Ursolic Acid, a Pentacyclin Triterpene, PotentiatesTRAIL-induced Apoptosis through p53-independentUp-regulation of Death ReceptorsEVIDENCE FOR THE ROLE OF REACTIVE OXYGEN SPECIES AND JNK*□S

Received for publication, September 10, 2010, and in revised form, November 17, 2010 Published, JBC Papers in Press, December 14, 2010, DOI 10.1074/jbc.M110.183699

Sahdeo Prasad, Vivek R. Yadav, Ramaswamy Kannappan, and Bharat B. Aggarwal1

From the Cytokine Research Laboratory, Department of Experimental Therapeutics, University of Texas M. D. Anderson CancerCenter, Houston, Texas 77030

Discovery of the molecular targets of traditional medicineand its chemical footprints can validate the use of such medi-cine. In the present report, we investigated the effect of ursolicacid (UA), a pentacyclic triterpenoid found in rosemary andholy basil, on apoptosis induced by TRAIL. We found thatUA potentiated TRAIL-induced apoptosis in cancer cells. Inaddition, UA also sensitized TRAIL-resistant cancer cells tothe cytokine. When we investigated the mechanism, we foundthat UA down-regulated cell survival proteins and induced thecell surface expression of both TRAIL receptors, death recep-tors 4 and 5 (DR4 and -5). Induction of receptors by UA oc-curred independently of cell type. Gene silencing of either re-ceptor by small interfering RNA reduced the apoptosisinduced by UA and the effect of TRAIL. In addition, UA alsodecreased the expression of decoy receptor 2 (DcR2) but notDcR1. Induction of DRs was independent of p53 because UAinduced DR4 and DR5 in HCT116 p53�/� cells. Induction ofDRs, however, was dependent on JNK because UA inducedJNK, and its pharmacologic inhibition abolished the inductionof the receptors. The down-regulation of survival proteins andup-regulation of the DRs required reactive oxygen species(ROS) because UA induced ROS, and its quenching abolishedthe effect of the terpene. Also, potentiation of TRAIL-inducedapoptosis by UA was significantly reduced by both ROSquenchers and JNK inhibitor. In addition, UA was also foundto induce the expression of DRs, down-regulate cell survivalproteins, and activate JNK in orthotopically implanted humancolorectal cancer in a nude mouse model. Overall, our resultsshowed that UA potentiates TRAIL-induced apoptosisthrough activation of ROS and JNK-mediated up-regulationof DRs and down-regulation of DcR2 and cell survivalproteins.

More than 80% of people around the world, for their day-to-day medicinal needs, rely on traditional medicine, whichhas been around for centuries. Even modern medicine in mostinstances relies on natural products, and 70% of anticancerdrugs have their roots in products derived from nature (1).The search for signature genes of different cancers has shownthat most cancers are due to dysregulation of multiple genesand multiple cell signaling pathways; thus, drugs that are mul-titargeted (once called “dirty drugs”) are needed. Compoundsfrom natural sources have an advantage in that they are usu-ally multitargeted. One such compound is ursolic acid (UA),2a pentacyclic triterpenoid that has been identified in a largevariety of medicinal herbs and other plants, including rose-mary (Rosemarinus officinalis), apples (Malus domestica),cranberries (Vaccinium macrocarpon), beefsteak (Perilla fru-tescens), pears (Pyrus pyrifolia), plum (Prunus domestica),bearberries (Arctostaphylos alpina), loquat (Eriobotrya japon-ica), scotch heather (Calluna vulgaris), basil (Ocimum sanc-tum), and jamun (Eugenia jambolana) (2). It has been shownthat UA can inhibit cell growth and induce apoptosis in vari-ous tumors (3–9). UA induces apoptosis through multiplepathways, such as inhibiting DNA replication (7, 10); inducingCa2� release (8); activating caspases (9, 11); activating JNK(12); down-regulating antiapoptotic genes (13, 14); inhibitingCOX2 and iNOS (15, 16); suppressing MMP-9 (17); and in-hibiting protein tyrosine kinase (10), STAT3 (18), and NF-�Bactivities (13). In animal studies, UA has been shown to bechemopreventive (19–21), to suppress tumor invasion (17),and to inhibit experimental metastasis of esophageal carci-noma (22). Whether UA can modulate the effect of apoptosis-inducing cytokines that are currently in clinical trial is notknown.TRAIL (tumor necrosis factor (TNF)-related apoptosis-

inducing ligand), a member of the TNF family, is one suchapoptosis-inducing cytokine that has shown promise as ananticancer agent (23). TRAIL selectively induces apoptosis intumor cells but not in normal cells (3). There are five differentreceptors that have been identified for TRAIL; however, onlydeath receptor 4 (DR4) (TRAIL-R1) and DR5 (TRAIL-R2)

* This work was supported, in whole or in part, by National Institutes ofHealth Grants CA-16672 and CA-124787-01A2. This work was also sup-ported by a grant from the Clayton Foundation for Research (to B. B. A.)and a grant from the Center for Targeted Therapy of M. D. Anderson Can-cer Center.

□S The on-line version of this article (available at http://www.jbc.org) con-tains supplemental Fig. 1.

1 The Ransom Horne, Jr., Professor of Cancer Research. To whom corre-spondence should be addressed: 1515 Holcombe Blvd., Unit 143, Hous-ton, TX 77030. Tel.: 713-794-1817; Fax: 713-745-6339; E-mail: aggarwal@mdanderson.org.

2 The abbreviations used are: UA, ursolic acid; DR, death receptor; DcR, de-coy receptor; ROS, reactive oxygen species; PARP, poly(ADP-ribose) po-lymerase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide; PI, propidium iodide; NAC, N-acetyl cystiene.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 7, pp. 5546 –5557, February 18, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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This article has been retracted by the publisher. Fig. 1B was assembled from a composite of many images. The actin immunoblot from Fig. 2A, right, was reused in Fig. 4A, left. Parts of the actin immunoblot from Fig. 2C, left panel, had been reused in supplemental Fig. 1A. The actin immunoblot from Figs. 2E and 4B, left panels; and supplemental Fig. 1B was reused in Fig. 5B, top panels. Parts of the JNK immunoblot

from Fig. 4C were reused in Fig. 7C, JNK.

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have cytoplasmic death domains that activate the apoptoticmachinery upon TRAIL binding (3). Other receptors, such asdecoy receptor 1 (DcR1) and DcR2, although membrane-bound, exhibit dominant negative effects by sequestering theligand (24). Osteoprotegerin, although it binds TRAIL, lacks atransmembrane domain and thus is a soluble receptor.Numerous reports have shown that various types of cancer

cells are resistant to the apoptotic effects of TRAIL (25–27).The exact mechanism of resistance to TRAIL is still not fullyunderstood; however, it can occur at different points inTRAIL-induced apoptotic signaling pathways. For instance,dysfunction of DR4 and DR5, overexpression of antiapoptoticproteins, and loss of proapoptotic proteins has all been linkedwith TRAIL resistance. Activation of different subunits ofmitogen-activated protein kinases and nuclear factor-�B arealso reported to develop TRAIL resistance in certain types ofcancer cells (28). Therefore, modulation of TRAIL-inducedapoptotic signaling molecules is an important strategy to sen-sitize cancer cells for effective cancer therapy.In the current report, we tested whether UA can potentiate

TRAIL-induced apoptosis and sensitize resistant cancer cellsto TRAIL. We found that this triterpene can indeed enhanceTRAIL-induced apoptosis through up-regulation of deathreceptors and down-regulation of antiapoptotic proteins viaproduction of reactive oxygen species (ROS) and activation ofJNK.

EXPERIMENTAL PROCEDURES

Reagents—A 50 mM solution of UA (from Sigma), with pu-rity greater than 90%, was prepared in DMSO, stored as smallaliquots at �20 °C, and then diluted further in cell culturemedium as needed. Further fractionation of UA revealed thatminor impurities had no activity (data not shown). Solublerecombinant human TRAIL/Apo2L was purchased fromPeproTech. Penicillin, streptomycin, RPMI 1640, and fetalbovine serum were purchased from Invitrogen. Anti-�-actinantibody was obtained from Sigma-Aldrich. Antibodiesagainst DR4, DR5, Bcl-xL, Bcl-2, Bax, cFLIP, poly(ADP-ri-bose) polymerase (PARP), and JNK1 and the annexin V stain-ing kit were purchased from Santa Cruz Biotechnology, Inc.(Santa Cruz, CA). Dichlorodihydrofluorescein diacetate waspurchased from Invitrogen.Cell Lines—HCT116, HT29, Caco2 (human colon adeno-

carcinoma), A293 (human embryonic kidney carcinoma), PC3(human prostate cancer), MDA-MB-231 and MCF-7 (humanbreast cancer), SCC4 (squamous cell carcinoma), and KBM-5(human chronic leukemia) cells were obtained from theAmerican Type Culture Collection. HCT116 variants withdeletion of p53 and Bax were kindly supplied by Dr. Bert Vo-gelstein (Johns Hopkins University, Baltimore, MD). The hu-man colon cancer HCT116 variant cell lines were cultured inMcCoy’s 5A medium. HCT116, A293, MDA-MB-231, SCC4,and MCF-7 cells were cultured in DMEM. Caco2 and HT29cell lines were cultured in RPMI1640. KBM-5 cells were cul-tured in Iscove’s modified Dulbecco’s medium. DMEM andRPMI were supplemented with 10% fetal bovine serum, 100units/ml penicillin, and 100 mg/ml streptomycin. Iscove’smodified Dulbecco’s medium was supplemented with 15%

fetal bovine serum 100 units/ml penicillin, and 100 mg/mlstreptomycin.Live/Dead Assay—To measure apoptosis, we used the Live/

Dead� assay (Invitrogen), which assesses intracellular esteraseactivity and plasma membrane integrity. This assay was per-formed as described previously (29).Cytotoxicity Assay—The effects of DBA on TRAIL-induced

cytotoxicity were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method(29).Propidium Iodide Staining for Apoptosis—To determine

the effect of DBA on the cell cycle, treated and untreated cellswere stained with PI as described earlier (18). A total of10,000 events were analyzed by flow cytometry using an exci-tation wavelength of 488 nm and emission wavelength of610 nm.Analysis of Cell Surface Expression of DR4 and DR5—

Treated and untreated cells were stained with phycoerythrin-conjugated mouse monoclonal anti-human DR5 or DR4(R&D Systems) for 45 min at 4 °C according to the manufac-turer’s instructions, resuspended, and analyzed by flow cy-tometry with phycoerythrin-conjugated mouse IgG2B as anisotype control.Annexin V Assay—The early indicator of apoptosis was de-

tected by using an annexin V/PI binding kit (Santa Cruz Bio-technology, Inc.) and then analyzed with a flow cytometer(FACSCalibur, BD Biosciences).Measurement of ROS—Intracellular ROS of cells were de-

tected as described elsewhere (29).Transfection with siRNA—HCT116 cells were plated in

each well of six-well plates and allowed to adhere for 12 h. Onthe day of transfection, 12 �l of HiPerFect transfection re-agent (Qiagen) was added to 50 nmol/liter siRNA in a finalvolume of 100 �l of culture medium. After 48 h of transfec-tion, cells were treated with 20 �M UA for 12 h and then ex-posed TRAIL for 24 h.Western Blot Analysis—To determine the levels of protein

expression, whole-cell extracts were prepared in lysis buffer asdescribed previously (13). In the in vivo case, colorectal tumortissues (75–100 mg/mouse) were minced and incubated onice for 30 min in 0.5 ml of ice-cold whole-cell lysate buffer(10% Nonidet P-40, 5 mol/liter NaCl, 1 mol/liter HEPES, 0.1mol/liter EGTA, 0.5 mol/liter EDTA, 0.1 mol/liter PMSF, 0.2mol/liter sodium orthovanadate, 1 mol/liter NaF, 2 �g/mlaprotinin, 2 �g/ml leupeptin). The minced tissue was homog-enized with a Dounce homogenizer and centrifuged at16,000 � g at 4 °C for 10 min. The extracted proteins werethen resolved on a 10% SDS gel, and Western blotting wasperformed as described previously (13).RNA Analysis and RT-PCR—DR5 mRNA was detected us-

ing RT-PCR as follows. Total RNA was isolated from cellsusing TRIzol reagent (Invitrogen) as instructed by the manu-facturer. One microgram of total RNA was converted tocDNA using Superscript reverse transcriptase and then am-plified by platinum Taq polymerase using the SuperscriptOne Step RT-PCR kit (Invitrogen). The total RNAs were thenamplified by PCR using the following primers: DR5 sense (5�-AAGACCCTTGTGCTCGTTGTC-3�), DR5 antisense (5�-

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GACACATTCGATGTCACTCCA-3�), DR4 sense (5�-CTGAGCAACGCAGACTCGCTGTCCAC-3�), DR4 antisense(5�-TCCAAGGACACGGCAGAGCCTGTGCCAT-3�),GAPDH sense (5�-GTCTTCACCACCATGGAG-3�), andGAPDH antisense (5�-CCACCCTGTTGCTGTAGC-3�). Thereaction sequence consisted of 50 °C for 30 min, 94 °C for 2min, and 94 °C for 35 cycles of 15 s each; 50 °C for 30 s; and72 °C for 45 s with an extension at 72 °C for 10 min. PCRproducts were run on 2% agarose gel and then stained withethidium bromide. Stained bands were visualized under UVlight and photographed.Animal Protocol—The HCT116 cells were orthotopically

implanted as described previously (30). One week after im-plantation, the mice were randomized into the followingtreatment groups (n � 6/group): (a) untreated control (cornoil, 100 �l daily) and (b) UA (250 mg/kg once daily orally).Therapy was continued for 4 weeks, and the animals wereeuthanized 1 week later. Primary tumors in the colon wereexcised, snap-frozen in liquid nitrogen, and stored at �80 °C.Our experimental protocol was reviewed and approved by theInstitutional Animal Care and Use Committee at M. D.Anderson Cancer Center.Statistical Analysis—All data are expressed as mean � S.E.

of three independent experiments. Statistical significance wasdetermined using unpaired Student’s t test, and a p value ofless than 0.001 was considered statistically significant.

RESULTS

The aim of this study was to investigate whether UA (Fig.1A) can enhance the sensitivity of tumor cells to TRAIL and,if so, through what mechanism. We used human colon cancerHCT116 cells for most of these studies, but other cell typeswere also used to determine the specificity of this effect.UA Enhances TRAIL-induced Apoptosis—We examined

first the effect of UA on TRAIL-induced cytotoxicity by usingthe MTT method, which detects mitochondrial activity. TheHCT116 cells were moderately sensitive to either UA orTRAIL alone. However, pretreatment with UA significantlyenhanced TRAIL-induced cytotoxicity (Fig. 1B, left).We also determined whether UA also enhances TRAIL-

induced apoptosis of colon cancer cells. We found that UAand TRAIL treatments alone induced 21 and 16% apoptosis,respectively, in HCT116 cells. Interestingly, combinationtreatment with UA and TRAIL enhanced apoptosis to 76%(Fig. 1B, right).To confirm the effect of UA on TRAIL-induced apoptosis,

we measured apoptosis by FACS analysis of the sub-G1 frac-tion. The results indicated that the UA and TRAIL treatmentalone induced 12 and 20% apoptosis, respectively. Combina-tion treatment with both UA and TRAIL enhanced apoptosisto 50% (Fig. 1C, top). When apoptosis was examined usingannexin V/PI staining, we found that apoptosis was inducedat 13% by UA, 7% by TRAIL, and 47% by the combination ofthe two (Fig. 1C, bottom).

Next, we examined the effect of UA, TRAIL, and their com-bination on the activation of caspase-8, caspase-3, and PARPcleavage. We found that although UA and TRAIL alone hadlittle effect on the activation of caspases and on PARP cleav-

age, the two together were highly effective (Fig. 1D). To-gether, our results indicate that UA can enhance TRAIL-in-duced apoptosis.UA Induces the Expression of TRAIL Receptors DR4 and

DR5 in Cancer Cell Lines—To explore the underlying mecha-nism that may be responsible for enhancement of TRAIL-induced apoptosis by UA, we examined the effect of UA onthe expression of death receptors. UA induced both DR4 andDR5 in a dose-dependent manner (Fig. 2A, left). Whether thisinduction of the DRs was dependent on time was also exam-ined. UA induced both DR4 and DR5 in a time-dependentmanner as well (Fig. 2A, right).We also investigated whether UA induces cell surface ex-

pression of TRAIL receptors. We found that UA increased thecell surface expression of both DR5 and DR4 in colon cancerHCT116 cells (Fig. 2B). The level of DR4 cell surface expres-sion induced by UA was almost equal to that for DR5.To determine whether up-regulation of TRAIL receptors

by UA was specific to HCT116 cells or also occurs in othercell types, we exposed the following cells to 20 �M UA for24 h: MCF-7, MDA-MB-231, PC3, SCC4, A293, HT29,Caco2, and KBM-5 cells. UA induced expression of both DR5and DR4 in almost all of these cell lines (Fig. 2C).We also examined the induction of DR5 and DR4 in other

colon cancer cell lines like HT29 and Caco2 cells in additionto HCT116 cells. The induction of DR5 or DR4 by UA inMDA-MB-231 cells was insignificant. These findings suggestthat the up-regulation of DR5 and DR4 by UA was not celltype-specific.UA Up-regulates DRs at Transcriptional Level—Whether

TRAIL receptors are induced by UA at the transcriptionallevel was investigated by RT-PCR. UA substantially up-regu-lated both DR4 and DR5 mRNA expression in a dose-depen-dent manner (Fig. 2D).UA Down-regulates Decoy Receptor—Next we investigated

whether UA can modulate the expression of decoy receptors.We found that although UA had no influence on the expres-sion of DcR1, it decreased the expression of DcR2 (Fig. 2E).Thus, inhibition of DcR2 expression by UA could also con-tribute to apoptosis by TRAIL.Gene Silencing of DRs Abolishes the Effect of UA on TRAIL-

induced Apoptosis—To determine whether up-regulation ofDR5 or DR4 is needed in TRAIL-induced apoptosis, we used agene-silencing approach to abolish UA-induced expression ofthese receptors. We found that transfection of cells with DR5siRNA but not with the control scrambled siRNA reduced theUA-induced up-regulation of DR5. Similarly, transfection ofcells with siRNA for DR4 reduced the UA-induced DR4 ex-pression (Fig. 3A).We next examined whether the suppression of DR5 or DR4

by siRNA could abolish the effects of UA on TRAIL-inducedapoptosis using the Live/Dead assay. We found that the si-lencing of DR5 reduced the UA-induced apoptosis from 22 to12%, whereas silencing of DR4 reduced apoptosis to 14%. Si-lencing of both receptors reduced UA-induced apoptosis to8%, whereas control scrambled siRNA had no effect. Theseresults indicate that DRs contribute to UA-induced apoptosisas well.

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The TRAIL-induced apoptosis was reduced from 17 to 8%by silencing of DR5, but silencing of DR4 had no significanteffect on TRAIL-induced apoptosis. When we examined theeffect of UA and TRAIL in combination, we found that apo-ptosis was reduced from 62 to 34% by DR5 siRNA, to 48% byDR4 siRNA, and to 28% by the two siRNAs together, whereaswith control scrambled siRNA, change in apoptosis was not

significant (Fig. 3B). Silencing of DR5 had a more dramaticeffect on TRAIL-induced apoptosis than silencing of DR4.The silencing of both receptors abolished the apoptosis asmuch as silencing of DR5 alone. Overall, these results suggestthat DR5 plays a major role in TRAIL-induced apoptosis andthat enhancement of apoptosis by UA is linked to up-regula-tion of the receptors.

FIGURE 1. UA potentiates TRAIL-induced apoptosis of HCT116 cells. A, chemical structure of UA. B, cells were pretreated with 20 �M UA for 12 h. The me-dium was removed, and the cells were then exposed to TRAIL (25 ng/ml) for 24 h. Cell viability was then analyzed by the MTT method as described under“Experimental Procedures” (left) and by the Live/Dead assay (right). *, p � 0.001. C, cells were treated with 20 �M UA for 12 h and washed with PBS to re-move UA. The cells were then treated with TRAIL (25 ng/ml) for 24 h. Cells were stained with PI alone (top) and PI/annexin V (bottom) separately and thenanalyzed by FACS. D, cells were pretreated with UA for 12 h, and then the UA was washed out. The cells were then treated with TRAIL for 24 h. Whole-cellextracts were prepared and analyzed by Western blot using antibodies against caspase-8, caspase-3, and PARP.

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UA Down-regulates the Expression of AntiapoptoticProteins—Various antiapoptotic proteins, including survivin(31), Bcl-xL (32), XIAP (33), and cFLIP (34), have been shownto induce resistance to TRAIL-induced apoptosis. We exam-ined whether UA sensitized the cells to TRAIL throughdown-regulation of the expression of these cell survival pro-teins. Results of Western blot showed that UA inhibited ex-pression of the antiapoptotic proteins survivin, XIAP, cFLIP,and Bcl-2 (Fig. 4A, left). These results indicate that down-regulation of antiapoptotic proteins by UA could be anothermechanism of potentiation of TRAIL-induced apoptosis.UA Enhances the Expression of Proapoptotic Proteins—

Whether UA can modulate the expression of proapoptoticproteins was also examined. We found that UA cleaved theproapoptotic protein Bid and induced the expression of Bax

(Fig. 4A, right). Up-regulation of Bax by UA suggests thatthese proteins may disrupt mitochondrial homeostasis, whichfurther leads to apoptosis.UA-induced Up-regulation of TRAIL Receptors is

p53-independent—Because p53 has been reported to inducedeath receptors (35), we investigated whether UA up-regu-lates DRs through up-regulation of p53. We found that UAdid not up-regulate p53; if anything, at a higher dose (30 �M),it down-regulated p53 (Fig. 4B, left). We also determinedwhether UA-induced induction of TRAIL receptors is medi-ated through p53 in HCT116 cell lines that lack p53. Wefound that UA induced DR5 and DR4 in p53 parental as wellas p53 knock-out HCT116 cells in a dose-dependent manner(Fig. 4B, right). These results indicate that induction of TRAILreceptors by UA is p53-independent.

FIGURE 2. UA induces DR5 and DR4 expression. A, HCT116 cells (1 � 106 cells/well) were treated with the indicated UA doses (left) and for the indi-cated times (right). Whole-cell extracts were then prepared and analyzed for DR5 and DR4 by Western blotting. B, HCT116 cells were treated with 20�M UA for 24 h and then harvested for analysis of cell surface DR4 and DR5 by immunofluorescent staining and subsequent flow cytometry. Filledgray peaks indicate cells stained with a matched control phycoerythrin-conjugated IgG isotype antibody. C, UA up-regulated DR5 and DR4 in varioustypes of cancer cells. Cells (1 � 106 cells) were treated with 20 �M UA for 24 h, after which whole-cell extracts were prepared and analyzed by West-ern blotting using antibodies against DR5 and DR4. D, UA induces mRNA expression for DR5 and DR4. HCT116 cells (1 � 106/ml) were treated withthe indicated concentration of UA for 24 h, and total RNA was extracted and examined for expression of DR4 and DR5 by RT-PCR. GAPDH was usedas an internal control to show equal RNA loading. E, HCT116 cells were pretreated with the indicated doses of UA for 24 h. Whole-cell extracts wereprepared and subjected to Western blotting for DcR1 and DcR2. The same blots were stripped and reprobed with �-actin antibody to verify equalprotein loading.

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UA-induced Up-regulation of TRAIL Receptors IsBax-independent—We found that UA induced Bax. WhetherUA up-regulates DRs through up-regulation of Bax was inves-tigated. For this, we used Bax knock-out HCT116 colon can-cer cells. UA induced expression of DR5 and DR4 in both Baxparental and Bax knock-out HCT116 cells (supplemental Fig.1A), indicating that induction of TRAIL receptors are inde-pendent of Bax expression.UA-induced Up-regulation of Death Receptor Is Not Medi-

ated through Activation of ERK1/2 and GSK-3�—Activationof ERK1/2 and GSK-3� has been linked with induction ofTRAIL-induced apoptosis (36, 37). We therefore investigatedwhether UA activates ERK1/2 and GSK-3�. Results showedthat UA activated neither ERK1/2 nor GSK-3� and had noeffect on the expression levels of these proteins (Fig. 4C, left).UA-induced Up-regulation of DRs Is Not Mediated through

Activation of CHOP and PPAR�—Next we determinedwhether UA modulates CHOP and PPAR�. We found thatDBA did not modulate either PPAR� or CHOP (supplemental

Fig. 1B), indicating that induction of DRs is not mediatedthrough PPAR� or CHOP.UA-induced Up-regulation of DRs Requires JNK Activation—

Activation of the TRAIL receptor by H2O2 (38) and by CDDO(39) requires activation of JNK.We investigated whether UA canactivate JNK by exposing the cells to different concentrations ofUA for 24 h and then examined the cells for activation of JNK.Western blotting results showed that UA induced JNK activationin a dose-dependent manner (Fig. 4C, right), but under the sameconditions it had no effect on total JNK protein level.Next, we investigated whether activation of JNK is

needed for UA-induced up-regulation of death receptors.For this we used SP600125, a specific pharmacologic inhib-itor of JNK. As shown in Fig. 4D (left), pretreatment of cellswith this JNK inhibitor significantly suppressed the UA-induced up-regulation of DR5 and DR4 expression. Theseresults suggest that JNK is involved in UA-induced up-reg-ulation of DRs. Suppression of JNK also leads to inhibitionof UA-induced cleavage of PARP (Fig. 4D, left).

FIGURE 3. Blockage of DRs induction reverses the ability of UA to augment TRAIL-induced apoptosis. HCT116 cells were transfected with DR5 siRNA,DR4 siRNA, and control siRNA alone or combined. After 48 h of transfection, cells were treated with 20 �mol/liter UA. A, after 24 h, whole-cell extracts wereprepared and analyzed by Western blotting. B, cells were exposed to 20 �mol/liter UA for 12 h, washed with PBS to remove UA, and then treated with 25ng/ml TRAIL. Cell death was determined using the Live/Dead assay. Error bars, S.E.

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Furthermore, we examined whether the suppression of JNKactivation by its inhibitor could abrogate the apoptosis in-duced by UA, TRAIL, and the combination of UA and TRAIL.We found that apoptosis induced by UA, TRAIL, and thecombination was reduced from 18 to 10%, from 24 to 13%,and from 64 to 42%, respectively (Fig. 4D, right). Thus, sup-pression of JNK activation substantially reduced the apopto-sis, although not completely.

UA-induced Up-regulation of DR5 Requires ROS—ThatROS is needed for induction of death receptors by certainagents has been demonstrated (29). To determine whetherUA has the ability to generate ROS, we treated HCT116 cellswith UA and used dichlorodihydrofluorescein diacetate as aprobe to measure the increase in ROS levels in the cells. Wefound that UA induced the production of ROS in a dose-de-pendent manner (Fig. 5A).

FIGURE 4. Effects of UA on antiapoptotic, proapoptotic, and kinase expression. A, HCT116 cells were pretreated with the indicated doses of UA for 24 h.Whole-cell extracts were prepared and analyzed by Western blotting using the antibodies against antiapoptotic (left) and proapoptotic (right) proteins. Thesame blots were stripped and reprobed with �-actin antibody to verify equal protein loading. B, HCT116 wild type (left) and p53 knock-out HCT116 (right)cells were pretreated with the indicated doses of UA for 24 h. Whole-cell extracts were prepared and subjected to Western blotting for p53, DRs proteins.C, HCT116 cells were pretreated with the indicated doses of UA for 24 h. Whole-cell extracts were prepared and subjected to Western blotting with thep-ERK1/2, p-GSK-3� (left), and p-JNK antibodies (right). D, HCT116 cells were treated with JNK inhibitor (SP600125) for 1 h and then exposed to 20 �M UA for24 h. Whole-cell extracts were prepared and analyzed for the expression of DR4, DR5, and PARP using relevant antibodies. Quantitation of each band isshown below the blots (left). Cells were seeded in chamber slides and exposed with JNK inhibitor for 1 h and then exposed to 20 �M UA. After 12 h, cellswere washed with PBS to remove UA and then treated with 25 ng/ml TRAIL for 24 h. Cell death was determined by the Live/Dead assay (right).

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Next we determined whether ROS production is needed forup-regulation of expression of DR5 and DR4 by UA.We foundthat pretreatment of cells with the ROS scavenger NAC blockedUA-induced up-regulation of DR5 and DR4 protein expressionsin a dose-dependent manner (Fig. 5B, top), indicating that ROSgeneration is critical for the effect of UA on TRAIL receptors.NAC Abrogates the Effect of UA in Suppression of Antiapo-

ptotic Proteins—Next we examined whether NAC abrogatesUA-induced inhibition of antiapoptotic proteins. The results

revealed that pretreatment of NAC effectively abolishedthe effect of UA in suppression of XIAP, cFLIP, and Bcl-2but not survivin (Fig. 5B, bottom). The effect of NAC ininhibiting the effect of UA on XIAP is more prominentthan others.UA-induced Potentiation of Apoptosis Induced by TRAIL Is

Reversed by Quenchers of ROS—We next examined whetherROS is needed for potentiation of TRAIL-induced apoptosisby UA. As shown in Fig. 5C, UA enhanced TRAIL-induced

FIGURE 5. Up-regulation of DR4 and DR5 by UA is mediated by ROS. A, we first determined whether UA induces production of ROS. HCT116 cells (1 �106 cells) were labeled with dichlorodihydrofluorescein diacetate (DCFDA), treated with the indicated concentrations of UA for 1 h, and then examined forROS production by flow cytometry. MFI, mean fluorescence intensity. B, HCT116 cells were pretreated with various concentrations of NAC for 1 h and thenwith 20 �M UA for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting using DR5 and DR4 antibodies (top) and antiapoptotic anti-bodies (bottom). C, NAC reversed cell death induced by the combination of UA and TRAIL. HCT116 cells were pretreated with NAC for 1 h and then treatedwith UA for 12 h. Cells were washed with PBS and treated with TRAIL for 24 h. Cell death was determined by the Live/Dead assay. D, NAC suppressedcaspase activation and PARP cleavage induced by the combination of UA and TRAIL. HCT116 cells were pretreated with NAC for 1 h and then treated withUA for 12 h. Cells were then washed with PBS and treated with TRAIL for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting usingrelevant antibodies. �-Actin was used as a loading control.

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apoptosis in HCT116 cells, and pretreatment of cells withNAC markedly reduced this UA-induced enhancement from68 to 34% (Fig. 5C).We also found that NAC reversed the effect of UA on TRAIL-

induced cleavage of procaspases and PARP (Fig. 5D), again sug-gesting the critical role of ROS in UA effects on TRAIL.UA Sensitizes TRAIL-resistant Colon Cancer Cells—It has

been shown that colon cancer HT-29 cells are completely re-sistant to TRAIL (40). We therefore investigated whether UAaffects TRAIL-resistant HT29 cancer cells. We found thatHT29 cells were moderately sensitive to UA but resistant to

TRAIL. However, pretreatment with UA enhanced TRAIL-induced apoptosis from 2% to 42% (Fig. 6A).Furthermore, we examined apoptosis by FACS analysis by

PI staining and found that UA alone induced 8.2% apoptosis,whereas TRAIL showed no cell death in HT29 cells. Interest-ingly, the pretreatment with UA sensitized the cells to TRAILand induced apoptosis of 24% (Fig. 6B). Results of the cyto-toxicity assay by MTT uptake also showed that HT29 cellswere moderately sensitive to UA but resistant to TRAIL.However, pretreatment of UA sensitized the HT29 cells toTRAIL and induced apoptosis (Fig. 6C).

FIGURE 6. UA sensitizes TRAIL resistance cells and induces apoptosis. A, HT29 cells were pretreated with 20 �M UA for 12 h. The medium was removed,and the cells were exposed to TRAIL for 24 h. Cell death was then analyzed by the Live/Dead assay. HT29 cells were treated with 20 �M UA for 12 h, washedwith PBS to remove UA, and then treated with 25 ng/ml TRAIL for 24 h. Cells were stained with PI for FACS analysis (B), and cell viability was determined byan MTT assay (C). *, p � 0.001. D, HT29 cells were treated with UA and TRAIL separately for 24 h. Whole-cell extracts were prepared and subjected to West-ern blotting using relevant antibodies. Error bars, S.E.

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We then determined whether UA up-regulates the expres-sion of DRs in HT29 cells. UA induced DR5 and DR4 (Fig.6D), suggesting that UA sensitized the HT29 cells to TRAIL-induced apoptosis.UA Up-regulates DR Expression, Down-regulates Cell Sur-

vival Proteins, and Activates JNK in Colorectal Tumors inVivo—Whether UA up-regulates the expression of DR4 andDR5 in vivo was examined in orthotopically implanted humancolorectal tumor from nude mice treated with UA. The West-ern blot analysis revealed that tumor tissues from UA-treatedanimals when compared with those from vehicle-treated ani-mals had up-regulated expression of both of the death recep-tors, DR4 and DR5 (Fig. 7A), had down-regulated expressionof cell survival proteins (survivin and Bcl-2) (Fig. 7B), and hadactivated JNK (Fig. 7C). These results in vivo are in agreementwith those in vitro.

DISCUSSION

TRAIL has recently been considered a highly promisingcandidate as an anti-cancer drug, because it induces apoptosisspecifically in malignant or transformed cells without any cy-totoxicity toward a variety of normal cells (41–43). A consid-erable number of cancer cells, however, are resistant to apo-ptosis induced by TRAIL (28). TRAIL induces apoptosis byinteracting with two different death-inducing receptors, DR4

and DR5. Therefore, targeting death receptors and their sig-naling molecules to trigger apoptosis in tumor cells is an at-tractive concept for cancer therapy.Several reports have demonstrated that chemotherapeutic

agents and ionizing radiation can sensitize cells to TRAIL-induced cytotoxicity (44, 45). In the present study, we demon-strate for the first time that UA, a pentacyclic triterpene, cansensitize cancer cells to TRAIL-induced apoptosis (Fig. 8).When we investigated the mechanism, we found that UA-induced up-regulation of death receptors and down-regula-tion of antiapoptotic proteins. In our study, UA treatmentinduced dose- and time-dependent increases in the proteinlevels of DR5 and DR4. We also demonstrated the up-regula-tion of expression of cell surface death receptors by UA. Si-lencing of DR5 and DR4 by their respective siRNAs effectivelyinhibited the cell death induced by the combination of UAand TRAIL, demonstrating the critical role of death receptorsin this event. Silencing of DR5, however, was more effectivethan silencing of DR4 in inhibiting UA and TRAIL-inducedapoptosis.The induction of death receptors by UA was not cell type-

specific because induction was observed in a wide variety ofcancer cell types, including breast, prostate, head and neck,kidney, leukemic, and colon cancer cells. Induction of TRAILreceptors in some cells, however, was much more pronouncedthan in other cell types. Thus, UA is likely to potentiate theanticancer effect of TRAIL in a wide variety of cells.Resistance to TRAIL-induced apoptosis has been reported

to be associated with overexpression of antiapoptotic pro-teins. Among Bcl-2 family proteins, Bcl-2 has been linkedwith suppression of apoptosis by TRAIL (46). In our study,Bcl-2 was suppressed by UA treatment. In addition, UA treat-ment also decreased the expression of Bcl-xL, survivin, andXIAP proteins but had no effect on c-FLIP expression, whichis also linked to TRAIL resistance (31, 32). When we lookedfor other potential mechanisms, we found that UA signifi-cantly up-regulated the expression of Bax and cleavage of Bidproteins. The former has been shown to be critical forTRAIL-induced apoptosis (47). However, our result also

FIGURE 7. UA up-regulates DRs, down-regulates cell survival proteins,and activates JNK in orthotopically transplanted human colorectal tu-mor in nude mice in vivo. Whole cell extracts of tumor tissues were sub-jected to Western blotting to analyze expression of DR4 and DR5 (A), ex-pression of cell survival proteins (B), and activation of JNK (C). V, vehicle(corn oil).

FIGURE 8. Schematic representation of the mechanism by which UA po-tentiates TRAIL-induced apoptosis.

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showed that UA-induced apoptosis mediated through expres-sion of DR is independent of Bax expression.It has been suggested that oxidative stress plays a major

role as a mediator of cell death (48). ROS generation has beenproposed to be involved in the up-regulation of DR5 by nu-merous cancer chemopreventive agents, including curcuminand sulforaphane (49, 50), zerumbone (51), and garcinol (29).In the current study, our data showed that UA induces up-regulation of DRs through production of ROS. The antioxi-dant NAC abolished the up-regulation of DR induced by UA.Therefore, ROS generation is critical for UA-induced DR-mediated apoptosis in cancer cells.The transcriptional regulation of DR5 is complex, and mul-

tiple potential binding sites of various transcription factors,including CHOP and p53, are present in the upstream regionof DR5 (52, 53). However, we found that the induction of DR5by UA occurs independently of p53 and CHOP. These resultsare consistent with those previously reported for the protea-some inhibitor MG132 (54) and for HDAC inhibitors (55).In addition to DR4 and DR5, other death receptors called

decoy receptors (DcRs) are also involved in the TRAIL-in-duced apoptotic signaling pathway. TRAIL can also bindDcR1 (TRAIL-R3) and DcR2 (TRAIL-R4). These latter recep-tors fail to induce apoptosis, because DcR1 lacks an intracel-lular domain and DcR2 has a truncated cytoplasmic deathdomain. In addition, DcR1 and DcR2 inhibit DR4- and DR5-mediated apoptosis by TRAIL. While DcR1 prevents the as-sembly of the death-inducing signaling complex by titratingTRAIL within lipid rafts, DcR2 is co-recruited with DR5within the death-inducing signaling complex, where it inhibitsinitiator caspase activation (56). Here we demonstrated thatUA reduced the expression of DcR2, which allow the avail-ability of ligand for DR4 and DR5 for induction of apoptosis.In contrast, no change in DcR1 occurred. The expression ofDcR2 has been shown to be regulated by p53 (57). Because wedid find down-regulation of p53 at higher doses, it is possiblethat this suppression of p53 mediates decrease of DcR2.Activation of stress-activated proteins such as JNK is

known to enhance TRAIL-induced apoptosis (58). Our find-ings provide evidence that activation of JNK by UA up-regu-lates DRs, which may further lead to an increase in TRAIL-induced apoptosis. UA was found to be ineffective inactivating ERK1/2 MAPK. Although ROS can lead to induc-tion of MAPK (59), in our study, UA induced TRAIL recep-tors independently of MAPK. In another study, quercetinaugmented TRAIL-induced apoptosis through the ERK-medi-ated down-regulation of the survivin signal transductionpathway (60). In our study, however, UA induced apoptosisthrough down-regulation of survivin but independently ofERK activation.We observed that UA sensitized the tumor cells that are

resistant to TRAIL. Although the mechanisms underlyingsensitization of TRAIL-resistant cells are not clear, some im-portant components, such as down-regulation of antiapop-totic proteins in signaling pathways, may be involved in thisprocess (28). Because Bcl-2, XIAP (inhibitor of caspase), andsurvivin (46, 61) are involved in TRAIL resistance, down-reg-ulation of these proteins by UA is the probable reason for the

sensitizing of TRAIL-resistant cells. In addition, induction ofdeath receptors could further contribute to the sensitivity.UA is a pentacyclic triterpene isolated from various tradi-

tional medicinal plants. Interestingly, CDDO, which is a syn-thetic analog designed based on ursolic acid and betulinic acidand which is also a pentacyclic triterpene, has been shown toinduce death receptors (39, 62). CDDO sensitized tumor cellsto TRAIL through up-regulation of death receptors anddown-regulation of cFLIP (62). In addition, Zou et al. (39)found that CDDO-Me induces DR5 up-regulation throughinduction of CHOP, and Hyer et al. (62) found that the cellsurface expressions of DR4 and DR5 were significantly up-regulated by CDDO or CDDO-Im but not by CDDO-Me.Why Zou et al. found up-regulation of DRs by CDDO-Me andHyer et al. did not is not clear. We showed, however, that UA-induced up-regulation of DR is independent of CHOP. All ofthese groups did show that activation of JNK is needed forup-regulation of the receptors.Whether our in vitro results have relevance to those in vivo

was also investigated. We found that UA up-regulated DRsexpression, down-regulated cell survival proteins, and acti-vated JNK in tumor tissue from animals treated with theagent in vivo. This indicates that UA-induced apoptosis couldbe due to up-regulation of DRs and activation of JNK in vivo.Overall, our results provide the first mechanistic evidence

that UA treatment results in sensitization of TRAIL-resistantcells and potentiation of TRAIL-induced apoptosis throughROS and JNK-mediated up-regulation of DR4 and DR5 anddown-regulation of antiapoptotic proteins (Fig. 8), thus ren-dering cancer cells more sensitive to the cytotoxic activities ofTRAIL. Considering that UA by itself is highly safe and exhib-its anticancer activities against a wide variety of tumors invitro (12, 13, 18) and in vivo (22, 63), its potential use in com-bination with TRAIL should be explored. Further studies inanimals are needed to investigate the anticancer potential ofUA in combination with TRAIL.

Acknowledgment—We thank Michael Worley from the Departmentof Scientific Publications for carefully editing the manuscript.

REFERENCES1. Butler, M. S., and Newman, D. J. (2008) Prog. Drug Res. 65, 3– 442. Liu, J. (1995) J. Ethnopharmacol. 49, 57– 683. Aggarwal, B. B. (2003) Nat. Rev. Immunol. 3, 745–7564. Es-saady, D., Simon, A., Ollier, M., Maurizis, J. C., Chulia, A. J., and

Delage, C. (1996) Cancer Lett. 106, 193–1975. Hsu, Y. L., Kuo, P. L., and Lin, C. C. (2004) Life Sci. 75, 2303–23166. Li, J., Guo, W. J., and Yang, Q. Y. (2002) World J. Gastroenterol. 8,

493– 4957. Kim, D. K., Baek, J. H., Kang, C. M., Yoo, M. A., Sung, J. W., Chung,

H. Y., Kim, N. D., Choi, Y. H., Lee, S. H., and Kim, K. W. (2000) Int. J.Cancer 87, 629 – 636

8. Baek, J. H., Lee, Y. S., Kang, C. M., Kim, J. A., Kwon, K. S., Son, H. C.,and Kim, K. W. (1997) Int. J. Cancer 73, 725–728

9. Harmand, P. O., Duval, R., Delage, C., and Simon, A. (2005) Int. J. Can-cer 114, 1–11

10. Choi, B. M., Park, R., Pae, H. O., Yoo, J. C., Kim, Y. C., Jun, C. D., Jung,B. H., Oh, G. S., So, H. S., Kim, Y. M., and Chung, H. T. (2000) Pharma-col. Toxicol. 86, 53–58

Ursolic Acid Promotes TRAIL-induced Apoptosis

5556 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 7 • FEBRUARY 18, 2011

by guest on April 21, 2018

http://ww

w.jbc.org/

Dow

nloaded from

11. Choi, Y. H., Baek, J. H., Yoo, M. A., Chung, H. Y., Kim, N. D., and Kim,K. W. (2000) Int. J. Oncol. 17, 565–571

12. Zhang, Y., Kong, C., Zeng, Y., Wang, L., Li, Z., Wang, H., Xu, C., andSun, Y. (2010) Mol. Carcinog. 49, 374 –385

13. Shishodia, S., Majumdar, S., Banerjee, S., and Aggarwal, B. B. (2003)Cancer Res. 63, 4375– 4383

14. Kassi, E., Sourlingas, T. G., Spiliotaki, M., Papoutsi, Z., Pratsinis, H., Ali-giannis, N., and Moutsatsou, P. (2009) Cancer Invest. 27, 723–733

15. Subbaramaiah, K., Michaluart, P., Sporn, M. B., and Dannenberg, A. J.(2000) Cancer Res. 60, 2399 –2404

16. Suh, N., Honda, T., Finlay, H. J., Barchowsky, A., Williams, C., Benoit,N. E., Xie, Q. W., Nathan, C., Gribble, G. W., and Sporn, M. B. (1998)Cancer Res. 58, 717–723

17. Cha, H. J., Park, M. T., Chung, H. Y., Kim, N. D., Sato, H., Seiki, M., andKim, K. W. (1998) Oncogene 16, 771–778

18. Pathak, A. K., Bhutani, M., Nair, A. S., Ahn, K. S., Chakraborty, A., Ka-dara, H., Guha, S., Sethi, G., and Aggarwal, B. B. (2007) Mol. Cancer Res.5, 943–955

19. Gayathri, R., Priya, D. K., Gunassekaran, G. R., and Sakthisekaran, D.(2009) Asian Pac. J. Cancer Prev. 10, 933–938

20. Singletary, K., MacDonald, C., and Wallig, M. (1996) Cancer Lett. 104,43– 48

21. Huang, M. T., Ho, C. T., Wang, Z. Y., Ferraro, T., Lou, Y. R., Stauber, K.,Ma, W., Georgiadis, C., Laskin, J. D., and Conney, A. H. (1994) CancerRes. 54, 701–708

22. Yamai, H., Sawada, N., Yoshida, T., Seike, J., Takizawa, H., Kenzaki, K.,Miyoshi, T., Kondo, K., Bando, Y., Ohnishi, Y., and Tangoku, A. (2009)Int. J. Cancer 125, 952–960

23. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl,J. K., Sutherland, G. R., Smith, T. D., Rauch, C., and Smith, C. A. (1995)Immunity 3, 673– 682

24. Pan, G., Ni, J., Wei, Y. F., Yu, G., Gentz, R., and Dixit, V. M. (1997) Sci-ence 277, 815– 818

25. Ozoren, N., Fisher, M. J., Kim, K., Liu, C. X., Genin, A., Shifman, Y.,Dicker, D. T., Spinner, N. B., Lisitsyn, N. A., and El-Deiry, W. S. (2000)Int. J. Oncol. 16, 917–925

26. Grotzer, M. A., Eggert, A., Zuzak, T. J., Janss, A. J., Marwaha, S., Wie-wrodt, B. R., Ikegaki, N., Brodeur, G. M., and Phillips, P. C. (2000) Onco-gene 19, 4604 – 4610

27. Shiiki, K., Yoshikawa, H., Kinoshita, H., Takeda, M., Ueno, A., Nakajima,Y., and Tasaka, K. (2000) Cell Death Differ. 7, 939 –946

28. Zhang, L., and Fang, B. (2005) Cancer Gene Ther. 12, 228 –23729. Prasad, S., Ravindran, J., Sung, B., Pandey, M. K., and Aggarwal, B. B.

(2010) Mol. Cancer Ther. 9, 856 – 86830. Kunnumakkara, A. B., Diagaradjane, P., Guha, S., Deorukhkar, A.,

Shentu, S., Aggarwal, B. B., and Krishnan, S. (2008) Clin. Cancer Res. 14,2128 –2136

31. Chawla-Sarkar, M., Bae, S. I., Reu, F. J., Jacobs, B. S., Lindner, D. J., andBorden, E. C. (2004) Cell Death Differ. 11, 915–923

32. Hinz, S., Trauzold, A., Boenicke, L., Sandberg, C., Beckmann, S., Bayer,E., Walczak, H., Kalthoff, H., and Ungefroren, H. (2000) Oncogene 19,5477–5486

33. Schimmer, A. D., Welsh, K., Pinilla, C., Wang, Z., Krajewska, M., Bon-neau, M. J., Pedersen, I. M., Kitada, S., Scott, F. L., Bailly-Maitre, B.,Glinsky, G., Scudiero, D., Sausville, E., Salvesen, G., Nefzi, A., Ostresh,J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25–35

34. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner,V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D.,French, L. E., and Tschopp, J. (1997) Nature 388, 190 –195

35. Wu, W. G., Soria, J. C., Wang, L., Kemp, B. L., and Mao, L. (2000) Anti-cancer Res. 20, 4525– 4529

36. Wang, Q., Wang, X., Hernandez, A., Hellmich, M. R., Gatalica, Z., andEvers, B. M. (2002) J. Biol. Chem. 277, 36602–36610

37. Belyanskaya, L. L., Ziogas, A., Hopkins-Donaldson, S., Kurtz, S., Simon,H. U., Stahel, R., and Zangemeister-Wittke, U. (2008) Lung Cancer 60,355–365

38. Shenoy, K., Wu, Y., and Pervaiz, S. (2009) Cancer Res. 69, 1941–195039. Zou, W., Liu, X., Yue, P., Zhou, Z., Sporn, M. B., Lotan, R., Khuri, F. R.,

and Sun, S. Y. (2004) Cancer Res. 64, 7570 –757840. Gliniak, B., and Le, T. (1999) Cancer Res. 59, 6153– 615841. Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A., Marsters,

S. A., Blackie, C., Chang, L., McMurtrey, A. E., Hebert, A., DeForge, L.,Koumenis, I. L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahr-okh, Z., and Schwall, R. H. (1999) J. Clin. Invest. 104, 155–162

42. Srivastava, R. K. (2000) Mol. Cell. Biol. Res. Commun. 4, 67–7543. Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin,

M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P.,Goodwin, R. G., Rauch, C. T., Schuh, J. C., and Lynch, D. H. (1999) Nat.Med. 5, 157–163

44. Martelli, A. M., Tazzari, P. L., Tabellini, G., Bortul, R., Billi, A. M., Man-zoli, L., Ruggeri, A., Conte, R., and Cocco, L. (2003) Leukemia 17,1794 –1805

45. Nagane, M., Cavenee, W. K., and Shiokawa, Y. (2007) J. Neurosurg. 106,407– 416

46. Fulda, S., Meyer, E., and Debatin, K. M. (2002) Oncogene 21, 2283–229447. Ravi, R., and Bedi, A. (2002) Cancer Res. 62, 1583–158748. Jacobson, M. D. (1996) Trends Biochem. Sci. 21, 83– 8649. Jung, E. M., Lim, J. H., Lee, T. J., Park, J. W., Choi, K. S., and Kwon, T. K.

(2005) Carcinogenesis 26, 1905–191350. Kim, H., Kim, E. H., Eom, Y. W., Kim, W. H., Kwon, T. K., Lee, S. J., and

Choi, K. S. (2006) Cancer Res. 66, 1740 –175051. Yodkeeree, S., Sung, B., Limtrakul, P., and Aggarwal, B. B. (2009) Cancer

Res. 69, 6581– 658952. Yamaguchi, H., and Wang, H. G. (2004) J. Biol. Chem. 279,

45495– 4550253. Takimoto, R., and El-Deiry, W. S. (2000) Oncogene 19, 1735–174354. Hetschko, H., Voss, V., Seifert, V., Prehn, J. H., and Kogel, D. (2008)

FEBS J. 275, 1925–193655. Insinga, A., Monestiroli, S., Ronzoni, S., Gelmetti, V., Marchesi, F., Viale,

A., Altucci, L., Nervi, C., Minucci, S., and Pelicci, P. G. (2005) Nat. Med.11, 71–76

56. Merino, D., Lalaoui, N., Morizot, A., Schneider, P., Solary, E., andMicheau, O. (2006) Mol. Cell. Biol. 26, 7046 –7055

57. Meng, R. D., McDonald, E. R., 3rd, Sheikh, M. S., Fornace, A. J., Jr., andEl-Deiry, W. S. (2000) Mol. Ther. 1, 130 –144

58. Corazza, N., Jakob, S., Schaer, C., Frese, S., Keogh, A., Stroka, D., Kas-sahn, D., Torgler, R., Mueller, C., Schneider, P., and Brunner, T. (2006)J. Clin. Invest. 116, 2493–2499

59. Huang, J., Wu, L., Tashiro, S., Onodera, S., and Ikejima, T. (2008)J. Pharmacol. Sci. 107, 370 –379

60. Kim, J. Y., Kim, E. H., Park, S. S., Lim, J. H., Kwon, T. K., and Choi, K. S.(2008) J. Cell. Biochem. 105, 1386 –1398

61. Ng, C. P., Zisman, A., and Bonavida, B. (2002) Prostate 53, 286 –29962. Hyer, M. L., Croxton, R., Krajewska, M., Krajewski, S., Kress, C. L., Lu,

M., Suh, N., Sporn, M. B., Cryns, V. L., Zapata, J. M., and Reed, J. C.(2005) Cancer Res. 65, 4799 – 4808

63. Kowalczyk, M. C., Walaszek, Z., Kowalczyk, P., Kinjo, T., Hanausek, M.,and Slaga, T. J. (2009) Carcinogenesis 30, 1008 –1015

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S1

DR5

DR4

0 5 10 20 30 0 5 10 20 30 UA (μM)Bax Parental Bax Knockout

β- actin

PPARγ

CHOP

β-actin

(A)

(B) 0 5 10 20 30 UA (μM)

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SUPPLEMENTARY FIGURE 1: Upregulation of DRs is independent of bax, CHOP and PPARγ expression. (A) To determine whether bax were involved in the induction of DR5/DR4, bax parental and bax knockout cells were treated separately with UA for 24 h, and whole-cell extracts were prepared for Western blotting for DR5 and DR4. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (B) HCT116 cells were pretreated with the indicated doses of UA for 24 h. Whole-cell extracts were prepared and subjected to Western blotting with the CHOP and PPARγ antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading.

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Sahdeo Prasad, Vivek R. Yadav, Ramaswamy Kannappan and Bharat B. AggarwalTHE ROLE OF REACTIVE OXYGEN SPECIES AND JNK

through p53-independent Up-regulation of Death Receptors: EVIDENCE FOR Ursolic Acid, a Pentacyclin Triterpene, Potentiates TRAIL-induced Apoptosis

doi: 10.1074/jbc.M110.183699 originally published online December 14, 20102011, 286:5546-5557.J. Biol. Chem. 

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Sahdeo Prasad, Vivek R. Yadav, Ramaswamy Kannappan and Bharat B. AggarwalTHE ROLE OF REACTIVE OXYGEN SPECIES AND JNK

through p53-independent Up-regulation of Death Receptors: EVIDENCE FOR Ursolic Acid, a Pentacyclin Triterpene, Potentiates TRAIL-induced Apoptosis

doi: 10.1074/jbc.M110.183699 originally published online December 14, 20102011, 286:5546-5557.J. Biol. Chem. 

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VOLUME 286 (2011) PAGES 5546 –5557DOI 10.1074/jbc.A110.183699

Ursolic acid, a pentacyclin triterpene, potentiatesTRAIL-induced apoptosis through p53-independentup-regulation of death receptors. EVIDENCE FOR THEROLE OF REACTIVE OXYGEN SPECIES AND JNK.Sahdeo Prasad, Vivek R. Yadav, Ramaswamy Kannappan, and Bharat B. Aggarwal

This article has been retracted by the publisher. Fig. 1B was assem-bled from a composite of many images. The actin immunoblot from Fig.2A, right, was reused in Fig. 4A, left. Parts of the actin immunoblot fromFig. 2C, left panel, had been reused in supplemental Fig. 1A. The actinimmunoblot from Figs. 2E and 4B, left panels; and supplemental Fig. 1Bwas reused in Fig. 5B, top panels. Parts of the JNK immunoblot from Fig.4C were reused in Fig. 7C, JNK panels.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 32, p. 16924, August 5, 2016© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

16924 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 32 • AUGUST 5, 2016

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