Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer · The current study demonstrates that pharmacologic ... ascorbate as a radiosensitizer in the treatment of pancreatic

Therapeutics, Targets, and Chemical Biology

Pharmacological Ascorbate RadiosensitizesPancreatic CancerJuan Du1, John A. Cieslak III2, Jessemae L.Welsh1, Zita A. Sibenaller2, Bryan G. Allen1,3,Brett A.Wagner2, Amanda L. Kalen2, Claire M. Doskey2, Robert K. Strother2,Anna M. Button3, Sarah L. Mott3, Brian Smith3, Susan Tsai4, James Mezhir1,3,Prabhat C. Goswami2,3, Douglas R. Spitz2,3, Garry R. Buettner2,3, and Joseph J. Cullen1,2,3,5

Abstract

The toxicity of pharmacologic ascorbate is mediated by thegeneration of H2O2 via the oxidation of ascorbate. Becausepancreatic cancer cells are sensitive to H2O2 generated byascorbate, they would also be expected to become sensitizedto agents that increase oxidative damage such as ionizingradiation. The current study demonstrates that pharmacologicascorbate enhances the cytotoxic effects of ionizing radiation asseen by decreased cell viability and clonogenic survival in allpancreatic cancer cell lines examined, but not in nontumori-genic pancreatic ductal epithelial cells. Ascorbate radiosensiti-

zation was associated with an increase in oxidative stress–induced DNA damage, which was reversed by catalase. In micewith established heterotopic and orthotopic pancreatic tumorxenografts, pharmacologic ascorbate combined with ionizingradiation decreased tumor growth and increased survival, with-out damaging the gastrointestinal tract or increasing systemicchanges in parameters indicative of oxidative stress. Our resultsdemonstrate the potential clinical utility of pharmacologicascorbate as a radiosensitizer in the treatment of pancreaticcancer. Cancer Res; 75(16); 3314–26. �2015 AACR.

IntroductionPharmacologic ascorbate induces cytotoxicity and oxidative

stress in pancreatic cancer cells, compared with normal cells(1). In the extracellular environment, pharmacologic concen-trations of ascorbate can oxidize to form hydrogen peroxide(H2O2; refs. 1–4). H2O2 will diffuse readily across the cellmembrane causing oxidative damage to cellular proteins,lipids, and DNA. The generation of H2O2 correlates with theconcentration of ascorbate in both a time- and dose-dependentmanners. Ascorbate has been shown to decrease viability in allpancreatic cancer cell lines studied, but has no effect on non-tumorigenic pancreatic ductal epithelial cells (1), and cytotox-icity was reversed with scavengers of H2O2. Furthermore, in vivotreatment with pharmacologic ascorbate inhibited tumorgrowth and prolonged survival. Thus, ascorbate has beenhypothesized to be a "prodrug" for formation of H2O2 inpancreatic cancer xenografts (1, 3). Therapeutic interventionsdesigned to increase oxidant stress (such as ionizing radiation,IR) in combination with pharmacologic ascorbate would be

predicted to preferentially sensitize tumor cells versus normalcells via metabolic oxidative stress (1, 5).

IR has long been known to induce DNA damage. In addition todirect damage, IR generates reactive oxygen species (ROS) that candamage proteins, lipids, and DNA, inducing both single- anddouble-strand DNA breaks (6). Formation of double-strandbreaks results in the rapid phosphorylation of histone H2AX(7). Mammalian-phosphorylated H2AX (g-H2AX) is believed tofacilitate the recruitment and retention of DNA repair and check-point proteins (8, 9). Radiosensitive tumor cells have been shownto retain g-H2AX for a longer duration after IR than radioresistantcells.

Pharmacologic ascorbate-mediated H2O2 formation alsocauses DNA damage, which involves transitionmetal ions such asFe2þ associatedwithDNA (10). Fe2þ reacts withH2O2, producingsite-specific hydroxyl radical (HO

*

), damaging DNA bases as wellas the sugar/phosphate backbone of DNA (11). The base excisionrepair pathway is themajor system for repair of oxidative-inducedDNA damage (12). Thus, DNA damage can be assessed bymeasuring g-H2AX, which is upregulated in the presence ofdouble-strand breaks.

Because both IR and pharmacologic ascorbate initiate DNAdamage, we hypothesize that pharmacologic ascorbate has poten-tial as a radiosensitizer in pancreatic cancer. Here, we demonstratethat pharmacologic ascorbate is a selective radiosensitizer inpancreatic cancer versus normal nontumorigenic pancreatic duc-tal epithelial cells. Pharmacologic ascorbate also enhanced IR-inducedDNAdamage in pancreatic cancer cells as well as enhanc-ing IR-induced inhibition of tumor growth in established humanpancreatic xenografts without causing systemic changes in para-meters indicative of oxidative stress or enhancing normal tissueinjury to the gut epithelium. These results demonstrate the poten-tial utility of pharmacologic ascorbate as an adjuvant topancreaticcancer radiotherapy.

1Department of Surgery, University of Iowa College of Medicine, IowaCity, Iowa. 2Department of Radiation Oncology, University of IowaCollege of Medicine, Iowa City, Iowa. 3Holden Comprehensive CancerCenter, Iowa City, Iowa. 4Medical College of Wisconsin, Milwaukee,Wisconsin. 5Veterans Affairs Medical Center, Iowa City, Iowa.

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

Corresponding Author: Joseph J. Cullen, The University of Iowa Carver Collegeof Medicine, 4604 JCP, 200 Hawkins Dr., Iowa City, IA 52242. Phone: 319-353-8297; Fax: 319-356-4609; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-1707

�2015 American Association for Cancer Research.

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Materials and MethodsCell culture

MIA PaCa-2, AsPC-1, and PANC-1 human pancreatic adeno-carcinoma cells were obtained from the American Type CultureCollection and passaged for fewer than 6 months after receipt.MIA PaCa-2 cells were maintained in DMEM supplemented with10% FBS and 1% penicillin–streptomycin. AsPC-1 cells weremaintained in RPMI 1640 with 10% FBS and 1% penicillin–streptomycin. PANC-1 cells were cultured in DMEM supplemen-ted with 10% FBS. Also, patient-derived pancreatic cancer celllines (403 and 339s) from the Medical College of Wisconsinsurgical oncology tissuebank (13, 14)were cultured inDulbecco'sModified Eagle's Media Nutrient Mixture F-12 with penicillin/streptomycin, human recombinant EGF, bovine pituitary extract,hydrocortisone, and human recombinant insulin. In addition tothe pancreatic cancer cell lines, we also used the nontumorigenicHPV16-E6E7 immortalized cell line derived from normal pan-creatic ductal epithelium (H6c7) with near normal genotype andphenotype of pancreatic duct epithelial cells (15). H6c7 cells weremaintained in keratinocyte serum-free media supplemented withepidermal growth factor (5 ng/mL) and bovine pituitary extract(50 mg/mL). The H6c7 cells were characterized by IDEXX-RADIL.Profile data were not available for comparison purposes for H6c7cells. However, the genetic profile for H6c7 cells was comparedwith the cell line genetic profiles available in the DSMZ STRdatabase and did not match any other reported profiles in theDSMZ database. The H6c7 cells alone do not form colonies sofeeder cells were used as described (15).

L-ascorbic acid was purchased from Macron Chemicals. Stocksolutions of ascorbate (1.0 mol/L, pH 7.0) were made as previ-ously described (1). For ascorbate treatments, cells were placed infresh media and treated with ascorbate for 1 hour at 37�C. Todetermine clonogenic survival, cells were treated, trypsinized,counted, diluted, and plated for clonogenic cell survival assay aspreviously described (1). Surviving colonies were fixed andstained with Coomassie blue after 10 to 14 days and countedunder an inverted light microscope. As another indicator of cellviability, an assay monitoring the reduction of MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) wasused. Cells were seeded in 60 mm2 dishes at 3 � 105 cells perwell and allowed to attach for 24 hours. Next, cells were irradiatedwith cesium-137 source, doses ranging from 0 to 10 Gy. Imme-diately prior and during IR, cells were treated with ascorbate (0.25mmol/L) in DMEMwith 10% FBS for 1 hour. After treatment, themedia were changed, and cells were allowed to recover for 72hours. Themedia were removed, and cells were then incubated ina solution ofMTT, 1mg/mL (Sigma-Aldrich), dissolved in serum-free DMEM at 37�C for 3 hours in the dark. At the end of theincubation time, themedia were removed, andDMSOwas addedto each plate to dissolve the precipitate. Samples were transferredto a 96-well plate for plate reader analysis and read at 590 nmon aTecan SpectraFluor Plus plate reader (Tecan).

Catalase treatmentTo determine whether H2O2 was responsible for the cytotoxic

effects of ascorbate and radiation, cells were treated with variousforms of catalase, including adenovirus catalase (AdCAT), bovinecatalase (100 U/mL), or catalase-polyethylene glycol (PEG-CAT;200 U/mL). Catalase and PEG-catalase were purchased fromSigma-Aldrich. The AdCAT construct used was a replication-

defective, E1- and partial E3-deleted recombinant adenovirus(16). Inserted into the E1 region of the adenovirus genome isthe human catalase gene, which is driven by a cytomegaloviruspromoter. For the adenovirus experiments, approximately 106

cells were plated in 10mL of complete media in a 100mm2 tissueculture dish and allowed to attach for 24 hours. Cells were thenwashed three times in serum- and antibiotic-free media. Theadenovirus constructs were applied to cells in 4 mL of serum-and antibiotic-free media. Control cells were treated with theempty adenovirus (AdEmpty) construct. Cells were incubatedwiththe adenovirus constructs for 24 hours. Media were replaced with4 mL of complete media for an additional 24 hours before cellswere harvested for Western blot or treated for clonogenic assay.

Cell cycle analysisCell viabilitywasmeasured byflow cytometry using propidium

iodide (PI). After treatment, cells were collected by trypsinization,centrifuged at 500 g, and resuspended in 500 mL of Hank'sBuffered Salt Solution. After addition of 5 mL of PI (50 mg/mL),cells were incubated in the dark at room temperature for 5minutes. PI fluorescence was analyzed by flow cytometry (exci-tation at 488 nm, emission at >550 nm). To analyze alterations incell cycle by quantitation ofDNA content, cells were collected andfixed in suspension with 70% ethanol for 4 hours at 4�C. Cellswere washed with 1mL PBS, centrifuged, and resuspended in 100mL RNase A (1 mg/mL in PBS). After 30-minute incubation atroom temperature, 500 mL PI (35 mg/mL in PBS) was added toeach sample. After 1-hour incubation in the dark at room tem-perature, PI fluorescence was analyzed by flow cytometry.

Determination of intracellular hydrogen peroxideIntracellular H2O2 concentrations were determined by analysis

of the rate of aminotriazole-mediated inactivation of endogenouscatalase activity (17). Catalase is irreversibly inactivated by ami-notriazole (3-AT, 3-amino-1,2,4-triazole; Sigma-Aldrich) in thepresence of H2O2. Cells grown in 150 mm2 culture dishes wereirradiated at 3 Gy and then treated with ascorbate (20mmol/L) inthe presence of 3-AT (20mmol/L) for 0, 5, 10, 20, 30, 60, and 120minutes at 37�C. Cells were washed with ice-cold PBS, harvested,and lysed by freeze/thaw at �80�C followed by room tempera-ture. To determine the amount of fully active cellular catalase, celllysates (2 mL) were introduced into the reaction chamber of anoxygen monitor (YSI model 5300, YSI Inc.). Then 333 mmol/L ofH2O2 was injected into the reaction chamber, and the rate ofproduction of oxygenwas continuously recorded for 5minutes oruntil the curve reached a plateau. The rate of appearance ofdioxygen reflects the amount of active catalase in the cell lysate.Cell protein was determined by Bio-Rad DC protein assay. Theintracellular steady-state concentration of H2O2 was calculatedfrom the equation [H2O2]ss ¼ kinactivation/k1, where kinactivation isthe experimental pseudo first-order rate constant of catalaseinactivation (s�1) and k1 ¼ 1.7 � 107 M�1 s�1, i.e., the rateconstant for the formation of catalase compound I.

The rate of extracellular production of H2O2 was determinedwith a Clark electrode as in refs. 1 and 5.

Intracellular GSH and GSSG measurementCells grown in 100mm2 dishes were treated with IR, ascorbate,

or in combination. After treatments, cells were washed with PBSand harvested by trypsinization (floating cells in culture mediaand PBS were collected and combined with trypsinized cells).

Ascorbate Radiosensitization

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After centrifugation, cell pellets were suspended in 100 mL per-chloric acid buffer [5%perchloric acid with 100 mmol/LDETAPAC(diethylenetriaminepentaacetic acid); Sigma-Aldrich ChemicalCo.] to precipitate protein. Samples were sonicated and centri-fuged at 18,500 g for 5 minutes at 4�C using an EppendorfMicrocentrifuge. Supernatants were collected and stored at�80�Cor analyzed immediately using HPLC (ESA CoulArray; Dionex/Thermo Scientific) with electrochemical detection (ECD) follow-ing theprotocol described inPark and colleagues (18). In addition,in separate groups of treated mice, red blood cells (RBC) wereharvested and centrifuged (500 g for 5 minutes). The plasma wasremoved from the sample, and the remaining pellet of intact RBCswas washed twice with cold isotonic saline. An aliquot wasremoved for counting by a hemocytometer. From the RBC pellet,100 mL of RBCs were lysed with 300 mL of perchloric acid bufferand then centrifuged to pellet the protein (500 g, 5 minutes). Theclean supernatant was stored at �80�C or immediately analyzedfor total glutathione (tGSH) using a plate-reader–based assay (19).

Immunoblot analysisProtein (10–40 mg) was electrophoresed in a 4% to 20% Bio-

Rad ready gel and then electrotransferred to an Immobilon PVDFmembrane (EMD Millipore). Membranes were blocked in 5%nonfat milk for 1 hour, then treated with anti–g-H2AX (Ser 139)antibody (1:1,000; EMD Millipore), or with anti-catalase anti-body (1:5,000 dilution; Cell Signaling Technology). Horseradishperoxidase (HRP)–conjugated goat anti-rabbit or goat anti-mouse (1:50,000; Chemicon International) was used as a sec-ondary antibody. Anti-GAPDH (1:1,000; EMDMillipore) or anti-actin (1:4,000; Sigma-Aldrich) was used as a loading control.Blots were treated with SuperSignal West Pico ChemiluminescentSubstrate (Thermo Fisher Scientific) and exposed to autoradiogra-phy film.

ImmunohistochemistryMIA PaCa-2 cells were seeded in 8-well (0.8 cm2/well) glass

Lab-Tek Chamber slides at a density of 30,000 cells/well. After 48hours, cells were treatedwith ascorbate prior to IR treatment. Cellswere fixed in 3%paraformaldehyde and rehydrated inDulbecco'sPBS (pH 7.4) 30 minutes after treatment and then incubated inRNAse (0.5 mg/mL) at 37�C for 30minutes, and 10%normal goatserumwas applied overnight for blocking. Cells were incubated inanti–g-H2AX primary antibody (1:200; Calbiochem) for 1.5hours. A goat anti-rabbit antibody conjugated to Alexa Fluor488 (1:500) was used as a secondary antibody to fluorescentlytag g-H2AX. Slides were mounted in Vectashield with DAPI as anuclear counterstain. Slides were imaged on a Bio-Rad Radiance2100 confocalmultiphotonmicroscopewith laser sharp software.Z-plane images of each treatment were captured, and the intensityof fluorescence was quantified on ImageJ using a maximumintensity Z-projection with identical threshold values to calculatethe mean fluorescence intensity for individual cells.

Animal experimentsThirty-day-old athymic nude mice were obtained from Harlan

Sprague-Dawley. The nude mice protocol was reviewed andapproved by the Animal Care and Use Committee of The Uni-versity of Iowa. The animals were housed in a cage and were fed asterile commercial stock diet and tap water, ad libitum. Animalswere allowed to acclimate in the unit for 1 week before anymanipulations were performed. Each experimental group con-

sisted of 9 to 12 mice. MIA PaCa-2 or PANC-1 human pancreatictumor cells (2 � 106) were delivered subcutaneously into theflank region of nude mice with a 1-mL tuberculin syringeequipped with a 25-gauge needle. The tumors were allowed togrow until they reached between 3 mm and 4 mm in greatestdimension (2 weeks), at which time the mice were randomizedand treatment was initiated. This was defined as day 1 of theexperiment. Before IR, the animals were anesthetized with 80 to100mg/kg ketamine and 10mg/kg xylazine i.p. and shielded in alead block such that only the tumor-bearing right hind flank wasirradiated. Tumor size was measured twice a week using a digitalcaliper, and tumor volume was estimated according to the fol-lowing formula: tumor volume ¼ p/6 � L � W2, where L is thegreatest dimension of the tumor, and W is the dimension of thetumor in the perpendicular direction (20). Animals were eutha-nized by CO2 asphyxiation when the tumors reached a predeter-mined size of 1,000 mm3. In the orthotopic pancreatic cancermodel, 6-week-old athymic nude mice were injected with thehuman pancreatic cancer cell line MIA PaCa-2, which had previ-ously been stably transfected with firefly luciferase. Ultrasoundguidance was used to identify the pancreas as described (21), and400,000 cells suspended in 20 mL of a 1:1 mixture of PBS andMatrigel were injected into the pancreas under direct visualiza-tion. Bioluminescence-imaging microscopy was used to identifymice with established tumors within the pancreas, and tumor-containing mice were randomly assigned and treatment wasinitiated. Tumor growth was monitored using biolumines-cence-imaging microscopy to determine the average radiance(photons/s/cm2) of the tumors.

In separate groups of treated animals, mouse tumors wereexcised and processed as described to determine 4-hydroxy-2-nonenal-(4HNE)–modified proteins (22). Briefly, tissues werehomogenized and 25 mg of protein was blotted onto PVDFmembranes. Blots were incubated with the primary antibodyrecognizing the Michael addition product of 4HNE-modifiedcellular proteins (23) diluted 1:2,000 overnight at 4 �C, followedby 2 hours in secondary antibody, HRP-conjugated goat anti-rabbit polyclonal antibody (1:20,000), and chemiluminescencedetectionwith X-rayfilm. Immunoreactive protein on the dot blotwas analyzed using integrated densities determined with ImageJsoftware.

In separate groups of mice, a crypt cell assay was performed.Mice were exposed to 10Gy or 13Gy of total abdominal radiationwith andwithout ascorbate (4 g/kg/d). After 48 hours, each animalwas sacrificed and sections were made of the jejunum and thenviewed under light microscopy and expressed as surviving cells/circumference. In addition, blood samples for TNFawere collectedinto 4.0 mL heparinized vial from control mice and mice treatedwith radiation (13 Gy) and/or ascorbate (4g/kg). Samples werecentrifuged at 200 g for 20minutes. Serum samples were collectedfrom each tube and stored at �80�C. TNFa levels in the serumwere measured using a Quantikine ELISA Kit (R&D Systems).

Statistical analysisA single factor ANOVA followed by the Tukey post hoc test was

used to determine statistical differences between mean for mul-tiple comparisons, or a Student t test was used when only twocomparisons. All mean were calculated from three experiments,with error bars representing the SEM. All Western blots wererepeated at least twice. All data are expressed as mean � SEM.A dose-modifying factor (DMF) was calculated for each cell line

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Figure 1.Ascorbate radiosensitizes pancreaticcancer cells. A, MIA PaCa-2 pancreaticcancer cells were irradiated(0–3 Gy) with and without ascorbate(0.25 mmol/L) and clonogenic survivaldetermined. The resulting dosemodification factor at 10% iso-survivalwas 2.5, indicating enhancedradiosensitivity (mean � SEM, n ¼ 3).Pharmacologic ascorbate (0.25mmol/L) produced a flux of H2O2 of 550amol�1 cell�1 s�1 under our experimentalconditions. This rate is considerablygreater than the rate of cellular oxygenconsumption by these cells, 57 amol�1

cell�1 s�1 (29). B, PANC-1 pancreaticcancer cells were irradiated (0–10 Gy)and treated with ascorbate(0.25 mmol/L) in a similar fashion.Clonogenic survival yielded a DMF of2.2, indicating enhanced radiosensitivity(mean � SEM, n ¼ 3). C, AsPC-1 humanpancreatic cancer cells were irradiated(0–10 Gy) with and without ascorbate(0.25 mmol/L) and clonogenic survivaldetermined. The resulting dosemodification factor at 10% iso-survivalwas 1.25, indicating enhancedradiosensitivity (mean� SEM, n¼ 3). D,H6c7 immortalized human pancreaticductal epithelial cells were irradiated(0–6 Gy) and treated with ascorbate(0.25 mmol/L) to determine clonogenicsurvival. The clonogenic survival assaydemonstrates no radiosensitizationafter IRwith orwithout ascorbate (mean� SEM; n¼ 3). E, MIA PaCa-2 pancreaticcancer cells were irradiated(0–4 Gy) and treated with ascorbate(0.25 mmol/L) to determine viabilityusing the MTT assay and demonstratedradiosensitization. Data werenormalized to drug or vehicle control(mean � SEM; n ¼ 3). F, H6c7immortalized human pancreatic ductalepithelial cells were irradiated (0–10Gy)and treated with ascorbate(0.25 mmol/L) to determine cellviability. The MTT assay demonstratesminimal changes in cell viability after IRwithorwithout ascorbate (mean� SEM,n ¼ 3). G, patient-derived pancreaticcancer cells (339) in 4% O2 wereirradiated (0–6 Gy) with and withoutascorbate (0.5 mmol/L) and clonogenicsurvival determined. The resulting dosemodification factor at 40% iso-survivalwas 1.4, indicating enhancedradiosensitivity (mean� SEM; n¼ 3). H,patient-derived pancreatic cancer cells(403) in 4%O2were irradiated (0–6Gy)with and without ascorbate(0.5 mmol/L) and clonogenic survivaldetermined. The resulting dosemodification factor at 40% iso-survivalwas 1.6, indicating enhancedradiosensitivity (mean � SEM; n ¼ 3).






based on clonogenic surviving fraction. For the in vivo studies, thestatistical analyses focused on the effects of different treatmentson tumor progression. The primary outcomes of interest weretime to death and tumor growth over time. The log-rank test wasused to compare the survival times between treatment groups.Kaplan–Meier survival plotswere constructed to estimate survival.Linear mixed effects regression models were used to estimate andcompare group-specific tumor growth curves. Tests of statisticalsignificance were two-sided and performed using the Systat andSAS software.

ResultsAscorbate enhances IR-induced cytotoxicity

Previous studies from our laboratory have demonstrated thatpharmacologic ascorbate is cytotoxic to pancreatic cancer cellswhile normal cells are resistant (1). To test the effect of ascorbateon radiation response in cancer versus noncancerous cell lines, weperformed clonogenic (Fig. 1A–D) and cell viability assays (Fig.

1E and F) using three pancreatic ductal adenocarcinomas [MIAPaCa-2, PANC-1 (Fig. 1A and B), and AsPC-1 (Fig. 1C)], com-pared with normal nontumorigenic H6c7 pancreatic ductal epi-thelial cells (Fig. 1D). The radiation survival curves were normal-ized to ascorbate- or sham-treated controls and fit with a linearquadratic model. Pharmacologic ascorbate (0.25 mmol/L)enhanced IR-induced decreases in clonogenic survival. The pan-creatic cancer cell lines showed dose modification factors at 10%iso-survival of 2.5 (MIAPaCa-2), 2.2 (PANC-1; Fig. 1A andB), and1.25 (AsPC-1; Fig. 1C). In contrast, no radiosensitization wasnoted with ascorbate in the normal nontumorigenic H6c7 pan-creatic ductal epithelial cells (Fig. 1D). Similar results were seenusing cell viability where there was radiosensitization in the MIAPaCa-2 cell line (Fig. 1E)butno radiosensitization in theH6c7 cellline (Fig. 1F). To determine if ascorbate radiosensitization occursin the physiologically relevant environment seen in pancreaticcancer, we treated patient-derived pancreatic cancer cell lines, 339(Fig. 1G) and 403 (Fig. 1H; refs. 13, 14) in 4% O2. Again, wedemonstrated ascorbate radiosensitization in the 339 line with a

Figure 2.Ascorbate radiosensitization is mediated by H2O2. A, MIA PaCa-2 cells were treated with 3-AT to determine intracellular H2O2. Cells treated with IR þ ascorbate(20 mmol/L) showed an increased concentration of intracellular H2O2 (n ¼ 3, mean � SEM, P < 0.01). Pharmacologic ascorbate at 20 mmol/L produced H2O2 ata flux of 6,200 amol�1 cell�1 s�1 in our experimental conditions. B, ascorbate (2 mmol/L) radiosensitization depletes GSH and increases the half-cell reductionpotential (Ehc). Ascorbate at 2 mmol/L produced H2O2 at a flux of 1,400 amol�1 cell�1 s�1 in our experimental conditions (mean � SEM; n ¼ 3). C, catalasepartially rescues ascorbate (2 mmol/L) radiosensitization. Western blot demonstrates increased catalase immunoreactive protein in cells treated with the AdCATvector compared with AdEmpty-treated cells. D, MIA PaCa-2 cells were transfected with AdEmpty (50 MOI) or AdCAT (50 MOI) for 48 hours, then subjectedto IR (2 Gy), ascorbate (2 mmol/L), or the combined treatment. Ascorbate radiosensitization was reversed with catalase overexpression (n ¼ 3; mean� SEM; P < 0.01).

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DMF of 1.4 at 40% iso-survival and a DMF of 1.6 in the 403 cellline at 40% iso-survival. The timing for the administration ofpharmacologic ascorbate is also important. Treating cells withascorbate for 1 hour prior to IR or 1 hour immediately after IRdemonstrated a similar decrease in clonogenic survival whencompared with IR or ascorbate alone. However, when ascorbatewas administered 6 hours after IR, the decrease in clonogenicsurvival was decreased (Supplementary Fig. S1). These datastrongly support the hypothesis that pharmacologic ascorbate isa selective radiosensitizer in pancreatic cancer cells versus normalnontumorigenic pancreatic ductal epithelial cells.

Cell cycle analysis of ascorbate-induced radiosensitizationHydrogen peroxide generation has been reported to induce cell

cycle arrest at G1 and G2 (24). IR-induced DNA damage alsotriggers G1 or G2 arrest, allowing time for cells to repair DNAdamage (25). In MIA PaCa-2 cells treated with ascorbate(2 mmol/L) or 2 Gy alone or combination for 6, 24, 48, and72hours, therewas no significant change in cell cycle distribution.However, the sub-G1 population increased in cells treated with

ascorbate (2 mmol/L) or combination of 2 Gy IR and ascorbate(2mmol/L) at 6 and 24 hours. At 24 hours, there was a significantsub-G1 fraction in the combination group compared with thecontrol, IR alone, and ascorbate alone groups (SupplementaryFig. S2). In addition, the PI viability assay at 24 hours aftertreatment demonstrated that there was no significant change inviability with IR (2 Gy) alone while pharmacologic ascorbatealone induced an 89% � 1% sub-G1 fraction that was increasedto 96% � 2% with the combination treatment. These data,taken together with the clonogenic survival and cell viabilityassays, strongly support the hypothesis that pharmacologic ascor-bate is a radiosensitizer in pancreatic cancer cells.

Ascorbate radiosensitization is mediated by H2O2

Ascorbate-induced cytotoxicity ismediated by the formation ofH2O2 during the oxidation of ascorbate. Similarly, IR generatesnumerous ROS, including H2O2 and hydroxyl radical from theradiolysis of water. We hypothesized that ascorbate-inducedradiosensitization is mediated by H2O2. To determine if intracel-lular levels of H2O2 change upon exposure to pharmacologic

Figure 3.Ascorbate radiosensitization induces DNA damage. A, in MIA PaCa-2 pancreatic cancer cells, both IR (2 Gy) and ascorbate (2 mmol/L) induced the formation ofg-H2AX as determined by Western blot, whereas the combination treatment further enhanced g-H2AX formation. B, in AsPC-1 pancreatic cancer cells, ascorbate(2 mmol/L) radiosensitization increased g-H2AX immunoreactive protein, which was reversed with catalase pretreatment. C, g-H2AX immunohistochemistrydemonstrated increased g-H2AX formation with the combination treatment, which was reversed with catalase pretreatment. Cells were treated with ascorbate(1 mmol/L) and IR (1 Gy) or catalase (100 U/mL). Ascorbate at 1 mmol/L produced H2O2 at a flux of 690 amol�1 cell�1 s�1 under our experimental conditions. D,quantification of g-H2AX immunohistochemistry in MIA PaCa-2 cells treated with ascorbate (1 mmol/L) and IR (1 Gy) demonstrated increased induction ofg-H2AX during ascorbate radiosensitization, which was reversed with catalase pretreatment (n ¼ 3; mean � SEM; P < 0.01).






Figure 4.Pharmacologic ascorbate radiosensitization in vivo. A, linear mixed effects regression models were used to estimate and compare group-specific tumor growthcurves. Tumor growth was significantly inhibited with the ascorbate þ IR treatment compared with control animals or animals that received ascorbate alone.Control was saline (1 mol/L NaCl i.p. daily, 22.7 mL/g); IR (7.5 Gy on days 5 and 8 and 1 mol/L NaCl i.p. daily); ascorbate (4 g/kg i.p. daily); or ascorbate þ IR(mean � SEM, n ¼ 9–11 animals/group). B, Kaplan–Meier survival plots demonstrating survival as a function of time. The log-rank test was used for pairwisetreatment group comparisons of survival between treatment groups, demonstrating significantly increased overall survival of animals receiving pharmacologicascorbate and IR. C, pharmacologic ascorbate alters the status of the GSH redox buffer of RBCs. Blood was collected from separate groups of mice aftertreatments and assayed for the intracellular concentration of GSH in the RBCs. (Continued on the following page.)

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ascorbate, intracellular concentrations of H2O2 were determinedby the rate of aminotriazole-mediated inactivationof endogenouscatalase activity. Cells were treated with IR (3 Gy), ascorbate (20mmol/L), or in combination, and catalase activity was assayed incells treated with 3-aminotriazole (3-AT). Using the stoichiomet-ric inactivation of catalase in the presence of H2O2 and excess 3-AT, baseline intracellular concentration of H2O2 6 hours aftertreatment was 53 � 6 pmol/L in pancreas cancer cells; thisincreased to 71 � 9 pmol/L with exposure to pharmacologicascorbate, and 79 � 14 pmol/L following IR. However, thecombination of IR þ ascorbate increased the concentration ofintracellular H2O2 to 105 � 2 pmol/L (Fig. 2A).

Under steady-state conditions, intracellular GSH is main-tained at millimolar concentrations, which keeps cells in areduced environment and serves as the principal intracellularredox buffer when cells are subjected to an oxidative stressorincluding H2O2 (26). Glutathione peroxidase (GPx) activitycatalyzes the reduction of H2O2 to water with the conversion ofGSH to glutathione disulfide (GSSG). Under steady-state con-ditions, GSSG is recycled back to GSH by glutathione disulfidereductase using reducing equivalents from NADPH. However,under conditions of increased H2O2 flux, this recycling mech-anism may become overwhelmed, leading to a depletion ofintracellular GSH (27, 28).

Because ascorbate combined with radiation significantlyincreases the intracellular H2O2, we next examined the effectsof the combination of ascorbate and IR on the GSH/GSSGintracellular redox couple. MIA PaCa-2 cells were treated withIR (2 Gy) and ascorbate (2 mmol/L) and harvested at varioustime points (1–24 hours). Cells treated with IR þ ascorbateshowed a significant decrease in intracellular GSH concentrationat 6 hours compared with untreated cells (2.5 � 0.1 mmol/L vs.2.1 � 0.1 mmol/L, respectively, mean � S.D., n ¼ 3, P < 0.05)and failed to recover after 24 hours (Fig. 2B). The half-cellreduction potential (Ehc) of the GSSG/2GSH couple, which isa marker for overall redox environment of the cell (26), wascalculated using the Nernst equation. Ehc increased (more pos-itive), indicating a more oxidized intracellular redox environ-ment. This increase mirrored the depletion of GSH (Fig. 2B). MIAPaCa-2 cells have a baseline oxygen consumption rate of 57 amolcell�1 s�1 (29); assuming 1% efficiency, this corresponds to ametabolic rate for the production of H2O2 of less than 1 amolcell�1 s�1. The flux of H2O2 in these experiments was 1,400 amolcell�1 s�1, considerably greater than the metabolic flux, thusrecycling of GSSG to GSH is rate-limiting and depletion of GSHand an increase (more positive) in Ehc would be expected (28).Changes in Ehc have been shown to correlate with biologicstatus of the cell with a change from �240 mV to �170 mV ascells shift from the state of proliferation to the onset of cell death(26). These results indicate that ascorbate radiosensitization cancreate an overwhelming oxidative stress to pancreatic cancer cells,resulting in oxidation/depletion of the GSH intracellular redoxbuffer, resulting in cell death.

To further determine whether H2O2 was responsible for thecytotoxic effects of ascorbate þ radiation, cells were pretreatedwith either AdEmpty (50 MOI) or AdCAT (50 MOI) adenoviralvectors. Figure 2C demonstrates that there was robust expressionof immunoreactive intracellular catalase in MIA PaCa-2 cellstreated with the AdCAT vector when compared with control- orAdEmpty-treated cells. IR decreased clonogenic survival to 50% ofAdEmpty alone values, whereas ascorbate (2 mmol/L) decreasedclonogenic survival to 26% � 2% (Fig. 2D). The combination of2 Gy IR and ascorbate (2 mmol/L) further decreased clonogenicsurvival to 6% � 1%. This decrease in clonogenic survival wassignificantly reversed,with overexpression of intracellular catalaseresulting in clonogenic survival of 41% � 1%, which was notsignificantly different from IR, suggesting that H2O2 mediatesascorbate radiosensitization (Fig. 2D).

Ascorbate radiosensitization induces DNA damageg-H2AX is a sensor of DNA strand breaks (30) and promotes

efficient double-strand break repair (31). As shown in Fig. 3A, 2Gy IR and ascorbate (2mmol/L) induced formation of g-H2AX inMIA PaCa-2 cells. However, the combination of IR and ascorbateincreased formation of g-H2AX, indicating a greater number ofdouble-strand breaks. Previous studies have demonstrated thatascorbate-induced cytotoxicity ismediated by generation ofH2O2

(1–3). To determine if H2O2 mediates the increase in g-H2AXduring ascorbate-induced radiosensitization, cells were treatedwith extracellular catalase. Figure 3B demonstrates that the com-bination treatment of IR and ascorbate induced increased forma-tion of g-H2AX in AsPC-1 cells compared with either treatmentalone. The increase in g-H2AX with the combination treatmentwas inhibited with catalase pretreatment, suggesting that H2O2

mediates the increase in g-H2AX during ascorbate-induced radio-sensitization. In addition to Western blot, we demonstratedsimilar increases in double-strand DNA breaks as seen by g-H2AXon immunohistochemistry (Fig. 3C). In cells treated with ascor-bate (1 mmol/L), IR alone, or the combination of the twotreatments, the increase in fluorescence was reversed with catalasepretreatment. Once again, quantification of these images dem-onstrated significant increases in fluorescence after ascorbatetreatment and after IR þ ascorbate combination treatment thatwas ameliorated with catalase pretreatment (Fig. 3D). Theseexperiments were repeated with varying doses of pharmacologicascorbate and varying doses of IR with similar results (Supple-mentary Fig. S3). Thus, H2O2 significantly contributes to theDNAdamage observed upon exposure to pharmacologic ascorbate andIR, as seen by g-H2AX.

Ascorbate-induced radiosensitization in vivoWe have previously demonstrated that ascorbate alone inhi-

bits tumor growth in a mouse xenograft model with intraper-itoneal administration of 4 g/kg twice daily (1). Pre-establishedMIA PaCa-2 tumors in nude mice were treated with either:saline (1 mol/L NaCl i.p. daily); IR (7.5 Gy on days 5 and 8 and

(Continued.) Both IR and ascorbate alone decreased intracellular GSH compared with controls. The combination of ascorbate and IR did not further decrease GSHwhen compared with IR alone. D, complete blood counts and differential in control mice and those treated with ascorbate, IR, and IR þ ascorbate. Whiteblood cell counts were decreased with IR, which was unchanged when ascorbate was added to the treatment regimen. E, initially, purified BSA was reacted withexcess purified 4-HNE to create a positively labeled protein control, which is seen in the top plot. The negative control was purified BSA without purified 4-HNE.Then, to determine 4-HNE–modified proteins in separate groups of mice, dot blots for 4-HNE–modified proteins in cardiac muscle showed no changes inimmunoreactive protein after any of the treatments compared with controls. F, dot blots for 4-HNE–modified proteins in heart, kidney, and liver demonstrated nochanges in immunoreactive protein after any of the treatments compared with controls.






1 mol/L NaCl i.p. daily); ascorbate (4 g/kg i.p. daily); orascorbate þ IR. Supplementary Table S1 provides statisticalsummaries of tumor volumes used in the mixed linear regres-sion analysis of growth curves. The sample sizes (N) given inthe table are the total number of measurements availablewithin each group. Pairwise comparisons were carried out toassess group differences. Treatment with the combination ofascorbate þ IR significantly delayed tumor growth comparedwith controls or ascorbate alone (Fig. 4A). Summary statisticsfor the survival analysis are presented in Supplementary TableS2. The estimated survival curve for the treatment groups isgiven by the Kaplan–Meier plots in Fig. 4B. Ascorbate þ IR alsosignificantly increased overall survival compared with controls,IR alone, or ascorbate alone (Fig. 4B). Most notably, 54% ofmice treated with the combination of IR þ ascorbate had nomeasurable tumors (Supplementary Table S2). Treatment waswell tolerated as evidenced by similar weight gain patterns in allgroups of mice (Supplementary Fig. S4).

Oxidative stress indicatorsGlutathione is a measurable marker indicative of the oxidation

state of the thiol redox buffer in cells. In severe systemic oxidativestress, the GSSG/2GSH couple may become oxidized, i.e., theconcentration of GSH decreases and GSSG may increase becausethe capacity to recycle GSSG to GSH becomes rate-limiting. Asseen in Fig. 4C, intracellular concentrations of GSH in red bloodcells of mice after ascorbate, IR, and ascorbate þ IR treatmentsdecreased. However, the addition of pharmacologic ascorbatewith IR did not significantly reduce GSH levels compared with5 Gy IR alone. This suggests that the very high levels of pharma-cologic ascorbate in these experiments may have a pro-oxidanttoward red blood cells as seen by a decrease in the capacity of theintracellular redox buffer. To further determine the effect of thesetreatments on hematological parameters, complete blood countswere determined in separate groups of mice. Although ascorbateand IR decreased intracellular GSH in red blood cells, there wereno significant changes in hemoglobin and/or hematocrits

between groups of mice. As seen in Fig. 4D, mice that receivedIR alone or IR þ ascorbate had a significant decrease in whiteblood cells, suggesting some immunosuppression with IR. How-ever, ascorbate did not add to further decreases in white bloodcells, and the percentage of neutrophils and lymphocyte countswere unchanged between groups.

Oxidation of lipids generates highly reactive aldehydes, suchas 4-hydroxynonenal (4-HNE), which can be used as a markerof oxidative damage (32, 33). To further examine systemicoxidative stress, we harvested heart, kidney, and liver frommice treated with the various combinations to determine 4-HNE–modified proteins. As seen in Fig. 4E, there were nodifferences in 4-HNE–modified proteins in heart tissue in micetreated with the various combinations. In addition, dot blotanalysis of the other organs demonstrated no differences in 4-HNE proteins after the various treatments (Fig. 4F). These datasupport the hypothesis that ascorbate radiosensitization doesnot cause an increase in oxidative damage from lipid-derivedaldehydes to other organs.

Orthotopic tumorswere established using ultrasound guidanceas described (Fig. 5A; ref. 21). After bioluminescence-imagingmicroscopy identified mice with established tumors within thepancreas (Fig. 5B), mice were randomly assigned to one of fourtreatment groups: controls received NaCl (1 mol/L) i.p. daily;ascorbate 4 g/kg i.p. daily; NaCl i.p. daily and two separatefractions of 5 Gy IR one week apart on days 4 and 11; a groupthat received both IR and ascorbate. Tumor sizes were measuredweekly throughout the experiments, resulting in repeated mea-surements for eachmouse. Linearmixed effects regressionmodelswere used to estimate and compare group-specific tumor growthcurves. Supplementary Table S3 provides statistical summaries oftumor volumes used in the linearmixed effects regression analysisof growth curves. The sample sizes (N) given in the table are thetotal number of measurements available within each group.Pairwise comparisons were carried out to assess group differences.P values for the comparisons are listed in Supplementary Table S3.Tumor growth as determined by bioluminescence-imaging

Figure 5.Pharmacologic ascorbate radiosensitization in an orthotopic model. A, ultrasound-guided pancreatic injections. MIA PaCa-2 cells (4 � 105) suspended ina 20 mL 1:1 mixture of PBS and Matrigel were injected into athymic nude mice who were sedated with Isoflurane. Under ultrasound guidance, injections wereperformed directly into the pancreas just medial and inferior to the spleen. The spleen and pancreas are labeled. The yellow arrow indicates the shadow of the needleduring injection. B, bioluminescence imaging microscopy. Four days after the initial injections, mice were imaged using the Xenogen IVIS 200 microscope10 minutes after injection with 200 mL of 15 mg/mL luciferin. Initial exposure time was 1 minute. Displayed is a color scale of photons/second emitted superimposedover a photograph of the mouse as viewed in the prone position. The bioluminescence was localized to the left quadrant central quadrant as would beexpected for a pancreatic tumor. C, linear mixed effects regression models demonstrated significant inhibition in tumor growth with the ascorbateþ IR treatmentcomparedwith control animals or animals that received IR alone. Control was saline (1mol/LNaCl i.p. daily, 22.7 mL/g); IR (7.5 Gy on days 4 and 11 and 1mol/LNaCl i.p.daily); ascorbate (4 g/kg i.p. daily); or ascorbate þ IR (mean, n ¼ 7–12 animals/group). � , P < 0.05, control versus ascorbate þ IR; #, P < 0.05, ascorbate þ IRversus IR alone.

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microscopy (mean radiance photons/s/cm2) demonstrated adecrease in tumor growth of animals receiving both IR andascorbate compared with controls and compared with IR alone(�, P < 0.05 control vs. Ascorbateþ IR; �#, P < 0.05 Ascorbateþ IRvs. IR alone; Fig. 5C; Supplementary Fig. S5). As seen in theprevious heterotopic study, weights of the animals did not changeduring the study (data not shown).

Radiation-induced gastrointestinal toxicity is highly relevant tothe treatment of pancreatic cancer with radiation. To determine ifpharmacologic ascorbate changes the response of the gastroin-testinal tract following radiation in a clinically meaningful way, acrypt cell assay was performed (Fig. 6). Control animals and those

treatedwith pharmacologic ascorbate (4 g/kg) had similar degreesof crypt cell generation. IR alone (10 Gy) decreased crypts to 28%of control values, while 13 Gy decreased crypts to 44% of controlvalues. Addition of pharmacologic ascorbate partially reversed thedecreases in both the 10 Gy and 13 Gy groups of mice, suggestingthat ascorbate may protect the gastrointestinal tract from thedamaging effects of IR. In addition, previous studies have dem-onstrated that radiation-induced jejunal toxicity was accompa-nied by increases in serum TNFa (34). TNFawas 7.5� 2.2 pg/mLin controls and was increased to 25.0 � 1.5 pg/mL after 13 Gy.Treatment with pharmacologic ascorbate combined with IRdecreased TNFa to 12.5 � 0.7 pg/mL (mean � SEM, n ¼ 3, P <

Figure 6.Pharmacologic ascorbate partially reverses IR-induced jejunal damage. A, crypt cell assay fromcontrol mice. Sections were made of the jejunumand then viewed under light microscopy andexpressed as surviving cells/circumference. B, micewere treated with ascorbate 4 g/kg i.p. for 5 daysand then sacrificed. C, mice were exposed to 10 Gyof total abdominal radiation. After 48 hours, eachanimal was sacrificed and sections were made ofthe jejunum as described. D, mice treated withpharmacologic ascorbate (4 g/kg for 5 dayswith 10Gy total abdominal radiation on day 3 and thensacrificed on day 5). E, mice were exposed to 13 Gyof total abdominal radiation and sacrificed 48 hourslater. Note marked decrease in number ofregenerating crypts/circumference of thesectioned jejunum. F, mice treated withpharmacologic ascorbate (4 g/kg for5 dayswith 13 Gy total abdominal radiation on day 3and then sacrificed on day 5). Note increase inregenerating crypts compared with E. G,quantification of crypt cell assay. Mean � SEM;n ¼ 8–10 samples/group. � , P < 0.01 versuscontrols and ascorbate-treated animals. #, P < 0.05versus control and ascorbate-treated animals.






0.05 IR alone vs. ascorbate þ IR). Taken together, these datasuggest that pharmacologic ascorbate may protect the gut locallyby decreasing IR-induced damage to the crypt cells, and system-ically, by ameliorating increases in TNFa.

The in vivo animal experiments were repeated using PANC-1tumors. In these sets of experiments, we added gemcitabine to theIR group as done in clinical practice (35). The mice were dividedinto four groups and treated with either: saline (1mol/L NaCl i.p.daily); IR/gemcitabine (5Gy on days 4 and 8with gemcitabine 60mg/kg i.p. every fourth day for 2 weeks and 1 mol/L NaCl i.p.daily); ascorbate (4 g/kg i.p. daily); or ascorbate þ IR/gemcita-bine. As seen in Fig. 7A, treatment with IR/gemcitabine delayedtumor growth compared with ascorbate alone (P < 0.05). How-ever, the combination of ascorbate þ IR/gemcitabine furtherdelayed tumor growth compared with ascorbate alone (P <0.001). Summary statistics for the survival analysis are presentedin Supplementary Table S4. The estimated survival curve for thetreatment groups is given by the Kaplan–Meier plots in Fig. 7B.Ascorbate þ IR/gemcitabine also significantly increased overallsurvival compared with ascorbate alone (Fig. 7B). Most notably,100% of the mice on day 43 that were treated with the combi-nation of ascorbate þ IR/gemcitabine were surviving (Fig. 7B).Treatment was well tolerated as evident by similar weight gainpatterns in all groups of mice (Fig. 7C).

DiscussionOur current study demonstrates the potential for pharmaco-

logic ascorbate as a radiosensitizer in the treatment of pancreaticcancer. IR is the standard of care in pancreatic cancer in a variety of

clinical scenarios, including (i) locally advanced pancreatic can-cer; (ii) positive margins or positive lymph nodes after pancreaticresection; or (iii) large tumors that obstruct the duodenum(36). Chemotherapy combined with radiotherapy has alsoshown to improve survival in pancreatic cancer when comparedwith single-modality therapy (37). In addition, radiotherapycan also provide palliation to patients with locally advanceddisease (38). Thus, pharmacologic ascorbate-induced radiosensi-tization may have clinical benefits.

In our present study, we have shown that pharmacologicascorbate significantly decreases clonogenic survival and inhibitsthe growth of all pancreatic cancer cell lines as a single agent, aswell as sensitizes cancer cells to IR. This corresponds well withother reports demonstrating that pharmacologic ascorbateenhances IR-induced cell killing and DNA fragmentation, leadingto induction of apoptosis in HL60 leukemia cells (39). In addi-tion, Hurst and colleagues demonstrated that pharmacologicascorbate combined with IR leads to increased numbers of dou-ble-strand DNA breaks and cell cycle arrest when compared witheither treatment alone (40). Our previous studies demonstratedthat pharmacologic ascorbate could serve as a "prodrug" for thedelivery of H2O2 to tumors (1–4). There was both a time- anddose-dependent increase in measured H2O2 production withincreased concentrations of ascorbate. Others have demonstratedincreased levels of double-strand breaks with pharmacologicascorbate andH2O2 treatment to tumor cells (41, 42). In addition,the double-strand breaks induced by H2O2 were more slowlyrepaired.

Previously, we demonstrated that the immortalized, nontu-morigenic pancreatic ductal epithelial cells H6c7 are resistant to

Figure 7.Pharmacologic ascorbateradiosensitization in vivo. A, linearmixed effects regression models wereused to estimate and compare group-specific tumor growth curves. Tumorgrowth was significantly inhibited inmice with pancreatic tumor xenograftstreated with IR/gemcitabine comparedwith controls and ascorbate alone.Tumor growth was further inhibited inmice that received the ascorbate þ IR/gemcitabine treatment compared withcontrol animals or animals that receivedascorbate alone (mean� SEM, n¼ 11–12mice/group). B, Kaplan–Meier survivalplots demonstrating survival as afunction of time. The log-rank test wasused for pairwise treatment groupcomparisons of survival betweentreatment groups, demonstratingsignificantly increased overall survivalanimals receiving pharmacologicascorbate and IR/gemcitabinetreatment on day 43 after the initiationof treatment. C,weight changes ofmiceduring treatment periods inmiceduringtreatments from day 1 through day 15.

Du et al.





ascorbate-induced toxicity (1). Consistent with this study, ourcurrent study demonstrates that H6c7 cells are also resistant to thecombination treatment of IR and ascorbate. The selective ascor-bate-induced cytotoxicity may be due to the combination of lowlevels of antioxidant enzymes and high endogenous levels of ROSin cancer cells (43–45). Moreover, tumor cells are often defectivein DNA repair, whereas normal cells are proficient (46). Thecombination of ascorbate and IR provides two distinct mechan-isms of action: ascorbate-induced toxicity due to extracellularproduction of H2O2 that then diffuses into cells and causesdamage to DNA, protein, and lipids; and radiation-inducedtoxicity as a result of ROS-induced damage to DNA. In addition,redox metals like Fe2þ may play an important role in ascorbate-induced cytotoxicity. By catalyzing the oxidation of ascorbate,labile iron can enhance the rate of formation of H2O2; labile ironcan also react with H2O2. Recently, our group has demonstratedthat pharmacologic ascorbate and IR increase the labile iron intumor homogenates from thismurinemodel of pancreatic cancer(47). In our present study using a pancreatic cancer xenograftmodel, we demonstrated that ascorbate or IR alone decreasedtumor growth, but the combination treatment further inhibitedtumor growth, indicating that pharmacologic ascorbate is aneffective radiosensitizer in vivo. In addition, all groups of micehad similar weight gain during treatment, suggesting lack ofnormal tissue toxicity with the combination treatment.

That approximately 30% of pancreatic patients receive adiagnosis of advanced locoregional disease underscores theimportance of radiotherapy for local disease control (48). Ourpreclinical model shows that the combination of IR and ascor-bate enhances toxicity in pancreatic cancer cells compared witheither treatment alone. In a phase I clinical trial that investi-gated the use of pharmacologic ascorbate as an adjuvant togemcitabine, the standard of care, in the treatment of pancreaticcancer, it was well-tolerated with few dose-limiting toxicities(49). The data reported here support testing of pharmacologicascorbate as an adjuvant treatment for radiotherapy in pancre-atic cancer patients after surgical resection or with locallyadvanced disease. Indeed, the preclinical translational studiesin this current study have led us to develop and implement aphase I clinical trial (www.clinicaltrials.gov; NCT01852890,Cullen PI) with FDA approval (IND 105715, Cullen spon-sor-investigator).

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

Authors' ContributionsConception and design: J. Du, J.L.Welsh, B.A.Wagner, G.R. Buettner, J.J. CullenDevelopment of methodology: J. Du, J.L. Welsh, B.G. Allen, B.A. Wagner, G.R.Buettner, J.J. CullenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Du, J.A. Cieslak III, J.L. Welsh, Z.A. Sibenaller,B.G. Allen, B.A.Wagner, A.L. Kalen,C.M.Doskey, R.K. Strother, S. Tsai, J.Mezhir,P.C. Goswami, G.R. Buettner, J.J. CullenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.A. Cieslak III, J.L. Welsh, B.A. Wagner, C.M. Doskey,R.K. Strother, A.M. Button, S.L. Mott, B. Smith, P.C. Goswami, D.R. Spitz,G.R. Buettner, J.J. CullenWriting, review, and/or revision of the manuscript: J. Du, J.L. Welsh,Z.A. Sibenaller, C.M. Doskey, S.L. Mott, B. Smith, D.R. Spitz, G.R. Buettner,J.J. CullenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.M. Doskey, J. Mezhir, P.C. Goswami,J.J. CullenStudy supervision: P.C. GoswamiOther (assisted with assays measuring DNA damage; provided radiationservice): P.C. Goswami

AcknowledgmentsThe authors thank The University of Iowa Central Microscopy Research

Facility for their guidance and technical advice. They also thank Dr. Luke I.Szweda from the Medical Research Foundation, Oklahoma City, Oklahoma.The University of Iowa ESR Facility and the Radiation and Free Radical ResearchCore in the Holden Comprehensive Cancer Center provided invaluable assis-tance in the execution of these studies.

Grant SupportThis study was supported by NIH grants RO1 CA184051, RO1 CA169046,

P30 CA086862, P42 ES013661, RO1 CA111365, RO1 CA182804, T32CA078586, T32 CA148062, the Medical Research Service, an ASTRO CareerDevelopment Award JF2014-1, and the Department of Veterans Affairs1I01BX001318-01A2.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 6, 2014; revised May 12, 2015; accepted May 20, 2015;published OnlineFirst June 16, 2015.

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2015;75:3314-3326. Published OnlineFirst June 16, 2015.Cancer Res Juan Du, John A. Cieslak III, Jessemae L. Welsh, et al. Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer

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Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer · The current study demonstrates that pharmacologic ... ascorbate as a radiosensitizer in the treatment of pancreatic