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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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