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Introduction
Prostate cancer is the most common cancer in males, and
the second leading cause of male cancer deaths in
developed countries[1]. Current prostate cancer therapies such as
surgery, chemotherapy, and radiation therapy are of limited
efficacy and may not avoid the significant side-effects.
Androgen reduction therapy is commonly used to control
hormone-sensitive tumor cells; however, hormone refractory
clones often emerge after hormonal
therapy[2]. Advanced hormone refractory prostate cancer is almost
incurable[3]. Therefore, novel effective therapies,
including biotherapy, are urgently needed to be developed.
In a search for alternative and preventive therapies for
prostate cancer, attention has been focused on plant
poly-phenolic compounds, which are rich in
nutritional supplements such as soybeans, garlic, and green tea, and have
been used to augment anticancer
therapies[4,5]. Curcumin, the active component of turmeric, is a dietary constituent
that has received a great deal of attention recently as a
chemoprotective agent[6,7]. Several recent observations have
shown that curcumin has antioxidant and anti-inflammatory
activities, as well as anticarcinogenic activity in colon cancer,
breast cancer[8,9], and
leukemia[10]. The molecular mechanisms of the actions of curcumin are beginning to be
elucidated, including the effects of suppressing tumor
initiation and promotion in animal models. Curcumin is a potent
inhibitor of cycloxygenase-2, lipoxygenase, ornithine
decarboxylase[11_13], c-Jun/AP-1, nuclear
factor-κB[14,15], c-Jun N-terminal kinase, and protein kinase
C[16,17]. Curcumin inhibited epidermal growth factor receptor activity in various
tumors, including prostate
carcinoma[18]. Curcumin can cause a marked decrease in cell proliferation and microvessel
density, and an increase in apoptosis in prostate
tumors[19]. By virtue of its multiple effects, curcumin has potential
clinical application in the prevention of prostate cancer.
NKX3.1 has been identified as the androgen-regulated
NK-class homeobox gene that is largely specific to prostate
for expression, and is thought to play an important role in
normal prostate organogenesis and
carcinogenesis[20_23]. Human
NKX3.1 has been mapped to human chromosome 8p21, a locus that experiences a frequent loss of
heterozygosity in human prostate cancer, raising the possibility that
NKX3.1 may be a tumor suppresser
gene[22_24]. It has been reported that the loss
of a single allele may predispose to prostate carcinogenesis.
As a gene regulated by androgen, the maintaining
expression of NKX3.1 in the prostatic epithelium is dependent
on androgen signaling[20,22_23]. However, it is not very clear
whether this regulation occurs directly through the
interaction of the androgen receptor (AR) and the
NKX3.1 promoter.
In this study, an experimental investigation was
undertaken to characterize the effect of curcumin on
NKX3.1 expression in the prostate cancer cell LNCaP, and to
investigate the mechanisms in which curcumin downregulates the
homeobox gene NKX3.1 in the prostate cancer cell.
Materials and methods
Cell culture and treatments The human prostate cancer
cell line LNCaP was obtained from the American Type
Culture Collection (Manassas, Virginia, USA). This cell line was
established from a lymph node metastasis of a prostate
cancer patient and expressed mutant, but functional on AR. The
LNCaP cells were seeded in 35 mm culture dishes in RPMI-
1640 medium supplemented with 10% fetal bovine serum
(FBS) and 5% CO2 at 37 °C until reaching approximately
50%_70% confluence. The cells were maintained in serum-free
RPMI 1640 medium for a further 24 h before the experiments.
The cells were then treated with designated curcumin, R1881,
or flutamide in RPMI 1640 medium containing 5% FBS.
Curcumin (No C1386, Sigma, St Louis, MO, USA) and
flutamide (No F9397-1G, Sigma, St Louis, MO,
USA ) were dissolved in dimethyl sulfoxide, which is also a control
vehicle.
RT-PCR analysis Total RNA was extracted from the
LNCaP cells with Trizol reagent (MBI Fermentas, Burlington,
ON, Canada) following the manufacturer's instructions, and
the expression of NKX3.1 mRNA was determined by RT-PCR
using M-MuL V reverse transcriptase in the presence of
random hexamer primers. The PCR primers for
NKX3.1 mRNA were as follows: 5'-GTACCTGTCGGCCCCTGAACG-3'
(sense), and 3'-GGACCAGAGGCACATA TTGTCG-5' (antisense). A 500 bp
β-actin mRNA was amplified and used to normalize the quantity of the
NKX3.1 mRNA in RT-PCR. The primers for the AR were as follows:
5'-TCTCAAGAG-TTTGGATGGCTCC-3' (sense) and
5'-TCACTGGGTGTGG-AAATAGATG -3'
(antisense)[25]. The PCR profiles consisted
of initial denaturation at 94 °C for 3 min, followed by 28 cycles
of denaturation at 94 °C for 40 s, primer-annealing at 61 °C for
40 s, and primer extension at 72 °C for 40 s. The final primer
extension was performed at 72 °C for 10 min. The PCR
products were analyzed by electrophoresis on a 1.5% agarose gel
containing ethidium bromide and photographed under UV
light.
SDS-PAGE and Western blot analysis The LNCaP cells
were harvested at designated times, and nuclear extraction
was prepared using the protocol described
by a nuclear extraction kit (Active Motif, Carlsbad, CA, USA ). Protein
concentrations of cell nuclear extracts were quantified by the
bicinchoninic acid method. For Western blot analysis, 40 µg
of protein was separated on 10% SDS-PAGE and then
transferred to the nitrocellulose membrane. After being blocked
and washed, the membrane was incubated with human
specific anti-NKX3.1 antibody or anti-AR antibody (gifts from
Dr Charles YOUNG, Department of Urology Research, Mayo
Clinic, Rochester, MN, USA) at 4 °C for 12 h, followed by
incubation with horseradish peroxidase-labeled second
antibody for 1 h at room temperature. Immunoreactive bands
were then visualized by enhanced chemiluminescence (Santa
Cruz, San Diego, CA, USA). β-tubulin or β-actin (Sigma, St.
Louis, MO, USA ) was used to normalize the quantity of the
protein on the blot.
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared from the LNCaP cells using a nuclear
extraction kit (Active Motif, Carlsbad, CA, USA ) according
to the manufacturer's instructions. Equal amounts of sense
and antisense oligonucleotides were mixed and annealed in
a buffer (10 mmol/L Tris-HCl, pH 8.0, 200 mmol/L NaCl, and 1
mmol/L EDTA) by heating to 95 °C for 5 min and slowly
cooling to room temperature. The corresponding
oligonucleotides were labeled with digoxigenin
(DIG). The following oligonucleotides were used for the EMSA experiments:
the ARE decoy based on the deduced ARE sequence at the
promoter region of the human prostate-specific antigen (PSA)
gene[26], and the E2F decoy which contained the 8 bp
cis-element (underlined) that was identified in the c-myc
promoter[27].
ARE: sense 5'-TGCAGAACAGCAAGTGCTAGC-3',
antisense 5'-GCTAGCACTTGCTGTTCTGCA- 3';
NF-κB: sense 5'-GCCTGGGAAAGTCCCCTCAACT-3',
antisense 5'-AGTTGAGGGGACTTTCCCAGGC-3';
E2F: sense 5'-TGATTTCCCGCGGAT-3',
antisense 5'-ATCCGCGGGAAATCA-3'.
Binding reactions were carried out at room temperature
for 30 min in a mixture containing 4% glycerol, 1 mmol/L
MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 50
mmol/L NaCl, 10 mmol/L Tris·HCl, 2 µg poly (dI-dC), 10 µg nuclear
extracts, and a DIG-labeled oligonucleotide probe. Then the
reaction mixtures were subjected to electrophoresis in 5%
non-denaturing polyacrylamide gels in
0.25×Tris/borate/EDTA buffer. Based on the instructions of the DIG Gel Shift
Kit (Roche Co, Basel, Switzerland), electroblotting and
chemiluminescent detection were performed. The specificity of
AR-ARE binding was confirmed by adding 125 fold excess
of unlabeled ARE\E2F\NF-κB probe to the assay.
Transient transfection assay The LNCaP cells were
plated in a 24 well plate until they reached
a confluency of 90%. The cells were transiently transfected with a 1040 bp
NKX3.1 promoter-luciferase reporter plasmids
pGL3-NKX3.1[28] using
LipofectamineTM 2000 reagent (Invitrogen, Carlsbad,
CA, USA), while pRL-TK (Promega, Madison, WI, USA)
plasmids were used as a internal control. Twenty-four hours
after the transfection, the cells were treated with different
concentrations of curcumin for an additional 12 h. Whole
cell extracts were prepared and a luciferase assay was
performed according to the manufacturer's
instructions (Pro-mega, USA) for the
NKX3.1 promoter/luciferase transfection. Each transfection
was done 3 times and standard deviations
were calculated.
Statistical analysis All the measurement data were
analyzed and expressed as the mean±SEM. Results were
considered significant if P<0.05 was obtained by an appropriate
ANOVA procedure and Student's t-test.
Results
Curcumin inhibited the expression of the NKX3.1
gene To determine whether NKX3.1 expression levels changed
with curcumin treatment in the androgen-sensitive prostate
cancer cell LNCaP, expression of NKX3.1 was determined in
serum-starved LNCaP cells exposed to varying
concentrations of curcumin for 24 h by RT-PCR and Western blotting.
As shown in Figure 1A, NKX3.1 mRNA expression was
significantly downregulated by 40 µmol/L of curcumin
treatment (by ~5 fold), and less effect was observed in the cells
treated with 10 and 20 µmol/L curcumin.
To further confirm the inhibitory effect of curcumin on
NKX3.1 protein expression, Western blotting was carried
out. Since NKX3.1 is a nuclear protein and functions in the
nucleus, we prepared the nuclear extracts for Western
blotting after the LNCaP cells were exposed to curcumin for 24 h.
The results in Figure 1B show that the expression of the
NKX3.1 protein dramatically decreased by curcumin in a
dose-dependent manner, similar to its mRNA as shown in
Figure 1A.
To test whether the inhibitory effect of curcumin on
NKX3.1 expression occurs at the transcription level, we transfected a
vector containing a 1040 bp NKX3.1 promoter fragment in
the upstream of a luciferase reporter gene into the LNCaP
cells. As seen in Figure 1C, for the LNCaP cells exposed to
varying amounts of curcumin for 24 h, the
NKX3.1 promoter gave a gradual inhibition of the luciferase activity
(represent-ing NKX3.1 promoter activity) in a dose-dependent manner.
Taken together, curcumin could inhibit the expression of
NKX3.1 at the promoter, mRNA, and protein levels.
Curcumin inhibited androgen-mediated induction of
NKX3.1 expression Because
NKX3.1 is known as an androgen upregulated gene, we examined the nonmetabolizable
androgen R1881 and the AR antagonist flutamide to see if
they can influence expression of the NKX3.1 protein in LNCaP
cells exposed to curcumin. In Figure 2, we show that the
expression of NKX3.1 could be enhanced by R1881 (lane 2),
which was partially inhibited by the AR antagonist flutamide
(lane 3) without curcumin treatment of LNCaP cells. The
results demonstrated that NKX3.1 protein expression was
upregulated by androgen and AR activity without curcumin
treatment. When the LNCaP cells were exposed to curcumin
(30 µmol/L), the NKX3.1 protein had a notable decrease
(lane 4), and R1881 no longer stimulated
NKX3.1 protein expression significantly (lane 5), which suggests that
curcumin may have disrupted the AR function so that R1881
lost its stimulation on NKX3.1 expression. Furthermore,
curcumin and flutamide together could further decrease the
NKX3.1 protein (lane 6). Our data suggests that the
expression of NKX3.1 was partially dependent on the AR signaling
pathway, and curcumin could repress androgen and
AR-mediated induction of NKX3.1 expression.
Curcumin inhibited expression of the AR
gene To further elucidate the role of AR in curcumin-induced
NKX3.1 depression, we evaluated the effect of curcumin on AR
expression in LNCaP cells directly. Both RT-PCR and
Western blot analysis gave very similar results (Figure 3),
indicating that AR expression significantly decreased in mRNA
(Figure 3A) and protein (Figure 3B) levels by curcumin in a
dose-dependent manner. Remarkably, the effect of curcumin
on AR expression was almost identical to that on
NKX3.1 expression, as shown in Figure 1.
Curcumin decreased ARE binding activity The above
results led us to further investigate whether the function of
the AR could be affected by curcumin. We used the gel
band-shift technique as an in vitro functional assay to
determine the AR DNA binding activity. The results in Figure 4
show that ARE binding activity was dramatically inhibited
after 24 h of treatment with 30 and 40 µmol/L of curcumin
when compared to the control. The bands were confirmed to
be a result of specific binding for ARE, because the
DNA-protein complex was competed out by a 125 fold molar
excess of unlabeled ARE and could not be blocked by a 125
fold excess of unlabeled E2F and NF-κB oligonucleotides.
Discussion
Curcumin, used as a food additive and a herbal medicine
in Asia, is associated with a plethora of beneficial effects on
human health, predominant among which are the
anti-inflammatory and cancer chemoprophylaxis activities. Early works
have shown that curcumin could suppress the activation of
novel eukaryotic transcriptional factors, such as AP-1,
NF-κB and AR in prostate cancer[29,30], and significantly inhibit
the growth of prostate cancer cells in
vivo[19] and in
vitro[18]. The AR is a member of the nuclear receptor
superfamily of transcription
factors[31]. It is activated by its androgen ligand
or by a ligand-independent
manner[32_34]. Subsequently, the activated receptor homodimerizes and interacts with
specific androgen response elements in the regulatory regions
of androgen target genes, resulting in the stimulation of gene
expression. NKX3.1 is an androgen-regulated homeobox
gene in the prostate[22]. In the present study, we first
demonstrated the inhibitory effects of curcumin on
NKX3.1 expression in LNCaP cells and investigated the mechanisms.
In this study, we used 10_40 µmol/L of curcumin to treat
the prostate cancer cell LNCaP that expresses both
NKX3.1 and the AR. With RT-PCR, Western blotting, and luciferase
reporter analysis, we found that curcumin downregulated
the expression of the NKX3.1 gene at both mRNA and
protein levels in a dose-dependent manner. To further
investigate whether or not this inhibitory effect is androgen-AR
mediated, the LNCaP cells were treated with curcumin only
or curcumin and synthetic androgen analog R1881. The
results in Figure 2 show that curcumin could decrease the
NKX3.1 protein and inhibit the stimulation of
NKX3.1 expression by androgen, suggesting that curcumin could
repress the androgen-mediated induction of
NKX3.1 expression. Furthermore, the effects of curcumin on AR
expression and its DNA binding activity were detected by
Western blotting and EMSA to demonstrate the mechanisms
in which curcumin inhibits androgen-mediated induction of
NKX3.1 expression. The results show that curcumin
decreased AR expression significantly at both mRNA and
protein levels, and blocked AR-ARE binding activity in
EMSA. Our data suggests that curcumin can repress the
androgen-mediated induction of NKX3.1 expression by
inhibiting AR gene expression and blocking its DNA binding
activity.
In the EMSA experiment, we used the known ARE
sequence in the upstream of the PSA gene to show that
curcumin could block AR DNA binding activity and make
AR lose its function, disrupting the androgen-AR signaling
pathway. NKX3.1 is proved to be a prostate-specific and
androgen-regulated gene, and it was reported that there are
several potential AR binding sites in the regulatory region of
the NKX3.1 gene[35,36], but so far, there is no identification of
functional ARE in NKX3.1 to be reported. Further
investigation will be required to detect functional ARE in the
regulatory regions of the NKX3.1 gene and to determine if the ARE
is the actual function target of curcumin.
The inhibition of NKX3.1 gene expression by curcumin
seems to be inconsistent with the classical idea that the
antitumor agent increases the expression of the tumor
suppressor gene. NKX3.1 is supposed to be a tumor
suppressor gene; however, its function is mainly related to the
development and differentiation of normal prostate.
Evidence of NKX3.1 being involved in the prostate tumor and
progression mainly come from animal
models[37_41]. The reports on the roles of NKX3.1 in human prostate cancer are
pendent, and some reports are inconsistent. Bowen
et al showed that loss of NKX3.1 expression in human prostate
cancers correlated with tumor
progression[27], while Aslant et
al reported that the decline of NKX3.1 expression was not
correlated with prostate cancer progression and was not
associated with advanced stage. Thus,
NKX3.1 expression is not a clinically-valuable prognostic
factor[42]. However, overexpression of
NKX3.1 in prostate cancer also has been
reported[43]. So the roles of
NKX3.1 on prostate cancer in clinics needs to be further studied.
In summary, our results demonstrate that curcumin, as
an anticancer agent, can downregulate the expression of
NKX3.1 which is a prostate-specific and androgen-regulated
homeobox gene, and can intervene the AR signaling
pathway as well as repress the androgen-mediated induction of
NKX3.1 expression by inhibiting AR gene expression and
blocking its DNA binding activity.
Acknowledgement
The authors are grateful to Dr Charles YOUNG
(Depart-ment of Urology Research, Mayo Clinic, Rochester, MN,
USA) for providing the anti-NKX3.1 antibody and anti-AR
antibody.
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