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,
From the * Department of Urology and the
Molecular Image Center, Chang Gung Memorial
Hospital, and the School of Nursing and the
Department of Anatomy, Chang Gung University,
Kwei-Shan, Tao-Yuan, Taiwan, Republic of China.
| Correspondence to: Dr Horng-Heng Juang, Department of Anatomy, Chang Gung University, 259 Wen-Hua 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan, Republic of China (e-mail: hhj143{at}mail.cgu.edu.tw). |
| Received for publication January 7, 2008; accepted for publication July 24, 2008. |
 |
Abstract |
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Key words: IL-6, androgen receptor, PSA, PDEF, R1881
Curcumin (diferuloylmethane), a yellow curry pigment obtained from the
plant Curcuma longa Linnaeus, has been well documented as a potent
anti-inflammatory, anticarcinogenic, and antioxidant agent
(Bemis et al, 2006). The
evidence from epidemiological and preclinical perspectives has been carried
out with respect to the effects of curcumin in cancer chemoprevention
(Thomasset et al, 2007).
Serious studies have revealed the therapeutic potential of curcumin in
androgen-dependent and androgen-independent prostatic carcinoma cells (Dorai
et al, 2000,
2001,
2004). Studies suggested that
curcumin, which acts as the inhibitor of nuclear factor kappa B (NF-
B)
and nuclear transcription factor activator protein-1 (AP-1), down-regulated
cell survival mechanisms in human prostate cancer in vitro
(Chen and Tan, 1998;
Mukhopadhyay et al, 2001;
Deeb et al, 2004). Other
studies indicated that curcumin and curcumin analogs down-regulated the gene
expression of androgen receptors in androgen-sensitive prostate carcinoma
LNCaP cells (Nakamura et al,
2002; Ohtsu et al,
2002).
Several in vitro studies have revealed factors including androgen,
interlukin-6 (IL-6), and adriamycin that modulated PSA gene expression
(Jia et al, 2004; Tsui et al,
2004a,b).
Some transcriptional factors such as the androgen receptor (AR), p53,
NF-B, prostate-derived Ets factor (PDEF), and Sp family genes are
involved in the gene modulation of PSA in LNCaP cells, and most of the
transcriptional factor interacted with androgen receptor in the process of PSA
promoter regulation (Oettgen et al,
2000; Chen and Sawyers,
2002; Tsui et al,
2004a).
Our objectives for this study were to determine the regulatory mechanisms of curcumin on gene expression of PSA with or without the stimulation of androgen. We also evaluated the mechanisms of curcumin on the gene expression of PSA by interacting with other transcriptional factors other than the androgen receptor in the human prostatic carcinoma LNCaP cells.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Proliferation Assay
Cell proliferation in response to curcumin was measured with a
nonradioactive assay as described before (Hsieh and Juang, 2006). Cells (5000
cells/well) were grown in 100 mL of RPMI-PRF medium with 5% CD-FCS for 2 days.
Cells were incubated with 100 mL of 0, 5, 10, 20, 40, and 80 µM curcumin in
the same medium for 24, 48, and 72 h. Cells were than incubated with freshly
prepared
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS)/phenazine methosulfate (ratio 1:1 by volume) solution (Promega
Biosciences, San Luis Obispo, California) for 3 hours at 37°C in a
humidified 5% CO2 atmosphere.
Immunoblot Assay
LNCaP cells were incubated in the RPMI-PRF medium with 5% CD-FCS and
different drugs for a period of 24 hours. Equal quantities of cell extract (40
mg) were loaded onto a 12% sodium dodecyl sulfate polyacrylamide gel and
analyzed by the electrochemiluminescent detection system as described before
(Tsui et al, 2006). The rabbit
anti-human PDEF serum was prepared from our laboratory. The pcDNA3-PDEF (see
below) was digested with EcoRI and NotI, and the linearized
plasmid DNA was ligated with the pET32a (Novogen, La Jolla, California). The
fusion protein, after lysis of the bacterial pellet in IB buffer (20 mM
Tris-HCl, 10 mM EDTA, 1% Triton X-100, and 100 µg/mL lysozyme), was
purified through Ni-NTA beads (Qiagen, Hilden, Germany) and eluted following
the manufacture's instruction. Rabbits (New Zealand White) were immunized with
the PDEF-His-Tag fusion protein (57 kDa). The fusion protein was emulsified
with an equal volume of complete Freund adjuvant for the first injection, and
with incomplete Freund adjuvant for 3 subsequent booster injections. The
results of immunoblot assay for the whole-cell extract of LNCaP cells revealed
1 major band at 37 kDa and 1 weak band at 50 kDa, which represented the
glycosylation form of PDEF from this rabbit polyclonal antibody raised against
the human PDEF. The blotting membranes were probed with 1:200 diluted
polyclonal PSA antiserum (DAKO), 1:500 diluted human androgen receptor
antiserum (N-19; Santa Cruz Biotechnology, Santa Cruz, California), 1:1000
diluted β-actin antiserum (C11, Santa Cruz Biotechnology), or 1:5000
dilute PDEF antiserum. The intensity of different bands were analyzed by
GeneTools of ChemiGenius (Syngene, Cambridge, United Kingdom).
Reverse Transcription Polymerase Chain Reaction
The total RNA was isolated with Trizol reagent, and cDNA was synthesized as
described before (Hsieh and Juang, 2006). The primers were used for the
amplification of sequences specific to the androgen receptor
(Juang et al, 2004) and PDEF
as shown in the Table. The cDNA
quality was verified by performing controlled reactions with primers derived
from β-actin-P and β-actin-R
(Table). The polymerase chain
reaction (PCR) was carried out in a thermal cycler (Thermolyne, Dubuque,
Iowa), and the parameters were 30 cycles of 94°C for 0.5 minutes, 55°C
for 1 minute, and 72°C for 1 minute. The PCR products were separated by 2%
agarose gel electrophoresis and visualized by ethidium bromide staining under
the image capture and analysis system of ChemiGenius (Syngene).
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Reporter Vector Constructs
The promoter of the PSA gene was isolated from the PAC clone (LLNLF-214C7;
Human Genome Mapping Project Resource Centre, Hinxton, Cambridge, United
Kingdom). The PAC clone was digested with HindIII, and one 6-kbp DNA
fragment was subcloned into the pGL3 reporter vector (Promega Bioscience). The
reporter vectors (pPSAH, –41 to –5874; pPSABHE, –4801 to
–3933 and –41 to –589; pPSAKH, –41 to –1557;
pPSABH, –41 to –589) containing the 5' flanking region of
the human PSA gene were cloned by 5' deletion or PCR as described before
(Tsui et al, 2004b). The
promoter of the PDEF gene was isolated from the BAC clone (RP11-375E1;
Invitrogen, Carlsbad, California). The BAC clone was digested with
SacI, and one 3.7-kbp DNA fragment was subcloned into the pGEM11
vector (pGEM11-PDEF; Promega Bioscience). The human PDEF promoter/enhancer DNA
fragment was synthesized by PCR with 2 primers (T7 and PDEFpromR
5'-AAGCTTAGCCGCGAGATGAAGAGTTGGC-3') from the pGEM11-PDEF vector.
The DNA fragment (–1 to –3280), which was digested with
HindIII and SacI, was ligated with the reporter vector pbGL3
(Promega Bioscience). Proper ligation and orientation were confirmed by
extensive restriction mapping and sequencing.
Expression Vector Constructs and Transient Overexpression
The full-length human PDEF cDNA vector (MGC: 4546840) was purchased from
Invitrogen. A DNA fragment containing the coding region of human PDEF cDNA was
synthesized by reverse transcription (RT)–PCR with 2 primers (PDEFP,
5'-GAATTCATGGGCAGCGCCAGCCCGGGTC-3', and PDEFR,
5'-GCGGCCGCTCAGATGGGGTGCACGAACTGG-3') from the PDEF cDNA vector.
The overexpression vector, pcDNA3 (Invitrogen), was digested with
EcoRI and NotI, and the linearized plasmid DNA was ligated
with the human PDEF cDNA fragment after digestion with EcoRI and
NotI, resulting in the insertion of the full-length PDEF cDNA into
the polyadenlyate region that was controlled by the CMV promoter
(pcDNA3-PDEF). Proper ligation and orientation were confirmed by extensive
restriction mapping and sequencing. The expression vector was introduced into
LNCaP cells by electroporation. Ten million cells were suspended in 20 µg
of pcDNA3-PDEF and pcDNA3, respectively, and in 500 µL of RPMI medium
without serum in a 4-mm gap cuvette. Electroporation was conducted with the
ECM 830 (BTX, San Diego, California) set at 170 V with a 70-ms pulse length
and with the use of a single pulse setting. Cells were maintained in RPMI
medium with 10% FCS for 3 days and then in RPMI-PRF medium with 5% CD-FCS for
another 2 days before further experiments.
Transient Transfections and Reporter Assay
LNCaP cells (1 x 104 cells/well) were plated onto 24-well
plates 1 day before transfection. Cells were transiently transfected with
TransFast transfection reagent as described before
(Tsui et al, 2006). To
eliminate the variable degrees of efficiency of reporter activity, cells were
transfected with same amount of plasmid in each well by adding the same
concentration of pcDNA3 vector. Reporter vector–transfected LNCaP cells
were then treated with different drugs in RPMI-PRF medium with 5% CD-FCS as
indicated for an additional 24 hours.
Enzyme-Linked Immunosorbent Assay
LNCaP cells were incubated with 1 mL of different concentrations of drugs
as indicated in RPMI-PRF medium with 5% CD-FCS in a 6-well plate (2 x
105 cells/well) for a period of 24 hours. After incubation, the
culture media from each well were collected for PSA assays. Cell pellets were
washed twice with ice-cold phosphate-buffered saline (PBS) and then dissolved
in 500 µL of PBS. After sonication for 10 seconds, the cell extracts were
centrifuged at 23 000 x g for 20 minutes. The PSA levels in 20
mL of the cell supernatant or culture media were measured by PSA enzyme-linked
immunosorbent assay (ELISA), as described previously
(Tsui et al, 2004a). The PSA
level in each sample was adjusted by the concentrations of protein in the
whole-cell extract, which was measured by a BCA protein assay kit.
Electrophoretic Mobility Shift Assay
An electrophoretic mobility-shift assay (EMSA) was performed as previously
described (Tsui et al, 2008).
The double strands of DNA fragment containing the putative androgen response
element (ARE) of the PSA gene (5'-TGCAGAACAGCAAGTGCTAGC-3') was
5' end-labeled with 
-P32–adenosine triphosphate
with the use of T4 polynucleotide kinase. The nuclear material of LNCaP cells
after treatment with different concentrations of curcumin for 12 hours was
extracted with the NE-PER nuclear and cytoplasmic extraction reagents as
described by the manufacturer (PIERCE). The 5' end-labeled ARE (ARE
probe; 5 nM) was incubated with 2 µg of nuclear extract (NE) from LNCaP
cells in 20 µL of binding buffer (25 mM HEPES buffer, pH 7.9, 50 mM KCl,
0.05 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and
10% glycerol) containing 0.5 µg of poly(dI-dC)-poly(dI-dC). Protein-DNA
complex formation was analyzed in 4% polyacrylamide gel by
autoradiography.
Statistical Analysis
Results are expressed as 
±
SE of at least 3 independent replications of each experiment. Statistical
significance was determined by a paired t test analysis with the
SigmaStat program for Windows version 2.03 (SPSS Inc, Chicago, Illinois).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Transient gene expression assay with the use of the PSA reporter vector (pPSABHE), which contained the ARE DNA fragment, indicated that 1 nM R1881 enhanced about 40-fold of the reporter activity; however, 20 µM curcumin treatment significantly blocked the stimulation of the androgen (Figure 2A). Results from 5' deletion reporter assays indicated that the effects of curcumin appeared to be mediated via a region located at –4801 to –3911 bp, a region containing the ARE, upstream of the translational starting point of the PSA gene. The EMSA assay indicated that curcumin treatments blocked the effect of androgen on the gene expression of PSA by decreasing the binding activity of the androgen receptor on the ARE of the PSA gene (Figure 2B).
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B, our study
also determined the effect of NF-B on the gene expression of PSA. Our
strategy was to use an inhibitor of the tumor necrosis factor
alpha–induced transcription factor NF-B (IKB)
overexpression vector to block NF-B nuclear translocation in the cells.
When LNCaP cells were cotransiently transfected with the inhibitor of kappa B
(IB) overexpression vectors and PSA reporter vector (pPSABHE), which
contains both the putative ARE and the NF-B response element, the
reporter assay indicated that overexpression of the NF-B inhibitor
alpha (IB
) vector decreased the promoter activities in a
dose-dependent manner. Moreover, adding 20 µM curcumin further inhibited
the promoter activities of PSA reporter vector
(Figure 2D). Transient gene expression assays revealed that forced overexpression of PDEF significantly enhanced the promoter activity with the use of a PSA reporter vector (pPSABHE) in LNCaP cells (Figure 3A). The results of RT-PCR and immunoblot assays indicated that transiently overexpressed PDEF induced PSA gene expression (Figure 3B). Results from ELISA revealed that transiently overexpressed PDEF significantly up-regulated both intracellular PSA levels and PSA secretion (Figure 3C). The results of immunoblot assays indicated that curcumin itself did not block gene expression of PDEF; moreover, the reporter assay with a PDEF reporter vector also confirmed that curcumin treatments did not affect the gene expression of PDEF in LNCaP cells (Figure 3D).
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Results from RT-PCR revealed that curcumin blocked the stimulation of 20 ng/mL of IL-6 treatment on the expression of the AR gene; moreover, results from the immunoblot assay also revealed that curcumin treatment blocked the stimulation of IL-6 on AR and PSA gene expression (Figure 4A). Results from ELISA indicated that when LNCaP cells were treated with 20 ng/mL of IL-6, the intracellular PSA levels increased about 2-fold; however, curcumin treatment (20 µM) blocked PSA biosynthesis by stimulation of IL-6 in LNCaP cells (Figure 4B). The transient gene expression assay also showed similar results, demonstrating that curcumin treatment blocked up-regulation of PSA promoter activity by IL-6 (Figure 4C).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Curcumin also was found to induce apoptosis in LNCaP cells through
suppression of NF-B activity
(Mukhopadhyay et al, 2001).
Therefore, it was speculated that curcumin might block gene expression of PSA
by suppressing NF-B activity. Previous studies have indicated that
NF-B activates PSA expression in LNCaP cells and NF-B binding
sites found in the PSA core enhancer, which is also a region containing the
ARE (Chen and Sawyers, 2002).
In our study, we used the superrepressor IB (S32A/S36A), a
dominant negative inhibitor of NF-B, to abolish NF-B activity,
indicating that overexpression of IB inhibited PSA promoter
activity in a dose-dependent way; however, even when NF-B activity in
the LNCaP cells was blocked by overexpression of IB, curcumin
treatment still down-regulated the PSA promoter activity. Therefore, it seems
that curcumin blocked PSA promoter activity through signal pathways more than
NF-B.
PDEF, a novel prostate epithelium-specific Ets transcription factor, not only acts as an androgen-independent transcriptional activator of the PSA promoter but also directly interacts with the DNA binding domain of androgen receptors and enhances androgen-mediated activation of the PSA promoter (Oettgen et al, 2000). Recent studies also indicated that silibinin and tectorigenin treatments decreased PSA secretion in LNCaP cells through down-regulation of the gene expression of PDEF (Thelen et al, 2004, 2005). Our study revealed that overexpression of PDEF enhanced PSA gene expression at transcriptional and translational levels under androgen-free conditions. However, curcumin treatments blocked PSA gene expression in the androgen bypass condition, but did not inhibit PDEF gene expression.
Several lines of evidence suggested that high levels of circulating IL-6 correlate with advanced prostate cancer. IL-6 signaling can activate androgen receptors in a ligand-independent manner and might play an important role in hormone-refractory prostate cancer progression (Hobisch et al, 1998; Jia et al, 2004; Edwards and Bartlett, 2005). IL-6 increases PSA and androgen receptor expression through a STAT3-dependent pathway in the absence of androgen in LNCaP cells (Chen et al, 2000; Lin et al, 2001). Our results agreed with those of an earlier study that indicated that IL-6 induced expression of the androgen receptor, which up-regulated PSA promoter activity in the androgen-independent pathway (Hobisch et al, 1998). Moreover, curcumin blocked stimulation of IL-6 on the androgen receptor, which attenuated PSA gene expression in a ligand-independent manner.
With this study, we demonstrated that curcumin inhibits the gene expression and activity of the androgen receptor that attenuates the stimulation of R1881- and IL6-mediated PSA gene expression in LNCaP cells. Overexpression of PDEF or IL-6 stimulated PSA gene expression in the androgen bypass pathways. Our results suggested that curcumin down-regulates PSA gene expression in androgen-dependent and androgen-independent pathways through blockage of gene expression of the androgen receptor but not of PDEF in LNCaP cells.
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Footnotes |
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