PANC-1 cells proliferative response to ionizingradiation is related to GSK-3� phosphorylation

Nora A. Mohamad, Graciela P. Cricco, Claudia M. Cocca, Elena S. Rivera,Rosa M. Bergoc, and Gabriela A. Martín

Abstract: Radiotherapy may be used to treat pancreatic cancer and relieve pain. We have previously reported thathistamine modulates pancreatic adenocarcinoma PANC-1 cell proliferation. This work was aimed to evaluate whetherhistamine improves radiosensitivity of PANC-1 cells in relation to phosphorylation/inhibition of glycogen synthase kinase-3� (GSK-3�). Immediately after � irradiation, intracellular hydrogen peroxide was markedly decreased together with arapid increase in catalase activity. Although histamine diminished catalase activity in nonirradiated cells, it only partiallyhindered the increase observed in irradiated cells and could not modify radiosensitivity. In control cells, a high expressionof total and a very low expression of phosphorylated/inactive GSK-3� were found. An increment in reactive oxygenspecies levels produced an augmentation in GSK-3� phosphorylation and suppressed cell proliferation. In both control andhistamine-treated irradiated cells, the rise in catalase activity lowered reactive oxygen species levels and only a smallincrease in phosphorylated GSK-3� was detected. Alternatively, 3-aminotriazole, an irreversible inhibitor of catalase,reduced the survival fraction in irradiated control cells along with an increment in phosphorylated GSK-3�. These resultssuggest that upon irradiation, early catalase activation may be responsible for keeping GSK-3� active conceding cells asurvival advantage toward cytotoxic effects of ionizing radiation.

Key words: PANC-1, ionizing radiation, GSK-3�, histamine.

Résumé : La radiothérapie peut être utilisée pour traiter le cancer du pancréas et soulager aussi la douleur. Nous avonsdéja rapporté que l’histamine module la prolifération des cellules de cancer pancréatique PANC-1. Ce travail visait aévaluer si l’histamine pouvait améliorer la radiosensibilité des cellules PANC-1 en lien avec la phosphorylation/inhibitionde la glycogène synthase kinase-3� (GSK-3�). Immédiatement après l’irradiation gamma, le peroxyde d’hydrogèneintracellulaire diminuait de façon marquée, parallèlement a une augmentation rapide de l’activité de la catalase. Même sil’histamine diminuait l’activité de la catalase chez les cellules non irradiées, elle freinait partiellement seulementl’augmentation observée chez les cellules irradiées et n’affectait pas la radiosensibilité. Chez les cellules contrôles, uneexpression élevée de GSK-3� totale et une très faible expression de GSK-3� phosphorylée/inactive étaient trouvées. Uneaugmentation des niveaux d’espèces réactives d’oxygène produisait une augmentation de la phosphorylation de GSK-3� etinhibait la prolifération cellulaire. Tant chez les cellules contrôles que chez les cellules irradiées traitées a l’histamine,l’augmentation de l’activité de la catalase abaissait les niveaux d’espèces réactives d’oxygène et seule une petiteaugmentation de GSK-3� phosphorylée était détectée. Sinon, le 3-aminotriazole, un inhibiteur irréversible de la catalase,réduisait la fraction survivante des cellules contrôles irradiées parallèlement a une augmentation de GSK-3� phosphorylée.Ces résultats suggèrent qu’a la suite de l’irradiation, l’activation précoce de la catalase pourrait être responsable de garderla GSK-3� active, conférant aux cellules un avantage de survie envers les effets cytotoxiques des radiations ionisantes.

Mots-clés : PANC-1, radiation ionisante, GSK-3�, histamine.

[Traduit par la Rédaction]

Introduction

Pancreatic cancer is a devastating disease with a poorprognosis because of its rapid systemic progression. Radio-therapy, in combination with chemotherapy, may be usedfor nonmetastasized pancreatic cancer that cannot be re-

moved by surgery, as a neoadjuvant before surgery, andalso to relieve pain.

It is well known that ionizing radiation (IR) inducesreactive oxygen species (ROS) and reactive nitrogen spe-cies (RNS) production, such as superoxide anion (SO),hydrogen peroxide (H2O2), and nitric oxide (NO), which

Received 27 January 2012. Revised received 16 May 2012. Accepted 9 June 2012. Published at www.nrcresearchpress.com/bcb on25 November 2012.

N.A. Mohamad, G.P. Cricco, and E.S. Rivera. Laboratorio de Radioisótopos. Facultad de Farmacia y Bioquímica. Universidad deBuenos Aires. Junín 956, C1113AAB, Buenos Aires, Argentina.C.M. Cocca, R.M. Bergoc, and G.A. Martín. Laboratorio de Radioisótopos. Facultad de Farmacia y Bioquímica. Universidad deBuenos Aires. Junín 956, C1113AAB, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas,Universidad de Buenos Aires. Junín 956, C1113AAB, Buenos Aires, Argentina.

Corresponding author: Gabriela A. Martín (e-mail: gamartin@ffyb.uba.ar).

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Biochem. Cell Biol. 90: 779–790 (2012) Published by NRC Research Pressdoi:10.1139/o2012-032

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are involved in radiation damage (Ward 1988). ROS con-centrations are tightly controlled by specific scavenging sys-tems comprising enzymatic and nonenzymatic antioxidants.The most important enzyme systems include catalase, super-oxide dismutases (SOD), and glutathione peroxidase (GPx).When the capacity of cell antioxidant defences is surpassed,excessive amounts of ROS and RNS may start toxic reactionsoxidizing and disabling structures required for cell survival(Sun et al. 1998). Alternatively, ROS and RNS depending ontheir concentration, timing, and location are important signal-ing molecules in biological processes such as gene expressionand cell growth, differentiation, and apoptosis (Thannickal andFanburg 2000).

Radiosensitizers, drugs that reduce the proportion of sur-viving cells exposed to IR, are used in radiotherapy to improvethe therapeutic response. They are intended to enhance tumor-cell killing while having less or null effects on normal tissues.We have determined that histamine (HA) acts as a radiosen-sitizer for human breast cancer cells but also as a radioprotec-tor for normal tissues. HA protects bone marrow againstcellular damage induced by IR and also prevents radiation-induced toxicity on small intestine by modulating antioxidantenzymes expression, suppressing apoptosis, and increasingproliferation of damaged intestinal mucosa (Medina et al.2007, 2010).

HA involvement in normal and neoplastic cell growth hasbeen extensively studied and a dose-dependent inhibitoryeffect on cell proliferation has been reported (Rivera et al.2000; Blaya et al. 2010). In previous work we demonstratedthat human pancreatic carcinoma cell line PANC-1 expressesH1, H2, H3, and H4 HA receptors and that HA differentiallymodulates cell growth (Cricco et al. 2006, 2008).

NO is synthesized from L-arginine by different nitric oxidesynthase isoenzymes (NOS). We have demonstrated thatPANC-1 cells express only the constitutive endothelial NOSisoform that is induced by high concentrations of HA (Criccoet al. 2007).

Glycogen synthase kinase-3� (GSK-3�) is a Ser/Thr proteinkinase that plays a major role in epithelial cell homeostasis andin diverse human diseases when deregulated (Frame andCohen 2001). GSK-3� is active in normal resting cells and itssubstrates are usually inhibited by phosphorylation, thereforeGSK-3� appears as a prevalent repressor. The promoting orsuppressing role in tumor-cell growth depends on the cell type(Mishra 2010). GSK-3� activity is inhibited by phos-phorylation at Ser9, which may be differentially regulated byROS according to concentration and cell type (Cannito et al.2008; Nair and Olanow 2008).

This work was aimed to characterize the radiobiological pa-rameters of PANC-1 cells and to evaluate if HA modifies clono-genic proliferation of irradiated cells in relation to ROS and NOintracellular levels modulation and the phosphorylation/inhibitionof GSK-3�.

Materials and methods

Cell linePANC-1 cells (ATCC No. CRL1469) were cultured in

RPMI 1640 (Cricco et al. 2006).

Clonogenic proliferation assaysThe clonogenic assay is an in vitro cell survival assay based

on the ability of a single cell to grow into a colony. A colonyis defined to consist of at least 50 cells. The assay essentiallytests every cell in the population for its ability to undergounlimited division. The clonogenic assay is the method ofchoice to determine cell reproductive death after treatmentwith IR and can also be used to determine the efficacy of otheragents (Franken et al. 2006).

Cells were seeded in 6-well plates as single-cell suspensions(3300 cells/well) and treated with different concentrations of3-morpholino-sydnonimine hydrochloride (SIN-1), N-acetyl-L-cysteine (NAC), sodium nitroprussiate (SNP), HA, catalase (allfrom Sigma-Aldrich, St Louis, Mo.), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), 3-amino-1,2,4-triazole (3-AT) (both from Calbiochem-EMDBiosciences,Darmstadt, Germany), and H2O2 (Merck, Darmstadt, Ger-many). After 8 days, cultures were fixed with 4% bufferedsaline formaldehyde, stained with 1% toluidine blue, andobserved using a light microscope. Clonogenic proliferationwas evaluated by counting colonies containing 50 cells ormore and expressed as a percent of the control (Medina etal. 2006).

Survival curvesCells were seeded in 6-well plates (3300 cell/well) and after

24 h were irradiated with 0–5 Gy with an IBL 437C H typeirradiator, 137Cs source, dose rate 7.1 Gy/min. HA (10 �mol/L)was added or not immediately after plating and continued up to24 h post irradiation. After 8 days, clonogenic proliferation wasevaluated. The survival fraction was calculated as the ratio be-tween the number of colonies counted in each dose of irradiationand the number of colonies in the corresponding nonirradiatedcultures (dose zero). Survival fraction was calculated for HA-treated cultures and for nonHA-treated cultures. Survival curves(survival fraction vs. dose (SF vs. D)) were plotted and fitted tothe linear-quadratic model, SF � exp – (�D � �D2), usingGraphPad Prism 5.0 software (Calif.). Curves were used to de-termine survival parameters �, �, �/�, and 2 Gy survival fraction(2 Gy SF).

Analysis of intracellular SO, H2O2, and NODihydroethidium (DH), dichlorofluorescein-diacetate (DCF-

DA), and diaminofluorescein-diacetate (DAF-2DA) are specificdyes (Sigma–Aldrich) used to monitor intracellular SO, H2O2,and NO, respectively (Nair and Olanow 2008; Ding et al. 2002).Cells were either treated with 10 �mol/L HA for 24 h or leftuntreated; then, cells were irradiated with 2 Gy or not. Cellswere incubated with 5 �mol/L of each dye (25 min at 37 °C)and subjected to flow-cytometric analysis using a FACSCali-bur instrument (Becton Dickinson, Franklin Lakes, N.J.). Forirradiated cells 2 experimental conditions were run by addingdyes immediately before or after irradiation.

ImmunoblottingCells (7 � 105) were incubated for 24 h in the presence or

absence of treatments and then irradiated with 2 Gy or not. Atdifferent times after irradiation immunoblots were performed(Medina et al. 2006). Specific antibodies anti-catalase(1:2000), anti-�-actin (1:5000), anti-p-Ser9 GSK-3� (1:1000),or anti-GSK-3� (1:1000) were used. Nuclear and cytosolicfractions were obtained following Abcam protocols (www.

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abcam.com). Anti-Cu/Zn superoxide dismutase (Cu/Zn SOD,1:1000) and anti-lamin B1 (1:1000) antibodies were used tocontrol cytosolic and nuclear fractionation, respectively(Ougolkov et al. 2006).

Indirect immunofluorescenceCells were incubated for 24 h in the presence or absence of

treatments and then irradiated with 2 Gy or not. One hour afterirradiation, indirect immunofluorescence was performed(Medina et al. 2006) using rabbit anti-p-Ser9 GSK-3� (1:100)or rabbit anti-GSK-3� (1:100). Fluorescence was visualizedwith a confocal microscope (Nikon CI, objective lenses Nikon60x/1.40, Plan Apo).

AntibodiesMouse IGg1 isotype anti-catalase, mouse IGg1 isotype anti-

�-actin, horseradish peroxidase-conjugated anti-mouse oranti-rabbit, and FITC-conjugated anti-rabbit antibody werefrom Sigma–Aldrich. Rabbit anti-p-Ser9 GSK-3� and rabbitanti-GSK-3� were from Cell Signaling Technology, Danvers,Mass.. Horseradish peroxidase-conjugated anti-sheep antibodywas from Chemicon International, Temecula, Calif. Rabbit poly-clonal anti-lamin B1 antibody was from Abcam, Cambridge,U.K. Sheep anti-Cu/Zn SOD antibody was from Calbiochem,Darmstadt, Germany.

Catalase activity assayCells were either treated with10 �mol/L HA for 24 h or left

untreated and irradiated with 2 Gy or not. Catalase activity wasevaluated immediately after irradiation or 24 h later (Medinaet al. 2006).

Results

Survival curvesIn radiotherapy biological death, incapacity of cells to di-

vide after irradiation is a major concern. To plot the cellsurvival curve, the fraction of cells capable of dividing andproducing colonies following a known dose of radiation (sur-vival fraction) is calculated and plotted against dose. Amongmathematical models used to fit the survival data the quadraticmodel (SF � exp – (�D � �D2)) is the best one that definesthe response for low doses (Joiner 1997) that are of interest inradiotherapy. Survival parameters determined from the sur-vival fraction vs. dose curve describe the shape of the curveand are a convenient way to compare the response of cells withIR. The � coefficient and 2 Gy SF are estimates of intrinsicradiosensitivity, while the � coefficient indicates the efficiencyof DNA repair. PANC-1 cells disclosed a relatively highradioresistance (Fig. 1) as evidenced by the determined coef-ficients (Tubiana et al. 1990). Furthermore, HA was not ef-fective in modifying the response of PANC-1 cells to IR asrevealed by the identical survival curves. The area under thesurvival curve (AUC) in a linear–linear plot (also called meaneffective dose) is another parameter representative of wholecell population behavior and defines the survival data withoutany assumed mechanism of death. When AUCs were deter-mined for control and HA-treated irradiated cells, no signficantdifferences were observed (1.51 � 0.04 vs. 1.61 � 0.02,respectively; p � 0.51, t test).

Role of ROS and NO in cell proliferationROS intracellular levels, evaluated by the fluorescent probe

DCF-DA, were increased in a concentration-dependent way(Fig. 2A) while a decrease in clonogenic proliferation wasobserved when PANC-1 cells were exposed to differentconcentrations of exogenous H2O2 (Fig. 2B). The addition of125 IU of catalase, an enzyme that metabolizes H2O2 to waterand oxygen, counteracted the inhibitory effect of exogenousH2O2 on proliferation and also produced a slight diminution inROS intracellular basal levels with no significant effect on cellgrowth. HA produced an increase in the intracellular concentra-tion of ROS (Table 1) and its effect on proliferation washindered by catalase, indicating that H2O2 is the main speciesinvolved, even if DCF-DA could detect other ones (Fig. 2C).

To analyze the role of NO in PANC-1 cell growth, cultureswere treated with SIN-1 or SNP, 2 NO donors, which increasedintracellular NO levels. They both led to inhibition of cell growth(Fig. 2C and Supplementary Fig. S11). Micromolar concentra-tions of HA decreased proliferation to a similar extent as10 �mol/L SIN-1, and the same doses caused an increase inH2O2 and NO intracellular levels analogous to that produced by theexternally added concentrations of H2O2 or NO donors. To furtherassess the role of H2O2 and NO in HA inhibition of cell proliferationwe tested the effect of adding catalase or PTIO (a NO scavenger) incombination with HA. In both cases, proliferation was partiallyrestored to control values (Fig. 2C).

Effect of irradiation and HA on intracellular steady statelevels of ROS and NO

Adjuvant or neoadjuvant chemoradiation for pancreatic can-cer consists typically of a split course of radiation therapygiven concomitantly with chemotherapy. Conventionally total

1Supplementary data are available with the article through the journal Web site (www.nrcresearchpress.com/doi/suppl/10.1139/o2012-032).

Fig. 1. Histamine (HA) did not affect the response of PANC-1cells to ionizing radiation. Control and HA-treated cells wereirradiated (0–5 Gy). HA (10 �mol/L) was added after plating andcontinued up to 24 h post-irradiation. Eight days post irradiation,survival curves (survival fraction vs. dose) were plotted, fitted tothe linear-quadratic model, SF � exp (�D � �D2), usingGraphPad Prism 5.0 software, and used to determine parameters �,�, �/�, and 2 Gy survival fraction (2 Gy SF). Results represent themean � SE of 2 independent experiments run in duplicate.

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radiation dose is delivered daily in 2 Gy fractions, thereforethis dose was used in the following experiments.

Steady states of NO and ROS were evaluated in PANC-1 cellsin response to irradiation. Specific dyes were used to assess thereactive species in 2 different experimental conditions as follows:dyes were added to cells immediately before irradiation to scav-enge all the reactive species produced during irradiation or dyeswere added 5 min after irradiation to appraise the species remain-ing in cells after that time. Increases in both NO and ROS wereobserved in control cells when dyes were added before irradia-tion. In this condition, HA treatment produced an increase in bothspecies, in irradiated and nonirradiated cells (Table 1). Alterna-tively, ROS levels were markedly lessened when evaluatedafter irradiation. A similar effect, though less pronounced, wasfound for NO levels. However, no significant differences weredetected in SO levels, indicating again that H2O2 may be themain ROS involved (Table 1). Moreover, HA treatment didnot affect the observed responses to irradiation.

Effect of irradiation and HA on expression and activityof catalase

Catalase expression was evaluated by immunoblotting imme-diately or 12, 24, and 48 h after irradiation. Basal protein levelswere not affected even if cells were HA treated (Fig. 3A).Catalase activity, in turn, was decreased by 24 h HA treatmentin nonirradiated cells while it was significantly augmentedimmediately after irradiation, being partially reversed for the24 h HA treatment (Fig. 3B). Enzymatic activities returned tononirradiated control cells levels 24 h after irradiation.

3-Aminotriazole (3-AT), an irreversible inhibitor of catalaseactivity, was used at a concentration of 10 mmol/L as previ-ously determined (data not shown). 3-AT significantly inhib-ited catalase activity, and when cells were irradiated, 3-ATmarkedly hindered the increase in catalase activity producedby IR. These effects remained evident 24 h later (Fig. 3B) inopposition to HA action, which was milder and short-lived.Protein expression was not modified (Fig. 3A).

The effect of 3-AT treatment on radiosensitivity was eval-uated by the 2 Gy SF. The 3-AT 2 Gy SF (0.18 � 0.04) wasreduced when compared with the irradiated control (0.28 �0.05). Additionally, when cells were treated with 10 �mol/LHA and irradiated in the presence of 3-AT, radiosensitivitywas further increased (Table 2). Changes in intracellular H2O2(Table 3) paralleled the variation in radiosensitivity.

Fig. 2. Role of nitric oxide (NO) and hydrogen peroxide (H2O2)intracellular levels in PANC-1 cell proliferation. (A) The effect ofexogenous H2O2 on the levels of intracellular H2O2 is reversed bycatalase. *, p 0.05 vs. control and 5 �mol/L H2O2 � cat (one-wayANOVA and Bonferroni post-test). (B) The effect of H2O2, 125 IU ofcatalase (cat), and a combination of catalase and 10 �mol/L H2O2 onclonogenic proliferation. ***, p 0.001 vs. control (one-wayANOVA and Dunnett’s post-test). (C) The effect of NO donors(10 �mol/L 3-morpholino-sydnonimine hydrochloride (SIN-1) and300 �mol/L sodium nitroprussiate (SNP)), 10 �mol/L histamine (HA),a NO scavenger (100 nmol/L 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)), catalase, and their combinations with HA onclonogenic proliferation. **, p 0.01 vs. control and *, p 0.05 vs. HA(one-way ANOVA and Bonferroni post-test). Results are expressed as themean � SE of 3 independent experiments run in triplicate.

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Effect of irradiation and HA on GSK-3�phosphorylation

We next evaluated GSK-3� expression, an enzyme in-volved in PANC-1 cell survival, which may be inactivated byphosphorylation at Ser9. Expression of total GSK-3� was notmodified by any treatment as assessed by immunoblotting.Phosphorylation at Ser9 was increased by the 10 �mol/L HA,10 mmol/L 3-AT, and 2 �mol/L H2O2 treatments, all of whichaugmented intracellular ROS levels and reduced cell prolifer-ation (Fig. 4A). When cells were irradiated, catalase activitywas immediately enhanced, ROS were distinctly decreasedand p-Ser9 GSK-3� levels were similar in control and HA-

Table 1. Reactive oxygen species (ROS), nitric oxide (NO), and superoxide intracellular levels in irradiated and nonirradiated, control andhistamine (HA)-treated PANC-1 cells.

ROS level (%) Nitric oxide level (%) Superoxide level (%)

Irradiated (2 Gy) Irradiated (2 Gy)Irradiated(2 Gy)

Nonirradiated Before After Nonirradiated Before After Nonirradiated Before After

Control 100 145�15** 41�8*** 100 140�5** 72�5* 100 81�7 85�5HA (10 �mol/L) 140�16* 135�11* 37�10��� 130�8* 145�8** 81�6��� 112�3 95�5 83�6

Note: Control and 10 �mol/L HA treated cells were incubated for 24 h and then irradiated with 2 Gy or not. After incubation with dyes, cells weresubjected to flow-cytometric analysis. Dyes were added immediately before irradiation (before) or dyes were added 5 min after irradiation (after). Thepercent of the respective nonirradiated control cells were calculated, and the results are expressed as the mean � SE of 3 independent experiments run induplicate. For ROS levels: *, p 0.05 and **, p 0.01 vs. nonirradiated control; and ***, p 0.001 vs. nonirradiated HA. For NO levels: *, p 0.05and ***, p 0.001 vs. nonirradiated control; and ���, p 0.001 vs. nonirradiated HA. For superoxide levels there were no significant differences betweenthe obtained results. Two-way ANOVA and Bonferroni post-test were used to determine statistical differences.

Fig. 3. Effect of histamine (HA), 3-aminotriazole (3-AT), andirradiation on expression and activity of catalase. Cells wereincubated in the presence or absence of 10 �mol/L HA for 24 hand 10 mmol/L 3-AT for 30 min prior to 2 Gy irradiation. (A)Immediately or 12, 24, and 48 h after irradiation cell lysates wereimmunoblotted. Catalase bands were densitometrically evaluatedand referred to the respective �-actin. A representative immunoblotis shown (24 h after irradiation). (B) Immediately after irradiation(time 0 h) or 24 h later catalase activity was determined in cellhomogenates. Results are expressed as the mean � SE of 3independent experiments run in duplicate. *, p 0.05 and**, p 0.01 vs. nonirradiated control 0 h; ***, p 0.001 vs.irradiated control 0 h; and #, p 0.01 vs. irradiated control 24 h(two-way ANOVA and Bonferroni post-test).

Table 2. Effect of a catalase inhibitor on the 2 Gy survivalfraction (2 Gy SF).

ControlHA(10 �mol/L)

3-AT(10 mmol/L)

HA (10 �mol/L) �3-AT (10 mmol/L)

2 Gy SF 0.28�0.05 0.27�0.06 0.18�0.04 0.12�0.03*

Note: Control and 10 �mol/L histamine (HA) treated PANC-1 cellswere � irradiated (0–5 Gy). 3-Aminotriazole (3-AT; 10 mmol/L) wasadded 30 min before irradiation and removed 60 min after. Eight days postirradiation survival curves were plotted and the survival parameter 2 Gy SFwas determined. Results represent the mean � SE of 3 independentexperiments run in duplicate. *, p 0.05 vs. control and vs. 10 �mol/LHA (one-way ANOVA and Bonferroni post-test).

Table 3. Hydrogen peroxide (H2O2) levels in 3-aminotriazole(3-AT)-treated cells.

H2O2 (% of nonirradiated control)

Nonirradiated Irradiated (2 Gy)

Control 100 50�510 �mol/L HA 52�10** 57�710 mmol/L 3-AT 124�5 223�15***10 �mol/L HA �

10 mmol/L 3-AT189�20** 265�14*,***

Note: Control and 10 �mol/L histamine (HA) treated PANC-1 cellswere � irradiated with 2 Gy. 3-AT (10 mmol/L) was added 30 minbefore irradiation and the specific dye DCF-2A was added 5 min after.Results represent the mean � SE of 3 independent experiments run induplicate. *, p 0.05 vs. irradiated 10 mmol/L 3-AT; **, p 0.01 vs.nonirradiated control; and ***, p 0.001 vs. nonirradiated control and10 �mol/L HA (two-way ANOVA and Bonferroni post-test).

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treated cells, since HA could not impede catalase activation. Incontrast, when catalase activity was further inhibited in thepresence of 3-AT, p-Ser9 GSK-3� levels were increased inirradiated cells treated or not treated with HA (Fig. 4B).

Almost undetectable levels of cytoplasmic p-Ser9 GSK-3�observed in control cells by indirect immunofluorescence wereaugmented by 3-AT, HA, and H2O2. Irradiation raised p-Ser9

GSK-3� expression in the cytoplasm (Fig. 5). Total GSK-3�was expressed in the nucleus and cytoplasm and treatmentsthat increased phosphorylation/inactivation also provoked de-pletion in the nuclear pool of total GSK-3�. When irradiatedcells were treated with 3-AT and HA, nuclear expression oftotal GSK-3� was found to be very low and cells further lostclonogenic capacity (Fig. 6; Table 2). NAC, a ROS scavenger,was used at a concentration of 10 mmol/L to evaluate theeffect of ROS concentration on the localization of total GSK-3�. When NAC was added previously to 2 Gy irradiation,there was an increase in the nuclear expression of totalGSK-3� (Fig. 6). To further demonstrate the role of ROSlevels on GSK-3� localization we carried out experiments ofsubcellular fractionation to isolate the cytosols and nuclei ofcontrol and irradiated cells treated with 3-AT. Nuclear andcytosolic fractions were immunoblotted using anti-GSK-3�

and anti-p-Ser9 GSK-3� antibodies. Data show an enhance-ment of total GSK-3� and p-Ser9 GSK-3� in the cytosolicpool in concordance with immunofluorescence results in3-AT-irradiated and nonirradiated cells and in control irradi-ated cells (Fig. 7A and 7B).

DiscussionExperimental and clinical results show that chemo and

radiotherapy may contribute to the local control of pancreaticadenocarcinoma but their combination is needed for a moreeffective treatment. However, due to radioresistance andchemoresistance, little effect has been achieved on the mor-tality rate of this malign illness that remains a therapeuticchallenge in human oncology.

In this study, we performed the radiobiological character-ization of PANC-1 cells by means of survival curves analyzedusing the linear quadratic model (Fig. 1). Very steep survivalcurves (almost straight) result from very radiosensitive cells,and the corresponding high � components dominate thesecurves. � coefficients represent the repair of sublethal DNAdamage and reflect the bending of the last portion of the curve(quadratic component). A wide range of � coefficients (initialslopes of the curves) between 0.1 and 1 Gy–1 has been reportedfor tumor cells after single doses of IR. The values obtainedfor PANC-1 cells (0.41 � 0.1 Gy–1) indicate intermediatelevels of resistance. The 2 Gy SF, the simplest of all param-eters, has definite advantages as 2 Gy per fraction is the mostcommon fractionation scheme used in clinical radiotherapy.The 2 Gy SF displays high values for radioresistant (�0.5) andlower values (0.2) for the more radiosensitive cell lines.Large � coefficients (�0.10 Gy–2) as those determined forPANC-1 cells signal a low efficient recovery of DNA damage.Low �/� quotients (5–7 Gy) as obtained for PANC-1 cellsdescribe curvy survival curves with relatively low � compo-nents and higher resistance, while greater ratios give lessconvex curves indicative of a considerable � component (moreradiosensitive cells). Cells displaying a low �/� ratio requirelarge doses per fraction for effective tumor treatment (Tubianaet al. 1990; Joiner and van der Kogel 1997; van den Aardweget al. 2003).

We have previously demonstrated that HA enhances sen-sitivity to IR of the breast cancer cell line MDA MB-231 bya downregulation of catalase activity and an enhancementin H2O2 intracellular levels, at the same doses that inhibitcell proliferation (Medina et al. 2006). In this work, weassayed the combination of HA and IR and found that theradiobiological parameters of pancreatic cancer cells PANC-1were not modified, even though HA can inhibit cell prolifer-ation (Fig. 1).

Tumor cells produce ROS and NO, which are involved insignal-transduction pathways that regulate multiple biologicalprocesses. In PANC-1 and other pancreatic cancer cell lines,serum and growth factors stimulate ROS generation, which actas prosurvival mediators (Vaquero et al. 2004). The intracel-lular concentration of hydrogen peroxide is so critical thattumor cell growth may be inhibited if there is a slight increaseover basal levels. (Nicco et al. 2005; Wang and Yi 2008;Sarsour et al. 2009). Accordingly, our experiments (Fig. 2)demonstrated that low doses of exogenous H2O2 exert a neg-ative action on PANC-1 cell proliferation that is reversed bycatalase addition. Additionally, NO donors also inhibited pro-

Fig. 4. Effect of histamine (HA), hydrogen peroxide (H2O2),3-aminotriazole (3-AT), and irradiation on total GSK-3� and p-Ser9

GSK-3� expression. Cells were incubated in the presence or absenceof 10 �mol/L HA or 2 �mol/L H2O2 for 24 h and 10 mmol/L 3-ATfor 30 min prior to 2 Gy irradiation. One hour after irradiation wholecell lysates were immunoblotted. (A) A representative immunoblotcorresponding to nonirradiated cells is shown. (B) A representativeimmunoblot corresponding to irradiated cells is shown. �-actin wasused as the control for protein levels in cell extracts.

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liferation indicating that high levels of NO are antiprolifera-tive. We have previously determined that micromolarconcentrations of HA decrease PANC-1 cell proliferation andthat the same concentrations produce an increase in NO intra-cellular levels (Cricco et al. 2007). Our current experimentsdemonstrated that catalase addition partially reversed HA in-hibition of cell proliferation as PTIO also did. These resultssuggest that to a certain extent the modification of intracellularreactive species concentrations mediates HA-induced inhibi-tion of cell proliferation.

It has been described in epithelial and nonepithelial celllines that radiation-stimulated ROS/RNS generation occurswithin seconds of starting radiation treatment and persists fora few minutes post irradiation when basal levels are restored(Leach et al. 2001; Spitz et al. 2004; Han et al. 2007). Increas-ing evidence suggests that this transient generation of ROS/RNS is linked to alterations in mitochondria metabolism(Leach et al. 2001; Zabbarova and Kanai 2008). The subse-quent metabolic pathways activated by this initial redox per-turbation have important implications in terms of the final

cellular response to IR and are dependent on cell type. Ourresults demonstrate that intracellular H2O2 concentration wasdrastically downregulated a few minutes after irradiation (Ta-ble 1). This important diminution below basal levels is relatedto the rapid increase in catalase activity, even if protein ex-pression remained unchanged; enzymatic activity returned tobasal levels past 24 h of irradiation (Fig. 3). Expression ofcatalase is known to be regulated at message, protein, andactivity levels (Reimer et al. 1994; Nishikawa 2008). Moststudies report signal regulation at all 3 levels, however in somesets of circumstances only activity is modified. For examplecytokines and NO have been demonstrated to inhibit activitywithout affecting protein or mRNA levels in RINm5F cells(Sigfrid et al. 2003). On the other hand, c-Abl and Arg nonre-ceptor tyrosine kinases activate catalase in response to oxida-tive stress. Elevated H2O2 levels induce binding of c-Abl andArg to catalase that is phosphorylated and activated (Cao et al.2003). In our earlier work in MDA MB-231 cells, we foundthat IR-induced downregulation of catalase activity is accom-panied by steady protein expression levels (Medina et al. 2006).

Fig. 5. Effect of histamine (HA), 3-aminotriazole (3-AT), and irradiation on p-Ser9 GSK-3� localization. Cells were incubated for 24 h inthe presence and absence of treatments. 3-AT (10 mmol/L) was added for 30 min before treatment and then cells were irradiated with 2 Gnot. One hour after irradiation indirect immunofluorescence was performed. Cells were observed using a confocal microscope. The bottomimages of both panels show immunopositivity for the antigens (green), and the top images show the bottom image merged with nuclearpropidium iodide fluorescence (orange).

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Additionally, NO intracellular concentration was also di-minished shortly after irradiation, but not as markedly as H2O2was (Table1). The short-lived activation of constitutive iso-forms of NOS by IR has been described and may be related toreversible depolarization of the mitochondrial membrane po-tential (Leach et al. 2002; Han et al. 2007; Lee et al. 2008).Moreover, we and others have demonstrated that NO levelscan negatively regulate constitutive NOS expression and ac-tivity (Hashimoto et al. 2006; Cricco et al. 2007).

HA-driven modest inhibition of catalase activity in irradi-ated cells was not enough to compensate the response in ROSlevels induced by IR (Fig. 3; Table1). In addition, even thoughHA increases the NO concentration by activating constitutiveNOS (Cricco et al. 2007), in irradiated cells NO levels werealso diminished. The low concentrations of NO generated byconstitutive NOS react with SO competing with endogenousSOD for substrate to form peroxynitrites that are highly un-stable and mostly convert to relatively inert nitrates and ni-trites (Mikkelsen and Wardman 2003). This may be one of thereasons for the observed decrease in NO levels, which couldalso contribute to the diminution of H2O2.

Prompt activation of scavenging mechanisms confers char-acteristics of radioresistance to cells (Fisher and Goswami2008; Diehn et al. 2009). To confirm the involvement ofcatalase activation in the response of PANC-1 cell to irradia-

tion we tested 3-AT and found that the 2 Gy SF was signifi-cantly reduced in 3-AT-treated cells (Table 2) when comparedwith control cells indicating a rise in radiosensitivity. Thiseffect paralleled an important and lasting inhibition of enzy-matic activity (Fig. 3B). In HA-treated cells, 3-AT increasedradiosensitivity to a larger extent indicating that early catalaseactivation is critical in the resistance of PANC-1 cells to IR.As we have not tested the activity of other enzymes that canaffect H2O2 levels, such as SOD, GPx, and peroxiredoxins,their possible involvement in the response of PANC-1 cells toirradiation cannot be disregarded. Nonetheless, the resultsobtained assign catalase a decisive role in the clonogenic survivalof irradiated PANC-1 cells. Accordingly, other authors havedemonstrated in colon and liver tumor cells that basal levels ofROS are mainly controlled by catalase (Laurent et al. 2005).

GSK-3� controls a broad range of cellular processes com-prising energy metabolism, transcription control, and cell fatedetermination by modulating cellular regulatory proteins andtranscription factors (Jope and Johnson 2004). In regard totumor biology, the role of GSK-3� is not univocal and oppositefunctions have been reported, from repression of Wnt/�-cateninsignaling and thus prevention of cell growth to maintenance ofcell survival and proliferation through the NF-�B pathway(Wilson and Baldwin 2008; Luo 2009). Diverse studies showedthat inhibition of GSK-3� results in decreased proliferation

Fig. 6. Effect of histamine (HA), hydrogen peroxide (H2O2), HA � 3-aminotriazole (3-AT), N-acetyl-L-cysteine (NAC), and irradiation onGSK-3� localization. Cells were incubated in the presence or absence of 10 �mol/L HA or 2 �mol/L H2O2 for 24 h and 10 mmol/L 3-ATor 10 mmol/L NAC for 30 min prior to 2 Gy irradiation. One hour after irradiation indirect immunofluorescence was performed. Thebottom images of both panels show immunopositivity for the antigens (green), and the top images show the bottom images merged withnuclear propidium iodide fluorescence (orange) and visible light.

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and (or) survival of chronic lymphocytic leukaemia (Ougolkovet al. 2006), pancreatic (Ougolkov et al. 2005), colorectal(Shakoori et al. 2005), ovarian (Cao et al. 2006), thyroid(Kunnimalaiyaan et al. 2007), brain (Kotliarova et al. 2008),and renal (Bilim et al. 2009) cancer cells.

In control proliferative PANC-1 cells, we found a high expres-sion of total GSK-3� associated with nuclear accumulation and avery low expression of cytoplasmic phosphorylated GSK-3�,indicating that the enzyme is predominately active (Fig. 5 and 6).Elevated expression of active GSK-3� has been reported inhuman pancreatic cancer cell lines and adenocarcinomas, withnuclear accumulation positively correlated with dedifferentia-tion. Inhibition of GSK-3� decreases pancreatic cancer cellsurvival and proliferation and retards growth in tumor xeno-

grafts (Ougolkov et al. 2006; Mamaghani et al. 2009). Ourresults reveal that treatments that cause an increment in intra-cellular ROS levels also produce GSK-3� phosphorylation atSer9, which hinders enzyme activity (Fig. 4). This increase inphosphorylation is related to inhibition of proliferation for alltreatments assayed in nonirradiated cells. ROS regulation ofGSK-3� phosphorylation mediated through PI-3k/Akt andERK kinases is reported in literature. Oxidative modificationof phosphatases by H2O2 causes their inhibition and is alsoimplicated in PI-3k/Akt signaling and GSK-3� phos-phorylation (Rhee et al. 2000; Clerkin et al. 2008). In PANC-1and other epithelial cancer cells, the rapid inactivation ofGSK-3� under hypoxic conditions mediated by the intracel-lular generation of ROS has been described to occur early as

Fig. 7. Effect of 3-aminotriazole (3-AT) and irradiation on GSK-3� localization and p-Ser9 GSK-3� cytosolic and nuclear expression.Cells were incubated in the presence or absence of 10 mmol/L 3-AT for 30 min prior to 2 Gy irradiation. (A) One hour after irradiationindirect immunofluorescence for GSK-3� was performed. Bottom images of each group show immunopositivity for the antigens (green),middle images show immunopositivity for antigens merged with nuclear propidium iodide fluorescence (orange), and top images show bothmerged with visible light. (B) One hour after irradiation subcellular fractionation to isolate the cytosols and nuclei of control and irradiatedcells treated or not treated with 3-AT was performed. Nuclear and cytosolic fractions were then immunoblotted using anti-GSK-3� andanti-p-Ser9 GSK-3� antibodies. Representative immunoblots corresponding to GSK-3� and p-Ser9 GSK-3� expression are shown. Proteinloading in lanes was verified by anti-�-actin antibody. Lamin B1 and Cu/Zn SOD were used to confirm that the nucleus and cytosol weresuccessfully extracted.

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a previous stage to epithelial-mesenchymal transition (Cannitoet al. 2008).

In irradiated control cells, the immediate rise in catalaseactivity lowered ROS levels (Table 1; Fig. 3B), and there wasonly a small increase in p-Ser9 GSK-3�.

Although HA could decrease catalase activity, induceGSK-3� inactivation, and prevent cell proliferation in nonir-radiated control PANC-1 cells, it could not potentiate irradi-ation effects. In irradiated cells, HA treatment only partiallyand transiently inhibited the increase in catalase activity(Fig. 3B), with detected ROS intracellular concentrations anal-ogous to irradiated control cells (Table 3), the 2 Gy SF beingsimilar in control and HA-treated irradiated cells (Table 2).Distinctly decreased ROS levels in irradiated cells did notseem to parallel GSK-3� phosphorylation in Ser9 (Fig. 4);however, an initial ROS burst is induced by IR as demon-strated by adding specific dyes before irradiation (Table 1).GSK-3� inactivation may be a downstream event of ROSinitially generated, and the low levels of p-Ser9-GSK-3� ob-served may be related to lower and transient elevations ofROS. This response is different to that observed when theirreversible inhibitor 3-AT was used and consequently theeffects on proliferation. The larger augmentation in p-Ser9

GSK-3� levels observed in irradiated cells treated with cata-lase inhibitor 3-AT (Fig. 4B) implies that GSK-3� inactivationis associated with higher and lasting levels of ROS. Theemployment of NAC confirmed the involvement of ROS lev-els to define the differential GSK-3� subcellular localization,a cell event mainly related to the PANC-1 proliferative re-sponse to IR (Fig. 6). Moreover, results from immunoblots ofsubcellular fractionation employing anti-p-Ser9 GSK-3� andanti GSK-3� antibodies (Fig. 7B) were in full concordancewith those observed by immunofluorescence (Fig. 5 and 7A).Data support our assumption that IR increases p-Ser9 GSK-3�levels in the cytoplasm and reinforce the association amongROS levels, GSK-3� phosphorylation, and the proliferativeresponse to IR in PANC-1 cells.

The mechanism involved in the regulation of catalase ac-tivity and ROS levels in HA-treated cells remains to be elu-cidated and may be more complex than a direct inhibition ofenzymatic activity.

All this evidence signals that upon irradiation, early catalaseactivation is related to keeping GSK-3� active, and hence toconferring PANC-1 cells a survival advantage toward cyto-toxic effects of IR.

AcknowledgementsThis work was supported by grants from the University of

Buenos Aires (UBACYT B029 and 20020100100799) andfrom ANPCYT (PICT 01022).

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