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Introduction
Maspin is a mammary serine protease inhibitor with
tumor suppressive activity for prostate
cancers[1_3], and its expression decreases with prostate cancer
progression[4]. Functional studies have demonstrated that maspin inhibits
tumor invasion and motility of human prostate cancer cells
in vitro[5], as well as tumor growth and metastasis in the
nude mice assay[6,7]. It is speculated that upregulation of
maspin in the prostate tumors may offer great hope for
reversing the tumor phenotypes[8,9]. Accumulating evidence
has shown that maspin regulation is controlled at the
transcriptional level, and some elements in the maspin promoter
are identified. In our previous studies, we characterized a
negative androgen-responsive element (ARE) element and a
positive Sp1 element in the maspin promoter in prostate
cancer cells[10]. In order to identify what agents can regulate
maspin expression, we treated prostate cancer cells with
different compounds to select the potential agent for prostate
cancer therapy. Gum mastic, a natural resin, has been shown
to dramatically increase maspin expression.
Known for centuries, gum mastic is a resinous exudate
obtained from the stem and the main leaves of
Pistacia lentiscus trees and is extensively used in Mediterranean and
Middle Eastern countries, both as a dietary supplement and
herbal remedy[11]. Medical trials have indicated that gum
mastic has no side effects, and shows its protective effects
on the gastrointestinal environment, such as relief of ulcers,
reducing the intensity of gastric mucosal damage caused by
anti-ulcer drugs and aspirin, and possessing anti-acid and
cytoprotective qualities[12_14]. Recently, gum mastic has
shown its antibacterial and antiviral action and antitumor
effect in many traditional Chinese
medicines[15,16]. In the present work, we ascertained the effect of gum mastic on
maspin expression in human prostate cancer cells and
discovered possible mechanisms involved in this regulatory
system.
Materials and methods
Cell culture and treatments The human prostate cancer
cell lines, LNCaP and DU-145, were obtained from the
American Type Culture Collection (Manassas, VA, USA). The
LNCaP cell line was established from a lymph node
metastasis of a prostate cancer patient and expressed
androgen-receptor (AR), and DU-145 was from a bone metastasis and
was absent of AR. 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 8 h before
experiments in order to synchronize cells. The cells were then
treated with gum mastic at indicated concentrations in RPMI-
1640 medium containing 5% FBS (GIBCO BRL Grand Island,
NY, USA). Gum mastic (Sigma, St Louis, MO, USA,
No G0878) was dissolved in DMSO which was also a control
vehicle.
Western blot analysis Treated LNCaP cells were
harvested and lysed as described
previously[4]. Cell extracts were quantified by BCA method. For the Western blot
analysis, 40 µg cell extracts were separated on 10%
SDS-PAGE and transferred to the nitrocellulose membrane, and
then the nitrocellulose membrane was immediately blocked
with 5% non-fat milk in PBS buffer for 1 h at room temperature.
After blocking, the membrane was incubated with human
specific anti-maspin antibodies (BD Biosciences, San Diego,
CA, USA) at 4 °C for 12 h, followed by the incubation with
peroxidase-labeled second antibody for 1 h, and
immunoreactive bands were visualized by enhanced
chemiluminescence (ECL, Santa Cruz Biotechnology, Inc, Santa Cruz, CA,
USA). β-tubulin (Sigma, USA) was used to normalize the
quantity of the protein on the blot. At least 3 independent
Western blots were performed.
RT-PCR analysis Total RNA was isolated from the treated
cells by TRIzol reagent (Invitrogen, Carlsbad, CA, USA ).
According to the manufacturer,s instructions, a portion of
total RNA (2 µg) was transcribed reversibly with the M-Mulv
reverse transcriptase in the presence of a random hexamer
primer. The resulting cDNA preparation was subjected to
PCR amplification using PCR kit from TaKaRa Biotech
(DaLian, China). The primers used for maspin gene were:
sense 5'-TGCTGCCTACTTTGTTGGCAAGT-3' and antisense 5'-TGATACTGTCAATGTTTCCCATACAGA-3', and for the
housekeeping gene β-actin: sense 5'-GTGGGGCGCCCA-GGCACCACGATG-3' and antisense
5'-CTCCTTAATGTCA-CGCACGATTT-3'. PCR profiles consisted of first initial
denaturation at 94 °C for 3 min, followed by 8 cycles of
denaturation at 94 °C for 40 s, primer-annealing at 53 °C for 40 s, and
primer extension at 72 °C for 40 s. Then the primers for
β-actin were added into the reaction complex, and the reaction
continued at the condition of denaturation at 94 °C for 40 s,
primer-annealing at 53 °C for 40 s, and primer extension at 72
°C for 40 s for 16 cycles. 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. At least 3
independent RT-PCR were performed.
Transient transfection assay pGL3-860 bp maspin
promoters were described previously. Transient transfection
was performed using Lipofectamine 2000 reagent (Invitrogen,
Carlsbad, CA, USA). Briefly, for the firefly Lucifer's reporter
assay, the cells were transfected with 1 µg of pGL3-860 bp in
combination with 0.05 µg pRL-TK (Promega, Madison, WI,
USA) for the internal control. In following transfections, the
cells were incubated with designated concentrations of gum
mastic additionally for 12 h in RPMI-1640 medium
containing 5% FBS, then the cell extracts were prepared and
luciferase assays were performed according to the
manufac-turer,s instruction of Dual-Luciferase Reporter Assay
System (Promega, Madison, WI, USA). At least 3 independent
transfections were performed, and standard deviations (SD)
were calculated.
Nuclear extracts LNCaP cells were grown in the same
conditions as described earlier, and cultured in RPMI-1640
with addition of gum mastic for 24 h.
2×109 cells were pelleted and resuspended in 1 mL cold hypotonic buffer (10 mmol/L
HEPES, pH 7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5
mmol/L DTT, and 0.5 mmol/L PMSF). Following 15 min incubation
on ice, the cells were lysed by adding 50 µL of 10% NP-40
and centrifuged at 6000×g for 5 min at 4 °C. Then the pellets
were resuspended in 70 µL of cold hypertonic buffer (20
mmol/L HEPES, pH 7.9, 1.5 mmol/L MgCl2, 420 mmol/L
NaCl, 0.2 mmol/L EDTA, 25% glycerol, 10 µg /mL aprotinin,
and 0.5 mmol/L PMSF). The resuspended cells were stirred
gently at 4 °C for 30 min and centrifuged at 12 000 rpm for 5
min at 4 °C. The supernatant was extensively dialyzed against
the dialysis buffer (20 mmol/L HEPES, pH 7.9, 50 mmol/L KCl,
25% glycerol, 0.5 mmol/L DTT, and 0.5 mmol/L PMSF) for 2 h
at 4 °C. The protein concentration of the dialyzed material
was determined by BCA method and stored at -80 °C in small
aliquots.
Electrophoretic mobility shift assay
(EMSA) Equal amounts of sense and antisense oligonucleotides were mixed
and annealed in 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 cooling
to room temperature for over 3 h. The corresponding
oligonucleotides were labeled with digoxin (DIG). The following
oligonucleotides were used for EMSA experiments: maspin
Sp1: sense 5'-TGCCGCCGAGGCGGGGCGGGGCGGGGCGT-GGAG-3' and
antisense 5'-GCTCCACGCCCCGCCCCGCCCC-GCCTCGGCGGCA-3'; maspin ARE: sense
5'-AAGAATGGA-GATCAGAGTACTT-3' and antisense
5'-AAGTACTCTGA-TCTCCATTCTT -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 DIG-labeled oligonucleotide probe. Then the
reaction mixture was 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, Palo Alto, CA, USA), electroblotting and
chemiluminescent detection were performed.
The specificity of ARE and Sp1 binding was confirmed
by adding 125-fold in excess of unlabeled DNA probe to the
assay. At least 3 independent EMSA were performed.
Results
Gum mastic increased maspin expression Maspin is a
tumor-suppressing gene for prostate cancer. The aim of this
study was to determine whether maspin protein levels change
with gum mastic treatment in prostate cancer LNCaP cells.
Western blotting was performed. As shown in Figure 1A,
when the LNCaP cells were exposed to different
concentrations of gum mastic for 24 h, gum mastic increased maspin
expression in a dose-dependent manner. Gum mastic 8
μg/mL increased maspin expression about 1.5-fold. To further
demonstrate the increased effect of gum mastic on the steady
levels of maspin mRNA, RT-PCR was used. The results in
Figure 1C and 1D show that maspin mRNA expression was
significantly increased by gum mastic, which was
consistent with the effect of gum mastic on maspin protein
expression. To understand the potential mechanism by which
the expression of maspin could be affected by gum mastic,
transient transfections with a maspin promoter-luciferase
construct was performed to determine if transcriptional
activity of the maspin gene was changed by gum mastic. As
shown in Figure 1E, the activity of the maspin promoter was
increased by gum mastic, consistent with the results of
Figure 1A_1D. Therefore, we may conclude that the increased
effects of gum mastic on maspin expression mainly occur at
the transcriptional level, which subsequently affects the
levels of maspin mRNA and protein. At the same time, we also
detected maspin protein levels in androgen-independent
prostate cancer DU-145 cells treated with gum mastic. As
illustrated in Figure 1F, gum mastic increased maspin
expression in DU-145 cells.
Gum mastic inhibited nuclear AR
expression It has been demonstrated that maspin is an AR-mediated gene. AR binds
to the ARE element in the maspin promoter to inhibit its
expression. To determine whether the increased effect of
gum mastic on maspin expression might be due to the
alteration of AR expression, the effect of gum mastic on AR
expression was studied by Western blotting. Since the AR
was a nuclear protein and functioned in the nucleus, we
prepared the nuclear extracts for Western blotting after the
LNCaP cells were exposed to gum mastic for 24 h. The
results from Figure 2 show that the expression of the AR
protein was dramatically decreased by gum mastic in a
dose-dependent manner.
Gum mastic inhibited AR binding to ARE in the
maspin promoter The above results led us to investigate whether
the AR binding to ARE could be inhibited by gum mastic.
We used the gel band-shift technique as an in
vitro functional assay to determine the binding activity of ARE in the
maspin promoter. The results in Figure 3 show that ARE
binding activity was largely decreased by 8 µg/mL gum
mastic after 24 h of treatment when compared with 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
oligonucleo-tides. Furthermore, the bands could be reduced by a
specific anti-AR antibody. Together with the earlier results, it
was implicated that gum mastic increased
maspin expression by inhibiting AR activity.
Gum mastic enhanced the binding activity of Sp1
element in the maspin promoter We have demonstrated that
the Sp1 element in the maspin promoter plays a positive role
in its transcription[10]. To further study the potential
molecular mechanisms of gum mastic on maspin expression, the
binding activity of the Sp1 element in the maspin promoter
was detected after the LNCaP cells were exposed to different
concentrations of gum mastic for 24 h. The results from
Figure 4 show that gum mastic enhanced the binding
activity of Sp1 element in the maspin promoter. The bands were
confirmed to be a result of specific binding for Sp1 because
the DNA-protein complex was competed out by a 125-fold
molar excess of unlabeled Sp1 oligonucleotides, but not by a
125-fold molar excess of unlabeled E2F oligonucleotides.
Discussion
We have demonstrated that AR binds to the ARE
element in the maspin promoter to inhibit its expression, and
Sp1 plays a positive role in maspin
transcription[10]. In this study, it was shown that gum mastic inhibited AR binding to
ARE, and enhanced Sp1 binding activity. Moreover, gum
mastic enhanced the activity of the maspin promoter, which
might finally contribute to its upregulation for maspin
expression.
Gum mastic is an affordable and safe natural supplement
that protects the digestive system, heals peptic and
duodenal ulcers, and eradicates Helicobacter
pylori from the gut, while H pylori is a primary agent to promote the
development of gastric cancer[17]. It is implicated that gum mastic
may effectively prevent gastric cancer. Recently, it was
reported that gum mastic induces the apoptosis of the
human colon cancer HCT116 cells by activating caspase-8 and
caspase-9[18]. Gum mastic has also been shown to inhibit
LNCaP cell growth by inhibiting AR
function[19]. AR is closely related to the development and progression of prostate
cancer[20,21]. Androgen-responsive prostate cancer LNCaP cells
require AR for continued proliferation and
survival[22]. AR blockade may be an effective strategy in the fight against
prostate cancer. Maspin is an AR target gene, specifically
targeting AR, or its downstream signaling molecules will be
potentially effective for achieving total AR blockade.
Maspin is often silenced in prostate cancer cells and
exhibits suppressing activity against tumor growth and
metastasis. It has also been shown to be involved in
processes that are important to both tumor growth and
metastasis such as cell invasion, angiogenesis, and more recently,
apoptosis[5_9]. Therefore, maspin can be taken as a potential
target for cancer therapy. Maspin expression is directly
regulated by the p53 gene. p53 induces maspin expression in
prostate cancer cells and suppresses tumor growth and
metastasis[23]. g-Linolenic acid, an essential fatty acid with
anticancer properties, is reported to induce maspin expression
and inhibit cell motility[24]. Peroxisome proliferator-activated
receptor-gamma, nitric oxide, and the
manganese-containing superoxide dismutase induced maspin expression in
breast and prostate cancer cells. This effect correlated with
a differentiated phenotype, decreased cell motility and
invasiveness, and increased the apoptotic
index[25_29]. Our study discovered another potential agent for inducing maspin
expression and inhibiting the growth of prostate cancer cells.
Gum mastic inhibited AR function and increased the
AR-mediated gene maspin expression in LNCaP cells. On the
other hand, gum mastic also has been shown to inhibit the
proliferation of androgen-refractory prostate cancer cells,
DU-145 (data not shown), which express little of the AR.
This implies that the inhibitory effect of gum mastic on
prostate cancer is not limited in an androgen-responsive manner,
and other signal pathways may be involved in the
mastic-mediated regulatory system. Sp1 is a ubiquitous
transcription factor that binds to consensus elements in the proximal
promoters of a wide variety of genes. It is involved in the
regulation of many aspects of physiological and
pathological conditions, including cell growth, apoptosis,
angio-genesis, and invasion[30,31]. Sp1 has 2 other closely related
members of a gene family encoding proteins with very
similar structures, but different molecular weights, Sp3 and Sp4.
Sp1, Sp3, and Sp4 are highly conserved and can recognize
the GC box with identical
affinities[32]. Gum mastic increased Sp1 binding activity, which may contribute to its increased
effect on maspin expression; the upregulation of maspin
leads to its inhibition on prostate cancer cells.
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