RelB Expression Determines the DifferentialEffects of Ascorbic Acid in Normal andCancer CellsXiaowei Wei1,2, Yong Xu1,2, Fang Fang Xu3, Luksana Chaiswing1, David Schnell1,Teresa Noel1, Chi Wang4, Jinfei Chen2, Daret K. St. Clair1, and William H. St. Clair2
Abstract
Cancer cells typically experience higher oxidative stress thannormal cells, such that elevating pro-oxidant levels can triggercancer cell death. Although pre-exposure to mild oxidativeagents will sensitize cancer cells to radiation, this pre-exposuremay also activate the adaptive stress defense system in normalcells. Ascorbic acid is a prototype redox modulator that wheninfused intravenously appears to kill cancers without injury tonormal tissues; however, the mechanisms involved remainelusive. In this study, we show how ascorbic acid kills cancercells and sensitizes prostate cancer to radiation therapy whilealso conferring protection upon normal prostate epithelial cellsagainst radiation-induced injury. We found that the NF-kBtranscription factor RelB is a pivotal determinant in the differ-ential radiosensitization effects of ascorbic acid in prostate
cancer cells and normal prostate epithelial cells. Mechanistical-ly, high reactive oxygen species concentrations suppress RelBin cancer cells. RelB suppression decreases expression of thesirtuin SIRT3 and the powerful antioxidant MnSOD, which inturn increases oxidative and metabolic stresses in prostatecancer cells. In contrast, ascorbic acid enhances RelB expressionin normal cells, improving antioxidant and metabolic defensesagainst radiation injury. In addition to showing how RelBmediates the differential effects of ascorbic acid on cancer andnormal tissue radiosensitivities, our work also provides a proofof concept for the existence of redox modulators that canimprove the efficacy of radiotherapy while protecting againstnormal tissue injury in cancer settings. Cancer Res; 77(6); 1345–56.�2017 AACR.
IntroductionProstate cancer is the most prevalent cancer in the United
States and is the second leading cause of cancer deaths in men(1). The predominant prostate cancer therapy, ionizing radia-tion (IR), is used to treat more than 750,000 patients per year(2). Unfortunately, the therapeutic efficacy of IR tends todecrease when cancer cells develop adaptive responses to resistit. Even with modern conformal radiation therapy, biochemicalfailure occurs in approximately 45% of patients with a locallyconfined disease (3). In addition, corresponding with the wide-
spread use of high-dose IR to treat prostate cancer, the incidenceof radiation-related genitourinary toxicity has increased (4).Therefore, novel therapeutic strategies to enhance both radio-sensitization in cancer cells and radioprotection in normaltissues are urgently needed.
Cancer cells are usually under higher oxidative stress thannormal cells, and cellular redox state is thought to be importantto cell fate. The level of oxidative stress is also critical to radiationresponse (5, 6). In addition to directly damaging DNA, IR canproduce a large amount of free radicals that cause cell death (5).Because of water radiolysis, IR causes excess superoxide genera-tion and allows leakage of electrons from the electron transportchain, resulting in mitochondrial dysfunction (7). However, IRalso tends to induce adaptive reactive oxygen species (ROS)defense systems in cancers, which may lead to radioresistance(8).While increasing radiation intensity can improve the ability tocontrol cancer growth, it presents the significant risks of increasingunwanted side effects, including injury to normal tissues andreduced quality of life for cancer survivors. Thus, an attractiveradiation therapy would be one that exploits the intrinsic differ-ences in the cellular redox statuses of normal cells and cancer cellsby selectively boosting ROS generation in cancer cells to pushthem into oxidative stress overload while stimulating adaptiveresponses in normal cells.
Ascorbic acid, better known as vitamin C, has a somewhatcontroversial history as a therapeutic drug for cancer treatment(9, 10). Emerging studies suggest that only intraperitoneal orintravenous ascorbic acid, and not orally administered ascorbicacid, can reach pharmacological concentrations that kill cancer(10, 11). However, although clinical trials of ascorbic acid have
1Department of Toxicology and Cancer Biology, Markey Cancer Center, Univer-sity of Kentucky, Lexington, Kentucky. 2Department of Oncology, Nanjing FirstHospital, Nanjing Medical University, Nanjing, China. 3Department of RadiationMedicine, Markey Cancer Center, University of Kentucky, Lexington, Kentucky.4Biostatistics Core, Markey Cancer Center, University of Kentucky, Lexington,Kentucky.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Authors: Daret K. St. Clair, Department of Toxicology andCancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536.Phone: 859-257-3956; Fax: 859-323-1059; E-mail: [email protected]; William H.St. Clair, Department of Radiation Medicine, University of Kentucky, 800 RoseStreet, Lexington, KY 40536. Phone: 859-323-6486; Fax: 859-257-4931; [email protected]; and Jinfei Chen, Department of Oncology, Nanjing First Hos-pital, Nanjing Medical University, 68 Changle Road, Nanjing, Jiangsu Province,PR China. Phone: 86-025-87726234; Fax: 86-025-87726234; E-mail:[email protected]
demonstrated the differences between administration routes andthe safety of intravenous ascorbic acid (12, 13), there is still a lackin understanding the mechanisms by which ascorbic acid killscancer cells. In general, the cytotoxicity induced by ascorbic acidseems to be primarily mediated by hydrogen peroxide (H2O2)generated in extracellular fluids (14). A recent elegant studydemonstrated that pharmacologic ascorbate enhances the cyto-toxic effects of IR in pancreatic cancer cell lines, but not innontumorigenic pancreatic ductal epithelial cells because pan-creatic cancer is sensitive to H2O2 (15). Thus, the cytotoxic effectof ascorbic acid may well be dependent on the local redoxenvironment of the cancer cells.
Mounting evidence suggests that ROS-mediated NF-kB activa-tion is involved in the radiation resistance of cancers (16). TheNF-kB family includes NF-kB 1 (p50/p105), NF-kB 2 (p52/p100), c-Rel, RelA (p65), and RelB. Recent studies have demonstrated thatRelB is uniquely expressed at a high level in prostate cancer withhigh Gleason scores, suggesting that RelB plays an important rolein the radioresistance of prostate cancer (17, 18). The presentstudy examined the effects of ascorbic acid on cell survival and onthe responses to radiation of prostate cancer cells and normalprostate epithelial cells in vitro and in vivo. We found that theintrinsic differences in the cellular redox state of normal andcancer cells account, in part, for the differential effects of ascorbicacid on themodulation of cellular redox status. The expression ofRelB in normal and cancer cells serves as a central regulator fortheir opposing responses to radiotherapy.
Materials and MethodsCell culture, cell transfection, and reagents
Human prostate cancer cell lines LNCaP and PC3, as well asnormal human prostate epithelial viral transformed PZ-HPV-7(PZ) cells, were obtained fromATCC. These cell lines are routinelychecked for morphologic and growth changes to probe for cross-contaminated or genetically drifted cells. All cell lines used havebeen reauthenticated using the short tandem repeat (STR) pro-filing service byATCC.Normal human epithelial cells (PrEC)werepurchased from Lonza. The cells were cultured andmaintained inthe manufacturer's suggested media. Plasmid-cloned RelB cDNAand siRNA for knocking down RelB were transfected into LNCaPand PC3 cells using a Lipofectamine 2000 kit (Life Technologies)according to the manufacturer's instructions. ascorbic acid (pow-der, USP/FCC grade, Fisher Chemical) was prepared as a 1 mol/Lstock solution in sterile water, with sodium hydroxide addeddropwise to adjust the pH to 7.0, as previously described. H2O2
(Sigma) solutions were prepared freshly prior to application tothe cells.
Treatment and cell survival analysisCell survival rates were quantified by colony survival and MTT
assays. For colony survival analyses, the cells were plated in 6-wellplates at low densities and then treated with 0 to 1.0 mmol/Lascorbic acid for 2 hours, which corresponds to the clinical timecourse of intravenous ascorbic acid administration (19). Theformed colonies were washed with 1� PBS and stained with acrystal violet dye for clonogenic assay. The surviving fraction wascalculated as the ratio of the number of colonies formed to thenumber of cells effectively plated.
For the MTT assay, cells were seeded in flat-bottomed96-well plates, treated with 0 to 64 mmol/L ascorbic acid for
2 hours, washed with fresh media, and incubated for 2 dou-bling times in the absence of ascorbic acid. Cell viability wasdetected by an MTT assay kit (Trevigen) following the standardprotocol. The IC50 for each cell line was calculated from adose–response curve using GraphPad Prism 6.0 software(GraphPad software). For evaluations of combination therapy,PC3 and PZ cells were treated with ascorbic acid, IR, or acombination of the two. IR was performed at 1 hour afterascorbic acid treatment by a 250-kV X-ray machine (FaxitronX-ray Corp.) with peak energy of 120 kV, 0.05-mm Al filter, at adose of 0 to 6 Gy. After a total 2-hour exposure to ascorbicacid, cells were incubated for 2 doubling times in the absenceof ascorbic acid and assessed by MTT assay. Combinationindex (CI) values were calculated by CompuSyn 1.0 (Compu-Syn), and the effects of combined IR and ascorbic acid treat-ment were evaluated according to the acknowledged range ofCI as published (20).
AnimalsFour- to 5-week-old male NCRNU (nu/nu athymic nude)
mice were obtained from Taconic (Hudson). For formation ofxenograft tumors, 1.8 � 106 cells mixed in Matrigel (BDBiosciences) were subcutaneously injected into the right flanksof the mice. Tumor volumes were routinely measured and theirsizes calculated on the basis of a protocol described elsewhere(21). Animals with an average tumor size of 350 mm3 wererandomized into 4 groups (n ¼ 10) and treatment commencedwith intraperitoneal injection as follows: (i) control, salineonce daily; (ii) ascorbic acid, 4.5 g/kg once daily; (iii) IR, 2 Gyonce every other day; and (iv) IR þ ascorbic acid. The dosage ofascorbic acid was determined by conversion of clinical trial dataand with reference to recent studies (22). For combinationtreatments, IR was performed at 1 hour after ascorbic acidinjection. After treatment, the mice were observed daily andhumanely killed when the tumor reached the maximum size of1,500 mm3. The tumor, prostate, and bladder tissues werecollected for protein and RNA analysis. All animal experimentalprocedures were approved by the Institutional Animal Careand Use Committee of the University of Kentucky (Lexington,KY), Approval Protocol No. 01077M2006.
Quantification of ROS levelAn Amplex Red assay was used to quantify the levels of
extracellular H2O2 after ascorbic acid and IR treatments. Briefly,cells were incubated with 50 mmol/L Amplex Red reagent(ThermoFisher) at 37�C for 10 minutes. Fluorescence wasdetected at ex/em 550/590 nm using a Gemini XPS MicroplateReader (Molecular Devices). The extracellular H2O2 level wascalculated by a standard curve and normalized to the cellnumber. MitoSox Red (ThermoFisher), a highly selective mito-chondrial superoxide indicator for live cells, was used toestimate the levels of superoxide, and Mitotracker green FM(ThermoFisher) was used to locate mitochondria. Rotenone(200 nmol/L, Sigma), known to be a mitochondrial superoxideinducer, was used as a positive control. To account for super-oxide-specific fluorescence, the cells were pretreated with 100units/mL PEG-SOD (Sigma) for 24 hours followed by thetreatment. In brief, cells were loaded with Mitotracker Greenat 100 nmol/L for 15 minutes followed by 5 mmol/L MitoSoxRed for 10 minutes at 37�C and rinsed 3 times with HBSSbefore measuring fluorescence. Cellular fluorescence intensity
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was detected using an Olympus IX71 fluorescence microscopeand a Gemini XPS Microplate Reader at ex/em 510/580 nm.
Catalase and MnSOD treatmentsTo further determine the specific high ROS level induced by
ascorbic acid and radiation, PC3 cells were treated with variousforms of catalase or MnSOD, including catalase (100 U/mL),PEG-CAT (200 U/mL), PEG-MnSOD (100 U/mL), or adenovi-rus MnSOD. Catalase, PEG-catalase (PEG-CAT), and PEG-MnSOD were purchased from Sigma-Aldrich. For the adenovi-rus experiments, viral vectors utilized included AdCMVEmpty(AdEmpty) and AdCMVMnSOD (AdMnSOD), manufactured byViraquest, Inc. as previously described (23, 24). Approximately106 PC3 cells were plated in a 100-mm2 tissue culture dish andincubated with the adenovirus constructs for 24 hours. Mediawere replaced with 5 mL of complete media for an additional24 hours before cells were harvested for MTT assay or treated forROS measurements.
Measurement of oxygen consumption rates, ATP, andlactate production
To determine whether ascorbic acid changes mitochondrialfunction in cancer and normal cells, a Seahorse BioscienceXF96 Extracellular Flux Analyzer was used to measure oxygenconsumption rates (OCR) after 2 hours of treatment with4 mmol/L ascorbic acid. The data were normalized with proteinlevels and expressed as the OCR in pmol/min/mg protein.Cellular ATP concentrations were measured using an ATP Assaykit (Biomedical Research Service Center). Extracellular andintracellular lactate levels were measured by a Lactate Assaykit (Biomedical Research Service Center). Data were normal-ized to the cell number, as indicated in each figure.
Western blotsLysates from homogenized cells and tumor tissues were elec-
trophoresed on a 10% to 12% (w/v) SDS-PAGE gel, transferredonto a nitrocellulose membrane, and subsequently incubatedwith primary antibodies against RelA, RelB, Bcl-xl, Bax, orGAPDHfrom Santa Cruz Biotech, or SIRT1 (Upstate Biotech), MnSOD(Upstate Biotech), SIRT3 (Cell Signaling), or b-actin (Sigma). Allsecondary antibodieswere obtained fromSantaCruz Biotech. Theblots were visualized using an enhanced chemiluminescencedetection system (Amersham Pharmacia Biotech).
Real-time PCRmRNA was isolated from cells and tissues using a MagNA
Pure Compact RNA Isolation Kit (Roche) and reverse-tran-scribed using a TaqMan reverse transcription kit (Thermo-Fisher) according to the manufacturer's instructions and thenanalyzed using a LightCycler 480 Real-Time PCR System (RT-PCR, Roche) with gene-specific primers. All primers were pur-chased from Invitrogen. Primer sequences for human geneswere as follows:
RELB 50-cacttcctgcccaaccac-30 (forward) and 50-gacacggtgc-cagagaaga-30 (reverse); Bcl-xl 50-agccttggatccaggagaa-30 (for-ward) and 50-gctgcattgttcccatagagt-30 (reverse); SIRT3 50-cttgctgcatgtggttgatt-30 (forward) and 50-cggtcaagctggcaaaag-30
(reverse); b-actin 50-ccaaccgcgagaagatga-30 (forward) and 50-ccagaggcgtacagggatag-30 (reverse). Primer sequences for mousegenes were as follows:
RelB 50-gtgacctctcttccctgtcact-30 (forward) and 50-tgtattcgtc-gatgatttccaa-30 (reverse); Sirt3 50- tcctctgaaaccggatgg-30 (for-ward) and 50-tcccacacagagggatatgg-30 (reverse); b-actin 50-ctggctcctagcaccatga-30 (forward) and 50-acagtgaggccaagatggag-30 (reverse).
NF-kB binding assayNuclear extracts from the treated and untreated PC3 and PZ
cells were prepared by a nuclear extract kit (ActiveMotif). Bindingactivities of RelA and RelB were measured using an ELISA-basedTransAM NF-kB Family kit (Active Motif) according to the man-ufacturer's protocol.
Chromatin immunoprecipitationA Pierce Agarose ChIP Kit (ThermoFisher) was used to study
RelB-mediated transcriptional regulation according to themanufacturer's protocol. A potential RelB binding site waspredicted in the promoter region of the human SIRT3 geneon the basis of a search of the Ensembl genome database and arecent study (25). Briefly, chromatin was pulled down usinga RelB antibody (Santa Cruz Biotech), and a DNA fragmentcontaining an NF-kB element located in the SIRT3 promoterregion was analyzed by quantitative PCR (qPCR) with Light-Cycler 480 SYBR Green I Master Kit (Roche). PCR primersequences for SIRT3 were 50- gaattatgaaatgagcacag-30 (forward)and 50-caggatagcaagaacgagca-30 (reverse). Rabbit IgG antibodywas used as a negative control. ChIP-qPCR data were normal-ized by input preparation.
Intracellular catalase, Gpx and MnSOD enzymatic assayThe activities of catalase and Gpx were measured by a Catalase-
Specific Activity Assay Kit (Abcam) and a Gpx Cellular activityassay Kit (Sigma) according to the manufacturers' protocols,respectively. MnSOD activities were measured by the nitrobluetetrazolium-bathocuproin sulfonate reduction inhibition meth-od. Sodium cyanide (2 mmol/L) was used to inhibit CuZnSODactivity as a previous study described (26).
Quantitative and statistical data analysesMultiple independent experiments were conducted for each set
of data presented. Image data were quantified using the quanti-tative imaging software Image-pro Plus 6.0 (Media Cybernetics).Toxicity comparisons of multiple groups were analyzed usingANOVA and a post-hoc test. Data represent the mean � SEM.Kaplan–Meier survival curves, and the log-rank test were per-formed for comparison of the survival curves in animal experi-ments. Statistical significances of other experimentswere analyzedusing one-way ANOVA and Tukey multiple comparison tests. Allanalyses were performed with IBM SPSS 21.0 software (Micro-soft). Differences with an associated P < 0.05 were considered tobe significant.
ResultsAscorbic acid enhances radiosensitivity in prostate cancer cellsbut protects normal cells from radiotoxicity
To determine the cytotoxicity of ascorbic acid in prostatecancer and normal cells, LNCaP, PC3, PrEC, and PZ cells wereplated for colony survival assays and MTT assays. As shownin Fig. 1A and B, high doses of ascorbic acid alone efficientlykilled cancer cells but exerted no or minimal effect on normal
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cells. Interestingly, ascorbic acid appears to be more efficient inkilling aggressive prostate cancer PC3 cells than LNCaP cells.On the basis of a dose–effect curve, the IC50 values for PC3,LNCaP, PrEC, and PZ cell lines were quantified as 3.96, 12.81,36.56, and 33.79 mmol/L, respectively, indicating that ascorbicacid has different cytotoxic effects on prostate cancer andnormal cells.
To determine the capacity of ascorbic acid to sensitize prostatecancer cells to IR, we used the protocol for radiosensitization ofPC3 cells described in our previous study (27), taking intoconsideration the relative ascorbic acid IC50 value in clinicalapplication (28). The total dose of ascorbic acid was kept at 2mmol/L (for 1/2 IC50) or 4 mmol/L (for the full IC50). Pretreat-mentwith ascorbic acid significantly increased the radiosensitivityof PC3 cells in a dose-dependentmannerwithin an IR range of 0.5to 6 Gy (Fig. 1C). Interestingly, pretreatment with ascorbic acidresulted in the opposite effect in PZ cells, indicating that ascorbicacid actually protects normal cells against the cytotoxicity inducedby IR (Fig. 1C). To assess the combined effects of ascorbic acid andIR, the CI value for each dose was calculated by CompuSynsoftware on the basis of the Chou–Talalay method (20). A dose
of 4 mmol/L ascorbic acid conformed to the Cl range descriptionin that it displayed a synergistic effect on IR from 1 to 6 Gy in PC3cells (Supplementary Table S1). On the contrary, both doses ofascorbic acid in PZ cells displayed antagonistic effects on IR over adose range of 0.5 to 6 Gy. These results suggest that equivalentpharmacological doses of AA can exert different radiosensitiza-tion effects in prostate cancer and normal cells.
Ascorbic acid differentially modulates cellular ROS levels incancer and normal cells
To determine the effect of ascorbic acid on redox homeo-stasis in cancer and normal cells, the levels of ROS andmitochondrial superoxide anion were measured with andwithout ascorbic acid treatment. As shown in Fig. 2A, the basallevel of extracellular H2O2 was slightly but significantly higherin prostate cancer cells than in normal cells. In comparison tothe result in PZ cells, ascorbic acid treatment induced a sig-nificant increase in ROS in prostate cancer cells, especially PC3cells (Fig. 2A). Pretreatment with PEG-CAT or catalase obvi-ously inhibited such extracellular H2O2 increases and weak-ened toxicity of ascorbic acid in PC3 cells (Supplementary
Figure 1.
The effect of ascorbic acid onproliferation and radiosensitivity ofprostate cancer and normal cells. A,Two prostate cancer cell lines (PC3 andLNCaP) and one prostate epithelial cellline (PZ) were treated with differentconcentrations of ascorbic acid. Cellsurvival fraction was determined bycolony survival analysis. � and #, P <0.001 comparing PZ cells with PC3 (�)and LNCaP (#) cells, respectively;@,P<0.001 comparing LNCaP and PC3 cells.B, Two prostate cancer cell lines (PC3and LNCaP) and two prostate epithelialcell lines (PZ and PrEC) were treatedwith different concentrations ofascorbic acid. Cell survival fraction wasdetermined by MTT assay. IC50 for eachcell line was calculated on the basisof the dose–response curve. � , #, and &,P < 0.001 comparing PC3 cells to PZ (�),LNCaP (#), and PrEC (&) cells,respectively. @ and $, P < 0.001comparing LNCaP cells to PZ (@) andPrEC ($) cells, respectively. C, Prostatecancer PC3 cells and prostate epithelialPZ cells were treated with IR andascorbic acid at indicated doses. Cellsurvival fraction was determined byMTT assay. � and#,P<0.001 comparingIR group with IRþ2 mmol/L (#) andIRþ4 mmol/L (�) groups, respectively.@, P < 0.001 comparing IRþ2 mmol/Land IRþ4 mmol/L groups. All theanalyses were performed using linearregression models and likelihood ratiotests with Bonferroni correction.
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Fig. S1A and S1B). These results are consistent with previousreports that ascorbic acid produces H2O2 by oxidative reac-tion with metal ions in extracellular fluid and exerts cytotoxiceffects (14, 15).
To evaluate intracellular redox status, superoxide levels inmitochondria, the primary source of cellular ROS, were quan-tified in cancer and normal cells. Mitotracker green stainingwith negligible stimulated ROS was used to normalize MitoSoxred to quantify mitochondrial superoxide anion induced byascorbic acid with rotenone as a positive control. Ascorbic acidinduced superoxide generation in PC3 cells, but the effect wasmitigated by adding PEG-SOD (Supplementary Fig. S1C).Consistent with the results for cytotoxicity in Fig. 1, ascorbicacid increased IR-induced superoxide generation in PC3 cellsbut decreased it in PZ cells (Fig. 2B). Increasing endogenousMnSOD expression in PC3 cells clearly decreased superoxide,especially IR-induced superoxide generation after ascorbic acidtreatment (Supplementary Fig. S1D). These results suggest thatascorbic acid amplifies IR-induced cellular ROS level in cancercells. They also suggest that ascorbic acid suppresses IR-inducedROS generation in normal cells.
Ascorbic acid differentially modulates mitochondrial functionin cancer and normal cells
To test whether altering cellular redox status is associatedwith mitochondrial function, the OCR in the ascorbic acid–treated cells was measured using a Seahorse Bioscience XF96
OxygenFlux Analyzer. As shown in Fig. 3A, ascorbic acid treat-ment decreased basal, ATP-linked, and maximal OCR but notreserve capacity in PC3 cells. In contrast, ascorbic acid treat-ment increased the maximal OCR and reserve capacity of PZcells. To test whether such effects modulate the energy produc-tion of cells, the cellular ATP and lactate levels were determinedwith and without treatment. As expected, pretreatment withascorbic acid significantly diminished intracellular ATP whencombined with IR, but increased both extracellular and intra-cellular lactate production in PC3 cells (Fig. 3B–D). In contrast,combined ascorbic acid and IR treatment increased ATP pro-duction but decreased lactate concentrations in normal PZcells (Fig. 3B–D). These results suggest that ascorbic acid–mediated alterations in cellular oxidative and metabolic stres-ses play pivotal roles in the radiation response of both prostatecancer and normal cells. Importantly, ascorbic acid exacerbatesmitochondrial dysfunction in cancer cells but alleviates radia-tion-induced mitochondrial dysfunction in normal cells.
Ascorbic acid inversely regulates RelB expression in cancerand normal cells
NF-kB signaling is involved in multiple biologic processesresponsive to ROS (29). The best known activator of NF-kB isRelA, which is associated with radioresistance in many types ofcancer. Our previous findings demonstrate that RelB is alsohighly expressed in prostate cancer cells and is a major con-tributor to radioresistance (18, 30). To probe which member of
Figure 2.
The effect of ascorbic acid on redoxhomeostasis in prostate cancer andnormal cells. A, Prostate cancer andnormal epithelial cells were treated withascorbic acid and then incubated withAmplex Red. The extracellular H2O2
concentration was calculated by astandard curve and normalized to the cellnumber. B, Concentrations ofmitochondrial superoxide in PC3 and PZcellsweremeasuredbyMitoSoxRed aftertreatment with ascorbic acid and IR asindicated. Cellular fluorescence intensitywas detected by fluorescencemicroscopy and a fluorescencemicroplate reader. Mitochondria werevisualized by staining with MitotrackerGreen. Microplate readings werenormalized by protein levels. n.s, nosignificance detected. All the analyseswere performed using one-way ANOVAwith post-hoc Tukey honest significantdifference test.
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the NF-kB family is affected by ascorbic acid, we first deter-mined whether ascorbic acid regulates the expression of RelAand RelB in prostate cancer and normal cells at the mRNA andprotein levels. While ascorbic acid slightly increased RelAprotein levels in both cancer and normal cells, ascorbic acidstrongly downregulated RelB protein and mRNA in cancer cellsand upregulated RelB in normal cells (Fig. 4A). To further verifythis finding, an NF-kB binding assay was performed to confirmascorbic acid activation or suppression of RelA and RelB. Asshown in Fig. 4B, RelB binding activity was strongly suppressedin PC3 cells but apparently activated in PZ cells after ascorbicacid treatment alone. Importantly, pretreatment with ascorbicacid significantly diminished IR-induced RelB activation in PC3cells. No significant effect of ascorbic acid was observed on RelAbinding activity, but a small but significant increase wasobserved with combination treatment in PZ cells.
To further confirm the transcription effect of RelB on itstarget genes, the NF-kB–regulated genes Bcl-xL and Bax werequantified to verify the regulation of the NF-kB pathway byascorbic acid in prostate cancer and normal cells. Consistently,ascorbic acid downregulated Bcl-xL but upregulated Bax in PC3cells, whereas the reverse effect of ascorbic acid was observed inPZ cells (Supplementary Fig. S2A). Finally, to confirm the roleof RelB in ascorbic acid–induced cell killing of prostate cancercells, the level of RelB was manipulated by either overexpres-sion of RelB in LNCaP cells or knockdown of RelB in PC3 cells.Ascorbic acid–mediated cytotoxicity was increased in RelB-silenced
PC3 cells but reduced in RelB-overexpressed LNCaP cells (Fig. 4C,Supplementary Fig. S2A).
RelB acts as a central regulator in response to oxidative andmetabolic stresses in cancer cells
Generally, the radiosensitization we find in cancer cells is dueto the oxidative and metabolic stresses induced by ascorbicacid. Because our data showed that RelB plays an importantrole in ascorbic acid–induced cell killing of prostate cancercells, it was necessary to verify the functions of RelB in oxidativeand metabolic stresses. As shown in Fig. 5A, knockdown of RelBreduced basal ATP production and increased intracellular lac-tate level. When treated with ascorbic acid, IR, or combinationtreatment, RelB-silenced PC3 cells displayed more aggravatedmetabolic stress compared with the control group (Fig. 5A).The OCR data further confirmed the effect of suppressing RelBon mitochondrial function in cancer cells, especially whentreated with ascorbic acid and IR (Fig. 5B). To confirm the roleof RelB in response to oxidative stress, the activities of ROS-related enzymes were evaluated in RelB-silenced PC3 cells. Asshown in Fig. 5C and D, knocking down RelB markedlyweakened the ability of ROS elimination in cancer cells, whichwas revealed by the reduction of catalase, GPX, and MnSODactivities. These results confirm the role of RelB in oxidative andmetabolic regulations. Furthermore, they confirm that RelBmay be a central regulator that adjudicates the differentialeffects of ascorbic acid in normal and cancer cells.
Figure 3.
The effect of ascorbic acid on metabolic homeostasis in prostate cancer and normal cells. A, After ascorbic acid treatment, OCR in PC3 and PZ cellswas measured by a Seahorse Bioscience XF96 OxygenFlux Analyzer. B, Intracellular ATP levels were measured after treatment with ascorbic acid andIR at the indicated doses. C and D, Extracellular and intracellular lactate levels. Two-tailed Student t test was performed for comparisons of treatedgroups to control groups in OCR test. One-way ANOVA with post-hoc Tukey honest significant difference test was performed for comparisons of multiplegroups in cells. n.s., nonstatistical significance.
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RelB transcriptionally regulates SIRT3 in response toascorbic acid treatment
SIRT3, a member of the sirtuin family of NADþ-dependentprotein deacetylases, is known to play a critical role in main-taining mitochondrial function, ROS response, and cell prolif-eration, as well as in inducing radioresistance in cancers.Recently, a sequential action of SIRT1–RelB–SIRT3 has beenreported in sepsis (31), but the related mechanisms remain tobe fully elucidated. To probe the relationship of SIRT3 withRelB in ascorbic acid–induced cytotoxicity in cancer cells andthe protective effect in normal cells, the levels of SIRT1 andSIRT3 relative to RelB were quantified in PC3 and PZ cells. Asshown in Fig. 6A, upregulation and downregulation of SIRT3correlated with RelB in the ascorbic acid–treated cells, but no
significant changes were observed in SIRT1. Interestingly,MnSOD, a typical NF-kB–regulated mitochondrial antioxidantenzyme, was suppressed in PC3 cells but enhanced in PZ cellstreated with ascorbic acid (Fig. 6A).
Manipulation of RelB expression in cancer cells altered SIRT3levels, indicating that SIRT3 is regulated by RelB (Fig. 6B).Subsequently, RT-PCR showed that RelB transcriptionally reg-ulated SIRT3 in the ascorbic acid–treated cells (Fig. 6C). Fur-thermore, chromatin was pulled down by a RelB antibody, anda promoter region of the human SIRT3 gene containing an NF-kB element was quantified by qPCR (Fig. 6D). The amount ofthe pulled down promoter fragment was reduced by ascorbicacid treatment in PC3 cells but increased in ascorbic acid–treated PZ cells. When combined with IR, ascorbic acid
Figure 4.
Differential regulation of RelB by ascorbic acid in prostate cancer and normal cells. A, After ascorbic acid treatment, the expression levels of RelA andRelB were quantified by Western blot analyses and RT-PCR. B, Nuclear extracts from the treated and untreated cells were subjected to the NF-kBbinding assay kit. Binding activities of RelA and RelB were determined by ELISA analysis. C, RelB was overexpressed in LNCaP cells and silenced inPC3 cells by cell transfection, and the cells were treated with different concentrations of ascorbic acid. Cell survival fraction was determined by MTTassay. �, P < 0.001 comparing LNCaP-RelB and LNCaP-vector cells; #, P < 0.001 comparing PC3-siRelB and PC3-vector cells based on linear regressionmodels and likelihood ratio tests with Bonferroni correction. Other data were analyzed using one-way ANOVA with post-hoc Tukey honest significantdifference test.
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The regulation of RelB on redox and metabolic homeostasis. A, Intracellular ATP levels, extracellular and intracellular lactate levels were measured aftertreatment with ascorbic acid and IR at the indicated doses in PC3 and in RelB-silenced PC3 cells. B, After ascorbic acid and IR treatment, OCR in PC3and in RelB-silenced PC3 cells was measured by a Seahorse Bioscience XF96 OxygenFlux Analyzer. C, Level of cellular ROS was estimated by the ratio ofH2DCFDA to Carboxy-DCFDA. PEG-CAT was used as a control to remove ROS generated by ascorbic acid. D, Catalase activity, Gpx activity, and MnSODactivity were measured after treatment with ascorbic acid and IR in PC3 and in RelB-silenced PC3 cells. Two-tailed Student t test was performed forcomparisons of RelB-silenced group to control group.
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consistently suppressed the activated RelB–SIRT3 signal incancer cells but further activated the RelB–SIRT3 signal innormal cells.
Ascorbic acid enhances radiosensitivity of prostate cancer invivo
To further confirm our findings in vitro, a tumor-bearing mousemodel was used to verify the effect of ascorbic acid on tumorgrowth. Mice were subcutaneously injected with PC3 cells fol-lowed by ascorbic acid and IR treatments. Mice were humanelykilled and tumors as well as prostate and bladder tissues werecollectedwhen the tumor volume reached 1,500mm3. The tumorgrowth rate of each group shown in Fig. 7A demonstrates theefficacy of ascorbic acid in sensitizing prostate cancer to radiationtherapy. There was a trend toward significance in the differencesbetween ascorbic acid–treated and untreated mice. The timeneeded for the tumors in each group to reach 1,500 mm3 wasindependently analyzed (Supplementary Fig. S3A).
mRNA and protein levels of RelB and its targets, SIRT3 andMnSOD, were quantified in the extracted tumor tissues. Consis-tent with the results obtained in vitro, IR increased the levels ofRelB, SIRT3, and MnSOD, but ascorbic acid eliminated theincreases (Fig. 7B). In addition, prostate and bladder tissues wereused to probe whether ascorbic acid activates a protectiveresponse against radiation injury in normal tissues. Ascorbic acidincreased levels of RelB and SIRT3 mRNA in normal prostate andbladder tissues (Fig. 7C).
Overall, ascorbic acid enhances the radiosensitivity of pro-state cancer cells but protects normal cells from radiotoxicity
through RelB-dependent transcriptional regulation, with con-sequences for downstream target genes, such as SIRT3 andMnSOD, that lead to distinct responses to radiation, as illus-trated in a working model based on the results obtained todate (Fig. 7D).
DiscussionThe problems of the radioresistance of cancer tissues and the
toxic side effects of IR in normal tissues have been extensivelyinvestigated in both scientific and clinical settings. However,traditional approaches have focused on ensuring that protectingnormal tissues from injury does not also reduce the therapeuticefficacy of radiation. Here, we demonstrate that ascorbic acid, aredox active agent, can enhance the therapeutic efficacy of radi-ation therapy while simultaneously protecting normal tissuesagainst the side effects of radiation therapy.
That ascorbic acid enhances traditional radiotherapy andchemotherapy of cancer has been reported since 1977 (14, 15,32–35). In the intervening decades, ascorbic acid has been usedto alleviate some of the side effects of radiation therapyduring cancer treatment (34, 36). A recent study by Du andcolleagues clearly shows the radiosensitization induced bypharmacologic ascorbic acid in pancreatic cancer cells (15). Theseauthors also observed that ascorbic acid also potentially pro-tects the gastrointestinal tract from IR in vivo. Although it wasnot a major focus of the study, the observation by Du andcolleagues is consistent with our finding that ascorbic acid indeedprotects normal prostate and bladder tissues from IR.
Figure 6.
Modulation of SIRT3 due to ascorbic acid-mediated RelB regulation. A, After ascorbicacid treatment, the expression of SIRT1, SIRT3,RelB, and MnSOD in PC3 and PZ cells wasmeasured by Western blot analyses. B, Proteinlevels of SIRT1 and SIRT3 after manipulation ofRelB expression in LNCaP and PC3 cells weredetermined by Western blot analyses. C, Afterascorbic acid treatment, mRNA levels of SIRT3in PC3 cells and PZ cells were determined byRT-PCR. D, Protein–DNA complexes wereextracted from the ascorbic acid–treated oruntreated PC3 and PZ cells and thenimmunoprecipitated using aRelB antibody. TheSIRT3 promoter fragment containing an NF-kBelement was amplified by qPCR with specificprimers, which were normalized by their inputcontrols containing relevant unprecipitatedchromatin. E, After ascorbic acid and radiationtreatment, the expression of RelB-SIRT3 in PC3and PZ cells was measured by Western blotanalyses. One-way ANOVA with post-hocTukey honest significant difference test wasperformed for comparison of multiple groupsin cells.
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Elevated ROS levels, which are essential for tumorigenesis andmetastasis, have been observed in many types of cancer. Highlevels of ROS may reveal a specific vulnerability of malignancythat can be used to selectively enhance cell death by furtherincreasing the level of cellular ROS. Here, we show that ascorbicacid acts as a pro-oxidant at pharmacologic doses and differen-tially modulates cellular responses to ROS in normal and cancercells. Our results are consistent with previous studies demonstrat-ing that the cytotoxicity induced by ascorbic acid is primarilymediated by H2O2 (14, 15, 19 and 22). However, the previousstudies did not take into consideration the respective distinctbasal redox state of normal and cancer cells. Our data suggest thatascorbic acid causes an increase in ROS in prostate cancer cells thatreaches a threshold level of cell death but only slightly increases
ROS in normal cells, resulting in an induction of antioxidant andmetabolic responses. These opposing effects of ascorbic acid innormal and cancer cells aremediated, in part, by the expression ofRelB, a member of the alternative pathway of the NF-kB family.
NF-kB is known to be an important ROS-responsive transcrip-tional factor involved in both tumor progression and tumorresistance to treatment (16). Previous studies have indicatedthat ascorbic acid can inhibit NF-kB activation by preventing thedegradation of IkBa and nuclear translocation of RelA (37, 38).Recent studies by our laboratory and others have demonstratedthat RelB contributes to radioresistance of prostate cancer cells bysustaining NF-kB activation (17, 18). The present study furtherestablishes the role of RelB as a critical redox signaling sensorthat regulates its downstream target genes in response to ascorbic
Figure 7.
Ascorbic acid–mediatedradiosensitization of prostate cancercells in vivo.A, Prostate cancer PC3 cellswere injected into the flanks of nudemale mice and the formed tumors weretreated with ascorbic acid and IR asindicated. Tumor volume was measuredand tumor growth rate was calculated.Kaplan–Meier survival curves and thelog-rank test were performed forcomparison of the survival curves. B,Levels of RelB, SIRT3, and MnSOD intumor tissue were measured byWestern blot analyses and RT-PCR. C,mRNA levels of RelB and SIRT3 inprostate and bladder tissues werequantified by RT-PCR. D, Proposedmechanistic model for the ascorbicacid–mediated differential response toradiation in prostate cancer and normalcells. Ascorbic acid sensitizes cancercells to radiation by downregulatingRelB-SIRT3 signal, which in turnaggravates oxidative and metabolicstresses. In contrast, in normal prostateepithelial cells, H2O2 generated from theredox reaction of ascorbic acidupregulates RelB, leading to increasedSIRT3 levels, which enhances cellularstress defense systems. Kaplan–Meiersurvival curves and the log-rank testwere performed for comparison of thesurvival curves in animal experiments.Other data were analyzed using one-way ANOVA with post-hoc Tukeyhonest significant difference test.
Wei et al.
Cancer Res; 77(6) March 15, 2017 Cancer Research1354
acid, leading to the observed opposing radiation responses inprostate cancer and normal cells. Repression of IR-induced RelBactivation in cancer cells results in diminished oxidative defensecapacity and subsequently enhances radiosensitivity throughmitochondrial dysfunction.On the contrary, upregulation of RelBserves as a major mechanism by which ascorbic acid protectsnormal tissues against radiotoxicity, through upregulation ofantioxidant enzymes and the mitochondrial function of scaveng-ing ROS. RelA has been reported to be involved in RelB transcrip-tional activation (39); further studies will be needed to determinewhether andhowRelAparticipates in ameaningful changeof RelBas it relates to ascorbic acid.
Recently, metabolic alterations in cancer cells due to ascorbicacid–induced oxidative stress have been the subject of intenseinvestigation (15, 40). Although a high rate of aerobic glycolysisin tumors, known as the Warburg effect, has been observed invarious types of cancer, cancers have functional mitochondria,and mitochondrial respiration is necessary for cancer cell prolif-eration and resistance to therapy (41, 42). The present studyshows that the mitochondria in prostate cancer cells becomedysfunctional, with downregulated MnSOD, after ascorbic acidtreatment. As a MnSOD transcriptional regulator, RelB modula-tion by ascorbic acid leads to the suppression and induction ofMnSOD in cancer and normal cells, respectively.
Sirtuins are NADþ-dependent histone deacetylases in mam-malian cells and are involved in an array of critical cellularfunctions (43–46). Of the 7 human sirtuins, SIRT3 is the bestcharacterized in its regulation of many aspects of mitochondrialfunction. Physiologically, SIRT3 interacts with subunits of com-plexes I and II of the electron transport chain to improve mito-chondrial respiration (47). SIRT3 also deacetylates and activatesMnSOD tomaintain the antioxidant defense system in cells (48).A recent study described the expression of SIRT3 as a sequentialaction of the SIRT1/RelB axis in a sepsis model (31). The presentstudy suggests that the RelB/SIRT3 signaling axis may play acritical role in ascorbic acid treatment independent of SIRT1levels. Our results demonstrate that RelB regulates SIRT3 expres-sion through binding to its promoter region. Repression of RelB-activated SIRT3 transcription by ascorbic acid aggravates meta-bolic stress in cancer cells. In contrast, upregulation of SIRT3improves the ability of mitochondria to defend against metabolicstresses in normal cells. These results suggest that RelB may be aunique target for treatment of radiation-resistant prostate cancer.
Considering the unsatisfactory results of clinical trials of highdoses of oral ascorbic acid, and the reported successes of highdoses of intravenous ascorbate (13, 49), the complexity of themechanisms involved in ascorbic acid treatment deserves fur-
ther investigation. The present study indicates a promisinganticancer effect of ascorbic acid that is dependent on cellproperties such as the basal redox state of the cancer andnormal cells. The present study also reveals cell-dependentROS generation in ascorbic acid treatment and identifies theRelB/SIRT3/MnSOD axis as a critical contributor to ascorbicacid–induced radiosensitization of cancer cells and radiopro-tection of normal cells (Fig. 7D). Thus, while additional mech-anistic studies are needed to fully understand the biologicfunction of ascorbic acid, we anticipate that other redox-basedanticancer therapeutics with protective properties against cyto-toxic therapy will be discovered and that they will have asignificant impact on the care of patients with cancer.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: X. Wei, Y. Xu, J. Chen, D.K. St. Clair, W.H. St. ClairDevelopment of methodology: X. Wei, Y. Xu, F.F. Xu, D. SchnellAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Wei, F.F. Xu, L. Chaiswing, W.H. St. ClairAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Wei, Y. Xu, F.F. Xu, C. Wang, D.K. St. ClairWriting, review, and/or revisionof themanuscript:X.Wei, Y. Xu, L. Chaiswing,J. Chen, D.K. St. Clair, W.H. St. ClairAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Noel, D.K. St. ClairStudy supervision: J. Chen, D.K. St. ClairOther (performed selected experiments): L. Chaiswing
AcknowledgmentsThe authors thank Dr. Mike Mitov and Michael Alstott of the Redox
Metabolism Service Facility at the University of Kentucky, who providedtechnical support for the seahorse experiments.
Grant SupportThis work was supported mainly by NIH grants CA 049797 and CA 143428
to D.K. St. Clair and W.H. St. Clair. Additional support was provided by grantsfrom the National Natural Science Foundation of China (Grant No. 81272469)and the Natural Science Foundation of Jiangsu Province (Grant No.BL2012016) to J. Chen. The research used service facilities funded by a CancerCenter support grant (P30 CA177558).
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 March 16, 2016; revised November 15, 2016; accepted November18, 2016; published OnlineFirst January 20, 2017.
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