainingwith SytoxGreen per themanufacturer'sinstructions (Invitrogen). Fluorescence intensities from cellsgrown in 96-well plate were acquired using a plate reader(Beckman Coulter DTX-880; Beckman-Coulter) equipped with485 nm excitation and 535 nm emission filters. Tomeasure thetotal cell number, half of the samples in each treatment groupwere permeabilized with digitonin (120 mmol/L) when stainingwith Sytox Green and fluorescence intensities in digitonin-treated cells were taken as 100%. Data are represented as apercentage of dead cells after normalization to total cellnumber for each group.

For clonogenic assay, MCF-7, MDA-MB-231, and MCF-10Acellswere seeded at 300 cells per dish in 6-cmcell culture dishesand treatedwith 2-DG in the presence and absence of 1mmol/LMTDs for 6 hours. After 7 to 14 days, the number of coloniesformed was counted. The cell survival fractions were calcu-lated according to a published protocol (33).

Xenograft experimentsAll protocols were approved by the Medical College of

Wisconsin Institutional Animal Care and Use Committee.MDA-MB-231-luc cells [5 � 105 cells in 200mL of a mixtureof 1:1 PBS/Matrigel (BD Biosciences)] were injected intothe right mammary fat-pad of 8-week-old female SHO mice(Charles Rivers). Tumor establishment and growth weremonitored by injecting D-luciferin per manufacturer'sinstructions (Caliper Life Sciences) and detecting biolumi-nescence using the Lumina IVIS-100 In Vivo Imaging Sys-tem (Xenogen Corp.; ref. 30). The light intensities emittedfrom regions of interest were expressed as total flux(photons/s).

Two days after injection of cells, the mice were imaged toverify tumor establishment. Mice were then orally gavagedwith either water (control), Mito-CP (40mg/kg), 2-DG (1 g/kg),or a mixture of Mito-CP (40 mg/kg, final concentration) and 2-DG (1 g/kg, final concentration) 5 times/wk (Monday throughFriday). This treatment protocol was selected based on recentstudies showing that Mito-CP is cleared from plasma of micewithin approximately 6 hours of injection (34). After 4 weeks oftreatment, the mice were sacrificed, and the tumor, kidney,heart, and liver were removed and formalin fixed. These tissueswere then paraffin embedded and stained with hematoxylinand eosin (H&E).

StatisticsAll results are expressed as mean � SEM. Comparisons

among groups of dataweremade using a one-wayANOVAwith

Tukey post hoc analysis. P value of less than 0.05 was consid-ered to be statistically significant.

ResultsEffects of Mito-CP or Mito-Q alone and in combinationwith 2-DG on bioenergetic function in MCF-7 and MCF-10A cells

The OCR and ECAR (as a surrogate marker for glycolysis)weremeasured in a Seahorse Bioscience XF24 extracellularfluxanalyzer. The bioenergetic profiles obtained under variousexperimental conditions following Mito-CP and 2-DG treat-ments were determined according to the procedures outlinedpreviously (31, 32). As shown in Fig. 2A and B, addition ofMito-CP (1 mmol/L) greatly decreased the OCR in both MCF-7 andMCF-10A cells. Notably, Mito-CP stimulated ECAR in bothMCF-7 and MCF-10A cells, signaling an increase in glycolysislikely to compensate for the loss of OCR. As expected, 2-DG(5mmol/L) that inhibits glycolysis decreased the ECAR by 40%(Fig. 2C and D). Under these conditions, individual treatmentwith either Mito-CP or 2-DG slightly, but significantly,decreased the intracellular ATP levels in MCF-7 cells, but notin MCF-10A cells (Fig. 2E and F).

The extent of relative increase in glycolytic activity aftertreatment with Mito-CP (1 mmol/L) was notably higher inMCF-10A cells as compared with MCF-7 cells. To identify thesource of the difference in ECAR stimulation between these celllines, we next examined the potential for glycolysis stimulationin each cell line. ECARwasmeasured inMCF-7 cells cultured inmedia containing 5.5 or 17.5 mmol/L glucose and in MCF-10Acells cultured in media containing 17.5 mmol/L glucose (Sup-plementary Fig. S1A). After baseline ECAR was established,oligomycin was injected to the indicated final concentration.Because oligomycin inhibits mitochondrial ATP productionand results in compensatory increases in glycolysis, the degreeto which ECAR is stimulated by oligomycin should correlatewith the cellular glycolytic potential. As shown in Supplemen-tary Fig. S1A, oligomycin caused a more robust stimulation ofECAR in MCF-10A cells than MCF-7 cells, regardless of theglucose concentration used to culture the MCF-7 cells. Toconfirm this and rule out other effects of culture mediadifferences, MCF-7 and MCF-10A cells were seeded as normalinto Seahorse Bioscience culture plates. One hour before thestart of the experiment, the media was changed in all wells toa specialized DMEM-based assay media lacking glucose andFBS. Baseline ECAR was measured and then glucose wasinjected to a final concentration of either 5.5 or 17.5 mmol/Lto match routine culture conditions for each cell type (Sup-plementary Fig. S1B). This resulted in a stimulation of ECARthat was not significantly different between cell types orglucose concentrations (Supplementary Fig. S1B). Oligomy-cin was subsequently injected to a final concentration of 1mg/mL in all wells, and stimulation of ECAR was monitored.MCF-10A cells had a significantly greater stimulation ofECAR than MCF-7 cells regardless of glucose concentration(Supplementary Fig. S1B and C). Reversal of ECAR stimula-tion due to both glucose and oligomycin administration wasfound to be dependent on glycolysis. Finally, 2-DG was

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Cancer Res; 72(10) May 15, 2012 Cancer Research2636




injected to a final concentration of 25 mmol/L in all wells. 2-DG–dependent inhibition indicates that glucose- and oligo-mycin-stimulated ECAR was reflecting cellular glycolyticactivity.We nextmeasured the intracellular ATP levels inMCF-7 and

MCF-10A cells treatedwith different concentrations of 2-DG inthe presence of Mito-CP (1 mmol/L) and other control com-pounds (CP, TPPþ, and Dec-TPPþ). As shown in Fig. 3A, Mito-CP and 2-DG together decreased the intracellular ATP levels inMCF-7 cells by 20% to 30% more than in MCF-10A cells.Treatment with Mito-Q or with TPPþ conjugated to a long-chain aliphatic hydrocarbon (Dec-TPPþ) and 2-DG enhancedATPdepletion in both cell lines (Fig. 3B andSupplementary Fig.S2). In contrast toMito-CP, treatmentwithCP orMe-TPPþ andvarying levels of 2-DG did not cause enhanced intracellularATP depletion (Supplementary Fig. S2A and B). Similar resultswere observed in another breast cancer cell line, MDA-MB-231(Supplementary Fig. S3A and B). Notably, the enhancement ofATP depletion is not related to the delocalized cation only, asMe-TPPþ did not sensitize the cells to 2-DG (SupplementaryFig. S2). These results indicated thatMTDs (Mito-CP andMito-Q) with an antioxidant moiety (nitroxide group and Co-Q)conjugated to the TPPþ group via a long aliphatic carbon chaingreatly potentiate ATP-depleting activity of 2-DG in MCF-7cells.

Cytotoxic effects of Mito-CP or Mito-Q in combinationwith 2-DG in MCF-7 and MCF-10A cells

We measured the cytotoxicity after a 6 and 24 hours oftreatment of MCF-7 and MCF-10A with either Mito-CP (1mmol/L), Mito-Q (1 mmol/L), or Dec-TPPþ (1 mmol/L) andvarying concentrations of 2-DG under the same experimentalconditions as in Fig. 3. The cytotoxicity data obtained bymonitoring Sytox Green dye uptake normalized to the totalnumber of cells and are shown in Fig. 4. All 3 compoundscaused a slight increase in 2-DG cytotoxicity in MCF-7 cellsafter a 6 hour-treatment (Fig. 4A) that was markedly increasedafter a 24-hour treatment (Fig. 4B). Combined treatment with2-DG and either Mito-CP or Mito-Q caused a more dramaticincrease in cytotoxicity in MCF-7 cells compared with MCF-10A cells (Fig. 4B and D). In contrast, Dec-TPPþ (that wasdevoid of nitroxide or Co-Qmoiety) was equally toxic inMCF-7and MCF-10A cells in the presence of 2-DG (Fig. 4B and D).Similar trends were observed with regard to cell viability, asmeasured by the ability of the cell to reduce resazurin to re-sorufin. There was a modest decrease in cell viability (30%–40%) after a 6-hour treatmentwithMito-CP (1mmol/L),Mito-Q(1 mmol/L), or Dec-TPPþ (1 mmol/L) with varying levels of2-DG in MCF-7 or MCF-10A cells (Supplementary Fig. S4A–D).However, after a 24-hour treatment, Mito-CP and Mito-Qdecreased the viability of MCF-7 cells to a much greater

Figure 2. Bioenergetic profile ofbreast cancer cells (MCF-7) andnontumorigenic mammary epithelialcells (MCF-10A) treated with Mito-CP or 2-DG. A to D, MCF-7 or MCF-10Acellswere seeded to30,000cellsper well in specialized V7 Seahorsetissue culture plates. Changes inOCR (A, MCF-7; B, MCF-10A) andECAR (C, MCF-7; D, MCF-10A) weremonitored at 37�C for 4 hours. Theresulting effects on OCR and ECARare shown as a percentage of thebaseline measurement for eachtreatment. Data shown are themeans � SEM (n ¼ 5 per treatmentgroup). E and F, MCF-7 and MCF-10A cells were treated with Mito-CP(1 mmol/L) or 2-DG (5 mmol/L) for 6hours. Intracellular ATP levels weremonitored using a luciferase-basedassay. Data shown are the means �SEM, n¼ 4 per treatment group. �, P< 0.05 versus control.






extent as compared with MCF-10A cells (Supplementary Fig.S4B and D). These results are consistent with the cytotoxicityresults (Fig. 4). The glucose concentration in themedia used forMCF-7 andMCF-10A cells was different (5.56 vs. 17.5 mmol/L).Therefore, we tested the effects of 2-DG at different concen-trations. Notably, 2-DG (5 and 20mmol/L) did not significantlyaffect the colony formation or cell death inMCF-10A cells (Figs.4 and 5). Furthermore, we tested the cytotoxicity inMCF-7 cellscultured in media containing 17.5 mmol/L glucose (same as inMCF-10A cells). Notably, combined treatment with 2-DG andMito-CP for 24 hours still caused a more dramatic increase incytotoxicity in MCF-7 cells compared with MCF-10A cells (Fig.4B and D and Supplementary Fig. S5). In addition, the com-bined treatment with Mito-CP and 2-DG elicited greater celldeath and decreased colony formation in MCF-7 cells, evenwhen 2-DG was used at recommended glucose concentrationsin the media for each cell type (5 in MCF-7 vs. 20 mmol/L inMCF-10A). Incubationwith the untargeted nitroxide CP orMe-TPPþ with different 2-DG levels did not significantly increasecytotoxicity of 2-DG in either cell line even after a 24-hourtreatment (Supplementary Fig. S6).

Effects of Mito-CP, Mito-Q, or Dec-TPPþ and 2-DG onMCF-7, MDA-MB-231, and MCF-10A cell proliferation:Clonogenic analyses

One of the hallmarks of tumor cells is the ability to formcolonies (33). As shown in Fig. 5, addition of varying levels of 2-DG alone did not significantly decrease the colony formation inMCF-7, MDA-MB-231, or MCF-10A cells. In contrast, there wasa decrease in colony formation in MCF-7 cells when treatedwith 2-DG in the presence of 1 mmol/L Mito-CP or Mito-Q.Mito-CP wasmore potent thanMito-Q in inhibiting the colonyformation (Fig. 5). Figure 5B also shows the survival fractions ofMCF-7, MDA-MB-231, and MCF-10A cells calculated from theclonogenic survival assay. Both Mito-CP and Mito-Q morepotently decreased the survival fraction in MCF-7 and MDA-MB-231 cells as compared with MCF-10A cells in the presenceof 2-DG. Importantly, incubation with the Dec-TPPþ anddifferent 2-DG levels for 6 hours did not significantly decreasethe survival fraction in either cell line (Fig. 5).

Effects of Mito-CP and Mito-Q alone and in combinationwith 2-DG on mitochondrial bioenergetic function inMCF-7 and MCF-10A cells

Although treatment with Mito-CP and Mito-Q in the pres-ence of 2-DG dramatically decreased intracellular ATP levels inboth MCF-7 and MCF-10A cells, the clonogenic survival anal-ysis showed that this treatment selectively inhibited survival inMCF-7 to a much greater extent than in MCF-10A cells. Thissuggested that MCF-10A cells were able to recover from thecombination treatment (mitochondrial and glycolytic inhibi-tors), whereas theMCF-7 cells failed to recover under the sameconditions. To investigate this process in more detail, wemonitored the mitochondrial bioenergetic function in MCF-7 andMCF-10A cells using the XF24 extracellular flux analyzer.The protocol for this experiment is shown in SupplementaryFig. S7. As shown, both cell lines were treated with Mito-CP,Mito-Q, and 2-DG for 6 hours followed by washout of the

Figure 3. MTDs synergize with 2-DG to decrease intracellular ATPlevels in MCF-7 and MCF-10A cells. A–C, MCF-7 and MCF-10A cellsseeded in 96-well plates were treated with 2-DG in the presenceand absence of 1 mmol/L of the indicated MTDs (Mito-CP or Mito-Q) orrelevant controls for 6 hours. Intracellular ATP levels were monitoredusing a luciferase-based assay. Data are represented as apercentage of control (nontreated) cells after normalization to totalprotein for each well. The calculated absolute after normalization tototal protein for each well values of ATP (nmol ATP/mg protein) forMCF-7 and MCF-10A control cells were 20.6 � 1.9 and 28.0 � 2.0,respectively. Data shown are the means � SEM, n ¼ 4.

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treatments and returned to fresh culturemedia. After 36 hours,OCRwasmeasured and the effects of adding oligomycin, FCCP,and antimycin Awere determined (Fig. 6A–D). The use of thesemetabolic modulators allows determination of multiple para-meters of mitochondrial function. Interestingly, the mostdramatic effect of Mito-CP was on basal OCR and the OCRlinked to ATP production (Fig. 6E). These parameters werecalculated from the traces in Fig. 6A–D (31, 32). Notably,inhibition of OCR was persistent 36 hours after removal ofMito-CP inMCF-7 cells, but not inMCF-10A cells.Mito-Q had asimilar effect on mitochondrial function, although the degreeof inhibition was markedly lower. The effects of Mito-CP andMito-Q were also examined immediately after 6 hours treat-ment with these compounds (Supplementary Fig. S8). Consis-tent with the data shown in Fig. 2, after a 6-hour exposure toeitherMito-CP orMito-Q, mitochondrial oxygen consumptionwas decreased. Curiously, the effect of these compounds wasmore dramatic in MCF-10A cells as compared with MCF-7cells. Paired with the data in Fig. 6, this suggested that MCF-10A cells are more adept at recovering from inhibition ofmitochondrial function than MCF-7 cells. These data alsoshowed that the effect of 2-DG is not persistent afterremoval. As shown in Fig. 6E, this inhibition was reversed36 hours after removal of the 2-DG. In addition, we foundthat inhibition of OCR persisted up to 60 hours after

washout of Mito-CP or Mito-Q. As shown in SupplementaryFig. S9, the mitochondrial function was similarly inhibitedafter 60 hours following washout. Inhibition of mitochon-drial function was persistent in MCF-7 cells but not in MCF-10A cells. Collectively, these data suggested that the mech-anism for selective killing of MCF-7 cells when cotreatedwith 2-DG and MTDs is through irreversible mitochondrialinhibition.

Effects of Mito-CP and 2-DG on tumor growth: breastcancer xenograft model

We investigated the effects of the combined use of Mito-CP and 2-DG in an in vivo breast tumor model. Adminis-tration of 2-DG or Mito-CP alone had little or no effect onthe rate of tumor growth in the xenograft mouse model (Fig.7A). In contrast, the combined treatment of Mito-CP and 2-DG led to a significant decrease in the bioluminescencesignal intensity (total flux) as compared with the controlmice after 3 and 4 weeks of treatment (Fig. 7A and B).Furthermore, this combined treatment (Mito-CP plus 2-DG)for 4 weeks significantly diminished tumor weight (Fig. 7C)without causing any significant changes in kidney, liver,and heart weight or other major morphologic changes (asdetermined by H&E staining in Supplementary Fig. S10 andTable S1.)

Figure 4. The effects of 2-DG in thepresence and absence of MTDs onthe extent of cell death in MCF-7 andMCF-10A cells. MCF-7 and MCF-10A cells seeded in 96-well plateswere treated with 2-DG in thepresence and absence of 1 mmol/LMTDs for 6 hours (A, C) or 24 hours(B, D), and cell death was monitoredby staining with Sytox Green. Dataare presented as a percentage ofdead cells after normalization to totalcell number for each group. Datashown are the means � SEM, n ¼ 5.






DiscussionPrevious research has shown thatmitochondrial targeting of

antioxidants could inhibit tumor cell proliferation (25, 26).Delocalized phosphonium cations were shown to exert anti-tumor activity in several tumor cell lines (35, 36). For example,it was recently reported that Mito-CP, but not CP, induced anincrease in phosphorylated ERK1/2 in colon cancer cells (25).Furthermore, the selective toxicity of mitochondria-targetedvitamin-E succinate conjugated to TPPþ in tumor cells wasattributed to enhancedmitochondrial localization and bindingto complex 2 of the mitochondrial respiratory chain (37). Inother studies, selective targeting of tumor-specific cellularenergy metabolism synergistically exacerbated cytotoxicity incancer cells treated with antiglycolytic agents or with inhibi-tors of fatty acid b-oxidation (38). A metabolic shift to glycol-ysis occurs during antiangiogenic therapy with drugs (e.g.,

bevacizumab) that block VEGF in glioblastoma (39). Upregula-tion of the PI3K/Akt pathway occurs during antiangiogenictherapy. Adjuvant therapy with drugs (MTDs/2-DG combina-tion) targeted to glycolytic and mitochondrial metabolismcould be beneficial in antiangiogenic therapy for treatmentof brain tumors. 2-DG was shown to potentiate cisplatin-dependent antiproliferative effects in ovarian carcinoma cellsexpressing low levels of b-F1-ATPase (40). The combinedtreatment with 2-DG (inhibitor of aerobic glycolysis) alsoeffectively enhanced the efficacy of doxorubicin-induced che-motherapy (41).

Thus, we surmised that mitochondria-targeted antioxi-dants and nitroxides (SOD mimetics) would preferentiallyinhibit tumor cell proliferation, but not normal cell prolif-eration. Several recent studies showed increased cancer celldeath in the presence of antioxidant compounds, and the

Figure 5. MTDs synergize with 2-DG to inhibit colony formation inMCF-7 andMDA-MB-231 cells butnot in MCF-10A cells. A, MCF-7,MDA-MB-231, and MCF-10A cellswere treated with 2-DG only (top,left), 2-DG in the presence of Mito-CP (1 mmol/L; top, right), 2-DG inthe presence of Mito-Q (1 mmol/L)for 6 hours (bottom, left), 2-DG inthe presence of Dec-TPPþ

(bottom, right), and the number ofcolonies formed was counted. B,the survival fraction was calculatedunder the same conditions as in A.Thecalculatedplating efficiency forMCF-7, MDA-MB-231, and MCF-10A cells was 55 � 6, 33 � 4, and34 � 8, respectively. Data shownrepresent the mean � SEM.�, P < 0.05 (n ¼ 5) comparingMCF-7 and MDA-MB-231 withMCF-10A under the sametreatment conditions.

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mechanism of action of Mito-CP and Mito-Q is presumed torequire this antioxidant capability. However, in addition totheir antioxidant properties, Mito-CP and Mito-Q mighthave other unique functions due to the presence of thealkyl chain tethered to the TPPþ group. Our results showthat the methyl-triphenylphosphonium cation alone was notsufficient to affect cancer cell proliferation. In contrast,Decyl-TPPþ did increase cell death (Fig. 4), though thespecificity for cancer cells was notably lost. The moleculartarget involved in enhancing the cytotoxic effects of TPPþ-

substituted compounds in tumor cells is presently un-known, nor do we fully understand the mechanism(s) bywhich Mito-CP and Mito-Q preferentially inhibit tumor cellproliferation.

The most salient difference between the response of MCF-7andMCF-10A cells toMitoCP is in the stimulation of glycolysisin response to inhibition of mitochondrial oxygen consump-tion (Fig. 2). MCF-10A cells have a higher apparent ability tostimulate ECAR, indicating increased glycolytic function inresponse to Mito-CP. It is likely that this stimulation of

Figure 6. The effects of 2-DG andMTDs on OCR and ECAR in MCF-7andMCF-10A cells. A–D,MCF-7 andMCF-10A cells (20,000 cells per well)seeded in V7 culture plates weretreated with the indicatedcompounds for 6 hours. The cellswere then washed with completemedia (MEM-a for MCF-7 andDMEM/F12 for MCF-10A) andreturned to a 37�C incubator for 36hours. The cells were then washedwith unbuffered media as described.Five baseline OCR and ECARmeasurements were then takenbefore injection of oligomycin(1 mg/mL), to inhibit ATP synthase,FCCP (1–3 mmol/L) to uncouple themitochondria and yield maximalOCR, and antimycin A (10 mmol/L) toinhibit complex III and mitochondrialoxygen consumption. The effects ofMTDs and 2-DG on basal OCR, ATP-linked OCR and ECAR are shown inE. �,P < 0.01 (n¼ 5) comparingMCF-7 with MCF-10A under the sametreatment conditions.






glycolysis is better able to support ATP production inMCF-10Acells, as compared with MCF-7 cells (Fig. 2). Maintenance ofATP levels early in the treatment with Mito-CP suggests that

the cells are better able to survive cotreatment with 2-DG.This may happen despite a loss of ATP in both cell lineswhen cotreated with Mito-CP and 2-DG (Fig. 3). It is alsoworth noting that mitochondrial oxygen consumption andglycolysis both recover more completely in MCF-10A cells ascompared with MCF-7 cells (Fig. 6). This resilience likelyunderpins the preferential cytotoxicity of MitoCP and 2-DGcotreatment in MCF-7 cells both in vitro (Fig. 5) and in vivo(Fig. 7).

MDR-1 or p-glycoprotein, the product of theMDR1 gene, is amultidrug transporter. Elevated levels ofMDR-1 in cancer cells,therefore, antagonize the proapoptotic effects of anticancerdrugs, rendering these cells resistant to apoptosis (42). Cur-rently, the affinity of Mito-CP to p-glycoprotein–mediatedMDR recognition is not known. Previously, it was reportedthat simple nitroxides (e.g., Tempol) inhibit efflux of p-glyco-protein–mediated MDR-1 substrates suggesting that nitrox-ides act as competitive inhibitors of MDR-1 p-glycoproteins,thereby abrogating resistance to doxorubicin (43). The presentresults strongly suggest that 2-DG and Mito-CP (1 mmol/L)synergistically induced cytotoxicity in MCF-7 and MDA-MB-231 cells. Previous EPR results indicate that mitochondrialMito-CP levels in MCF-7 and MCF-10A cells were nearlyidentical (44). Several cancer cell lines in the National CancerInstitute panel of 60 tumor cell lines, including MCF-7 andMDA-MB-231, exhibit only very low levels of p-glycoproteinexpression (45). However, chemotherapy (i.e., doxorubicin)could induce MDR through phenotypic modification leadingto elevated p-glycoprotein levels (46). Under these conditions,the intracellular levels Mito-CP could decrease via the pump-ing mechanism of p-glycoprotein; however, with decreasedintracellular ATP caused by Mito-CP and 2-DG, the p-glyco-protein-mediated pump activity is hindered, leading toenhanced accumulation of cationic drugs (47). A recent pub-lication suggests that intracellular ATP levels are a key deter-minant of chemoresistance in colon cancer cells (48). Thisrepresents one hypothetical mechanism whereby inhibition ofATP levels by Mito-CP and 2-DG may further synergize withother classical antineoplastic drugs to enhance tumor celldeath.

Bioluminescence imaging is one of the most widely usedmethods to monitor tumor growth and its therapeuticresponse (49). The substrate luciferin is oxidized by lucifer-ase-expressing cancer cells while concomitantly emittinglight (50). Thus, the signal intensity generally reflects thenumber of cancer cells and tumor size. The oxidation ofluciferin by luciferase requires ATP as a cosubstrate. ATPdepletion should lead to diminished bioluminescence fromluciferin, independent of the tumor cell number. In thisstudy, the depletion of ATP caused by Mito-CP and 2-DGin cultured MDA-MB-231 cells (Supplementary Fig. S3) wascorrelated to the decrease of bioluminescence signalinduced by the same treatment in MDA-MB-231-luc cells(Supplementary Fig. S11). The decrease in the signal inten-sity observed in the in vivo experiments may, therefore, bedue to inhibition of cell growth or decrease in intracellularATP levels in the tumor cells or both. The confirmation ofthe antiproliferative efficiency of MTDs in vivo stems from

ControlA

B

C

Mito-CP2-DG

100

75

50

25

0

Control

(n = 10

)Mito

-CP

(n = 11

)2-

DG

(n = 9)

Mito-C

P + 2-DG

(n = 8)

Tu

mo

r w

eig

ht

(mg

)

Weeks

To

tal f

lux

(ph

oto

ns/

s)

MitoCP + 2-DG

3.0 × 109

2.5 × 109

2.0 × 109

1.5 × 109

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5.0 × 108

0.0

0 1 2 3

140

120

100

80

60

40

20

×106

4

*

*

*

*

Figure 7. Combined treatment with Mito-CP and 2-DG inhibits MDA-MB-231-luc tumor xenograft growth. Tumor growthwasmonitoredweekly for 4weeks following injection of MDA-MB-231-luc cells into the rightmammaryfat pad. Luminescence was monitored as described in the Materials andMethods and plotted as the light flux in photons/s (A). Representativeimages are shown inB.At 4weeks, tumorswere excised.Wet tumorweightwasmeasured and is plotted in C. Data shown represent the mean� SEM(n � 8 per group). �, P < 0.05 and ��, P < 0.01 versus control group.

Cheng et al.





the tumor weight data, indicating a significantly lower tumorweight in xenograft mice treated with Mito-CP and 2-DG.The possibility of noninvasive monitoring of ATP levelsduring treatment with MTDs and/or antiglycolytic agentsin the in vivo xenograft breast cancer model is currentlyunderway.In summary, we report in this study that the combined

use of a mitochondria-targeted antioxidant that is rela-tively nontoxic to normal cells and an antiglycolyticagent (e.g., 2-DG) synergistically enhanced the cytotoxicpotential in breast tumor cells. As has been shown inearlier publications (26, 28), an added value of this adju-vant therapy is the potential cytoprotective ability ofMTDs (e.g., Mito-CP and Mito-Q) against conventionalchemotherapy-induced mitochondrial oxidative damagein normal cells, thereby increasing the overall therapeuticindex.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G. Cheng, J. Zielonka, B.P. Dranka, B. KalyanaramanDevelopment of methodology: G. Cheng, J. Zielonka, B.P. Dranka, J. Joseph,B. KalyanaramanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Cheng, J. Zielonka, B.P. Dranka, D. McAllister,A.C. MackinnonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Cheng, J. Zielonka, B.P. Dranka, A.C. MackinnonWriting, review, and/or revision of themanuscript:G. Cheng, J. Zielonka, B.P. Dranka, A.C. Mackinnon, B. KalyanaramanAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):G.Cheng, J. Zielonka, D.McAllister, A.C.Mackinnon, B. KalyanaramanStudy supervision: G. Cheng, B. Kalyanaraman

Grant SupportThis research was funded by the National Cancer Institute (grant R01

CA152810).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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 5, 2011; revised February 29, 2012; acceptedMarch 9, 2012;published OnlineFirst March 19, 2012.

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2012;72:2634-2644. Published OnlineFirst March 19, 2012.Cancer Res Gang Cheng, Jacek Zielonka, Brian P. Dranka, et al. Trigger Breast Cancer Cell DeathMitochondria-Targeted Drugs Synergize with 2-Deoxyglucose to

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