Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines

Cancer Letters 175 (2002) 165–173 www.elsevier.com/locate/canlet

Radiosensitizing and anti-proliferative effects of resveratrol in two human cervical tumor cell lines Imran Zoberi a,1, C. Matthew Bradbury b,1, Heather A. Curry a, Kheem S. Bisht b, Prabhat C. Goswami c, Joseph L. Roti Roti a, David Gius b,* a

Section of Cancer Biology, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO, USA b Radiation Oncology Branch, Radiation Oncology Sciences Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Building 10 Room B3B69, Bethesda, MD 20892-1002301, USA c Free Radical and Radiation Biology Program, Department of Radiation Oncology, B180 Medical Laboratories, University of Iowa, Iowa City, IA 52242, USA Received 11 May 2001; received in revised form 10 July 2001; accepted 7 August 2001

Abstract Resveratrol is a polyphenol isolated from the skins of grapes that has been shown to significantly alter the cellular physiology of tumor cells, as well as block the process of initiation and progression. At least one mechanism for the intracellular actions of resveratrol involves the suppression of prostaglandin (PG) biosynthesis. The involvement of PGs and other eicosanoids in the development of human cancer is well established. PGs are synthesized from arachidonic acid via the cyclooxygenase pathway and have multiple physiological and pathological functions. In addition, evidence has arisen suggesting that PGs may be implicated in the cytotoxic and/or cytoprotective response of tumor cells to ionizing radiation (IR). As such, we hypothesized that tumor cells may exhibit changes in the cellular response to IR following exposure to resveratrol, a naturally occuring compound that inhibits cyclooxygenase-1 (COX-1) activity. Thus, clonogenic cell survival assays were performed using irradiated HeLa and SiHa cells pretreated with resveratrol prior to IR exposure, and resulted in enhanced tumor cell killing by IR in a dose-dependent manner. Further analysis of COX-1 inhibition indicated that resveratrol pretreatment: (1), inhibited cell division as assayed by growth curves; and (2), induced an early S phase cell cycle checkpoint arrest, as demonstrated by fluorescence-activated cell sorting, as well as bromodeoxyuridine pulse-chase analysis. These results suggest that resveratrol alters both cell cycle progression and the cytotoxic response to IR in two cervical tumor cell lines. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cytotoxicity; Cyclooxygenase; Cyclooxygenase-1; Cell cycle; Ionizing radiation

1. Introduction Resveratrol (3,5,4 0 -trihydroxy-trans-stibene), is a phytoalexin present in grapes that appears to have multiple intracellular properties, including antioxi* Corresponding author. Tel.: 11-314-362-9781; fax: 11-314362-9790. E-mail address: [email protected] (D. Gius). 1 These two authors contributed equally to this manuscript.

dant and anti-inflammatory activities [1–3]. It also appears to inhibit cellular events associated with tumor initiation, promotion and progression [3,4]. At least one mechanism explaining the effects of resveratrol involves the suppression of prostaglandin (PG) biosynthesis [3,5,6]. PGs are arachidonate metabolites that have been implicated in the development, growth, proliferation, and overall regulation of malignant tumors, an association that has provided for expansive investigations into the mechanisms through which

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00719-4

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PGs adopt these roles in the cell [7,8]. In the course of studying the known roles of PGs as cellular regulators, evidence has arisen suggesting that PGs may also be implicated in the response of tumor cells to ionizing radiation (IR) and other cytotoxic insults [9,10]. This evidence is based, in part, on the observation that the addition of exogenous PGs to immortalized cultured cells can serve either to protect or to sensitize these cells to IR, with the protective or sensitive response depending on the PG that is used [9–11]. Several groups have published extensively on the role of PGs in the cellular and cytotoxic response to IR [8,11,12]; the mechanistic pathway, however, is less clearly understood [10]. PGs are synthesized from arachidonic acid by either of two enzymes, cyclooxygenase-1 (COX-1) or cyclooxygenase-2 (COX-2) [12,13]. COX-1 is normally a constitutively expressed enzyme, while the expression of COX-2 is inducible by cytokines or multiple other stimuli, a similarity shared with a particular class of genes referred to as immediate early genes [14,15]. Furthermore, elevated COX-2 expression and PG levels have been observed in a variety of tumors, including cancers of the colon, breast, prostate, and head and neck [14–15]. It has been shown that non-steroidal anti-inflammatory agents, such as ibuprofen and indomethacin, inhibit cyclooxygenase (COX) activity and sensitize tumor cells to the cytotoxic effects of IR, suggesting a role for COX activity in the cellular response to IR [16,17]. This observation is of interest because it suggests that the regulation or altered regulation of COX-2 and PGs by tumor cells may play a role in carcinogenesis [3–6] and, perhaps, the susceptibility of malignant cells to the cytotoxic and genotoxic effects of radiation [8–12,16,17]. Considering that greater than half of all cancer patients receive radiation therapy as part of their treatment regimens [18], it is possible that an increased understanding of PGs, the COX enzymes, and their roles in the cellular and molecular events following cultured tumor irradiation can eventually be applied toward efficacious radiation treatment modalities. Resveratrol is a natural compound that has been shown to inhibit COX-1 activity, and as such, it seemed logical to determine if resveratrol would also alter how tumor cells respond to the cytotoxicity of IR. Using Western blotting analyses, we determined that COX-1 is constitutively expressed in the cervical

carcinoma cell lines HeLa, CaOv3, Me180, A2780, and SiHa, as well as diploid NIH 3T3 cells. Pretreatment with resveratrol enhanced tumor cell killing by IR in HeLa and SiHa tumor cells. Further analysis demonstrated that resveratrol pretreatment inhibited cell division as assayed by growth curves and induced an early S cell cycle checkpoint arrest, as is demonstrated by fluorescence-activated cell sorting (FACS) and bromodeoxyuridine (BrdU) pulse-chase analysis. The results of these experiments suggest that resveratrol alters the cellular response to IR and also inhibits cell cycle progression. 2. Materials and methods 2.1. Cell culture, drug treatment, and IR exposure The human cervical carcinoma cell lines, HeLa, Me180, A2780, and SiHa, and the mouse normal fibroblast cell line, NIH 3T3, were grown in Eagle’s minimum essential medium alpha supplemented with 10% heat-inactivated calf serum, penicillin (100 U/ ml), and streptomycin (100 mg/ml) in a humidified, 95% air, 5% CO2 incubator at 378C. Cells were seeded at a density of 2 £ 10 5 cells/100-mm tissue culture dish and grown to 55–65% confluence (4–5 £ 10 6 cells/100-mm dish) prior to treatment with resveratrol (Cayman Chemical, Ann Arbor, MI). Stock solutions of resveratrol (3 mM) were prepared in 100% ethanol. To eliminate a sham response induced by the ethanol alone, volumes of the ethanol corresponding to the highest molar concentration of drug being administered in each experiment were added to control samples. Cells treated with resveratrol were incubated for 4 h at 378C in the presence of the drug prior to further treatment or harvest. Cells were exposed to doses of IR, ranging from 2 to 8 Gy, using a General Electric Maxitron 250 kV X-ray machine that contains an exposure chamber supporting a 378C, 5% CO2 atmosphere. Control, non-irradiated cells were placed into a similar chamber next to the Xray machine. Following IR exposure, cells were returned to a 378C incubator. 2.2. Preparation of cellular extracts and subcellular fractions Nuclear and cytoplasmic subcellular fractions and

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extracts from whole cells were prepared for analysis via a previously described method [18,19]. After preparation of whole cell extracts or subcellular fractions, protein quantifications within each sample were assessed via Bradford analysis (BioRad Laboratories, Hercules, CA) on a Beckman (Fullerton, CA) DU-640 spectrophotometer to ensure equal protein loading. 2.3. Denaturing polyacrylamide–sodium dodecyl sulfate gel electrophoresis and Western blot assays Equal amounts of protein were mixed with Laemmli lysis buffer [19] and boiled for 5 min. Protein samples were then separated on a denaturing polyacrylamide–sodium dodecyl sulfate (SDS) gel and transferred to a nitrocellulose membrane using a semi-dry transfer apparatus (Owl Scientific, Portsmouth, NH). The membrane was blocked and hybridized overnight at room temperature with a polyclonal antibody against COX-1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1:1000 in 2.5% milk in a PBS–Tween (0.05%) solution, after which the membrane was washed and incubated with an antirabbit IgG-HRP secondary antibody (Santa Cruz Biotechnology). The membrane was analyzed via an enhanced chemiluminescence (ECL) method (Amersham-Pharmacia Biotech, Newark, NJ) as per the manufacturer’s instructions and resolved on radiographic film (Eastman-Kodak, Rochester, NY). 2.4. Clonogenic cell survival assays HeLa cells were plated at a density of 2 £ 10 5 cells/ 100-mm tissue culture dish and grown to 70–75% confluency (4–5 £ 10 6 cells/100-mm dish) in a humidified 378C, 5% CO2 incubator. After attaining sufficient growth, the cells were pretreated with resveratrol (10 or 25 mM) for 4 h, incubated at 378C for predesignated time periods, and exposed to IR at doses of 2, 4, 6, or 8 Gy [20,21]. Immediately following irradiation, cells were trypsinized and quantified via a Coulter counter. Plating efficiencies were measured and similar for all samples. Dilutions of the treated cells were prepared, and duplicate 60-mm tissue culture dishes were seeded with 200–20,000 cells each. Colonies were allowed to form in an undisturbed, humidified, 378C, 5% CO2 environment for 10–12 days, after which they were fixed with 70% ethanol, stained with crystal violet, and counted. Only those colonies

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containing .50 cells were considered to be a viable colony or survivor (S). Survival curves are shown plotted as log S/S0 (where S0 is the total number of cells plated per 60-mm dish multiplied by the plating efficiency) as a function of the dose of IR (Gy) and curves were plotted and visually fitted as described [21]. The method to calculate the dose modification factor (DMF) at 10 or 50% isosurvival [6,21] where dose to reach 10% (or 50%) survival in control HeLa cells treated with resveratrol, is shown in Eq. (1): DMFð10;50Þ ¼

Dose to reach 10% ðor 50%Þ survival in treated cells Dose to reach 10% ðor 50%Þ survival in control cells

ð1Þ

2.5. Cellular growth assays and FACS analysis HeLa cells were seeded at a density of 2 £ 10 4 cells/ 35-mm dish into medium containing an established concentration of resveratrol (10 or 25 mM) and were returned to a humidified 378C, 5% CO2 incubator for growth (Day 0). For the following 8 days, three plates from each treatment condition were trypsinized and quantified via a Coulter counter, with fresh medium being added every 3 days. Plating efficiencies were measured and similar for all samples. Growth curves are plotted as the mean number of cells/dish as a function of time (days). For FACS analysis, HeLa cells were seeded into 100-mm tissue culture dishes at a density of 1 £ 10 5 cells/dish and incubated at 378C in the presence of resveratrol (25 mM) for prescribed time periods of 4–48 h [21,22]. After treatment with resveratrol, cells were harvested by trypsinization, centrifuging at 1000 revs./min for 5 min, washing twice sequentially in PBS, and fixing in 70% ethanol. The fixed samples were stored at 48C until analysis and then washed and centrifuged for two more cycles. The samples were incubated in RNase (0.2%) for 30 min at 48C, labeled with propidium iodide, and analyzed using CyCLOPS e (version 3.14) software. The cell cycle distribution was assessed by determining BrdU incorporation versus DNA content. Asynchronously growing HeLa cells were pulselabeled with BrdU and cultured in absence or presence of 25 mM resveratrol. Cells were harvested by trypsinization and fixed in 70% ethanol. Fixed cells were treated with HCl, and indirect immunostaining was performed using anti-BrdU monoclonal antibody [23]. Nuclei were then treated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse anti-

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body and counterstained with propidium iodide. FACS analysis was performed following previously published assays [23]. 2.6. COX inhibitor screening assay Inhibition of COX-1 enzyme activity following treatment with resveratrol was determined via a screening kit obtained from Cayman Chemical and performed according to the manufacturer’s instructions. By using an antibody to prostanoids, this immunoassay (EIA) directly measures PGs produced as a result of the COX pathway. COX-1 activity with or without resveratrol (10–25 mM) was determined by incubating COX-1 enzyme and heme with a reaction buffer consisting of 100 mM Tris–HCl (pH 8.0), 5 mM EDTA, and 2 mM phenol. The assay was initiated by the addition of arachidonic acid, incubated at 378C for 2 min, and stopped by the addition of hydrochloric acid. The samples were added to a 96-well mouse anti-rabbit IgG coated plate, incubated for 18 h at room temperature, developed by adding Ellman’s reagent (5,5 0 -dithiobis(2-nitrobenzoic acid)), and scanned on a microplate reader (Bio-Tek Instruments, Winooski, VT) at 405 nm. Ellman’s Reagent is an aromatic disulfide that reacts with aliphatic thiol groups to form mixed protein disulfides at 1:1 molar bases. When Ellman’s reagent reacts with ZSH groups on proteins, an intense yellow color is detected at 412 nm and measured. Average absorbance readings were calculated and normalized for autofluorescence and non-specific antibody–antigen binding, and then plotted by the percentage sample bound/maximum sample bound (%B/B0) as a function of inhibitor dose received. The standard curves showed expected relationships between bound sample and PG dose (data not shown). 3. Results 3.1. COX-1 is constitutively expressed in cervical tumor cell lines cells In order to assess the effects of COX activity on the cellular response to IR, the basal expression of immunoreactive COX protein levels was initially established in multiple human cervical carcinoma cell lines. Whole cell extracts from HeLa, NIH 3T3,

Me180, A2780, and SiHa cells were prepared for Western analysis as previously described [12,13] and were hybridized with polyclonal antibodies against COX-1. These results demonstrate that COX-1 immunoreactive protein levels are constitutively expressed in multiple different cultured cell types (Fig. 1A).

3.2. Resveratrol is a dose-dependent radiation sensitizer Resveratrol has been shown to inhibit COX-1 activity with a minimal effect on the activity of COX-2 [14,15]. With this perspective in mind, the cell biological effects of resveratrol on HeLa and SiHa cell survival after exposure to IR were investigated. HeLa and SiHa cells were treated with 10 or 25 mM of resveratrol and returned to a 378C incubator for 4 h, after which the cells were exposed to 2, 4, 6, or 8 Gy of IR. Duplicate 60-mm dishes were plated from dilutions of the treated cells and incubated for a 10–12day period, after which they were fixed, stained, counted, and plotted as previously described. Both the slope and initial shape of the curve reflect the radioresponse of the cells. When plotted as a function of the IR dose, a dose-dependent increase in sensitization of HeLa (Fig. 1B, left panel) and SiHa (right panel) cells to IR following pretreatment with resveratrol can be observed. The DMF at 50% survival for 25 mM of resveratrol in HeLa cells is 1.35 and 2.07 in SiHa cells. We also estimated the values of the a and b survival curve parameters (Table 1). These values were estimated by linear regression analysis of the transformed survival curve: 2lnðS=S0 Þ=D ¼ a 1 bD, where D is the radiation dose, S/S0 is the surviving fraction and a and b are the survival equation constants. Interestingly, the a parameter increased with the resveratrol dose, while the b parameter showed little or no change. The R value in Table 1 shows the quality of fit and indicates that all of the data (except for the control HeLa cells) were statistically consistent with the linear fit. The poor fit for the control HeLa data appeared to be due to a slightly low survival value at 4 Gy. The results of these experiments indicate that pretreatment with resveratrol results in greater susceptibility of these tumor cells to IR-induced cytotoxicity.

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Fig. 1. (A) COX-1 expression in several cervical tumor cell lines. Whole cell extracts from several different cervical tumor cell lines were isolated as previously described and Western blot analyses of COX-1 protein levels were determined using an anti-COX-1 antibody (Santa Cruz Biotechnology, Inc.). Equal protein loading was determined using a Bradford protein assay. HeLa-1 (lane 1), HeLa-2 (lane 2), NIH 3T3 (lane 3), Mel80 (lane 4), A2780 (lane 5), and SiHa cells (lane 6) are shown. (B) Resveratrol is a dose-dependent radiation sensitizer. Clonogenic cell survival curves of HeLa cells treated with 10 or 25 mM resveratrol for 4 h prior to exposure to 2, 4, 6, or 8 Gy IR. After exposure, cells were trypsinized and plated at multiple densities. After roughly 14 days, colonies were fixed, stained with crystal violet, and counted. By definition, a colony must contain .50 cells to be considered a survivor (S). Survival fractions are normalized to non-irradiated controls.

3.3. Resveratrol, causes an early S phase cell cycle arrest To further investigate the role of resveratrol in the cellular response to IR, the effect of resveratrol on cell growth and cell cycle progression of HeLa cells was addressed. This was done for two reasons. First, it has been clearly established that the cytotoxicity induced

by IR varies widely through the different phases of the cell cycle [5]. Second, resveratrol has previously been shown to alter the cell cycle progression of tumor cells [5,6]. To address the possible effect of COX-1 inhibition on cellular growth and proliferation via exposure to resveratrol, HeLa cells were exposed to 10 or 25 mM of resveratrol for periods of up to 8 days. Untreated cells and cells treated with ethanol only

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Table 1 a , b , DMF, R, and probability values a

Control 10 mM Res 25 mM Res

HeLa cells Alpha

Beta

R

SiHa cells Alpha

Beta

R

0.080 ^ 0.041 0.165 ^ 0.020 0.226 ^ 0.012

0.019 ^ 0.007 0.016 ^ 0.003 0.014 ^ 0.041

0.88 0.95 0.98

0.089 ^ 0.007 0.168 ^ 0.003 0.221 ^ 0.005

0.008 ^ 0.001 0.006 ^ 0.001 0.010 ^ 0.001

0.88 0.95 0.98

DMF for 25 mM ¼ 1.35

P ¼ 0.05

DMF for 25 mM ¼ 2.07

P ¼ 0.02

a Data shown represent the a and b components and the uncertainty of the cell survival curves presented Fig. 1A, as well as the R and probability values.

were analyzed as controls. Each day, the number of cells/plate for each treatment condition was assessed, and the results were plotted as the number of cells/ plate and as a function of time (days). The results of these experiments demonstrate a cell growth inhibition in HeLa cells that correlates with progressively increasing doses of resveratrol (Fig. 2A), indicating an inhibition of cell growth and proliferation and suggesting a possible induction of a cell cycle delay. To determine the phase of the cell cycle progression that is arrested by exposure to resveratrol, FACS analyses were performed. HeLa cells were treated with 25 mM of resveratrol and harvested as previously described [14] at time points of 4–28 h. The results of the FACS analyses demonstrated a considerable accumulation of cells in S phase after treatment with resveratrol as compared with control cells treated with ethanol alone, or control, untreated HeLa cells (Fig. 2B). Similar effects on cell cycle progression were also observed in SiHa cells (data not shown). The results of these experiments demonstrate that resveratrol induces an S phase cell cycle checkpoint. Resveratrol-induced delay in S phase was further verified by determining the cell cycle phase distribution in BrdU incorporated cells. Asynchronously growing HeLa cells were pulse-labeled with BrdU and cultured in the presence or absence of resveratrol. Cell cycle phase distributions were assayed by flow cytometry. Results from Fig. 3A showed that following the resveratrol treatment, the fraction of S phase cells increased to 60% at 20 h and continued to increase to 80% at 28 h. The majority of the cells within the S phase were blocked in early S during the 20–28 h of treatment (see arrow in Fig. 3A), suggesting that resveratrol induces an early S phase block in HeLa cells.

Fig. 2. (A) Cell growth-inhibitory effect of resveratrol on HeLa cells. HeLa cells were plated at 2 £ 10 4 cells/plate and treated with 10 or 25 mM resveratrol. Cells were harvested at 1, 2, 3, 4, 5, 6, 7, and 8 days and counted as described. The numbers of HeLa cells/plate (three plates/condition) were determined and plotted as a function of days. (B) Resveratrol arrests HeLa cells in the S phase. HeLa cells were plated at 2 £ 10 4 cells/plate and treated with 25 mM resveratrol or an identical volume of ethanol and harvested at 4, 8 (data not shown), 12, 16, 20, 24, and 28 h. The percentages of cells in G1, S, and G2/M phases of the cell cycle were determined via FACS analysis.

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4. Discussion

Fig. 3. (A) Resveratrol induces an early S phase cell cycle arrest. Asynchronously growing HeLa cells were pulse-labeled with BrdU and cultured in the presence or absence of 25 mM resveratrol. Cell cycle phase distribution was assessed by indirect immunostaining of BrdU-labeled cells using flow cytometry. The fraction of cells was calculated using Cyclops software. The arrow indicates the accumulation of resveratrol treated cells in the early S phase of cell cycle. (B) Effects of resveratrol on Cox-1 and Cox-2 activity. Cox-1 (open circle) and Cox-2 (open square) activity were determined via a screening kit obtained from Cayman Chemical (Ann Arbor, MI) and performed according to the manufacturer’s instructions. Results were obtained using an antibody to prostanoids; this immunoassay (EIA) directly measures PGs produced as a result of the COX pathway.

3.4. Resveratrol inhibits COX-1 activity To confirm that resveratrol inhibits COX-1 activity, the enzymatic COX-1 activity was determined in the presence of resveratrol (Cayman Chemical). These results demonstrated that resveratrol inhibits COX-1 activity at 10 and 25 mM (Fig. 3) with little or no effect on COX activity. These results are similar to those observed by others [5,6].

PGs formed along a dysregulated COX pathway have been shown to mediate tumor promotion in animal experiments and may play a role in other processes involved in tumor growth, such as angiogenesis, metastasis, and immunosuppression [16,17,24]. The results presented here suggest that resveratrol, a polyphenol found in the skin of grapes, acts as an agent that induces both an early S phase cell cycle check point and an increase in cellular sensitivity to the cytotoxicity of IR. Interestingly, analysis of the survival data suggest that resveratrol increased the a parameter of the linear quadratic survival curve, but had little effect on the b parameter. The conventional, dual action interpretation of the linear quadratic survival equation assumes that complex DNA lesions, such as multiple damaged sites or double strand breaks, are responsible for cell lethality and that these lesions arise either by a single radiation interaction (related to the a parameter) or the combination of two radiation interactions (related to the b parameter) [25]. Therefore, the increase in the a parameter suggests that resveratrol exposure causes cells to be more susceptible to complex DNA lesions arising by single radiation interactions [25]. Single strand breaks in such regions will act as double strand breaks, leading to an increased lethality by single ‘hit’ radiation interactions. The ability of resveratrol to induce an S phase cell cycle arrest has been demonstrated by several other investigators, and these results further suggest a role for resveratrol in the regulation of cell cycle progression [5,6]. While it is tempting to suggest a functional relationship between the inhibition of cell cycle progression and the increased radiosensitivity effect of resveratrol, this appears not to be the case. The radiosensitizing effect of resveratrol slowly declines with exposure times exceeding 8 h (data not shown), while the inhibition of cell cycle progression increases after 8 h. As such, the preliminary results presented above suggest a role for resveratrol as a radiosensitizing agent and a tumor cell growth inhibitor, but the mechanisms for these two effects may be separate. The results of the experiments overviewed in this manuscript are consistent with the observations that PGs alter the cellular and cytotoxic response to IR [7– 12]. Moreover, genotoxic byproducts, such as organic free radicals, reactive oxygen species, and malondial-

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dehyde, produced in the course of prostanoid biosynthesis may contribute to genetic instability (mutator phenotype) of neoplastic cells, thereby promoting malignant progression [4,16,17,24,26– 29]. The role of COX in the cellular and cytotoxic response to IR appears to be a complicated one, and its effect as a radioprotector and/or radiosensitizing agent may depend on multiple cellular, genetic, and epigenetic factors, as well as other intracellular alterations. While resveratrol inhibits COX-1 activity, it clearly has other effects that may account for the effects on radiosensitization and inhibition of cell cycle progression [4,5]. The most significant nonCOX effects may be due to alterations in the intracellular oxidation/reduction state or cellular redox status. Such a possible redox sensitive, non-COX process would be consistent with several recent publications suggesting that resveratrol alters intracellular redox status [30–32]. While this work clearly shows that resveratrol alters how tumor cells respond to IR and inhibits cell cycle progression in two cervical tumor cell lines, the specific mechanism is unclear.

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Acknowledgements D.G. was supported by grants from the National Institute of Health (1 K08 CA72602-01), the American Cancer Society (RPG TBE-100674 and PO1 CA75556). P.C.G. was supported by NIH grant CA69593. I.Z. was supported by a grant from the American Society of Therapeutic Radiology and Oncology. J.R.R. was supported by grants from the National Institute of Health (5 P01CA75556-05 and 5 R01CA43198-15).

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