KEAP1 Is a Redox Sensitive Target That Arbitrates the ......2013/07/05 · Therapeutics, Targets, and Chemical Biology KEAP1 Is a Redox Sensitive Target That Arbitrates the Opposing

Therapeutics, Targets, and Chemical Biology

KEAP1 Is a Redox Sensitive Target That Arbitrates theOpposing Radiosensitive Effects of Parthenolide in Normaland Cancer Cells

Yong Xu1, Fang Fang2, Sumitra Miriyala1, Peter A. Crooks3, Terry D. Oberley4, Luksana Chaiswing4, TeresaNoel1, Aaron K. Holley1, Yanming Zhao1, Kelley K. Kiningham5, Daret K. St. Clair1, and William H. St. Clair2

AbstractElevated oxidative stress is observed more frequently in cancer cells than in normal cells. It is therefore

expected that additional exposure to a low level of reactive oxygen species (ROS) will push cancer cells towarddeath, whereas normal cells might maintain redox homeostasis through adaptive antioxidant responses. Wepreviously showed that parthenolide enhances ROS production in prostate cancer cells through activation ofNADPH oxidase. The present study identifies KEAP1 as the downstream redox target that contributes toparthenolide's radiosensitization effect in prostate cancer cells. In vivo, parthenolide increases radiosensitivity ofmouse xenograft tumors but protects normal prostate and bladder tissues against radiation-induced injury.Mechanistically, parthenolide increases the level of cellular ROS and causes oxidation of thioredoxin (TrX) inprostate cancer cells, leading to a TrX-dependent increase in a reduced state of KEAP1, which in turn leads toKEAP1-mediated PGAM5 and Bcl-xL (BCL2L1) degradation. In contrast, parthenolide increases oxidation ofKEAP1 in normal prostate epithelial cells, leading to increased Nrf2 (NFE2L2) levels and subsequent Nrf2-dependent expression of antioxidant enzymes. These results reveal a novel redox-mediated modification ofKEAP1 in controlling the differential effect of parthenolide on tumor and normal cell radiosensitivity. Further-more, they show it is possible to develop a tumor-specific radiosensitizing agent with radioprotective propertiesin normal cells. Cancer Res; 73(14); 1–12. �2013 AACR.

IntroductionIt is well documented that cancer cells are usually under

more oxidative stress than normal cells are, in part, due to ahyperactive metabolism that fuels their rapid growth (1, 2).Thus, a therapy designed to increase reactive oxygen species(ROS) to a level above the threshold for cancer cell death, but toan adaptable level for normal cells, would be an attractivestrategy to selectively kill cancer cells (3, 4). Redox homeostasisis thought to regulatemany cellular processes that are essentialfor maintenance of normal physiologic conditions but isaberrantly modulated in cancers (5, 6). The functions ofROS are both beneficial and deleterious due to their dual rolein the prosurvival and antisurvival pathways. As a secondarymessenger in cell signaling, ROS are required for normaldevelopment and can initiate adaptive responses in cellular

defense (7, 8). On the other hand, ROS cause structural damageand functional decline in DNA, proteins, and lipids, andconsequently act as an antitumorigenic factor by inducingcell senescence and apoptosis (9, 10). Indeed, ROS-mediatedcell death is an important basis for radiotherapy and manychemotherapeutic treatments (11, 12). Currently, these ther-apeutic strategies are being used to kill cancer cells withoutbenefit of a rational design that exploits the intrinsic differ-ences in the cellular redox status of normal cells and cancercells.

Antioxidant defense systems are essential for the regula-tion of ROS levels, which have an important function in themaintenance of cellular redox hemostasis. Mounting evi-dence shows that a decline in antioxidant function may beinvolved in tumorigenesis due to prooxidant conditions thatresult from ROS accumulation. For example, manganesesuperoxide dismutase (MnSOD) is downregulated in manytypes of cancer and overexpression of MnSOD resultsin suppression of tumorigenesis (13–15). High levels ofantioxidants caused by therapy-mediated activation ofprosurvival pathways, such as NF-kB and Nrf2, are thoughtto protect cancer cells against treatment (16–18). Thus,inhibition of prosurvival pathways has been a traditionalstrategy to enhance therapeutic efficacy.

Increasing evidence shows that certain mild prooxidantcompounds derived from natural herbal medicines mightenhance some anticancer treatments by modulating the

Authors' Affiliations: 1Graduate Center for Toxicology, 2Department ofRadiationMedicine, University of Kentucky, Lexington, Kentucky; 3Depart-ment of Pharmaceutical Sciences, UAMSCollege of Pharmacy, Little Rock,Arkansas; 4Department of Pathology, University of Wisconsin, Madison,Wisconsin; and 5Department of Pharmaceutical Sciences, Belmont Uni-versity School of Pharmacy, Nashville, Tennessee

Corresponding Authors: William H. St. Clair, Department of RadiationMedicine, University of Kentucky, College of Medicine, Lexington, KY40536. Phone: 859-257-4931; Fax: 859-323-6486; E-mail:[email protected]; and Daret K. St. Clair, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-4297

�2013 American Association for Cancer Research.

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redox state of cancer cells to high prooxidant levels (19, 20).Parthenolide, an active ingredient derived from the tradi-tional anti-inflammatory medical plant feverfew (Tanacetumparthenium), belongs to the family of sesquiterpene lactonescontaining an a-methylene-g-lactone moiety and an epoxidegroup, which is able to conjugate thiols of proteins through aMichael addition reaction (21). In addition to its anti-inflammatory effect, parthenolide seems to be toxic to avariety of cancer cells (22–25). Importantly, parthenolide hasno cytotoxic effect on normal cells (25). Mechanistically,parthenolide has been shown to increase apoptosis in cancercells through inhibition of multiple prosurvival pathways,such as NF-kB and PI3K-AKT (19, 26). However, thesefindings do not explain why parthenolide is not toxic tonormal cells.

Posttranslational modification is a keymechanism by whichproteins dramatically increase their functional diversity.Reversible redox modification of protein cysteine residuesplays an important role in vital cell signaling pathways relatedto many physiologic and pathogenic processes (27, 28). TheKeap1-Nrf2 pathway is one of the main cellular defensemechanisms against oxidative stress (29, 30). The present studyexamines the role of cysteine modifications in modulatingradiation responses in prostate cancer cells versus normalprostate epithelial cells. It elucidates the functional linkbetween redox modulation and cell signaling transductionpathways, and it provides evidence for the differential effectof parthenolide on cellular redox status in normal and cancercells. The results reveal that parthenolide-mediated redoxmodification of Keap1 serves as a central regulator of differ-ential responses to radiotherapy in normal and tumor cells.The present study provides a proof-of-concept for using anintrinsic difference in cellular redox conditions to kill tumorcells while protecting normal cells from the unwanted sideeffects of radiation.

Materials and MethodsCell culture, cell transfection, treatment, and cellsurvival analysis

Human prostate carcinoma/adenocarcinoma LNCaP,PC3, and DU145 cell lines as well as human prostate epi-thelial viral transformed PZ-HPV-7 (PZ) and RWPE-1 celllines were obtained from American Type Culture Collection.Normal prostate epithelial PrEC cells were purchased fromLonza. All cell lines were cultured and maintained in themedia recommended in the manufacturer's protocols.Plasmid-cloned Keap1 cDNA and Bcl-xL cDNA (OriGene)and siRNAs for knocking down Keap1, Nrf2, thioredoxin(TrX), PGAM5, and Bcl-xL (Dharmacon) were transfectedinto cultured cells before treatment. Parthenolide andits water-soluble prodrug, dimethylamino-parthenolide(DMAPT), were synthesized as previously described (31).The cells were treated with 0 to 5 mmol/L parthenolidefollowed by irradiation by a 250 kV X-ray machine (FaxitronX-ray Corp.) with peak energy of 130 kV, 0.05 mm Al filter, ata dose of 0 to 6 Gy. Cell survival rates were quantified bycolony survival fraction, Trypan blue exclusion assay, andMTT assay, as previously described (25, 32).

AnimalsFour- to five-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 flankof themice. Tumor volumeswere routinelymeasured and theirsizes calculated on the basis of a protocol described elsewhere(33). Animals with an average tumor size of 500 mm3 wererandomized into several groups for DMAPT and radiationtreatments. The tumors were treated 5 times with 10 mg/kgDMAPT and 3 Gy IR. The tumor tissues were collected, and 100mg of tissue was lysed to quantify amounts of oxidized orreduced Keap1 and levels of downstream proteins. To deter-mine the protective effect of parthenolide against radiationdamage, mice were pretreated with DMAPT followed by radi-ation treatment (5 � 3Gy). Prostate and bladder tissues werefixed, embedded, and processed for routine electron micros-copy. The embedded blocks were sectioned and transferred tocopper grids and counterstained with uranyl acetate, followedby lead citrate. Grids were observed in an electron microscope(Hitachi H-600) operated at 75 kV. Mitochondrial damage wasquantified by a pathologist (T.D. Oberley) using identifiedultrastructural changes including swelling, vacuolization,myelination, disorganization, and loss of cristae, lysosomaldegradation, and membrane disruption. Animal experimentalprocedures were approved by the Institutional Animal Careand Use Committee of the University of Kentucky (Lexington,KY), Approval Protocol No. 01077M2006.

Quantification of intracellular superoxide andprooxidant

Dihydroethidium (DHE, Invitrogen), which exhibits blue fluo-rescence in the cytosol until oxidized, was used to estimate thelevels of superoxide after parthenolide treatment. To confirm thelevel of superoxide induced by parthenolide, the cells were pre-treated with 50 mg PEG-SOD (Sigma) for 1 hour followed byparthenolide treatment. Antimycin (Sigma) was used as a posi-tive control because it has been shown to increase superoxide inall tested cell lines. A dichlorofluorescein (DCF) assay was usedto quantify the levels of intracellular ROS after parthenolidetreatment. The cells were labeled by both carboxy-H2DCFDA(sensitive to oxidation, Invitrogen) and carboxy-DCFDA (insen-sitive to oxidation, Invitrogen). The H2DCFDA:DCFDA ratio wasused to optimize the controls of cell number, dry uptake, andester cleavage. The procedures for both DHE and DCF wereconducted by theUniversity of Kentucky FlowCytometer Facilityusing a FACScan protocol provided by Dr. Douglas R. Spitz(Professor of Radiation Oncology at the University of Iowa; 34).

Measurement of oxygen consumption ratesTo determine how parthenolide changes mitochondrial

function in cancer and normal cells, a Seahorse BioscienceXF24 Extracellular Flux Analyzer was used to measure oxygenconsumption rates (OCR) after parthenolide treatment. TheXF24 creates a transient, 7 mL chamber in specialized micro-plates, which allows determination of oxygen and protonconcentrations in real time. To allow comparison betweenexperiments, data are expressed as the OCR in pmoL/min or

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the rate of extracellular acidification in mpH/min. Reservecapacity, an important index that indicates capacity of mito-chondrial respiration, is calculated by subtracting baselineOCR from maximal OCR.

Detection of oxidized and reduced forms of Keap1protein3-N-maleimido-propionyl biocytin was used to selectively

label sulfhydryl (SH) and then was detected by biotin–strep-tavidin integration on the blots, as previously described (35).To quantify disulfide (S-S) bonds, the SH formwas stabilized bytreating with N-ethylmaleimide and then the S-S bonds werereduced by treating with 2-mercaptoethanol. To identify SHand S-S moieties of Keap1 protein, the labeled proteins wereimmunoprecipitated by Keap1 antibody (Abcam) and sub-jected to SDS-PAGE, followed by detection with horseradishperoxidase-conjugated streptavidin (Sigma).

Immunoblots and immunoprecipitationHomogenized cells and tumor tissues were electrophoresed

on an 8% (w/v) SDS-PAGE gel, transferred onto a nitrocellulosemembrane, and subsequently incubated with primary antibo-dies against Keap1 (Abcam), Nrf2 (Abcam), MnSOD (UpstateBiotech), CuZnSOD (eBiosci), Gpx (Abcam), catalase (Milli-pore), TrX (BD Sciences), PGAM5 (Santa Cruz Biotech.), Bcl-xL(Santa Cruz Biotech.), LC3B (Cell Signaling), b-actin (Sigma),and pCNA (Santa Cruz Biotech.). All secondary antibodieswereobtained from Santa Cruz Biotech. Immunoblots were visual-ized using an enhanced chemiluminescence detection system(Amersham Pharmacia Biotech.). For immunoprecipitation,cell extracts were incubated overnight with one primary anti-body at 4�C and integrated with a protein A/G agarose (SantaCruz Biotech). Immunocomplexes were precipitated and frac-tionated on an SDS-PAGE gel. Interacting proteins weredetected by immunoblotting with their primary antibodies.

SOD enzymatic assayMnSOD activities were measured by the nitroblue tetrazo-

lium-bathocuproin sulfonate reduction inhibition method.Sodium cyanide (2 mmol/L) was used to inhibit CuZnSODactivity (36).

Statistical data analysesMultiple independent experiments were conducted for each

set of data presented. Images in Immunoblots were quantifiedusing Carestream Molecular Imaging software (CarestreamHealth Inc.). Statistical significance was analyzed using one-way ANOVA and Tukey multiple comparison test, followed bydata analysis with GraphPad Prism.

ResultsParthenolide enhances radiosensitivity of prostatetumors but protects normal tissues from radiationOur previous studies showed that parthenolide is able to

selectively enhance the radiosensitivity of prostate cancer cellswithout injury to normal prostate epithelial cells (24, 25). Todetermine whether parthenolide enhances radiotherapy invivo, human prostate cancer PC3 cells were subcutaneously

implanted into the right flank of nudemale mice. When tumorsize reached a volume of 500 mm3, animals were randomizedinto groups according to the treatment consisting of saline or10 mg/kg DMAPT. One hour after saline or DMAPT wasadministered, the tumors were treated with fractionated radi-ation of 1 Gy or 2 Gy per day for 5 days followed by routinemeasurement of tumor volume. The tumor growth curves areshown in Fig. 1A. Mice were humanely killed when a tumorreached the maximum size of 2,000 mm3. Tumor growth wasclearly delayed in the treatment groups, particularly when thedrug and radiation were combined, compared with growth inthe untreated group. The tumor growth rates after treatmentwere compared according to the days needed for tumorvolume to reach 2,000 mm3. DMAPT significantly enhancedradiotherapeutic efficiency compared with the effects of radi-ation treatment alone (Fig. 1B). A separate group of nude malemice that had no cancer cell implantation was treated withDMAPT and radiation to determine the toxicity of DMAPT toorgans that can be affected by radiotherapy of prostate cancer.Prostates and bladders of the animals were examined by lightand electron microscopy. At 60 days after irradiation, no grosspathology was observed (data not shown). However, ultra-structural damage was clearly observed by electron micros-copy (Fig. 1C). Mitochondrial damage was most pronouncedand this was morphometrically analyzed. The number ofdamaged mitochondria in prostate and bladder was propor-tional to radiation exposure. Pretreatment with DMAPT sig-nificantly reduced the number of damaged mitochondria inboth organs compared with the group without DMAPT treat-ment (Fig. 1C and D). These results indicate that DMAPT, thewater-soluble prodrug of parthenolide, is a promising agent forselectively enhancing the sensitivity of prostate cancer cells toradiation while protecting normal tissues from damage causedby radiation.

Parthenolide differentiallymodulates cellularROS levelsin cancer and normal cells

To determine the effect of parthenolide on redox homeo-stasis in cancer and normal cells, the levels of superoxide andtotal ROS after parthenolide treatment were measured usingflow cytometry. The mean fluorescence intensity of DHE andtheH2DCFDA:DCFDA ratiowere higher in prostate cancer PC3cells than in normal prostate PZ and PrEC cells, indicatinghigher basal levels of superoxide and total ROS, respectively(Fig. 2A and B). Following parthenolide treatment, DHE andDCF levels increased further in PC3 cells but declined slightly inboth types of noncancerous cells. Combining PEG-SOD withparthenolide treatment restored the basal level of DHE fluo-rescence. As positive controls, addition of equivalent concen-trations of the ROS-stimulating agents antimycin (Fig. 2A) andphorbol-12-myristate-13-acetate (PMA; Fig. 2B) caused highlevels of ROS in all the tested cell lines.

The levels of antioxidant proteins were also quantified(Fig. 2C). Parthenolide altered the protein level of the antiox-idant enzymes, in particular mitochondria-localized antioxi-dant enzymes. MnSOD and glutathione peroxidase (GpX)were significantly reduced in all 3 parthenolide-treated pros-tate cancer cell lines. Intriguingly, parthenolide had the

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opposite effect in the 3 normal prostate cell lines. However,neither cancer nor normal cells showed any obvious changesafter parthenolide treatment for the major cytosolic superox-ide removal protein, copper, and zinc-containing SOD (CuZn-SOD). This observation was confirmed by quantification of thecorresponding enzyme activity (Fig. 2D). These results suggestthat parthenolide-mediated alteration of cellular redox statusis mediated, at least in part, by changing the activities ofantioxidant enzymes in mitochondria.

To probe whether altering cellular redox status is associatedwith a change in mitochondrial respiration, the OCR in theparthenolide-treated cells was measured using a SeahorseBioscience FX OxygenFlux Analyzer. The basal and maximalOCR in normal cells was higher than in cancer cells (Fig. 2E).Importantly, parthenolide was able to increase the OCR andreserve capacity in PZ cells, whereas parthenolide had no effecton PC3 OCR. Finally, the cytotoxicity of parthenolide was

tested in all the cell lines using an MTT assay, which requiresactive mitochondria. As shown in Fig. 2F, parthenolide wastoxic to all the cancer cells but not to the normal cell lines.Taken together, these results suggest that changes in cellularredox status andmitochondrial functionmay be a cause for thedifferential biologic effects of parthenolide on cancer andnormal cells.

Keap1 is susceptible to parthenolide-mediated redoxmodification

Keap1, a redox-sensitive protein, has been reported to playan important role in cell survival under oxidative stress (29). Toinvestigate whether parthenolide modifies Keap1 function, aKeap1 antibody linked to biotin was used to immunoprecip-itate redox-modified Keap1 protein and the presence of oxi-dized (-S-S-) and reduced (-SH) cysteine residues was detectedusing a secondary antibody linked to streptavidin. In the 3

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Figure 1. The effect of parthenolide on radiosensitivities of prostate cancer and normal cells. A, prostate cancer PC3 cells were injected into the flanks of nudemale mice. The resulting tumors were treated with DMAPT and IR. Tumor volume was measured and tumor growth was calculated. B, time needed fortumor growth to reach 2,000mm3 volume after treatment was calculated and plotted. C, mice without cancer were treated with radiation alone (5� 3 Gy) andDMAPT (10 mg/kg) with radiation. Prostate and bladder tissues were removed for pathologic analysis using electron microscopy. Arrows indicate normalmitochondria and asterisks indicate mitochondria with myelin figures. M, normal mitochondria; Ly, lysosome; and V, mitochondria with vacuoles. D,quantification of mitochondrial damage in mice prostate and bladder tissues.

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normal cell lines, parthenolide increased the oxidized form ofKeap1 but decreased the reduced form of Keap1 (Fig. 3A).Interestingly, the results from the 3 cancer cell lines seemed tobe completely opposite to results observed in normal cellstreated with parthenolide: the level of the oxidized form wasdecreased, but the level of the reduced form was increased

(Fig. 3B). To verify that the observed increase in reduced Keap1also occurred in vivo, mouse xenograft tumor tissues with andwithoutDMAPT treatmentwere also used for determination ofKeap1 redox status. Consistent with data obtained from cul-tured tumor cells treated with parthenolide, the in vivo resultsshow that parthenolide decreased the oxidized form of Keap1

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Figure 2. The effect of parthenolide on redox homeostasis in prostate cancer and normal cells. A–B, cells were treated with parthenolide and then labeledwith DHE or DCF. Themean fluorescence intensity of DHE (A) and the ratio of H2DCFDA to DCFDA (B) were determined using flow cytometry. Concentrationsof cellular superoxide and total ROS were estimated by quantification of fluorescence intensity. Antimycin and PMA were used as positive controls forgeneration of ROS. PEG-SOD was used as a control to remove superoxide generated by DHE. PN, parthenolide. �, #, $ indicate the significances oftreatments compared to DMSO untreated control in PC3 (�), PZ (#), and PrEC ($) cells. C, the levels of antioxidant proteins were quantified by Western blots.D, the activities of the corresponding enzymes were measured. E, quantification of the basal oxygen consumption, ATP-linked oxygen consumption, themaximal OCR after the addition of FCCP, and the reserve capacity of the cells. NS, not significant. F, three cancer cell lines and 3 noncancer cell lines weretreated with parthenolide (PN) at the indicated concentrations. Cell survival fraction was determined by MTT assay.






but increased the reduced form of Keap1 in the tumors (Fig.3C). Changes in antioxidant proteins inmouse xenograft tumortissues treated with DMAPT are also consistent with the resultobtained from in vitro studies (Fig. 3D), indicating that parthe-nolide decreases the level of mitochondrial antioxidant pro-teins in prostate tumors.

Oxidization of Keap1 leads to activation of the Nrf2prosurvival pathway in normal cells

Activation of the Nrf2 signaling pathway through dissocia-tion with Keap1 resulting in Nrf2 nuclear translocation isconsidered to be a primary prosurvival pathway in responseto oxidative stress (30, 37). To examine whether parthenolidechanges Nrf2 nuclear translocation, the levels of Nrf2 in nucleiwere measured. As shown in Fig. 4A, the nuclear levels of Nrf2were increased in the 3 normal cell lines treated with parthe-nolide, but no changes were observed in the 3 cancer cell lines.To examine whether activation of the Nrf2 pathway is a majormechanism by which parthenolide protects normal cellsagainst radiation injury, Keap1 and Nrf2 were silenced in PZcells by transfecting their siRNA (Fig. 4B, left). Cell survivaldecreased when Nrf2 was silent. IR significantly reduced cellsurvival but the cell survival was restored when Keap1 wassilenced (Fig. 4B, right panel). These results suggest that

oxidation of Keap1 and subsequent activation of Nrf2 byparthenolide is essential for normal cell survival after radiationtreatment.

Thioredoxin is necessary for parthenolide-mediatedreduction of Keap1 in cancer cells

TrX is highly expressed in cancer cells and stimulates cellgrowth. We previously reported that parthenolide decreasesthe reduced form of TrX but increases the oxidized form of TrXin prostate cancer cells (25). In the present study, we verify thatTrX was expressed at a high level in all 3 cancer cell lines,whereas a low level was observed in the 3 noncancer cell lines(Fig. 5A). Immunoprecipitation of Keap1 protein from PC3 cellextracts using a TrX antibody suggests an interaction betweenKeap1 and TrX that is increased by parthenolide (Fig. 5B). Todetect whether the parthenolide-influenced reduction ofKeap1 in cancer cells is dependent on TrX, we selectivelysilenced TrX by transfecting its siRNA before parthenolidetreatment (Fig. 5C, left). As expected, the reduced form ofKeap1 was decreased, but the oxidized form of Keap1 wasincreased when TrX was silent (Fig. 5C, middle and right). Theresults suggest that TrX is interacting with Keap1 to keepKeap1 in a reduced state in parthenolide-treated cells. Tofurther confirm that the function of Keap1 leads to cell death

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Figure 3. Keap1 redox modification by parthenolide. A and B, 3 normal cell lines (A) and 3 prostate cancer cell lines (B) were treated with saline orparthenolide (PN). Keap1was immunoprecipitated with its antibody and SH- and S-Smoieties of Keap1were shown by ECL detection. C, tumor tissueswerehomogenized and incubated with MPB. Reduced (SH-) and oxidized (S-S) forms of Keap1 in the tissues were detected. D, tumor tissues were homogenizedand antioxidant proteins were quantified by Western blots. Right, representative blots. Left, the average of multiple blots.

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in parthenolide-treated cancer cells, a Keap1 expression con-struct was transfected into PC3 cells, followed by parthenolideand IR treatments. Overexpression of Keap1 resulted inincreases in cell death in both treated and untreated cells (Fig.5D, top). The levels ofmitochondrial phosphoglyceratemutase5 (PGAM5), a protein serine/threonine phosphatase that inter-acts with Bcl-xL in the mitochondrial membrane (38), and Bcl-xLwere clearly decreased in the Keap1-transfected cells, but nochanges were observed in Nrf2, Ikka, and IkBa (Fig. 5D,bottom). These results suggest that the parthenolide-increasedreduced form of Keap1 facilitates Keap1-mediated ubiquitin/proteasome-dependent degradation of PGAM5 and Bcl-xL,which is an established mechanism for parthenolide-mediatedcell death in cancer cells.

Keap1 triggers PGAM5-mediated Bcl-xL ubiquitindegradation in parthenolide-treated cancer cellsTo further investigate themechanismbywhich parthenolide

enhances the radiosensitivity of prostate cancer cells, wedetermined the interactions between Keap1, PGAM5, andBcl-xL. The results show that a reduced form of Keap1, whichis increased in parthenolide-treated PC3 cells, enhanced inter-action between Keap1 and PGAM5, as detected by immuno-precipitation using a PGAM5 antibody (Fig. 6A). Bcl-xL, aprosurvival mitochondrial protein, was also increased in thepulled down complex (Fig. 6A). Interestingly, the proteins thatare associated with Keap1 were decreased in whole-cellextracts (Fig. 6B). A time course of parthenolide treatmentshows that PGAM5 and Bcl-xL proteins were slightly increasedat 12 hours but decreased at 24 and 48 hours after treatment(Fig. 6C). Proteins in different cellular fractions were alsoquantified (Fig. 6D). The mitochondria-associated proteinsPGAM5 and Bcl-xL were reduced by the parthenolide treat-ment, but no change was observed in Hsp75, a control formitochondrial protein. Parthenolide had nomajor effect on the

levels of Nrf2 and Ikka in treated cells. These results suggestthat parthenolide enhances Keap1-mediated ubiquitin/pro-teasome-dependent degradation of PGAM5 and Bcl-xL (39).In addition, parthenolide increased the level of mitochondria-associated autophagic protein LC3B, suggesting that parthe-nolidemay enhance the radiation sensitivity of prostate cancercells partially through triggering the autophagy pathway.

Because Keap1 interacts with PGAM5/Bcl-xL/Nrf2, wedecided to determine the effect of PGAM5/Bcl-xL/Nrf2 inmediating parthenolide's effect on cancer cells. PGAM5, Bcl-xL, and Nrf2 were silenced using their siRNAs, followed byparthenolide treatment (Fig. 6E, bottom). The cell survivalfraction was decreased when PGAM5 or Bcl-xL was silent,which is similar to the effect of parthenolide (Fig. 6E, top). Nosignificant additive effects were observed when parthenolidewas combined with PGAM5 or Bcl-xL siRNA. In contrast, asignificant effect was observed when Nrf2 was silent. Theseresults suggest that Keap1-mediated PGAM5/Bcl-xL degrada-tion, but not Nrf2 degradation, is important for parthenolide-induced cancer cell death.

To further determine whether the function of Bcl-xL plays amajor role in protecting cancer cells against parthenolide-induced cell death, a plasmid carrying Bcl-xL cDNA wastransfected into PC3 cells followed by parthenolide treatment(Fig. 6F, bottom). The results show that expression of Bcl-xLefficiently protects cells from cytotoxicity caused by parthe-nolide (Fig. 6F, top). Taken together, these results suggest thatparthenolide enhances the radiosensitivity of prostate cancercells, in part, by triggering ubiquitin/proteasome-based deg-radation of Bcl-xL.

In summary, parthenolide provides radiosensitization inprostate cancer cells but radioprotection in normal cells, andthe observed differential effects are mediated, in part, by redoxmodification of Keap1, i.e., reducing Keap1 in cancer cellsbut oxidizing Keap1 in normal cells. The distinct redox

A

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Figure 4. Activation of Keap1-Nrf2pathway by parthenolide in normalcells. A, parthenolide (PN)increases nuclear levels of Nrf2 innormal cells but not in cancer cells.Nuclear proteins extracted fromthe parthenolide-treated cell lineswere immunoblotted to quantifynuclear levels of Nrf2 using PCNAas loading control. B, the effect ofKeap1 and Nrf2 in normal cells. PZcells were transfected with siRNAsto knock down Keap1 or Nrf2,respectively. After treatment withparthenolide and IR, the cellsurvival fraction was quantifiedusing Trypan blue exclusion assay(right) and the knocked downKeap1 and Nrf2 were confirmed byWestern blots (left).






modification of Keap1 initiates different signaling pathwaysthat affect mitochondrial function, leading to cell survival orcell injury in response to radiation, as illustrated in Fig. 7.

DiscussionThe majority of anticancer therapies fail because cancers

develop phenotypes that are treatment resistant and becausetreatments cause unwanted and/or detrimental side effects tonormal cells or to untargeted tissues. While conventionaladjuvant therapies improve tumor response to radiotherapy,they generally cause additional damage to normal tissues.Thus, the focus of the present study is to identify adjuvanttherapeutics that can reduce the side effects of radiotherapy.Our study provides a proof-of-concept for improving theefficacy of radiotherapy while protecting against injury to

normal tissues. It has been shown that parthenolide, theanti-inflammatory phytochemical, is able to suppress tumorgrowth in many organs (22–25). In addition, parthenolideseems to synergically enhance chemotherapeutic efficiencywhen it is combined with taxol or cisplatin to treat lung andgastric cancer cells (23, 40). Parthenolide also sensitizes radio-resistant osteosarcoma cells to radiotherapy (41). Here, weshow that DMAPT, a parthenolide prodrug, sensitized prostatecancer cells to radiotherapy in vivo and protected normalprostate and bladder against radiation-induced tissue injury.These results extend our previous survival studies in prostatecancer cell lines and normal prostate epithelial cells.

ROS, as products of cell metabolism, play a dual role intumorigenesis and tumor suppression. The "two-faced" char-acter of ROS has emerged as a potential source for discoveringanticancer drugs. Redox homeostasis is frequently deregulated

β-Actin

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Figure 5. TrX-dependent Keap1 reduction by parthenolide in prostate cancer cells. A, the levels of TrX in normal and cancer cells before and after treatmentwith parthenolide (PN) were detected by Western blots. B, after the indicated treatment, Keap1 was immunoprecipitated using a TrX antibody. C, PC3cells were transfectedwith TrX siRNA before the indicated treatment (top). SH- and S-S bands in Keap1 protein were detected as described in Fig. 3 (bottom).D, a Keap1 cDNA construct was transfected into PC3 cells. The levels of related proteins were detected by Western blots (bottom). The effect of Keap1 onradiosensitivity was analyzed using a Trypan blue exclusion assay (top).

Xu et al.





in cancers, as it is constantly exposed to high levels of ROScompared with normal counterparts. Our data show thatconstitutively elevated levels of oxidative stress in cancer cellsrepresent a specific vulnerability that can be selectively tar-geted by direct- or indirect-acting prooxidants and antioxi-dants or redoxmodulators. Theoretically, the differential redoxstatus of cancer cells compared with normal cells shouldprovide a therapeutic window for selective redox interventionvia additional increases in ROS. In this context, normal andcancer cells should respond differently to the same level ofprooxidant action generated either by direct production ofoxidative species or by modulation of specific cellular targetsinvolved in redox regulation. In this study, we show thatparthenolide serves as a prooxidant and displays a selectiveredox modification capability that differentially modulatescellular redox signals and targets. The Michael acceptor reactswith a thiol group of target proteins through covalent adduc-

tion (21). Parthenolide contains electrophilic a-methylene-g-lactone, a bisfunctional Michael acceptor, and displays apotential for bifunctional target alkylation and crosslinking.The present study shows the inverse effects of parthenolide onredoxmodification in cancer cells comparedwith normal cells.Remarkably, observations of the cytotoxic and cytoprotectiveeffects of parthenolide are consistent with its action in themodulation of ROS levels in both cancer and normal cells.Alteration of cellular ROS by parthenolide is attributed tofunctionally up- or downregulating antioxidant enzymes inmitochondria, which consequently regulates mitochondrialrespiration. Parthenolide is able to selectively reduce theactivity of several enzymes involved in oxidative stress removalin cancer cells, which in turn can cause ROS levels to rise abovethe threshold for cell death. This finding predicts that antiox-idant proteins and mitochondria are feasible therapeutictargets.

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Figure 6. Degradation of PGAM5-Bcl-xL caused by parthenolide-mediated reduction of Keap1 in prostate cancer cells. A, PC3 cells were treated withparthenolide (PN). Keap1 and Bcl-xL were immunoprecipitated using a PGAM5 antibody. B, the total levels of Nrf2, PGAM5, and Bcl-xL werequantified by Western blots. C, the levels of mitochondria- associated proteins after parthenolide treatments. D, proteins in various cellular fractionswere identified with antibody specific for each protein. E, PC3 cells were transfected with siRNA to knockdown PGAM5, Bcl-xL, and Nrf2, respectively(bottom). Cell survival fraction was quantified by Trypan blue exclusion assay (top). F, a Bcl-xL expression construct was transfected into PC3 cells and theexpression of Bcl-xL was monitored by Western blots (bottom). Cell survival fraction was quantified by Trypan blue exclusion assay (top).






It has been reported that parthenolide is a potent inhibitor ofNF-kB, which is a ROS-responsive transcriptional factorinvolved in both tumor progression and tumor resistance totreatment through upregulation of antiapoptotic genes, suchas Bcl-2, Bcl-xL, survivin, and XIAP (42). We previously showedthat NADPH oxidase-mediated inactivation of the Foxo 3signaling pathway is involved in the parthenolide-enhancedradiosensitivity of prostate cancer (25). However, previousstudies did not explain how parthenolide exerts such anopposing effect in tumor and normal cells. The present studyidentifies Keap1 as a redox signaling sensor that plays a pivotalrole in the differential regulation of the downstream signalingtargets in response to radiation-mediated cytotoxicity in pros-tate cancer and normal cells. Keap1, an adaptor protein forubiquitin-based processing by the CUL3/RBX1-dependent E3ubiquitin ligase complex, functions as a sensor for thiol-reac-tive redox modification (43). The present study shows thatstabilization of Nrf2 by oxidation of Keap1 serves as a majormechanism by which parthenolide protects normal tissuesagainst radiotoxicity through upregulation of antioxidantenzymes in mitochondria. However, Nrf2 transcriptional acti-vation did not play a major role in parthenolide-treatedprostate cancer cells. Thus, it is interesting to note that unliketraditional chemotherapeutic agents, parthenolide is unable toenhance resistance of prostate cancer to radiation treatmentby stimulating Nrf2 target genes.

In addition to regulating the Nrf2 signaling pathway, Keap1is able to bind other proteins such as p62 and PGAM5 (44).Interaction between Keap1 and p62 facilitates release of Nrf2from the complex, which is considered to be a noncanonicalcysteine-independent mechanism for the autophagy deficien-

cy–activated Nrf2 pathway (45). The N-terminus of PGAM5interacts with the Kelch domain of Keap1 and its C-terminusbinds to Bcl-xL. Keap1-dependent ubiquitination results inproteasome-dependent degradation of PGAM5 and Bcl-xL(38). Bcl-xL, an important member of the Bcl-2 family, is apotent antiapoptotic factor that plays a crucial role in cellsurvival by maintaining the electrochemical and osmotichomeostasis of mitochondria (46). The present study showsthat parthenolide increases the level of reduced Keap1 andconsequently induces Keap1-dependent degradation ofPGAM5 and Bcl-xL in cancer cells, suggesting that formationof the Keap1-PGAM5-Bcl-xL complex is a mechanism under-lying the effect of parthenolide on radiosensitization of pros-tate cancer cells.

Although a high rate of aerobic glycolysis in tumors, knownas the Warburg effect, has been observed in various types ofcancer, cancers have functional mitochondria, and mitochon-drial respiration is necessary for cancer cell proliferation (47).Cancer cells depend on a hyperactive metabolism to fuel theirrapid growth and also on antioxidative enzymes to quenchpotentially toxic ROS generated by such a high metabolicdemand (48). Our results show that parthenolide not onlysuppresses MnSOD and GpX, 2 major antioxidant enzymes inmitochondria, but also activates Bcl-xL degradation in cancercells, which suggests thatmitochondria are a feasible target foranticancer treatment. The present study also shows thatparthenolidemaymaintain normal cell survival through induc-tion ofMnSODandGpXactivity. Thus, amore efficient and safetherapymay involvemodification of cellular redox signaling byalteration of the antioxidant response coupled to selectivedegradation of prosurvival members of the Bcl2 family in

Cell defenseProtection

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Figure 7. A proposed mechanisticmodel for parthenolide-mediatedinverse therapeutic effects onradiosensitivity of prostate cancerand radioresistance of normalcells. Parthenolide sensitizescancer cells to radiation, in part, bymaintaining Keap1 in a reducedstate and enhancing its interactionwith PGAM5 and Bcl-xL, resultingin degradation of Bcl-xL inmitochondria. In contrast,parthenolide protects normal cellsagainst radiation via oxidation ofKeap1 and release of the Nrf2transcription factor for activationofmitochondrial antioxidantenzymes.

Xu et al.





cancer cells, as conventional anticancer therapy mainly causescell growth arrest or cell death by raising cellular ROS, whichoxidizes and damages DNA, proteins, and lipids. Optimizingprototype redox chemotherapeutics from natural sources pro-vides an exciting opportunity to further develop even bettercandidates to enhance therapeutic efficacy with less off-targettoxicity.

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

Authors' ContributionsConception and design: Y. Xu, P.A. Crooks, D.K. St. Clair, W.H. St. ClairDevelopment of methodology: Y. Xu, F. Fang, S. MiriyalaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Xu, F. Fang, L. Chaiswing, A.K. Holley, Y. Zhao, K.K.Kiningham, D.K. St. Clair, W.H. St. ClairAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Xu, F. Fang, S. Miriyala, P.A. Crooks, T.D. Oberley,D.K. St. Clair, W.H. St. Clair

Writing, review, and/or revision of the manuscript: Y. Xu, S. Miriyala, P.A.Crooks, D.K. St. Clair, W.H. St. ClairAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): Y. Xu, T. NoelStudy supervision: Y. Xu, D.K. St. Clair, W.H. St. Clair

AcknowledgmentsThe authors thank Yulan Sun, a previous graduate student, who initiated the

work.

Grant SupportThis work was supported by NIH grants CA49797, CA115801, and CA143428

(to D.K. St. Clair and William St. Clair). Additional support was provided by theEdward P. Evans Foundation and by the resources and facilities of the WilliamS. Middleton Veterans Administration Hospital (Madison, WI).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 27, 2012; revised March 18, 2013; accepted April 5, 2013;published OnlineFirst May 14, 2013.

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Published OnlineFirst May 14, 2013.Cancer Res Yong Xu, Fang Fang, Sumitra Miriyala, et al. and Cancer CellsOpposing Radiosensitive Effects of Parthenolide in Normal KEAP1 Is a Redox Sensitive Target That Arbitrates the

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