Ácido ursólico induz apoptose ,independente do p53, através do do JNK e aumento da

atividade dos receptores da morte potenciando o TRAIL

Ursolic acid, a pentacyclin triterpene, potentiates TRAIL-

induced apoptosis through p53-independent up-regulation of

death receptors: evidence for the role of reactive oxygen

species and JNK.

J Biol Chem. 2011 February 18; 286(7): 5546–5557.

Published online 2010 December 14. doi: 10.1074/jbc.M110.183699

PMCID: PMC3037668

Copyright © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

EVIDENCE FOR THE ROLE OF REACTIVE OXYGEN SPECIES AND JNK*

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 Cancer Center, Houston, Texas 77030

1 The Ransom Horne, Jr., Professor of Cancer Research. To whom correspondence should be

addressed: 1515 Holcombe Blvd., Unit 143, Houston, TX 77030., Tel.: Phone: 713-794-1817;

Fax: 713-745-6339; E-mail: aggarwal@mdanderson.org .

Abstract

Discovery of the molecular targets of traditional medicine and its chemical footprints can

validate the use of such medicine. In the present report, we investigated the effect of ursolic

acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by

TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA

also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the

mechanism, we found that UA down-regulated cell survival proteins and induced the cell

surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of

receptors by UA occurred independently of cell type. Gene silencing of either receptor by small

interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA

also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was

independent of p53 because UA induced DR4 and DR5 in HCT116 p53−/− cells. Induction of

DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic

inhibition abolished the induction of the receptors. The down-regulation of survival proteins

and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS,

and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced

apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In

addition, UA was also found to induce the expression of DRs, down-regulate cell survival

proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude

mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis

through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of

DcR2 and cell survival proteins.

Keywords: Apoptosis, Cancer Therapy, Cytokine, JNK, Reactive Oxygen Species (ROS)

Introduction

More than 80% of people around the world, for their day-to-day medicinal needs, rely on

traditional medicine, which has been around for centuries. Even modern medicine in most

instances relies on natural products, and 70% of anticancer drugs have their roots in products

derived from nature (1). The search for signature genes of different cancers has shown that

most cancers are due to dysregulation of multiple genes and multiple cell signaling pathways;

thus, drugs that are multitargeted (once called “dirty drugs”) are needed. Compounds from

natural sources have an advantage in that they are usually multitargeted. One such compound

is ursolic acid (UA),2 a pentacyclic triterpenoid that has been identified in a large variety of

medicinal herbs and other plants, including rosemary (Rosemarinus officinalis), apples (Malus

domestica), cranberries (Vaccinium macrocarpon), beefsteak (Perilla frutescens), pears (Pyrus

pyrifolia), plum (Prunus domestica), bearberries (Arctostaphylos alpina), loquat (Eriobotrya

japonica), scotch heather (Calluna vulgaris), basil (Ocimum sanctum), and jamun (Eugenia

jambolana) (2). It has been shown that UA can inhibit cell growth and induce apoptosis in

various tumors (3,–9). UA induces apoptosis through multiple pathways, such as inhibiting

DNA replication (7, 10); inducing Ca2+ release (8); activating caspases (9, 11); activating JNK

(12); down-regulating antiapoptotic genes (13, 14); inhibiting COX2 and iNOS (15, 16);

suppressing MMP-9 (17); and inhibiting protein tyrosine kinase (10), STAT3 (18), and NF-κB

activities (13). In animal studies, UA has been shown to be chemopreventive (19,–21), to

suppress tumor invasion (17), and to inhibit experimental metastasis of esophageal carcinoma

(22). Whether UA can modulate the effect of apoptosis-inducing cytokines that are currently in

clinical trial is not known.

TRAIL (tumor necrosis factor (TNF)-related apoptosis-inducing ligand), a member of the TNF

family, is one such apoptosis-inducing cytokine that has shown promise as an anticancer agent

(23). TRAIL selectively induces apoptosis in tumor cells but not in normal cells (3). There are

five different receptors that have been identified for TRAIL; however, only death receptor 4

(DR4) (TRAIL-R1) and DR5 (TRAIL-R2) have cytoplasmic death domains that activate the

apoptotic machinery upon TRAIL binding (3). Other receptors, such as decoy receptor 1 (DcR1)

and DcR2, although membrane-bound, exhibit dominant negative effects by sequestering the

ligand (24). Osteoprotegerin, although it binds TRAIL, lacks a transmembrane 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 fully

understood; however, it can occur at different points in TRAIL-induced apoptotic signaling

pathways. For instance, dysfunction of DR4 and DR5, overexpression of antiapoptotic proteins,

and loss of proapoptotic proteins has all been linked with TRAIL resistance. Activation of

different subunits of mitogen-activated protein kinases and nuclear factor-κB are also reported

to develop TRAIL resistance in certain types of cancer cells (28). Therefore, modulation of

TRAIL-induced apoptotic signaling molecules is an important strategy to sensitize cancer cells

for effective cancer therapy.

In the current report, we tested whether UA can potentiate TRAIL-induced apoptosis and

sensitize resistant cancer cells to TRAIL. We found that this triterpene can indeed enhance

TRAIL-induced apoptosis through up-regulation of death receptors and down-regulation of

antiapoptotic proteins via production of reactive oxygen species (ROS) and activation of JNK.

EXPERIMENTAL PROCEDURES

Reagents

A 50 mm solution of UA (from Sigma), with purity greater than 90%, was prepared in DMSO,

stored as small aliquots at −20 °C, and then diluted further in cell culture medium as needed.

Further fractionation of UA revealed that minor impurities had no activity (data not shown).

Soluble recombinant human TRAIL/Apo2L was purchased from PeproTech. Penicillin,

streptomycin, RPMI 1640, and fetal bovine serum were purchased from Invitrogen. Anti-β-

actin antibody was obtained from Sigma-Aldrich. Antibodies against DR4, DR5, Bcl-xL, Bcl-2,

Bax, cFLIP, poly(ADP-ribose) polymerase (PARP), and JNK1 and the annexin V staining kit were

purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Dichlorodihydrofluorescein

diacetate was purchased from Invitrogen.

Cell Lines

HCT116, HT29, Caco2 (human colon adenocarcinoma), A293 (human embryonic kidney

carcinoma), PC3 (human prostate cancer), MDA-MB-231 and MCF-7 (human breast cancer),

SCC4 (squamous cell carcinoma), and KBM-5 (human chronic leukemia) cells were obtained

from the American Type Culture Collection. HCT116 variants with deletion of p53 and Bax were

kindly supplied by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD). The human

colon cancer HCT116 variant cell lines were cultured in McCoy's 5A medium. HCT116, A293,

MDA-MB-231, SCC4, and MCF-7 cells were cultured in DMEM. Caco2 and HT29 cell lines were

cultured in RPMI1640. KBM-5 cells were cultured in Iscove's modified Dulbecco's medium.

DMEM and RPMI were supplemented with 10% fetal bovine serum, 100 units/ml penicillin,

and 100 mg/ml streptomycin. Iscove's modified Dulbecco's medium was supplemented with

15% fetal bovine serum 100 units/ml penicillin, and 100 mg/ml streptomycin.

Live/Dead Assay

To measure apoptosis, we used the Live/Dead® assay (Invitrogen), which assesses intracellular

esterase activity and plasma membrane integrity. This assay was performed 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 cells were stained with

PI as described earlier (18). A total of 10,000 events were analyzed by flow cytometry using an

excitation wavelength of 488 nm and emission wavelength of 610 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 manufacturer's

instructions, resuspended, and analyzed by flow cytometry with phycoerythrin-conjugated

mouse IgG2B as an isotype control.

Annexin V Assay

The early indicator of apoptosis was detected by using an annexin V/PI binding kit (Santa Cruz

Biotechnology, Inc.) and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences).

Measurement of ROS

Intracellular ROS of cells were detected 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. On the

day of transfection, 12 μl of HiPerFect transfection reagent (Qiagen) was added to 50

nmol/liter siRNA in a final volume of 100 μl of culture medium. After 48 h of transfection, cells

were treated with 20 μm UA for 12 h and then exposed TRAIL for 24 h.

Western Blot Analysis

To determine the levels of protein expression, whole-cell extracts were prepared in lysis buffer

as described previously (13). In the in vivo case, colorectal tumor tissues (75–100 mg/mouse)

were minced and incubated on ice 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, 0.1 mol/liter EGTA, 0.5 mol/liter EDTA, 0.1

mol/liter PMSF, 0.2 mol/liter sodium orthovanadate, 1 mol/liter NaF, 2 μg/ml aprotinin, 2

μg/ml leupeptin). The minced tissue was homogenized with a Dounce homogenizer and

centrifuged at 16,000 × g at 4 °C for 10 min. The extracted proteins were then resolved on a

10% SDS gel, and Western blotting was performed as described previously (13).

RNA Analysis and RT-PCR

DR5 mRNA was detected using RT-PCR as follows. Total RNA was isolated from cells using

TRIzol reagent (Invitrogen) as instructed by the manufacturer. One microgram of total RNA

was converted to cDNA using Superscript reverse transcriptase and then amplified by platinum

Taq polymerase using the Superscript One Step RT-PCR kit (Invitrogen). The total RNAs were

then amplified by PCR using the following primers: DR5 sense (5′-AAGACCCTTGTGCTCGTTGTC-

3′), DR5 antisense (5′-GACACATTCGATGTCACTCCA-3′), DR4 sense (5′-

CTGAGCAACGCAGACTCGCTGTCCAC-3′), DR4 antisense (5′-

TCCAAGGACACGGCAGAGCCTGTGCCAT-3′), GAPDH sense (5′-GTCTTCACCACCATGGAG-3′), and

GAPDH antisense (5′-CCACCCTGTTGCTGTAGC-3′). The reaction sequence consisted of 50 °C for

30 min, 94 °C for 2 min, and 94 °C for 35 cycles of 15 s each; 50 °C for 30 s; and 72 °C for 45 s

with an extension at 72 °C for 10 min. PCR products were run on 2% agarose gel and then

stained with ethidium bromide. Stained bands were visualized under UV light and

photographed.

Animal Protocol

The HCT116 cells were orthotopically implanted as described previously (30). One week after

implantation, the mice were randomized into the following treatment groups (n = 6/group): (a)

untreated control (corn oil, 100 μl daily) and (b) UA (250 mg/kg once daily orally). Therapy was

continued for 4 weeks, and the animals were euthanized 1 week later. Primary tumors in the

colon were excised, snap-frozen in liquid nitrogen, and stored at −80 °C. Our experimental

protocol was reviewed and approved by the Institutional 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

was determined using unpaired Student's t test, and a p value of less 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 cancer

HCT116 cells for most of these studies, but other cell types were also used to determine the

specificity of this effect.

FIGURE 1.

UA potentiates TRAIL-induced apoptosis of HCT116 cells. A, chemical

structure of UA. B, cells were pretreated with 20 μm UA for 12 h. The medium

was removed, and the cells were then exposed to TRAIL (25 ng/ml) for 24 h.

Cell viability was then (more ...)

UA Enhances TRAIL-induced Apoptosis

We examined first the effect of UA on TRAIL-induced cytotoxicity by using the MTT method,

which detects mitochondrial activity. The HCT116 cells were moderately sensitive to either UA

or TRAIL alone. However, pretreatment with UA significantly enhanced TRAIL-induced

cytotoxicity (Fig. 1B, left).

We also determined whether UA also enhances TRAIL-induced apoptosis of colon cancer cells.

We found that UA and TRAIL treatments alone induced 21 and 16% apoptosis, respectively, in

HCT116 cells. Interestingly, combination treatment 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 fraction. The results indicated that the UA and TRAIL treatment alone

induced 12 and 20% apoptosis, respectively. Combination treatment with both UA and TRAIL

enhanced apoptosis to 50% (Fig. 1C, top). When apoptosis was examined using annexin V/PI

staining, we found that apoptosis was induced at 13% by UA, 7% by TRAIL, and 47% by the

combination of the two (Fig. 1C, bottom).

Next, we examined the effect of UA, TRAIL, and their combination on the activation of caspase-

8, caspase-3, and PARP cleavage. We found that although UA and TRAIL alone had little effect

on the activation of caspases and on PARP cleavage, the two together were highly effective

(Fig. 1D). Together, our results indicate that UA can enhance TRAIL-induced apoptosis.

UA Induces the Expression of TRAIL Receptors DR4 and DR5 in Cancer Cell Lines

To explore the underlying mechanism that may be responsible for enhancement of TRAIL-

induced apoptosis by UA, we examined the effect of UA on the expression of death receptors.

UA induced both DR4 and DR5 in a dose-dependent manner (Fig. 2A, left). Whether this

induction of the DRs was dependent on time was also examined. UA induced both DR4 and

DR5 in a time-dependent manner as well (Fig. 2A, right).

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 indicated times (right).

Whole-cell extracts were then prepared and analyzed for DR5 and DR4 by

Western blotting. (more ...)

We also investigated whether UA induces cell surface expression of TRAIL receptors. We found

that UA increased the cell surface expression of both DR5 and DR4 in colon cancer HCT116

cells (Fig. 2B). The level of DR4 cell surface expression 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 other cell types, we exposed the following cells to 20 μm UA for 24 h: MCF-7,

MDA-MB-231, PC3, SCC4, A293, HT29, Caco2, and KBM-5 cells. UA induced expression of both

DR5 and 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 addition to HCT116 cells. The induction of DR5 or DR4 by UA in MDA-MB-231

cells was insignificant. These findings suggest that the up-regulation of DR5 and DR4 by UA was

not cell type-specific.

UA Up-regulates DRs at Transcriptional Level

Whether TRAIL receptors are induced by UA at the transcriptional level was investigated by RT-

PCR. UA substantially up-regulated both DR4 and DR5 mRNA expression in a dose-dependent

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 expression of DcR1, it decreased the expression of

DcR2 (Fig. 2E). Thus, inhibition of DcR2 expression by UA could also contribute to apoptosis by

TRAIL.

Gene Silencing of DRs Abolishes the Effect of UA on TRAIL-induced Apoptosis

To determine whether up-regulation of DR5 or DR4 is needed in TRAIL-induced apoptosis, we

used a gene-silencing approach to abolish UA-induced expression of these receptors. We

found that transfection of cells with DR5 siRNA but not with the control scrambled siRNA

reduced the UA-induced up-regulation of DR5. Similarly, transfection of cells with siRNA for

DR4 reduced the UA-induced DR4 expression (Fig. 3A).

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 μmol/liter (more ...)

We next examined whether the suppression of DR5 or DR4 by siRNA could abolish the effects

of UA on TRAIL-induced apoptosis using the Live/Dead assay. We found that the silencing of

DR5 reduced the UA-induced apoptosis from 22 to 12%, whereas silencing of DR4 reduced

apoptosis to 14%. Silencing of both receptors reduced UA-induced apoptosis to 8%, whereas

control scrambled siRNA had no effect. These results indicate that DRs contribute to UA-

induced apoptosis as well.

The TRAIL-induced apoptosis was reduced from 17 to 8% by silencing of DR5, but silencing of

DR4 had no significant effect on TRAIL-induced apoptosis. When we examined the effect of UA

and TRAIL in combination, we found that apoptosis was reduced from 62 to 34% by DR5 siRNA,

to 48% by DR4 siRNA, and to 28% by the two siRNAs together, whereas with control scrambled

siRNA, change in apoptosis was not significant (Fig. 3B). Silencing of DR5 had a more dramatic

effect on TRAIL-induced apoptosis than silencing of DR4. The silencing of both receptors

abolished the apoptosis as much as silencing of DR5 alone. Overall, these results suggest that

DR5 plays a major role in TRAIL-induced apoptosis and that enhancement of apoptosis by UA is

linked to up-regulation of the receptors.

UA Down-regulates the Expression of Antiapoptotic Proteins

Various antiapoptotic proteins, including survivin (31), Bcl-xL (32), XIAP (33), and cFLIP (34),

have been shown to induce resistance to TRAIL-induced apoptosis. We examined whether UA

sensitized the cells to TRAIL through down-regulation of the expression of these cell survival

proteins. Results of Western blot showed that UA inhibited expression 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 another mechanism of potentiation of

TRAIL-induced apoptosis.

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 (more ...)

UA Enhances the Expression of Proapoptotic Proteins

Whether UA can modulate the expression of proapoptotic proteins was also examined. We

found that UA cleaved the proapoptotic protein Bid and induced the expression of Bax (Fig. 4A,

right). Up-regulation of Bax by UA suggests that these proteins may disrupt mitochondrial

homeostasis, which further leads to apoptosis.

UA-induced Up-regulation of TRAIL Receptors is p53-independent

Because p53 has been reported to induce death receptors (35), we investigated whether UA

up-regulates DRs through up-regulation of p53. We found that UA did not up-regulate p53; if

anything, at a higher dose (30 μm), it down-regulated p53 (Fig. 4B, left). We also determined

whether UA-induced induction of TRAIL receptors is mediated through p53 in HCT116 cell lines

that lack p53. We found that UA induced DR5 and DR4 in p53 parental as well as p53 knock-

out HCT116 cells in a dose-dependent manner (Fig. 4B, right). These results indicate that

induction of TRAIL receptors by UA is p53-independent.

UA-induced Up-regulation of TRAIL Receptors Is Bax-independent

We found that UA induced Bax. Whether UA up-regulates DRs through up-regulation of Bax

was investigated. For this, we used Bax knock-out HCT116 colon cancer cells. UA induced

expression of DR5 and DR4 in both Bax parental and Bax knock-out HCT116 cells (supplemental

Fig. 1A), indicating that induction of TRAIL receptors are independent of Bax expression.

UA-induced Up-regulation of Death Receptor Is Not Mediated through Activation of ERK1/2

and GSK-3β

Activation of ERK1/2 and GSK-3β has been linked with induction of TRAIL-induced apoptosis

(36, 37). We therefore investigated whether UA activates ERK1/2 and GSK-3β. Results showed

that UA activated neither ERK1/2 nor GSK-3β and had no effect on the expression levels of

these proteins (Fig. 4C, left).

UA-induced Up-regulation of DRs Is Not Mediated through Activation of CHOP and PPARγ

Next we determined whether UA modulates CHOP and PPARγ. We found that DBA did not

modulate either PPARγ or CHOP (supplemental Fig. 1B), indicating that induction of DRs is not

mediated through PPARγ 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 can activate JNK by exposing the cells to different concentrations of

UA for 24 h and then examined the cells for activation of JNK. Western blotting results showed

that UA induced JNK activation in a dose-dependent manner (Fig. 4C, right), but under the

same conditions 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 inhibitor of JNK. As

shown in Fig. 4D (left), pretreatment of cells with this JNK inhibitor significantly suppressed the

UA-induced up-regulation of DR5 and DR4 expression. These results suggest that JNK is

involved in UA-induced up-regulation of DRs. Suppression of JNK also leads to inhibition of UA-

induced cleavage of PARP (Fig. 4D, left).

Furthermore, we examined whether the suppression of JNK activation by its inhibitor could

abrogate the apoptosis induced by UA, TRAIL, and the combination of UA and TRAIL. We found

that apoptosis induced by UA, TRAIL, and the combination was reduced from 18 to 10%, from

24 to 13%, and from 64 to 42%, respectively (Fig. 4D, right). Thus, suppression of JNK activation

substantially reduced the apoptosis, although not completely.

UA-induced Up-regulation of DR5 Requires ROS

That ROS is needed for induction of death receptors by certain agents has been demonstrated

(29). To determine whether UA has the ability to generate ROS, we treated HCT116 cells with

UA and used dichlorodihydrofluorescein diacetate as a probe to measure the increase in ROS

levels in the cells. We found that UA induced the production of ROS in a dose-dependent

manner (Fig. 5A).

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 (more ...)

Next we determined whether ROS production is needed for up-regulation of expression of DR5

and DR4 by UA. We found that pretreatment of cells with the ROS scavenger NAC blocked UA-

induced up-regulation of DR5 and DR4 protein expressions in a dose-dependent manner (Fig.

5B, top), indicating that ROS generation is critical for the effect of UA on TRAIL receptors.

NAC Abrogates the Effect of UA in Suppression of Antiapoptotic Proteins

Next we examined whether NAC abrogates UA-induced inhibition of antiapoptotic proteins.

The results revealed that pretreatment of NAC effectively abolished the effect of UA in

suppression of XIAP, cFLIP, and Bcl-2 but not survivin (Fig. 5B, bottom). The effect of NAC in

inhibiting the effect of UA on XIAP is more prominent than others.

UA-induced Potentiation of Apoptosis Induced by TRAIL Is Reversed by Quenchers of ROS

We next examined whether ROS is needed for potentiation of TRAIL-induced apoptosis by UA.

As shown in Fig. 5C, UA enhanced TRAIL-induced apoptosis in HCT116 cells, and pretreatment

of cells with NAC markedly reduced this UA-induced enhancement from 68 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 suggesting 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 resistant to TRAIL (40). We

therefore investigated whether UA affects TRAIL-resistant HT29 cancer cells. We found that

HT29 cells were moderately sensitive to UA but resistant to TRAIL. However, pretreatment

with UA enhanced TRAIL-induced apoptosis from 2% to 42% (Fig. 6A).

FIGURE 6.

UA sensitizes TRAIL resistance cells and induces apoptosis. A, HT29 cells were

pretreated with 20 μm 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 (more ...)

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. Interestingly, the

pretreatment with UA sensitized the cells to TRAIL and induced apoptosis of 24% (Fig. 6B).

Results of the cytotoxicity assay by MTT uptake also showed that HT29 cells were moderately

sensitive to UA but resistant to TRAIL. However, pretreatment of UA sensitized the HT29 cells

to TRAIL and induced apoptosis (Fig. 6C).

We then determined whether UA up-regulates the expression 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 Survival Proteins, and Activates JNK in

Colorectal Tumors in Vivo

Whether UA up-regulates the expression of DR4 and DR5 in vivo was examined in

orthotopically implanted human colorectal tumor from nude mice treated with UA. The

Western blot analysis revealed that tumor tissues from UA-treated animals when compared

with those from vehicle-treated animals had up-regulated expression of both of the death

receptors, DR4 and DR5 (Fig. 7A), had down-regulated expression of cell survival proteins

(survivin and Bcl-2) (Fig. 7B), and had activated JNK (Fig. 7C). These results in vivo are in

agreement with those in vitro.

FIGURE 7.

UA up-regulates DRs, down-regulates cell survival proteins, and activates JNK

in orthotopically transplanted human colorectal tumor in nude mice in vivo.

Whole cell extracts of tumor tissues were subjected to Western blotting to

analyze expression of (more ...)

DISCUSSION

TRAIL has recently been considered a highly promising candidate as an anti-cancer drug,

because it induces apoptosis specifically in malignant or transformed cells without any

cytotoxicity toward a variety of normal cells (41,–43). A considerable number of cancer cells,

however, are resistant to apoptosis induced by TRAIL (28). TRAIL induces apoptosis by

interacting with two different death-inducing receptors, DR4 and DR5. Therefore, targeting

death receptors and their signaling molecules to trigger apoptosis in tumor cells is an attractive

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 demonstrate for

the first time that UA, a pentacyclic triterpene, can sensitize 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-regulation of antiapoptotic proteins. In our study, UA

treatment induced dose- and time-dependent increases in the protein levels of DR5 and DR4.

We also demonstrated the up-regulation of expression of cell surface death receptors by UA.

Silencing of DR5 and DR4 by their respective siRNAs effectively inhibited the cell death induced

by the combination of UA and TRAIL, demonstrating the critical role of death receptors in this

event. Silencing of DR5, however, was more effective than silencing of DR4 in inhibiting UA and

TRAIL-induced apoptosis.

FIGURE 8.

Schematic representation of the mechanism by which UA potentiates TRAIL-

induced apoptosis.

The induction of death receptors by UA was not cell type-specific because induction was

observed in a wide variety of cancer cell types, including breast, prostate, head and neck,

kidney, leukemic, and colon cancer cells. Induction of TRAIL receptors in some cells, however,

was much more pronounced than in other cell types. Thus, UA is likely to potentiate the

anticancer effect of TRAIL in a wide variety of cells.

Resistance to TRAIL-induced apoptosis has been reported to be associated with overexpression

of antiapoptotic proteins. Among Bcl-2 family proteins, Bcl-2 has been linked with suppression

of apoptosis by TRAIL (46). In our study, Bcl-2 was suppressed by UA treatment. In addition, UA

treatment also decreased the expression of Bcl-xL, survivin, and XIAP proteins but had no

effect on c-FLIP expression, which is also linked to TRAIL resistance (31, 32). When we looked

for other potential mechanisms, we found that UA significantly up-regulated the expression of

Bax and cleavage of Bid proteins. The former has been shown to be critical for TRAIL-induced

apoptosis (47). However, our result also showed that UA-induced apoptosis mediated through

expression 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 been proposed to be involved in the up-regulation of DR5 by numerous

cancer chemopreventive agents, including curcumin and 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 antioxidant 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 multiple potential binding sites of various

transcription factors, including CHOP and p53, are present in the upstream region of DR5 (52,

53). However, we found that the induction of DR5 by UA occurs independently of p53 and

CHOP. These results are consistent with those previously reported for the proteasome

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-induced apoptotic signaling pathway. TRAIL can also bind DcR1 (TRAIL-

R3) and DcR2 (TRAIL-R4). These latter receptors fail to induce apoptosis, because DcR1 lacks an

intracellular domain and DcR2 has a truncated cytoplasmic death domain. In addition, DcR1

and DcR2 inhibit DR4- and DR5-mediated apoptosis by TRAIL. While DcR1 prevents the

assembly of the death-inducing signaling complex by titrating TRAIL within lipid rafts, DcR2 is

co-recruited with DR5 within the death-inducing signaling complex, where it inhibits initiator

caspase activation (56). Here we demonstrated that UA reduced the expression of DcR2, which

allow the availability of ligand for DR4 and DR5 for induction of apoptosis. In contrast, no

change in DcR1 occurred. The expression of DcR2 has been shown to be regulated by p53 (57).

Because we did find down-regulation of p53 at higher doses, it is possible that 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 findings provide evidence that activation of JNK by UA up-regulates DRs,

which may further lead to an increase in TRAIL-induced apoptosis. UA was found to be

ineffective in activating ERK1/2 MAPK. Although ROS can lead to induction of MAPK (59), in our

study, UA induced TRAIL receptors independently of MAPK. In another study, quercetin

augmented TRAIL-induced apoptosis through the ERK-mediated down-regulation of the

survivin signal transduction pathway (60). In our study, however, UA induced apoptosis

through down-regulation of survivin but independently of ERK activation.

We observed that UA sensitized the tumor cells that are resistant to TRAIL. Although the

mechanisms underlying sensitization of TRAIL-resistant cells are not clear, some important

components, such as down-regulation of antiapoptotic proteins in signaling pathways, may be

involved in this process (28). Because Bcl-2, XIAP (inhibitor of caspase), and survivin (46, 61)

are involved in TRAIL resistance, down-regulation of these proteins by UA is the probable

reason for the sensitizing of TRAIL-resistant cells. In addition, induction of death receptors

could further contribute to the sensitivity.

UA is a pentacyclic triterpene isolated from various traditional medicinal plants. Interestingly,

CDDO, which is a synthetic analog designed based on ursolic acid and betulinic acid and which

is also a pentacyclic triterpene, has been shown to induce death receptors (39, 62). CDDO

sensitized tumor cells to TRAIL through up-regulation of death receptors and down-regulation

of cFLIP (62). In addition, Zou et al. (39) found that CDDO-Me induces DR5 up-regulation

through induction of CHOP, and Hyer et al. (62) found that the cell surface 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 and Hyer et al. did not is not clear. We showed,

however, that UA-induced up-regulation of DR is independent of CHOP. All of these groups did

show that activation of JNK is needed for up-regulation of the receptors.

Whether our in vitro results have relevance to those in vivo was also investigated. We found

that UA up-regulated DRs expression, down-regulated cell survival proteins, and activated JNK

in tumor tissue from animals treated with the agent in vivo. This indicates that UA-induced

apoptosis could be 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-resistant cells and potentiation of TRAIL-induced apoptosis through ROS

and JNK-mediated up-regulation of DR4 and DR5 and down-regulation of antiapoptotic

proteins (Fig. 8), thus rendering cancer cells more sensitive to the cytotoxic activities of TRAIL.

Considering that UA by itself is highly safe and exhibits anticancer activities against a wide

variety of tumors in vitro (12, 13, 18) and in vivo (22, 63), its potential use in combination with

TRAIL should be explored. Further studies in animals are needed to investigate the anticancer

potential of UA in combination with TRAIL.

Supplementary Material

Supplemental Data

Click here to view.

Acknowledgment

We thank Michael Worley from the Department of Scientific Publications for carefully editing

the manuscript.

*This work was supported, in whole or in part, by National Institutes of Health Grants CA-

16672 and CA-124787-01A2. This work was also supported 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 Cancer Center.

The on-line version of this article (available at http://www.jbc.org) contains supplemental

Fig. 1.

2The abbreviations used are:

UA ursolic acid

DR death receptor

DcR decoy receptor

ROS reactive oxygen species

PARP poly(ADP-ribose) polymerase

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PI propidium iodide

NAC N-acetyl cystiene.

Other Sections▼

Abstract

Introduction

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Supplementary Material

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