Antagonist Effect of Triptolide on AKT Activation by
Truncated Retinoid X Receptor-alpha
Na Lu1., Jinxing Liu1., Jie Liu1, Chunyun Zhang1, Fuquan Jiang1, Hua Wu1, Liqun Chen1, Wenjun Zeng1,
Xihua Cao2, Tingdong Yan1, Guanghui Wang1, Hu Zhou2, Bingzhen Lin2, Xiaomei Yan3, Xiao-
kun Zhang1,2*, Jin-Zhang Zeng1*
1School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, Xiamen, China, 2Cancer Center, Sanford-Burnham Medical Research
Institute, La Jolla, California, United States of America, 3The Key Laboratory of Analytical Science, The Key Laboratory for Chemical Biology of Fujian Province, Department
of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China
Background: Retinoid X receptor-alpha (RXRa) is a key member of the nuclear receptor superfamily. We recently
demonstrated that proteolytic cleavage of RXRa resulted in production of a truncated product, tRXRa, which promotes
cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRa-mediated
signaling pathway in cancer cells is regulated remains elusive.
Methodology/Principal Findings: We screened a natural product library for tRXRa targeting leads and identified that
triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRa-
mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell
apoptosis dependent on intracellular tRXRa expression levels, demonstrating that tRXRa serves as an important intracellular
target of triptolide. We show that triptolide selectively induces tRXRa degradation and inhibits tRXRa-dependent AKT
activity without affecting the full-length RXRa. Interestingly, such effects of triptolide are due to its activation of p38.
Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRa degradation and AKT
activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRa interaction
with p85a leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and
AKT through tRXRa mediation. We finally show that targeting tRXRa by triptolide strongly activates TNFa death signaling
and enhances the anticancer activity of other chemotherapies
Conclusions/Significance: Our results identify triptolide as a new xenobiotic regulator of the tRXRa-dependent survival
pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide
represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with
unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.
Citation: Lu N, Liu J, Liu J, Zhang C, Jiang F, et al. (2012) Antagonist Effect of Triptolide on AKT Activation by Truncated Retinoid X Receptor-alpha. PLoS ONE 7(4):
Editor: Jean-Marc A. Lobaccaro, Clermont Universite ´, France
Received November 30, 2011; Accepted March 20, 2012; Published April 24, 2012
Copyright: ? 2012 Lu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Natural Science Foundation of China (NSFC: 30971445, 90913015 and 91129302), NSFC/Hong Kong Research
Grants Council (NSFC/RGC: 30931160431/N_HKU 735/09) and Natural Science Foundation of Fujian Province (2009J01198). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (JZ); firstname.lastname@example.org (XK)
. These authors contributed equally to this work.
Retinoid X receptor-a (RXRa) is a unique member of the
nuclear receptor superfamily [1,2]. In addition to forming
homodimer, RXRa also heterodimerizes with many other nuclear
receptors such as retinoic acid receptor (RAR), peroxisome
proliferator-activated receptor (PPAR), vitamin D3 receptor
(VDR), thyroid hormone receptor (TR) and Nur77 orphan
nuclear receptor [1,2]. Thus, RXRa plays critical roles in
regulating numerous cellular processes including cell growth,
differentiation and apoptosis [1,2], and the synthetic RXR ligand
TargretinH/Bexarotene has been approved for treating cutaneous
T-cell lymphoma . Consistent to its profound effects, altered
RXR expression and function are implicated in the pathogenesis
of diseases and cancer. Diminished RXRa expression is associated
with the development of certain malignancies, such as thyroid
carcinoma , prostate cancer  and non-small-cell lung cancer
. RXRa ablation in adult tissues results in preneoplastic lesions
in skin  and prostate . In addition to reduced levels of RXRa
protein, altered RXRa function by phosphorylation is associated
with the development of human hepatocellular carcinoma
[9,10,11] and colon cancer . RXR binding to PML/RAR is
essential for the development of acute promyelocytic leukemia
[13,14], further demonstrating the oncogenic potential of this
protein when it acts inappropriately. Altered RXRa function can
also be resulted from its proteolytic cleavage of the receptor
PLoS ONE | www.plosone.org1 April 2012 | Volume 7 | Issue 4 | e35722
protein, which is frequently observed in various human tumors
[15,16,17,18]. We recently reported our identification of an N-
terminally truncated tRXRa protein in various cancer cells and in
primary tumors but not in tumor surrounding or normal tissues
. Unlike full-length RXRa that resides in the nucleus, tRXRa
is cytoplasmic and interacts with the p85a subunit of phosphati-
dylinositol-3-OH kinase (PI3K) to activate the PI3K/AKT
pathway , a major survival pathway important for uncon-
trolled growth of tumor and its progression as well as drug
resistance . Thus, tRXRa acquires new function that is
different from RXRa. Since tRXRa is often elevated in cancer
cells, it is expected that targeting tRXRa represents a more
effective and specific strategy for developing RXR-based antican-
cer drug. Thus, we show that non-steroidal anti-inflammatory
drug sulindac and analogs bind to tRXRa and inhibit tRXRa-
mediated PI3K/AKT activation in vitro and in animals .
Triptolide, a diterpene triepoxide, is a major active component
of extracts derived from the medicinal plant Tripterygium wilfordii
Hook F (TWHF) . Triptolide has multiple pharmacological
activities including anti-inflammatory, immune modulation, anti-
proliferative and proapoptotic activity [20,21,22]. It has been
widely used to treat inflammatory diseases, autoimmune diseases,
organ transplantation and even tumors [20,23,24,25]. Despite its
potent apoptotic effect, the underlying mechanisms by which
triptolide induces apoptosis remain largely unclear. Triptolide has
been found to activate p53 apoptotic pathways [26,27,28], to
induce Bcl-2 cleavage and mitochondria dependent apoptosis ,
and to reduce the expression of cell cycle regulators  and
survival genes such as cyclin D1 and Bcl-x . In addition,
Triptolide has been described to decrease the expression of heat
shock proteins such as Hsp70, molecular chaperones associated
with oncogenesis, by inactivation of heat shock transcription factor
(HSF) [32,33], and to inhibit transcription of numerous pro-
inflammatory mediators [27,34]. Interestingly, triptolide was
shown to cooperate with tumor necrosis factor-a (TNFa) to
induce apoptosis in tumor cells . Here, we report that the
apoptotic effect of triptolide is partially mediated by intracellular
tRXRa expression in cancer cells. In addition, we show that
triptolide selectively induces tRXRa degradation in cancer cells
grown in vitro and in animals through its activation of p38 mitogen-
activated protein kinase (p38 MAPK or p38). Furthermore, our
results show that triptolide-induced p38 activation impairs tRXRa
interaction with p85a, leading to inhibition of tRXRa-mediated
AKT survival pathway. Our findings also demonstrate that
triptolide enhances the apoptotic effect of chemotherapeutic
agents and when used together with TNFa it strongly activates
death receptor-mediated apoptotic pathway, showing a novel
mechanism for shifting TNFa signaling from survival to death.
Triptolide induces cancer cell apoptosis dependent on
intracellular tRXRa expression
We recently reported that tRXRa, an N-terminally truncated
form of RXRa, could strongly promote cancer cell growth
through activation of PI3K/AKT pathway . To further
characterize the tRXRa-regulating pathway, we screened a
natural product library of Chinese herbs for potential regulators.
Our results show that triptolide strongly induces cancer cell
apoptosis by regulating tRXRa expression and function.
We demonstrated that triptolide strongly induced growth
inhibition in some cancer cell lines such as MCF-7 breast cancer
cells, but with much less effect in others like SW480 colon cancer
cells. Fig. 1B showed that MCF-7 cells significantly responded to
triptolide at concentrations as low as 20 nM after 12 h treatment,
while much higher concentrations (.80 nM) of triptolide were
required to inhibit the growth of SW480 cells. Fig. 1C further
showed that triptolide could dose-dependently induce apoptosis
(PARP cleavage) in MCF-7 cells between 20 and 100 nM.
Interestingly, triptolide-induced cancer cell apoptosis was closely
associated with its decreasing tRXRa expression, while the levels
of the full-length RXRa remained largely unaffected (Fig. 1C).
The proteasome inhibitor MG132 was then used to evaluate the
effect of triptolide on modulating tRXRa stability. Fig. 1D showed
that triptolide-induced tRXRa reduction was greatly prevented by
MG132, indicating that triptolide induces proteasome-mediated
tRXRa degradation. To determine the role of tRXRa in
regulating the apoptotic effect of triptolide, various cancer cell
lines were recruited. Fig. 1E showed that tRXRa was highly
expressed in QGY-7703 and HepG2 liver cancer cells, MCF-7
breast cancer cells, and HeLa cervical cancer cells, while level of
tRXRa in SW480 colon cancer cells was hardly detectable. When
the apoptotic effect of triptolide was examined, we found that the
levels of tRXRa expression in these cancer cell lines were
associated with their responses to the killing effect of triptolide.
Triptolide-induced PARP cleavage was seen in the tRXRa-
expressing cells but not in SW480 cells lacking tRXRa (Fig. 1E).
In addition, triptolide showed no cytotoxic effect in non-cancerous
HEK293T cells, which did not express tRXRa, even at high
concentration of 100 nM (data not shown). To determine whether
the intracellular tRXRa expression was essential for the death
effect of triptolide, we transfected HeLa and MCF-7 cancer cells
with RXRa siRNA, which effectively reduced the expression of
both tRXRa and the full-length RXRa. Although the contribu-
tion of downregulation of the full-length RXRa to the apoptotic
effect of triptolide was unknown, siRNA-mediated inhibition of
tRXRa expression greatly impaired the effect of triptolide on
inducing PARP cleavage in both HeLa and MCF-7 cancer cells
(Fig. 1F). Consistently, when the apoptotic cells detected by DAPI
staining were quantified , we found that treatment of MCF-7
cells with 50 nM for 12 h resulted in 48% cell death, while siRNA-
mediated inhibition of tRXRa reduced this effect to about 23%.
Our results clearly demonstrate that triptolide-induced cancer cell
apoptosis is at least partially mediated by tRXRa.
Triptolide suppresses tRXRa expression and tumor
growth in animals
To further study the effect of triptolide on modulating tRXRa
expression in vivo, mice with HepG2 tumor xenografts were treated
with triptolide for 12 days. Administration of triptolide caused a
53.7% reduction of tumor volume (Fig. 2A) and extensive tumor
cell apoptosis as indicated with brown TUNEL staining (Fig. 2B).
Consistent with our in vitro observation, we showed that triptolide-
induced tumor growth inhibition was closely associated with its
inducing downregulation of tRXRa in the tumors (Fig. 2C). Our
results demonstrate that tRXRa in cancer cells is a potential
molecular target for the anticancer activity of triptolide in vivo.
Triptolide induces tRXRa-mediated AKT inactivation and
We previously reported that the oncogenic activity of tRXRa
was due to its activation of the AKT survival pathway . We
then investigated whether triptolide could inhibit tRXRa-
dependent AKT activation. Indeed, treatment of HepG2 liver
cancer cells with triptolide resulted in a sustained inhibition of
AKT phosphorylation from 6 h after treatment, which was closely
associated with its inducing tRXRa degradation (Fig. 3A). To
Triptolide Inhibits AKT Activation by tRXRa
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study the role of tRXRa in triptolide inactivation of AKT, HepG2
cells were transfected with RXRa siRNA. Fig. 3B showed that
treatment of HepG2 cells with 50 nM triptolide for 9 h completely
inhibited AKT phosphorylation, while knocking down tRXRa
expression by siRNA greatly impaired triptolide on inducing AKT
dephosphorylation. These studies demonstrate that tRXRa
expression is required for triptolide to inactivate AKT. Our results
showed that triptolide-induced tRXRa degradation and AKT
inactivation were closely associated with its apoptotic effect
(Fig. 3A). To determine whether triptolide inhibition of AKT
activity was responsible for its induction of apoptosis in cancer
cells, HepG2 cells transfected with a constitutive-active form of
Figure 1. Triptolide induces cancer cell apoptosis dependent on intracellular tRXRa expression. (A) The chemical structure of triptolide.
(B) Growth inhibitory effect. MCF-7 and SW480 cells were treated with various concentrations of triptolide as indicated. Cell viability was measured by
the MTT colorimetric assay. *, P,0.05; **, P,0.01 (vs respective controls). (C) The effect of triptolide on tRXRa expression and PARP cleavage was
examined in MCF-7 cells. The cells were treated with vehicle or increasing concentrations of triptolide for 9 h. (D) Triptolide induced proteasome-
mediated tRXRa degradation. MCF-7 cells were treated with 50 nM triptolide with or without 10 mM MG132, a specific proteasome inhibitor. The
impact of MG132 on tRXRa turnover was evaluated. (E) tRXRa expression was determined in various cancer cells as indicated. The apoptotic effects of
triptolide in different cells were compared. The cells were treated with vehicle or 50 nM triptolide for 9 h. (F) HeLa and MCF-7 cells were transfected
with scramble or RXRa siRNA and incubated with vehicle or 50 nM triptolide for 12 h. Triptolide-induced PARP cleavage was compared between
control and RXRa siRNA transfections. (G) MCF-7 cells transfected with scramble or RXRa siRNA were treated with 50 nM triptolide for 12 h and
subjected to DAPI staining. The apoptotic cells induced by triptolide were quantified and expressed as percentage of the counted cells.
Triptolide Inhibits AKT Activation by tRXRa
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AKT (CA-AKT) were treated with triptolide and the apoptotic
effect of triptolide was assayed. Fig. 3C showed that triptolide-
induced nuclear condensation and fragmentation frequently found
in untransfected cells were inhibited in CA-AKT transfected cells.
Consistently, triptolide-induced Bax activation as revealed by
immunostaining of cells with conformation-sensitive Bax/6A7
antibody  was also inhibited by CA-AKT expression (Fig. 3C).
Triptolide-induced AKT inactivation and apoptosis were also
reproducible in several other cancer cell lines including MCF-7
breast cancer cells and A549 lung cancer cells (data not shown).
Triptolide inhibits TNFa-induced AKT activation
TNFa is known to induce both apoptotic and survival pathways
. We previously showed that one of the survival signaling
pathways of TNFa was mediated by tRXRa-dependent AKT
activation . Interestingly, triptolide was shown to sensitize
tumor cells to TNFa-induced apoptosis . To investigate
whether triptolide could inhibit TNFa-induced AKT activation,
MCF-7 cells were treated with vehicle or 10 nM TNFa in the
presence or absence of triptolide. In agreement with previous
results , immunoblotting assays showed that TNFa strongly
induced AKT activation in these cells, which was inhibited by
triptolide in a dose-dependent manner (Fig. 4A). Consistently, the
inhibitory effect of triptolide on AKT activation was associated
with decrease of tRXRa expression (Fig. 4A). Such effects of
triptolide were also observed in A549 lung cancer cells (Fig. 4B).
The role of tRXRa in triptolide inhibition of TNF-induced AKT
activation was then determined by studying the effect of triptolide
on TNFa-induced tRXRa interaction with p85a, an event that
leads to activation of the PI3K/AKT pathway . Co-
immunoprecipitation assays showed that endogenous p85a in
MCF-7 cells could be immunoprecipitated together with tRXRa
by nN197 anti-RXRa antibody but not by IgG (Fig. 4C).
Interaction of p85a with tRXRa was enhanced by TNFa. When
cells were treated with triptolide, both basal and TNFa-induced
tRXRa interaction with p85a was strongly inhibited (Fig. 4C),
demonstrating that triptolide-induced inhibition of AKT activa-
tion is due to its inhibition of tRXRa interaction with p85a.
Triptolide induces mitochondrial-mediated caspase 9-
dependent apoptosis and activates caspase 8-dependent
apoptotic pathways by TNFa
To further determine the apoptotic effect of triptolide, we
examined caspase 8, 9 and PARP cleavages in MCF-7 cells.
Fig. 5A showed that triptolide strongly increased caspase 9 and
PARP cleavages, while it failed to activate caspase 8, indicating
that triptolide can alone induce mitochondrial-activated apoptosis.
Consistently, triptolide has been shown to be inefficient for
apoptosis induction in caspase 9 knock-out cells but remains
sensitive in caspase 8 deficient cells . TNFa is known to induce
not only cell survival and proliferation through its activation of
PI3K/AKT and IKK/NF-kB pathways [15,40] but also cell death
through its activation of death receptor-dependent apoptotic
pathway . We then determined whether the ability of
triptolide to inhibit TNFa activation of AKT could result in
TNFa activation of caspase 8-dependent apoptotic pathway .
Fig. 5A showed that TNFa alone could not induce PARP cleavage
and had no appreciable effect on caspases 8 and 9, consistent with
the notion that the apoptotic pathway of TNFa is usually
inactivated in cancer cells . However, when MCF-7 cells
were co-treated with triptolide and TNFa, we observed proteo-
lytical cleavage of caspase 8 into p43, p41, and p18 active forms,
suggesting that triptolide is able to activate TNFa-dependent
apoptosis pathway. Induction of TNFa-dependent apoptosis by
triptolide contributed to overall death effect of triptolide as TNFa
and triptolide combination resulted in synergistic apoptotic effect.
This was also illustrated by our observation that knocking down
caspase 8 expression by siRNA transfection impaired the
synergistic effect of triptolide and TNFa. Consistently, triptolide
was described to sensitize lung cancer cells to TNF-induced
(TRAIL) . Thus, these results demonstrate that the death
effect of TNFa can be induced by triptolide.
We then showed that targeting tRXRa by triptolide could also
significantly enhance the apoptotic responses of other chemother-
apies such as 5-Fu in HepG2 liver cancer cells (Fig. 5B) and
camptothecin in MCF-7 breast cancer cells (Fig. 5C). Both 5-Fu
and camptothecin could not alter the basal and triptolide-reducing
p38 is involved in triptolide inhibition of tRXRa-
dependent AKT activation
One way that triptolide-induced tRXRa degradation is through
its binding to the receptor protein. However, our classical ligand
competition binding assays failed to detect any binding of triptolide
to purified RXRa protein (data not shown). We then reasoned that
Figure 2. Triptolide induces tumor growth inhibition and
tRXRa degradation in vivo. (A) Nude mice with HepG2 heptoma
xenografts were intraperitoneally injected (i.p.) daily with saline or
0.2 mg/kg triptolide for 12 days. Tumor sizes and weights in control and
triptolide-treated mice were compared. **, P,0.01 (vs control). (B)
Tumor sections were stained for TUNEL by immunohistochemistry to
show the apoptotic effect of triptolide. (C) The whole lysates prepared
form HepG2 xenografts treated with triptolide or vehicle were
subjected to Western blotting assays for detecting tRXRa expression.
**, P,0.01 (vs control).
Triptolide Inhibits AKT Activation by tRXRa
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triptolide might act indirectly to modulate the stability of tRXRa
the effect of triptolide on MAPK signal transduction pathways in
HepG2 cells revealed that triptolide could strongly activate Erk1/2,
p38, and JNK1/2 (Fig. 6A). The time course assays showed that
activity and PARP cleavage. To study the causal role of p38 in
triptolide modulation of tRXRa-dependent AKT activity, HepG2
p38 inhibitor SB203580, while JNK inhibitor SP600125 and
ERK1/2 inhibitor PD98059 were similarly used for comparison.
Fig. 6B showed that triptolide-induced tRXRa degradation and
SP600125 and PD98059, demonstrating that p38 is involved in
regulating tRXRa turnover and apoptosis by triptolide. Consistent-
ly, knocking-down p38 by siRNA transfection reduced the
inhibitory effects of triptolide on tRXRa stability, AKT activation
and PARP cleavage (Fig. 6C). Furthermore, we observed that
presence of SB203580 (Fig. 6D). Together, our results demonstrate
that p38 activation by triptolide is essential for its inactivation of
tRXRa-dependent AKT pathway and its apoptotic effect.
We recently demonstrated that truncated RXRa, tRXRa,
resulted from limited proteolytic cleavage of RXRa in several
human tumors as well as in a number of cancer cell lines, confers
tumor growth advantage due to its activation of PI3K/AKT
survival signaling . Here, we report that triptolide isolated
from Chinese medicinal herb Trypterygium wilfordii Hook F is a new
regulator of tRXRa-mediated signaling pathway.
We show that the levels of tRXRa in cancer cells determine
their apoptotic responses to triptolide (Fig. 1E and F). Triptolide
strongly induces PARP cleavage in tRXRa-expressing cells
including QGY-7703 and HepG2 liver cancer cells, MCF-7
breast cancer cells, and HeLa cervical cancer cells, while it has
little effect in SW480 colon cancer cells and HEK293T non-
cancerous cells that express trace amount of tRXRa (Fig. 1E and
Figure 3. Triptolide inhibits tRXRa-dependent AKT activity and induces cancer cell apoptosis. (A) HepG2 cells were treated with vehicle
or 50 nM triptolide for various time intervals as indicated. Time-dependent effects of triptolide on AKT activity, tRXRa degradation and PARP cleavage
were examined. (B) Effect of RXRa siRNA. HepG2 cells transfected scramble or RXRa siRNA were treated with vehicle or 50 nM triptolide for 9 h. The
effect of siRNA-mediated knocking down tRXRa expression on triptolide-inducing AKT dephosphorylation was studied. (C) Effect of CA-AKT. HepG2
cells were transiently transfected with active form of AKT expression vector (GFP-CA-AKT) and treated with 80 nM triptolide for 12 h. Apoptotic cells
(condensed and fragmentated) induced by triptolide were recognized by DAPI staining, while Bax activation was detected by conformation-sensitive
Triptolide Inhibits AKT Activation by tRXRa
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data not shown). Knocking down tRXRa expression by siRNA
greatly impairs the death effect of triptolide in cancer cells (Fig. 1F
and G). These findings suggest that tRXRa protein serves as one
of the important targets of triptolide action.
Our results reveal that the apoptotic effect of triptolide in cancer
cells is closely associated with its inducing tRXRa degradation in
vitro (Fig. 1) and in vivo (Fig. 2). Targeting tRXRa for degradation
by triptolide results in reduction of AKT activity (Fig. 3A). In
addition, triptolide strongly inhibits basal and TNFa-induced
AKT activity through disrupting the interaction between tRXRa
and p85a (Fig. 4C). Triptolide inactivation of tRXRa-dependent
AKT is critical for its apoptotic induction, which is illustrated in
Fig. 3D showing that triptolide-induced cancer cell apoptosis and
activation of pro-apoptotic molecule Bax are inhibited by
transfection of constitutive-active AKT.
Interestingly, the effect of triptolide on tRXRa stability and
AKT inactivation is due to its activation of p38 rather than
through directly binding to tRXRa. Our time-course assays show
that AKT inactivation by triptolide is closely correlated with its
activation of p38 (Fig. 6A). Inhibition of p38 by p38 siRNA
transfection or treatment with the p38 inhibitor SB203580
diminishes the effects of triptolide on inducing tRXRa degrada-
tion and inhibiting tRXRa-mediated AKT activation (Fig. 6B and
C). In addition, triptolide-induced inhibition of tRXRa interaction
with p85a is blocked by SB203580 (Fig. 6D). Although triptolide
also strongly activates JNK and Erk1/2, inhibition of both kinases
does not exert significant effect on tRXRa stability and cancer cell
apoptosis (Fig. 6B and C). p38 is typically a stress-activated kinase
Figure 4. Triptolide inhibits TNFa-induced AKT activation. (A, B)
The effect of triptolide on TNFa-induced AKT phosphorylation was
determined in MCF-7 cells (A) and A549 cells (B). Cells were treated with
vehicle or 10 ng/ml TNFa in the absence or presence of increasing
concentrations of triptolide for 12 h. (C) Co-immunoprecipitate assays
were carried out in MCF-7 cells to determine tRXRa interaction with
p85a. The cells were treated with vehicle or 50 nM triptolide in the
absence or presence of 10 ng/ml TNFa for 6 h. Cell lysates were
immunoprecipitated with DN197 anti-RXRa antibody. The coimmuno-
precipitates were then subjected to Western blotting analysis for tRXRa
expression and its co-precipitated p85a by nN197 anti-RXRa and anti-
p85a antibodies respectively.
Figure 5. Triptolide enhances the apoptotic effect of TNFa and
other chemotherapies. (A) MCF-7 cells were transfected with caspase
8 siRNA to evaluate whether triptolide could activate TNFa-dependent
death effect. Untransfected and transfected cells were treated with
vehicle or 50 nM triptolide with or without 10 ng/ml TNFa for 12 h.
Expression and cleavages of caspase 8, 9 and PARP were analyzed. (B, C)
Triptolide-enhanced the apoptotic effect of 5-Fu and camptothecin was
examined in HepG2 (B) and MCF-7 cells (C) respectively. Cells were
treated with 50 nM triptolide alone or in combination with 10 mM 5-Fu
or 10 mM camptothecin for 9 h.
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that promotes inflammation, and is frequently deregulated in
cancers, in which it exerts both tumor suppressive and promoting
effects [42,43]. Interestingly, the apoptotic effect of p38 is often
antagonized by AKT, and it is suggested that the cell fate is often
determined by the balance of AKT and p38 activities . Our
findings reveal that activation of p38 by triptolide results in
suppression of AKT activity and cancer cell apoptosis through
mediation of tRXRa, a novel mechanism for balancing the
activities of p38 and AKT.
We also demonstrate that targeting tRXRa by triptolide
strongly activates TNFa death signaling. TNFa is a multifunc-
tional cytokine that plays roles in diverse cellular events such as cell
survival and death [38,40]. Although TNFa can be a potent
death-inducing factor of cancer cells, its killing effects are often
Figure 6. Triptolide induces tRXRa degradation and AKT inactivation through activation of p38. (A) Triptolide induced activation of
several MAPK pathways. HepG2 cells were treated with vehicle or 50 nM triptolide for various time intervals as indicated. Triptolide-induced time-
dependent phosphorylation of p38, JNK and Erk1/2 was compared to its effect on decreasing AKT phosphorylation and PARP cleavage. (B) HepG2
cells were treated with 50 nM triptolide for 9 h with or without p38 inhibitor SB203580 (10 mM), JNK inhibitor SP600125 (10 mM) or Erk1/2 MAPK
inhibitor PD98059 (10 mM). The impact of inhibition of the individual pathways on tRXRa degradation and PARP cleavage was determined. (C) HepG2
cells transfected with scramble or p38 siRNAs were treated with vehicle or 50 nM triptolide for 9 h. The effect of siRNA-mediated p38 inhibition on
triptolide inactivation of AKT and tRXRa degradation was assayed. (D) HepG2 cells were treated with vehicle or 50 nM triptolide for 9 h in the
presence or absence of SB203580. The lysates were immunoprecipitated with DN197 anti-RXRa antibody and analyzed for its co-immunoprecipitated
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antagonized by its survival function that is mainly mediated by
activation of the NF-kB and PI3K/AKT pathways .
Triptolide was shown previously to sensitize cancer cells to
TNFa-induced apoptosis . We show here that triptolide
activates TNFa-dependent caspase 8-mediated apoptosis through
targeting tRXRa oncogenic protein (Fig. 4 and 5). The
combination of TNFa and triptolide results in stimulation of both
extrinsic and intrinsic apoptotic pathways, thus contributing to
greater apoptotic effect in cancer cells. The tRXRa-dependent
apoptotic effect of triptolide also significantly promotes the
anticancer activity of other chemotherapies such as 5-Fu, which
is shown to use Fas/FasL pathway [44,45] and requires thymine
DNA glycosylase for its anticancer activity , and camptothe-
cin, which potently disrupts DNA processing by inhibition of
topoisomerase I .
In summary, we demonstrate that triptolide serves as an
important regulator of tRXRa-mediated cancer cell survival
pathway by targeting the tumor-specific tRXRa protein through
an interesting novel signaling interplay between p38 and AKT.
. However, their significant side effects still limit these
compounds for clinical use. Thus, our findings provide useful
molecular basis for developing improved triptolide-based cancer
Materials and Methods
Lipofectamin 2000 was purchased from Invitrogen. Goat anti-
rabbit and anti-mouse secondary antibodies conjugated to
horseradish peroxidase and enhanced chemilumienescence(ECL)
reagents were from Thermo. Polyclonal antibodies against RXRa
(DN197), AKT1/2/3 (H-136), Cyclin D1 (H-295), and monoclo-
nal antibodies against Bax (6A7), GFP (B-2), c-Myc (9E10), GFP-
and FITC-labeled anti-rabbit IgG were from Santa Cruz
Biotechnology. Polyclonal antibodies against p38 and PARP,
and monoclonal antibodies against p-AKT (D9E), cleaved caspase
8 (Asp391), p-p38 (3D7), Erk1/2 (C-16), p-Erk1/2 (D13.14.4E), p-
JNK (81E11), and JNK (2C6) were from Cell Signaling
Technology. Polyclonal p85a antibody was from Millipore and
anti-mouse IgG conjugated with Cy3 from Chemicon. Monoclo-
nal antibodies against glyceraldehyde-3-phosphatedehydro-genase
(GAPDH) and b-actin, and chemicals including tripotide,
SB203580, TNFa, epidermal growth factor (EGF) were from
Sigma. Protein A beads were from GE Healthcare and
polyvinylidene difluoride (PVDF) membrane from Millipore.
TUNEL kit was from Roche. The cocktail of proteinase inhibitors
were from Amersham.
HepG2 (ATCC HB-8065), MCF-7 (ATCC HTB-22), HeLa
(ATCC CCL-2), A549 (ATCC CCL185), SW480 (ATCC CCL-
228), HEK293T (ATCC CRL-11268) and QGY-7703 (from
Institute of Biochemistry and Cell Biology, SIBS, CAS) .
Several siRNA oligos were synthesized (Ribobio Co, Guangz-
hou, China). siRNA sequence for p38 used in this study is: 59-
GGAATTCAATGATGTGTAT-39, while ERK1/2 siRNAs in-
clude a mixture of the following sequences: 59-CGTCTAATA-
TATAAATATA-39, 59-CCCTGACCCGTCTAATATA-39, 59-
TAACATA-39. The sequences for RXRa siRNA (M-003443-
02), caspase 8 siRNA (J-003466-14), and control siRNA (D-
001206-09-05) were described previously .
Cell Culture and Transfection
(FBS) in a humidified atmosphere containing 5% CO2at 37uC.
Subconfluent cells with exponential growth were used throughout
the experiments. Transfections were carriedout byusing Lipofecta-
mine 2000 according to the instructions of the manufacturer.
Confluent cells cultured in 96-well dishes were treated with
various concentrations of triptolide for 12 h. The cells were then
incubated with 2 mg/ml MTT for 1 h at 37uC and dissolved with
1 ml of dimethyl sulfoxide. Cell viability was measured based on
MTT dye conversion at 570 nm.
MCF-7 cells grown on 35-mm culture dishes were transfected
with RXRa siRNA or scramble siRNA. After 48 h of transfection,
cells were incubated with vehicle or with 50 nM triptolide in
serum-free medium for 12 h. Detached and attached cells were
collected for DAPI staining. Apoptotic cells were counted as
previously described .
Nude mice (BALB/c, SPF grade, 16–18 g, 4–5-week old) were
housed at 28uC in a laminar flow under sterilized conditions. Mice
were subcutaneously implanted with 200 ml HepG2 cell suspen-
sion (56106cells/per mouse). Mice were intraperitoneally injected
with 0.2 mg/kg triptolide or vehicle daily after 7 days of
transplantation. Food consumption, body weight and tumor sizes
of mice were measured every other day. Mice were scarified after
12-day drug treatment and the tumors removed for various
assessments. The study was approved by the ethics committee of
Tumor sections of HepG2 xenografts were stained with TUNEL
instructions (In situ Cell Death Detection Kit; Roche). The effect of
AKT on modulating the apoptotic effect of triptolide was
determined in HepG2 liver cancer cells transfected with GFP-CA-
80 nM triptolide for 12 h. The slides were incubated with anti-Bax
(6A7, 1:100) antibody and detected by anti-mouse IgG conjugated
with Cy3 (1:100). Cells were co-stained with 4969-diamidino-2-
phenylindole (DAPI) to visualize nuclei. The images were taken
under a fluorescent microscope (Carl Zeiss).
Cells were lysed in buffer containing 50 mM Hepes-NaOH
(pH 7.5), 2.5 mM EDTA, 100 mM NaCl, 0.5% NP40, and 10%
glycerol, with 1 mM DTT and proteinase inhibitor cocktail.
Whole cell lysates were subjected to immunoprecipitation with
anti-RXRa (DN197) as described .
A cocktail of proteinase inhibitors were included in all protein
purification. Equal proteins were electrophoresed on an 8% SDS-
PAGE gel and transferred onto PVDF membranes. The
membranes were incubated with primary and secondary antibod-
Triptolide Inhibits AKT Activation by tRXRa
PLoS ONE | www.plosone.org8April 2012 | Volume 7 | Issue 4 | e35722
ies as indicated and detected using ECL system. The antibodies
used in these assays included: RXRa (DN197; 1:1000), PARP
(1:1000), b-actin (1:10000), GAPDH (1:1000), Myc (1:2000), GFP
(1:1000), AKT (1:1000), p-AKT (1:500), p85a (1:1000), cyclin D1
(1:1000), caspase 8 (1:500), p38 (1:1000), p-p38 (1:500), Erk
(1:1000), p-Erk1/2 (1:2000), JNK (1:1000) and p-JNK (1:1000). All
data provided in the results are representative of at least three
Isolation and purification of triptolide
Triptolide was isolated from the roots of Chinese herb
Tripterygium wilfordii Hook F (TWHF) and its structure was
identified using a combination of chromatographic techniques
and nuclear magnetic resonance analysis. The purity of triptolide
used in this study was more than 98%. Triptolide was dissolved in
DMSO and stored as a stock at 1022M at 280uC. The working
concentrations of triptolide and the vehicle controls used in this
study contained 0.1% DMSO, a concentration which did not alter
Data were expressed as mean 6 SD from three or more
experiments. Statistical analysis was performed using Student’s t-
test. Differences were considered statistically significant with
Conceived and designed the experiments: NL JXL JL XKZ JZZ.
Performed the experiments: NL JXL JL CZ FJ HW LC WZ XC GW
TY HZ BL XY. Analyzed the data: NL JXL XKZ JZZ. Wrote the paper:
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