Overexpression of the orphan receptor Nur77 and its translocation induced by PCH4
may inhibit malignant glioma cell growth and induce cell apoptosis
Li-Fu Chang1#, Po-Cheng Lin1#, Li-Ing Ho2, Po-Yen Liu3, Wan-Chen Wu4, I-Ping Chiang5, Hui-Wen Chang5, Shinn-Zong Lin6, Yeu-Chern Harn7, Horng-Jyh Harn5*, and Tzyy-Wen Chiou1*
1
Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University, Hualien, Taiwan, R.O.C.
2
Department of Respiratory Care, Veterans General Hospital-Taipei, Taipei, Taiwan, R.O.C. 3
Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan, R.O.C. 4
Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, R.O.C. 5
Department of Pathology, China Medical University Hospital, Taichung, Taiwan, R.O.C. 6
Center for Neuropsychiatry, China Medical University and Hospital and Beigang Hospital, Taichung and Yun-Lin, Taiwan, R.O.C.
7
Graduate Institute of Networking and Multimedia, National Taiwan University, Taipei, Taiwan, R.O.C.
# The authors contribute equally in this study.
*Corresponding authors:
Dr. Tzyy-Wen Chiou, Ph.D.
Department of Life Science and Graduate Institute of Biotechnology, National Dong Hwa University
No. 1, Sec. 2, Da Hsueh Rd., Shoufeng, Hualien, Taiwan, Republic of China. Tel: 886-3-8633638
Fax: 886-3-8630262
E-mail address: twchiou@mail.ndhu.edu.tw
Dr. Horng-Jyh Harn
China Medical University & Hospital
2 Yuh-Der Road, Taichung, Taiwan, 40447, ROC
Tel: 886-4-22052121 ext. 2661; Fax: 886-4-22052121 ext. 2566
E-mail: dukeharn@www.cmuh.org.tw
Abstract
Background
In previous study, n-butylidenephthalide (BP), a natural compound from Angelica
sinensis, has anti-glioblastoma multiform (GBM) cell effects. In this study, we modified BP
structure to increase anti-GBM cell effects. The anti-GBM cell effects of one derivative of BP,
(Z)-N-(2-(dimethylamino)ethyl)-2-(3-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)phenoxy)a
cetamide(PCH4) were tested in vitro and in vivo.
Methods
MTT assay and PI/Annexin V assay were performed to evaluate the anti-GBM effects of PCH4. The Nur77 expression and translocation were assayed by RT-PCR and western blot. The
Nur77 siRNA was used to down-regulate the Nur77 expression. The JNK inhibitor (SP600125)
was used to block the JNK pathway
Results
The anti-GBM effect of PCH4 is four times more than BP. The IC50 of PCH4 on
DBTRG-05MG cells was 50 g/ml. Nur77 expression and translocation from the nucleus to
the cytoplasm were important in PCH4-induced apoptosis. Furthermore, the down-regulation
of PCH4-induced Nur77 expression by Nur77 siRNA reduced PCH4-induced apoptosis. In
addition, PCH4-induced apoptosis was associated with the JNK pathway. The JNK inhibitor,
Conclusions
In conclusion, PCH4, a derivative of BP, induced Nur77-mediated apoptosis via the JNK
pathway and this mechanism, which is different from that of BP, may explain the increase in
the antitumor effects on GBM.
Key words: glioblastoma multiform, the derivative of n-butylidenephthalide,
(Z)-N-(2-(dimethylamino)ethyl)-2-(3-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)phenoxy)a
Introduction
Glioblastoma multiform (GBM) is the most aggressive type of CNS gliomas, accounting
for 53.8% of all CNS gliomas cases (Porter et al., 2010). In our previous study,
n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica
sinensis, has antitumor effects in vitro and in vivo (Tsai et al., 2006). In vitro, GBM cells
treated with BP undergo cell cycle arrest at the G0/G1 phase and Nur77-mediated apoptosis,
which occurs via the protein kinase C (PKC) pathway (Tsai et al., 2006; Lin et al., 2008a). In
hepatocellular carcinoma cells, BP also induces apoptosis by inhibiting protein kinase B
(AKT) and the activation of the cAMP response element binding protein (CREB) pathway
(Chen et al., 2008a). In addition, Nur77-mediated apoptosis is associated with c-Jun
N-terminal kinases (Han et al., 2006).
The transcription factor Nur77 is a member of the nuclear hormone receptor family and
plays a role in apoptosis (Liang et al., 2007; Lee et al., 2009; Yang et al., 2009). In T cells, the
expression of Nur77 is induced by T cell receptor activation and increases T cell apoptosis (Li
et al., 2006). In addition, in prostate cancer cells, GBM cells, hepatocellular carcinomas, and
lung cncer cells, Nur77 expression also induces apoptosis (Li et al., 1998; Chintharlapalli et
al., 2005; Chen et al., 2008a; Lin et al., 2008b). The apoptotic effects of Nur77 were
discovered during the study of CD437, a retinoid-related molecule that induces apoptosis (Li
Apoptosis induced by Nur77 is due to its translocation from the nucleus to the
mitochondria through the activation of the JNK, PKC, and CREB pathways or the inhibition
of the AKT pathway. Nur77 then interacts with Bcl-2 to form a pro-apoptosis complex.
Finally, cytochrome c is released from the mitochondria and apoptosis occurs (Moll et al.,
2006; Chen et al., 2008b; Lin et al., 2008a). In addition, activation of the ERK pathway also
plays an important role in cadmium-induced Nur77 expression and apoptosis in lung cancer
A549 cells (Shin et al., 2004).
To synthesize the most effective and least toxic derivative of BP, we examined the
relationship between the structure and the corresponding activity. Among 32 synthetic
derivatives, the antitumor activity of PCH4 is four times than BP and PCH4 is more soluble.
Thus, we examined the role of Nur77 in the antitumor effects of PCH4.
The aim of this study was to elucidate the relationship between the structure and the
corresponding activity of one synthetic derivative of BP. The synthetic derivative of BP,
PCH4, had the most powerful antitumor effect of the 32 compounds, and thus, we assessed
the role and mechanism of Nur77-induced apoptosis after GBM cells were treated with PCH4.
Materials and Methods
Cell line and cell culture
Bioresources Collection and Research Centerin the Food Industry Research and Development
Institute (BCRC, Hsin Chu, Taiwan). The cells were maintained in RPMI-1640 medium
(Gibco, California, USA) containing 10% FBS, 0.01 M HEPES, and 1 mM sodium pyruvate
in a 37ºC incubator containing 5% CO2.
Chemicals and Reagents
Derivatives of BP were dissolved in DMSO at a concentration of 50 mg/ml and stored at
-20ºC. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in
phosphate buffer at a concentration of 5 mg/ml and stored at 4ºC. Carmustine, MTT, DMSO,
and cadmium acetate were purchased from Sigma (St. Louis, USA).
MTT assay
In this study, cell viability was determined by an MTT assay as previously described (Lin
et al., 2008b). Briefly DBTRG-05MG or GBM 8401 cells were seeded at 3 × 103 cells/well
for 24 hrs. Cells were then treated with different concentrations of PCH4 (12.5, 25, 50, 75,
100, 125, 150 g/ml) for 24 hr. Finally, the purple crystals were dissolved in DMSO, and the
absorbance was detected with an ELISA Reader at an absorption wavelength of 570 nm.
After treatment with the various chemicals, the cells were harvested and resuspended in
binding buffer (Becton, Dickinson and Company,NJ, USA). The PCH4 treated
DBTRG-05MG cells were harvested and labeled with 10 mg/ml Annexin V-FITC and 20
mg/ml propidium iodide (PI) (Becton, Dickinson and Company,NJ, USA). After labeling,
cells were analyzed with flow cytometry (FACScan; BD Biosciences).
RNA extraction
For analysis of mRNA expression, RT-PCR was used. DBTRG-05MG and GBM 8401 cells
were treated with 50 g/ml of PCH4 for 0.5, 1, 3, or 6 hr. Total RNA was extracted and
purified using an RNeasy mini kit (Qiagen, California, USA). RNA quality was confirmed
with electrophoresis.
RT-PCR
cDNAs were produced from 1 μg of total RNA using a Reverse-iT first strand
synthesis kit (ABgene, Epsom, UK). The cDNA was amplified with Nur77, Nurr1, NOR1,
and GAPDH primers using polymerase chain reaction. The primers are as follows: Nur77;
(Forward) 5′-CGACCCCCTGACCCCTGAGTT-3′ and (Reverse)
5′-GCCCTCAAGGTGTTGGAGAAGT-3′.
5′-GGTAAAGTGTCCAGGAAAAG-3′ NOR1; (Forward)
5′-TCTGCCTTCCAAACCAAAG-3′ and (Reverse) 5′-TGATGGAAAGTCTGAGGAC-3′.
glyceraldehyde-3-phosphate dehydrogenase; (Forward)
5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ and (Reverse)
5′-CATGTGGGCCATGAGGTCCACCAC-3′. The PCR conditions were: initial denaturation
at 95ºC for 10 min, 35 cycles of denaturation at 95ºC for 30 sec, annealing at 60ºC for 30 sec,
extension at 72ºC for 1 min, and a final extension step at 72ºC for 10 min. The PCR products
were then analyzed on a 2% agarose gel.
Cell fractionation
Nuclear, cytoplasmic, and mitochondrial fractions were separated using a mitochondrial
fractionation kit (Active Motif, California, USA). GBM 8401 cells were treated with 50 μg/ml
PCH4 for 6, 12, 24, or 48 hr. The cells were detached with 0.05% trypsin, washed once with
PBS, and resuspended in cold 1× cytosolic buffer for 15 min on ice. Cells were homogenized
with a homogenizer on ice for 30-50 strokes and passed through a 22-gauge needle. The
nuclei were recovered by centrifugation at 800 × g for 20 min at 4ºC. After the centrifugation,
the supernatant were mitochondria and cytoplasm fractions. The nuclear pellet was lysed in
lysis buffer (Intron biotechnology, joongang induspia, korea) on ice for 30 min. After the
mitochondrial and cytoplasmic fractions were separated. The mitochondria fraction was the
pellet and the supernatant was the cytoplasmic fraction. Finally, the mitochondrial fraction
was lysed in mitochondria buffer (Active Motif, California, USA).
Western blot analysis
Total cell protein was extracted using PRO-PREPTM protein extraction solution (INtRON,).
Briefly, the cell pellets were lysed with protein extraction solution and incubated at -20ºC for
20 min. Then, the cell lysates were centrifuged at 15000 × g for 5 min, and total protein was
collected. The protein concentration was measured using a protein assay kit (Strong Biotech,
Taipei, Taiwan). 20 μg of total protein were separated on a 10% SDS-PAGE gel and
transferred to PVDF membrane. Non-specific binding was blocked with 5% skim milk.
Proteins of interest were detected with primary antibodies to Nur77, phospho-ERK, ERK,
phospho-p38, p38, phospho-SAPK/JNK, SAPK/JNK, phospho-AKT, AKT, phospho-PKC,
cytochrome c, and β-actin (Cell signaling, Danvers, USA). The primary antibodies were
detected with horseradish peroxidase–conjugated anti-mouse, anti-rabbit, or anti-goat as
appropriate for 1 hr at 25ºC. Bound HRP–conjugated secondary antibody was visualized with
an enhanced chemiluminescence (ECL) Plus system.
siRNA specific for Nur77 (5′-CAGUCCAGCCAUGCUCCUCUU-3′) was purchased from
Santa Cruze. GBM 8401 cells were transfected with siRNA (25, 50, and 100 nM) using
lipofectamine. After 48 hr, the expression of Nur77 was measured using RT-PCR.
Transfection of the luciferase reporter system
GBM 8401 cells were co-transfected with 4 μg of plasmid consisting of the Nur77 promoter
linked to luciferase and 0.4 μg of plasmid pRL-TK using lipofectamine 2000. After 48 hr, the
transfected cells were treated with 60 μg/ml PCH4 or 1 μM CD437 for 6 hr. Promoter activity
was assayed using a dual luciferase assay kit (promega, WI, USA). Luciferase activity was
normalized to renilla luciferase expressed by the plasmid pRL-TK.
Results
PCH4 inhibits the growth of GBM cells in vitro and induces apoptosis
The MTT assay revealed that PCH4 inhibited the growth of DBTRG-05MG and GBM 8401
cells in a dose-dependent manner in vitro. The IC50 of PCH4 on DBTRG-05MG and GBM
8401 cells was 50 g/ml and 65 g/ml, respectively, after 24 hr of PCH4 treatment (Fig. 1).
Cells treated with PCH4 exhibited shrinkage and fragmentation of chromosomes. The
apoptotic effects of PCH4 were evaluated with PI/Annexin V staining and flow cytometry.
(Fig. 2). When DBTRG-05MG cells were treated with different concentration of PCH4 for 24
hrs, the percentage of apoptotic cells were increased with the increased concentration of
83.9% respectively.
PCH4 induces mRNA expression of Nur77, Nurr1, and NOR1
We examined mRNA expression of Nur77 in PCH4-treated DBTRG-05MG cells by using
RT-PCR. Nur7 was induced by treatment with PCH4. Nur77 was clearly upregulated 3 hr and
30 min, respectively, after PCH4 treatment. NOR1 was upregulated slightly 3 hr after PCH4
treatment (Fig. 3). Because Nur77 is implicated in apoptosis and growth inhibition,
upregulation of Nur77 mRNA may be associated with PCH4-induced apoptosis.
PCH4 induces apoptosis and Nur77 expression in DBTRG-05MG cells via the JNK pathway
To determine whether MAPK, JNK, or CREB play a role in PCH4-induced apoptosis,
DBTRG-05MG cells were treated with 75 g/ml of PCH4 for 0, 15, 30, 60, or 180 min and
then analyzed using western blotting. We found that pJNK and pERK were upregulated
following PCH4 treatment but the expression of pPKC, pAKT, and AKT were not changed
(Fig. 4).
Next, we used a JNK inhibitor (SP600125) or an ERK inhibitor (U0126) to examine
whether the PCH4-induced Nur77 expression occurred via the JNK or ERK pathways. To
confirm that SP600125 inhibited the JNK pathway, we performed western blotting for
SP600125 or U0126. When we used SP600125 in PCH4-treated DBTRG cells, pJNK and
pcJun were downregulated in a dose-dependent manner (Fig. 4b). In addition, PCH4-induced
Nur77 expression was decreased when cells were pre-treated with SP600125 (Fig. 4c).
Further, PCH4-induced apoptosis was decreased when PCH4-treated DBTRG cells were
pre-treated with 10-50 M SP600125 (Fig. 4d). However, PCH4-induced apoptosis was not
blocked when we used U0126 (data not shown).
PCH4-induced Nur77 migrates from the nucleus to the cytoplasm
We examined whether Nur77 migrated from the nucleus to the cytoplasm. Localization of
Nur77 was observed by immunofluorescence microscopy. In control cells, Nur77 was mainly
located in the nucleus. In PCH4-treated cells, Nur77 translocated from the nucleus to the
cytoplasm (Fig. 5a). To confirm the translocation of Nur77, cytosolic and nuclear fractions
were examined with western blot analysis. Nur77 was principally located in the nuclear
fraction before PCH4 treatment (Fig. 5b). However, Nur77 was progressively increased in the
cytoplasmic fraction during PCH4 treatment (Fig. 5b).
PCH4-induced apoptosis in DBTRG cells is triggered by Nur77 expression
To determine whether Nur77 is crucial in PCH4-induced apoptosis, Nur77 siRNA was used to
dose-dependent manner (Fig. 6a). Next, to determine the role of Nur77 in PCH4-induced
apoptosis, we used PI and Annexin V staining to identify apoptotic cells after treatment with
Nur77 siRNA and PCH4. Fewer apoptotic cells were observed after PCH4 treatment of
DBTRG cells transfected with Nur77 siRNA, and these effects were time dependent. When
100 nM Nur77 siRNA was used, the percent of PCH4-induced apoptotic cells was
significantly decreased from 36% to 16% (p<0.05) (Fig. 6b). Thus, PCH4 induced apoptosis
via Nur77 in GBM 8401 cells.
PCH-4-induced Nur77 expression occurs via the AP-1 binding site in the Nur77 promoter
Sequence analysis of the Nur77 promoter suggested that this gene may contain cis-acting
elements and an AP-1 binding site, which may play an important role in PCH4-induced
apoptosis. To determine the role of the AP-1 binding site in PCH-4-induced Nur77 expression,
plasmids containing mutations in nucleotides -496/+67 in the Nur77 promoter and a wild-type
control were constructed (Fig. 7a). The promoters were placed upstream of luciferase cDNA.
These constructs were transfected into DBTRG cells, which were treated with vehicle or
PCH4. The plasmid pRL-TK was used as an internal control for adjusting transfection
efficiency. Transfection of the wild-type plasmid (pNur77 496/+67) and PCH4 treatment
resulted in a 2.5-fold induction of luciferase activity (Fig. 7b). To examine the role of the
mutated AP-1 site. Significantly lower luciferase activity was observed following PCH4
treatment (~91%) (Fig. 6b). This result suggested that AP-1 may play an important role in
PCH4-induced Nur77 expression.
PCH4 inhibits the growth of xenografted DBTRG cells in nude mice associated with Nurr77
expression
To evaluate the anti-malignant glioma activity of PCH4 in vivo, 2 × 106 DBTRG cells were
injected subcutaneously into the dorsal subcutaneous tissue of nude mice. After the tumor size
reached about 80~120 mm3, mice were randomly divided into three groups: control, PCH4 50
mg/kg, and PCH4 100 mg/kg (n = 5 per group). Control mice were subcutaneously injected
with vehicle (DMSO) for five successive days. Mice in the PCH4 50 mg/kg group and PCH4
100 mg/kg group were subcutaneously injected with the indicated dose of PCH4 for five
successive days. Tumor growth was inhibited in the PCH4 50 mg/kg group and the PCH4 100
mg/kg group (Fig. 8a). Human DBTRG tumors treated with 50 mg/kg or 100 mg/kg PCH4
showed an upregulation of Nur77 and caspase 3 expression 5 days after treatment (Fig. 8b). In
addition, western blot analysis showed that Nur77 and cleaved caspase 3 protein were
Discussion
Alkylating agents such as carmustine (BCNU) and temozolomide are clinically available for
malignant glioma therapy. Their mechanism of action against malignant glioma is DNA
methylation on guanine base. Because the mechanism is non-specific, these drugs are
cytotoxic to normal cells. In addition, the usage of these alkylating drugs is sometimes
limited due to the drug resistance resulting from the expression of O6-methylguanine
methyltransferase (MGMT) in gliomas (Sharma et al., 2009). Although it is previously
reported that the MGMT expression of 30-60% gliomas is lowered by epigenetic silence and
is consequently sensitive to the alkylating drugs (Weller et al., 2010), there is still a
considerable portion of gliomas which can not be effectively treated by these alkylating drugs.
The development of a new targeted drug is therefore urgent.
Traditional Chinese herbs, which contain many unique and biomedically powerful compounds,
are a rich source of therapeutic candidates. Recent examples include paclitaxel and
camptothecin. n-butylidenephthalide, which is extracted from A. sinensis, is a natural
compound that has been investigated for its antitumor effect on GBM cells both in vitro and
in vivo. However, synthesis of a rationally designed n-butylidenephthalide derivative is
necessary to increase the cytotoxic effects on GBM cells.
Nur77 is a unique orphan member of the nuclear receptor family and is the most potent
n-butylidenephthalide induces cell apoptosis in GBM cells via targeting of Nur77 to
mitochondria (Lin et al., 2008b). This phenomenon was also investigated in other types of
cancer cells including lung, prostate, ovary, colon, and stomach tumor cells (Liu et al., 1994;
Uemura et al., 1995; Stocco et al., 2002; Shin et al., 2004). We showed that apoptosis of
GBM cells induced by n-butylidenephthalide requires Nur77 expression and that Nur77
translocates from the nucleus to the cytoplasm where it interacts with Bcl-2. This interaction
results in a conformational change in Bcl-2 that converts it into a cytotoxic molecule. These
findings suggest that n-butylidenephthalide derivatives that induce cytoplasmic localization of
Nur77 and its interaction with Bcl-2 may preferentially kill cancer cells.
In this study, we synthesized several new n-butylidenephthalide derivatives (Table 1) to
increase the solubility of the drug, increase the cytotoxicity on GBM cells, and identify
compounds that modulate the Nur77-mediated apoptotic pathway. Among these derivatives,
we found that the n-butylidenephthalide derivative, PCH4, was more soluble than
n-butylidenephthalide. PCH4 inhibited proliferation and induced apoptosis of GBM cells. In
addition, Nur77 was involved in the PCH4-induced apoptosis. Therefore, PCH4 may be a new
potential targeting drug in malignant glioma therapy.
In other studies, Nur77 was shown to be an oncogenic survival factor expressed in the nucleus
of cancer cells. However, following apoptotic stimulation, Nur77 translocates from the
apoptosis (Li et al., 2000).
In our study, we found that PCH4-induced Nur77 expression was upregulated in a
time-dependent manner using an RT-PCR assay. GBM cells underwent apoptosis after PCH4
treatment. We next examined PCH4-induced expression of Nur77 and the translocation of
Nur77 in apoptotic GBM cells (Figs. 2 and 4). PCH4-induced Nur77 expression was
increased 1 hr after PCH4 treatment and translocation of the Nur77 protein was observed at 6
hr.
We compared PCH4 and BP induced-Nur77 expression, which was previously studied.
BP-induced Nur77 expression was highest at 3 hr and was decreased at 6 hr. PCH4-induced
Nur77 expression was also highest at 3 hr, but it was still high at 6 hr, unlike BP-induced
Nur77 expression. Because Nur77 was associated with tumor cell apoptosis, we hypothesize
that PCH4 was more effective than BP because of the prolonged Nur77 expression at 6 hr.In
the animal study, we used PCH4 to treat nude mice with a GBM xenograft and the anti-GBM
activity of PCH4 is dose dependent. In addition, we also observed that the Nur77 and cleaved
caspase3 expression were accompanied with increasing dosage of PCH4. Interestingly, the
tumor size in PCH4-treated animals did not increase substantially during the first 12 days.
However, the tumor size began to increase after first 12 days. This phenomenon may be due
to the metabolism time of PCH4. Thus prolonging PCH4 treatment may be required in further
Because Nur77 was associated with tumor cell apoptosis, we decreased Nur77 expression
using Nur77 siRNA. When we used 100 nM Nur77 siRNA to downregulate Nur77 expression,
PCH4-induced apoptosis was reduced by 78.1%. This result demonstrates that Nur77 is a
major pathway in PCH4-induced GBM cell apoptosis.
The induction of Nur77 expression has been studied in several cell types. Nur77 expression
was rapidly induced by nerve growth factor in PC12 cells via calcium ions (Milbrandt, 1988),
by cadmium in human lung cancer cell lines via extracellular signal-regulated kinase and
protein kinase A (Shin et al., 2004), and by PGF2a and butaprost in human embryonic kidney
293/EBMA cells via protein kinase C (Liang et al., 2004). In addition, a recent report
indicated that activation of JNK or inhibition of the AKT pathway induces the translocation of
Nur77 from the nucleus to the cytoplasm in other cancer cells (Han et al. 2006). Thus,
regulation of Nur77 expression may involve a variety of intracellular signaling pathways that
depend on different stimuli.
In this study, we found that PCH4-induced apoptosis was also associated with the JNK
pathway. When GBM cells were treated with PCH4, we found that components of the JNK
pathway were upregulated at 15 min. After the JNK pathway was upregulated, Nur77
expression was increased at 1 hr and then GBM cells underwent apoptosis. When we used a
JNK inhibitor (SP600125), PCH4-induced apoptosis was reduced by about 60%, and Nur77
signaling pathway in PCH4-induced apoptosis. In our previous study (Lin et al., 2008b), BP
induced Nur77 expression through the PKC pathway, but in this study, we found that PCH4
induced Nur77 expression through the JNK pathway. PCH4 may bind to a different receptor
than BP and lead to Nur77 expression that lasts longer than the expression that occurs through
the PKC pathway.
There are potential cis-acting elements in the Nur77 promoter region. A region from -496 to
-334 was identified that contains enhancers that are responsive to prostaglandin F2 and
butaprost via the PKC pathway (Liang et al., 2004). The major PKC signal response element
in the Nur77 promoter is an AP-1-like element (Kim et al., 2005), whereas a portion of the
Nur77 promoter (-496 to -67) containing four AP-1 motifs has also been reported (Uemura et
al., 1995). In this study, we examined whether the AP-1 motif is involved in PCH4-induced
apoptosis, and studied the transcriptional mechanisms of PCH4-induced Nur77 mRNA
expression. We used a Nur77 promoter (-496 to +67) containing these four AP-1 motifs,
which was subcloned into a luciferase reporter plasmid. Following transfection with a plasmid
containing the wild-type Nur77 promoter, PCH4 treatment resulted in a 2.5-fold increase in
luciferase activity, compared with vehicle control (Fig. 7). To investigate whether these AP-1
motifs are functional during PCH4 treatment, two AP-1 motifs (-197 to -192; -179 to -173)
were mutated in the Nur77 promoter using the protocol of Kim et al. Mutated AP-1 motifs
suggests that PCH4 treatment led to an increase in Nur77 expression through AP-1 motifs.
In summary, the mechanisms of the antitumor activity of PCH4 were studied. We found that
PCH4 induced Nur77-mediated apoptosis in GBM cells. The JNK signaling pathway was
implicated in the regulation of PCH4-induced apoptosis via AP-1 motifs in the Nur77
promoter. These results suggest that Nur77 may be a target gene for PCH4 and that this drug
may be useful for targeting malignant glioma in the clinic.
Acknowledgements
This work was supported by a grant from National Science Council of the Republic of China
(96-2320-B-039-044-MY3).
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