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Advances in Brief |
and Inhibits Proliferation in PC3 Prostate Carcinoma Cells1
Department of Pathology [S. B. S., S. M., J. P., G. S. J.], Vanderbilt Prostate Cancer Center [S. B. S., S. M., R. J. M.]; Vanderbilt-Ingram Cancer Center [S. B. S., R. W., R. J. M., A. R. B., R. N. D.]; Departments of Cell Biology [R. A. G., R. J. M., R. N. D.], Medicine, Division of Gastroenterology [R. A. G., R. W., R. N. D.], Pharmacology, Division of Clinical Pharmacology [W. E. B., C. S., A. R. B.], and Urology [T. C., R. J. M.], Vanderbilt University Medical Center, Nashville, Tennessee 37232; Department of Pathology and Laboratory Medicine, Veterans University Medical Center, Nashville, Tennessee 37212 [J. P.]; and Departments of Pathology and Urology, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030 [T. M. W.]
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ABSTRACT |
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in
benign and malignant prostate tissues and the ability of 15S-HETE to
activate PPAR-dependent transcription and modulate proliferation of
the Pca cell line PC3. In contrast to benign prostate and similar to
most Pca tissues, 15-LOX-2 mRNA was not detected in PC3 cells, and they
did not produce detectable 15-HETE from [14C]AA. By
reverse transcription-PCR, PPAR mRNA was present in 18 of 18 benign
and 9 of 9 tumor specimens. The PPAR ligand BRL 49653 and 15S-HETE
caused a dose-dependent inhibition of PC3 proliferation in a 14-day
soft agar colony-forming assay (IC50 of 3 and 30
µM, respectively). 15S-HETE (10 µM) caused
greater inhibition than 10 µM 15R-HETE. At 3 days,
BRL 49653 and 15S-HETE caused a slight increase in cells in
G0-G1 and a corresponding decrease in cells in
S phase. In PC3 cells transiently transfected with a luciferase
reporter linked to a PPAR response element, 1 µM
BRL 49653 and 10 µM 15S-HETE caused approximately
threefold and greater than twofold induction of PPAR-dependent
transcription, respectively. By quantitative real-time reverse
transcription-PCR and Northern analysis, 3-day treatment with BRL 49653
and 15S-HETE caused a reduction of PPAR expression but a marked
up-regulation of the PPAR response element containing adipocyte type
fatty acid binding protein. These results support the hypothesis that
15-LOX-2-derived 15S-HETE may constitute an endogenous ligand for
PPAR in the prostate and that loss of this pathway by reduced
expression of 15-LOX-2 may contribute to increased proliferation and
reduced differentiation in prostate carcinoma. |
Introduction |
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We have previously demonstrated that the recently described 15-LOX-2
(7)
is uniformly expressed in the differentiated apical or
secretory cells of benign prostate (8)
. Benign prostate
tissue samples synthesize 15S-HETE as the major eicosanoid product from
exogenous AA. In contrast, by immunostaining and enzyme activity
assays, 15-LOX-2 and 15S-HETE formation were reduced in Pca
(8)
. More recently, using paired snap-frozen benign and
malignant prostate tissue obtained intraoperatively (9)
,
we have shown that 15-LOX-2 mRNA is reduced in tumor compared with
benign in the majority (>80%) of patients and confirmed that reduced
15S-HETE formation in tumor is indeed a common alteration (>60%) in
Pca.4
In addition, by immunostaining, reduced 15-LOX-2 correlated inversely
with the degree of tumor differentiation, with retained expression in
the majority of Gleason score 5 tumors compared with a statistically
significant reduction in Gleason 6, 7, and 8–10 tumors
(10)
. 15-LOX-2 was reduced in high-grade prostatic
intraepithelial neoplasia compared with benign glands, indicating that
this may be an early alteration in Pca development (10)
.
The goal of the present study was to examine if loss of 15-LOX-2
expression may contribute to the malignant phenotype in Pca, by
examining its effects on Pca cell proliferation. Studies examining
oxidized low-density lipoprotein and foam cell formation from
macrophages have shown that 15-HETE may activate transcription by the
nuclear receptor PPAR (11)
, and other studies have
shown inhibition of Pca cell line PC3 proliferation by synthetic
PPAR agonists (12)
. Therefore, we compared the effects
of 15S-HETE to known PPAR agonists on PC3 cell proliferation and
investigated the ability of 15S-HETE to activate PPAR-dependent
transcription in Pca cell lines.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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1–1.5 cm
in length, which are immediately placed in liquid nitrogen. Two to
three mm from one end of the cores were processed for RNA, and the
immediately adjacent 1–2 mm was processed for histology to be
representative of the portion processed for RNA. The 1- to 2-mm-thick
transverse section for histology was immediately placed in 10%
buffered formalin for routine processing and paraffin embedding.
Five-µm cross sections were stained with H&E and assessed as benign,
tumor, or mixed, with 
85% benign glands considered benign and 85%
tumor glands considered tumor. BRL 49653 was synthesized by Glaxo Wellcome. (PPRE)3-tk-luciferase (13) was provided by Dr. Ron Evans (The Salk Institute, San Diego, CA). The 15S-HETE was prepared using the soybean lipoxygenase, as described previously (14) . The 15R-HETE was prepared from 15S-HETE methyl ester by the following sequence: (a) stirring with activated manganese dioxide (10 mg/ml) in methylene chloride at room temperature for 2 h, producing the 15-keto analogue in a 20–30% yield; (b) filtration through a silica column in 5% methanol in methylene chloride and isolation of the 15-ketoeicosatetraenoate product by normal phase HPLC (Alltech; 5-µm silica column, solvent hexane/isopropanol, 100:1, by volume); (c) reduction of the 15-keto derivative using sodium borohydride in methanol; (d) isolation of the 15R and 15S enantiomers by chiral column chromatography using a Chiralpak AD column (Chiral Technologies, Inc., Exton, PA) and a solvent of hexane/ethanol (100:2, by volume); (e) saponification in aqueous 1 M KOH/methanol (1:1) for 2 h at room temperature, followed by (f) repurification of the 15R-HETE by normal-phase HPLC (hexane/isopropanol/glacial acetic acid; 100:1:0.1, by volume). The 15R- and 15S-HETEs were quantified by UV spectroscopy using a molar extinction coefficient of 23,000 at 235 nm and stored in ethanol at -20°C at a concentration of 10 or 100 mM.
Northern Blots and RT-PCR for 15-LOX-2 and PPAR mRNA.
Subconfluent or confluent cultured cells were examined for 15-LOX-2
mRNA by Northern blot and RT-PCR (with benign prostate as positive
control). Benign and malignant prostate tissues from RP specimens and
cell lines were examined for PPAR mRNA by RT-PCR. For RNA isolation
from prostate tissues, 50 mg were diced with fine scissors in 1 ml
Tri Reagent (Molecular Research Center, Inc., Cincinnati, OH).
Approximately 100 µl of autoclaved 200-µm glass beads (BioSpec
Products, Bartlesville, OK) were added, and the tissue was further
disrupted by two 20-s periods of agitation on a Mini-Beadbeater
(BioSpec Products), placing the specimen on ice between cycles. RNA was
extracted from these homogenates and from cultured cells in T75 flasks
using the Triazol reagent (Molecular Research Center, Inc.), according
to the manufacturer’s instructions. For Northern blots, 20 µg total
RNA was used, and blots were hybridized overnight at 42°C with
1 x 106 cpm/ml each of
32P-labeled cDNA probes for 15-LOX-2 and GAPDH
(prostate tissues and PC3 cells) or a-FABP and GAPDH (untreated or
treated PC3 cells) in ULTRAhyb (7)
. The 15-LOX-2 probe was
a 1088-bp cDNA amplified by PCR from a partial cDNA in pcDNA3
(Stratagene, La Jolla, CA), with primers
5'-TGC-CTC-TCG-CCA-TCC-AGC-T-3' and 5'-TGG-GAT-GTC-ATC-TGG-GCC-TGT-3'.
The GAPDH probe was an 1100-bp cDNA (Clontech, Palo Alto, CA).
The a-FABP probe was a 500 bp fragment released by BamHI and
XhoI digestion of a cDNA cloned into pBlueScript SK(-)
(Stratagene; Ref. 15
). Blots were exposed to Kodak X-OMAT
film at -80°C for 1–3 days or to a phosphorimaging screen (Super
resolution screen; Packard Instrument Co., Inc., Meriden, CT) for 1 to
4 h at room temperature and imaged on a Cyclone Storage Phosphor
System (Packard Instrument Co.).
One µg total RNA was used for conventional RT-PCR reactions with the
Promega Access RT-PCR System (Promega, Madison, WI), generally
according to the manufacturer’s instructions. For 15-LOX-2, the
primers used were 5'-GCC-TCT-CGC-CAT-CCA-GCT-3' (forward) and
5'-TGC-CGA-GTT-CTC-CTT-CCA-TGA-3' (reverse), which gives a 126-bp
amplified product. For PPAR, the primers were
5'-GAG-TTC-ATG-CTT-GTG-AAG-GAT-GC-3' (forward) and
5'-CGA-TAT-CAC-TGG-AGA-TCT-CCG-CC-3' (reverse), which generate a 233-bp
amplimer corresponding to portions of exons 2 and 3 contained in both
PPAR1 and PPAR2 isoforms (16)
. Primers to amplify
both isoforms were chosen because the relative expression in prostate
is not definitively established (17)
, and both contain the
same ligand-binding domain that would potentially bind 15-HETE
(16)
. 15-LOX-2 and PPAR primers span intron-exon
boundaries (7
, 16)
, obviating the need for inclusion of
DNase treatment during RNA extraction protocols. Thirty cycles of
reaction at 94°C for 30 s, 60°C for 30 s, and 72°C for
60 s, respectively, were carried out on a thermal cycler
(Perkin-Perkin-Elmer Corp.).
Quantitative Real Time RT-PCR for PPAR and a-FABP.
PPAR and a-FABP mRNA copy numbers in untreated and treated PC3 cells
were determined by real-time quantitative RT-PCR using a Lightcycler
fluorescence temperature rapid-air cycler (Roche Molecular
Biochemicals, Indianapolis, IN) with cDNA standard curves and the
double-stranded DNA-binding fluorescent probe SYBR Green
(18, 19, 20, 21)
. Amplifications were done in glass capillary
tubes using a 20-µl reaction of 100 ng total PC3 RNA, 5
mM magnesium chloride, 11 ng/µl Taq-start
antibody (Clontech Laboratories), 2.0 µl 1 x SYBR
Green RNA master-mix (Roche Molecular Biochemicals), 0.4 µl reverse
transcriptase enzyme (Roche Molecular Biochemicals), and 1.0
µM each primer. The cDNA template for PPAR
consisted of a 761-bp fragment inserted into PCRII (Stratagene). The
primers were identical to those listed above. The a-FABP template for
standard curves consisted of a 500-bp fragment, released by
BamHI and XhoI digestion from a full-length (619
bp) cDNA inserted into pBlueScript SK(-) (Stratagene). The primers
were 5'-TCA-GTG-TGA-ATG-GGG-ATG-TGA-3' (forward) and
5'-TCA-ACG-TCC-CTT-GGC-TTA-TGC-3' (reverse), which generate a 288-bp
amplimer. The one-step real-time RT-PCR reactions consisted of the
following steps: reverse transcription at 55°C for 15 min,
denaturation at 95°C for 1 min, amplification for 45 cycles, and
melting curve analysis from 95°C to 65°C at a rate of 0.1°C/s
under continuous fluorescence monitoring. The amplification programs
consisted of heating at 20°C/s to 95°C, cooling at 20°C/s to
53°C, annealing at 53°C for 5 s, heating at 20°C/s to
72°C, elongation at 72°C for either 12 s (for PPAR) or
16 s (for a-FABP), and heating at 5°C/s to either 84°C
(PPAR) or 83°C (a-FABP) for fluorescence acquisition. The
specificity of the amplimer in each reaction was confirmed by the
melting curve analysis, with initial gel confirmation that this large
peak corresponded to the expected amplimer (18
, 21, 22, 23)
.
The contribution to fluorescence signal of any nonspecific products
and/or primer dimers was eliminated by increasing the temperature to
2° below the melting temperature of the specific product, which
eliminated any other minor cDNAs (which have lower melting
temperatures; Ref. 19
). Copy numbers of mRNA were
calculated from serially diluted standard curves generated from
purified cDNA template (24)
. Serial dilutions (1:10) over
a range of four to five orders of magnitude were used to generate the
standard curves (1010–106
copies for PPAR;
1010–107 copies for
a-FABP). The serially diluted standards were simultaneously amplified
with the unknown samples to generate a linear standard curve using the
fit points method of analysis with four points. Standard curves for
both PPAR and a-FABP had correlation coefficients of 1.00. Control
samples run in triplicate had a variance of 10%. All of the
biological samples fell on the standard curves, and copy numbers of the
unknown samples were calculated using the Lightcycler software (version
3).
Enzyme Assay for 15S-HETE Formation.
Control samples of benign prostate were homogenized as described
previously (8)
. For determination of possible 15-HETE
formation from cell lysates, cultured cells that were 80–100%
confluent in T75 flasks were removed by trypsinization, pelleted, and
washed. The entire pellet was resuspended in 100 µl Dulbecco’s PBS
(pH 7.4) and sonicated for 3 s. One hundred µl (equal or greater
to the protein amount routinely incubated from positive prostate
tissues) were transferred to a new tube and incubated with 50
µM [1-14C]AA for 45 min. Two
hundred fifty µl methanol were added, followed by addition of 125
µl dichloromethane, vortex-mixing, and evaporation under nitrogen.
After resuspension in 50 µl methanol, the sample was collected on a
C18 Bond Elute cartridge. One-fifth and subsequent four-fifth fractions
were analyzed by reverse-phase HPLC, with radioactivity monitored with
an on-line Radiomatic Instrument Flo-One detector as described
previously, using a solvent system of methanol:water:acetic acid
(80:20:0.01) at a flow rate of 1.1 ml/min and addition of cold HETE
standards to monitor retention times (8)
. In additional
experiments, formation of possible AA metabolites was assessed using
intact monolayers incubated with labeled AA and calcium ionophore.
Eighty to 100% confluent PC3 cells in T75 flasks were incubated in 5
ml PBS, with 2 µCi [14C]AA with or without
cold AA (50 µM total) and 5 µM calcium
ionophore A23187 for 15 min at 37°C.
Soft Agar Colony Assay.
Effects of BRL 49653 and 15-HETE on PC3 proliferation were assessed
using an agar cloning technique. An underlay of 0.5% agar in Ham’s
F12 containing 5% FCS was prepared by mixing equal volumes of 1% agar
and 2x Ham’s F12 plus 10% FCS. Two ml of this mixture were pipetted
into the wells of six-well plates and allowed to set. PC3 cells,
70–100% confluent, were trypsinized, and the cells were resuspended
in growth medium and counted with a hemocytometer. The cells were then
diluted to a final concentration of 2000/ml in a mixture of 0.7% agar
and 2x Ham’s F12. BRL 49653 and 15-HETE were added from appropriate
stock solutions in 50% Etoh/50% DMSO to achieve indicated final
concentrations and a final solvent concentration of 0.5%. Vehicle
controls received similar volumes of solvent alone. Two ml of the cell
suspension were aliquoted into each well. The agar was allowed to set,
and the plates were incubated in a humidified chamber at 37°C for 14
days. Colonies were counted in a blinded manner using a 4x objective
on a Zeiss inverted microscope. Data are expressed as percent of
control.
Cell Cycle Analysis.
PC3 cells (5 x 105
) were seeded
into T75 flasks, and 24 h later, media was replaced with 10 ml
fresh media (Ham’s F12K with 10% fetal bovine serum), with or without
indicated final concentrations of BRL 49653 or 15S-HETE added in 50%
Etoh/50% DMSO (final solvent concentration < 1%).
Vehicle controls received equal volumes of solvent alone. After 72 h, cells were removed by trypsin-EDTA, washed, pelleted, and
resuspended in 300 µl of 1x PBS with subsequent dropwise addition of
700 µl ice-cold 100% Etoh (final concentration, 70%). Fixed cells
were stored at -20°C until stained with propidium iodide with
RNase, using standard methods. Cell cycle analysis was performed using
a Becton Dickinson FACScan and linked Modfit cell cycle analysis
and Winlist software (Verity Software House, Topsham, ME).
Cell Transfections and Luciferase Assays.
PC3 and DU-145 cells (5.0 x 105
)
were transfected using FUGENE 6 at a lipid:DNA ratio of 3:1. Cells were
exposed to a mix containing 150 ng/ml of (PPRE)3-tk-luciferase
(a gift from R. Evans, Salk Institute, La Jolla, CA), 150 ng/ml
pCDNA3.1, 1.0 ng/ml pRL-SV40 in Opti-MEM (Life Technologies). The
transfection mix was replaced after 5 h with 10%
charcoal-stripped fetal bovine serum-containing media (Hyclone)
supplemented with either 0.1% vehicle (DMSO or ethanol) or the
indicated concentrations of BRL 49653 and 15S-HETE. After 24 h,
cells were harvested in 1x luciferase lysis buffer. Relative light
units from firefly luciferase activity were determined using a
luminometer (MGM Instruments) and normalized to the relative light
units from renilla luciferase using the Dual Luciferase kit (Promega).
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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mRNA and
protein and the susceptibility to growth inhibition by known PPAR
agonists (12)
. Because 15-LOX-2 mRNA and enzyme activity
are reduced in most Pcas compared with benign tissue, we investigated
whether this is a property of PC3 cells and if these cells are a valid
model for 15-LOX-2-negative prostate tumors. Compared with the usual
benign prostate tissue, 15-LOX-2 mRNA was not detected in PC3 cells by
either Northern blots or RT-PCR (Fig. 1)
. Similarly, compared with the usual presence of 15-LOX-2 catalytic
activity in benign prostate (8)
, 15-HETE formation from
exogenous AA was not demonstrated in similar incubations of PC3 cells
(Fig. 1)
. DU-145 cells were also negative for 15-LOX-2 mRNA by Northern
blot and RT-PCR. LNCaP cells were negative for 15-LOX-2 mRNA by
Northern blot and showed only a faint band by RT-PCR compared with
benign prostate. Neither DU-145 nor LNCaP cells synthesized detectable
15-HETE from exogenous AA (not shown).
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mRNA. by RT-PCR. PPAR mRNA was detected in all of the benign and
malignant prostate tissues examined, as well as in PC3 carcinoma cells
(Fig. 2)
. PPAR was also detected in DU-145 and LNCaP cells (not shown).
PPAR
and PPARß(
) were also detected by RT-PCR in all of the
benign and malignant prostate tissues. PPARß() was also detected
in PC-3, DU-145, and LNCaP cells, whereas PPAR mRNA was detected in
LNCaP but not in PC-3 or DU-145 cells (not shown).
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agonist BRL 49653 caused inhibition of PC-3 cell
proliferation in a soft agar colony-forming assay. A dose-dependent
effect was achieved over the 10 nM to 10 µM
range tested, with an IC50 of 3
µM (Fig. 3A)
. Similarly, exogenous 15S-HETE caused a dose-dependent
inhibition of PC3 colony formation, with an IC50
of 30 µM (Fig. 3A)
. These levels
are similar to those that have achieved PPAR-mediated effects in
other cell types (25)
and were clearly not toxic to cells,
based on flow cytometric and ultrastructural analyses (not shown).
Inhibition was clearly evident at 10 µM
15S-HETE and was essentially complete at 100
µM. In addition to fewer colonies, formed
colonies were discernibly smaller at the higher concentration ranges
(not shown). Possible stereospecificity of the 15-HETE effect was
examined by comparing exogenous 15S- versus 15R-HETE. In
each of four different experiments, the inhibition of colony formation
was greater for 15S- than for 15R-HETE (Fig. 3B)
.
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. Thirty µM 15S-HETE (which causes a similar degree of
growth inhibition in the soft agar assay) produced a similar slight
increase in the G0-G1
fraction (Fig. 4)
, whereas 3 and 10 µM were without
discernible effect (not shown).
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-dependent Transcription in
PC3 Cells. agonists has been reported (12
, 17)
, the
ability of these agents to actually activate PPAR-dependent
transcription in these cells, which supports mechanism of action, has
not been reported. The functional status of endogenous PPAR
in PC3 cells was determined using luciferase reporter assays.
Concentrations of BRL 49653 inhibiting PC3 proliferation caused
PPAR-dependent transcription in PC3 cells transiently transfected
with a PPRE-luciferase reporter construct (Fig. 5A)
. Similarly, concentrations of 15S-HETE inhibiting PC3
proliferation also caused PPAR-dependent transactivation (Fig. 5A)
. BRL 49653 (1 µM) and 15S-HETE
(10 and 30 µM) caused an approximately
threefold and a greater than twofold increases in luciferase activity,
respectively (n = 3). Similarly, BRL 49653
caused PPAR-dependent transactivation in DU-145 cells. Ten
µM 15S-HETE caused a >2.5-fold increase in
luciferase activity in similarly transfected DU-145 cells
(n = 3, not shown).
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was investigated. The gene for a-FABP contains PPRE
elements in its promoter and is up-regulated by PPAR agonists in
adipocytes (26)
. Although a-FABP mRNA was not detected by
Northern blots in untreated or vehicle-treated control cells at 3 days,
concentrations of BRL 49653- and 15S-HETE-inhibiting proliferation of
PC3 cells caused up-regulation of a-FABP mRNA expression at 3 days
(Fig. 5B)
.
Treatment of the breast cancer cell line 21PT with synthetic PPAR
agonists increased expression of PPAR mRNA (27)
, and
the PPAR agonist
15-deoxy-
12,14-PGJ2
increased PPAR2 but not PPAR1 protein in Pca cell lines
(17)
. Whether changes in a-FABP in BRL 49653- and
15S-HETE-treated cells may be attributable in part to increases in
PPAR was investigated. PPAR mRNA was not detected by Northern
analysis in control or treated cells (not shown). By quantitative
real-time RT-PCR, we observed a reduction in PPAR mRNA in PC3 cells
treated for 3 days with either BRL 49653 or 15S-HETE, using primers
detecting both isoforms. PPAR mRNA copy numbers were 3.2- and
2.7-fold greater in vehicle control than in 3 µM BRL
49653- and 30 µM 15S-HETE-treated PC3 cells,
respectively. In contrast, this was accompanied by a marked
up-regulation in a-FABP mRNA copy number, which was 19.5- and 2.7-fold
higher in 3 µM BRL 49653- and 30 µM
15S-HETE-treated cells, respectively, compared with vehicle controls.
These results further support that a-FABP up-regulation was
attributable to ligand-dependent activation of PPAR-mediated
transcription, rather than attributable to increases in PPAR itself.
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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mRNA is uniformly present in benign and malignant
prostate and that 15S-HETE, similar to synthetic PPAR agonists,
activates PPAR-dependent transcription and inhibits
proliferation of PC3 Pca cells. 15S-HETE also activated
PPAR-dependent transcription in DU-145 cells, and others have
recently reported inhibition of DU-145 proliferation by multiple
PPAR agonists (17
, 28)
. These results support the
hypothesis that reduced 15-LOX-2 may etiologically contribute to Pca
development or progression by reduced expression of PPAR-regulated
genes.
PPAR agonists have been shown to inhibit proliferation and
potentially induce differentiation in carcinoma cell lines from
multiple organs, including breast (27)
, colon
(13)
, and bladder (15)
, in addition to their
ability to promote adipocytic differentiation in benign adipocytes
(26)
and liposarcomas (29)
. Although these
studies suggest PPAR agonists as potential therapeutic agents in
these malignancies, possible endogenous ligands in many, if not all, of
these other organs have not been identified. The uniform expression of
15-LOX-2 and formation of 15S-HETE in benign prostate and the ability
of 15S-HETE to activate PPAR-dependent transcription in Pca cells
indicates that 15S-HETE may represent a true endogenous ligand for
PPAR in prostate. Although concentrations of 15S-HETE added
exogenously in the present study and in a previous study showing
PPAR activation (25)
represent essentially
pharmacological doses, the actual concentrations reaching the nucleus
under these conditions or formed intracellularly in vivo are
unknown.
The mechanisms whereby 15S-HETE activation of PPARdependent
transcription leads to growth inhibition remain to be characterized.
PPAR-mediated terminal differentiation in adipocytes leads to cell
cycle arrest (30)
. In the present studies, BRL 49653 and
15S-HETE caused a slight delay in cell cycle progression in liquid
cultures. The degree of these changes may be sufficient to account for
the prominent effect observed in soft agar colony-forming assays. In
previous studies, inhibition of PC3 proliferation by synthetic PPAR
agonists was not accompanied by detectable effects on the cell cycle at
4 days in liquid culture (12)
, whereas Butler et
al. recently reported that
15-deoxy-12,14-PGJ2
induced cell death in Pca cell lines with accumulation in S phase at
48 h (17)
. Whether this result reflects an
agonist-specific effect or some other mechanism besides PPAR
activation for
15-deoxy-12,14-PGJ2 is
not clear. The activation of PPAR by PGJ2 in
Pca cell lines has not been demonstrated in prior studies (12
, 17)
, and a more direct effect at the plasma membrane of added
exogenous oxidized lipids cannot be excluded.
PGJ2 is not a natural PG product, such that any
demonstrated effects are more pharmacological that potentially
physiological in vivo. This is in contrast to 15-HETE,
because there is a high level of 15-LOX-2 catalytic activity in the
benign prostate (8
, 31)
. In contrast, inhibition of colon
cancer cell lines in soft agar was accompanied by a more prominent
increase in the G0-G1
fraction than observed herein (13)
. Inhibition of bladder
cancer cell lines by troglitazone in mitogenic assays was
accompanied by possible G1 arrest, as indicated
by increased expression of cyclin-dependent kinase
inhibitors, p21WAF1/CIP1 and
p16INK4, and decreased expression of cyclin D1
(15)
. The differential effects of PPAR agonists on cell
cycle and possible induction of differentiation are likely dependent on
the specific tumor cell lines studied.
The function of 15-HETE formed from the previously characterized
15-LOX-1 has been uncertain. 15-LOX-1 is able to oxygenate
polyunsaturated fatty acids esterified to phospholipids in cell
membranes and to catalyze oxygenation at other positions, with
formation of 12-hydroperoxyeicosatetraenoic acid and other lipid
hydroperoxides. This has led to the speculation of its function in
degradation of cell organelles by initiating lipid peroxidation as in
maturation of reticulocytes and in oxidation of low-density lipoprotein
during atherogenesis (32)
. However, this latter function
may also depend at least in part on activation of PPAR in
macrophages (11
, 25)
. We believe that the highly specific
formation of 15S-HETE from phospholipase
A2released AA by 15-LOX-2 is in keeping with
a ligand function dependent on structure and stereoconfiguration
(7)
. Previous stereospecificity has been demonstrated for
8S- versus 8R-HETE activation of PPAR (33)
.
In the present study, 10 µM 15S-HETE
demonstrated a modestly greater ability to inhibit PC3 proliferation
than 10 µM 15R-HETE. However, the
pharmacological concentrations used in the present study are the final
concentrations added to the media. Concentrations actually achieved at
the nucleus, where PPARs are located, are unknown, such that
stereospecificity of PPAR activation may be greater than indicated
in the present studies. This remains to be demonstrated in cell-free
systems. There are inherent limitations of adding exogenous lipophilic
agents to cells in culture. 15S-HETE made by benign prostate epithelial
cells (8
, 31)
could be secreted and function in a
paracrine manner. However, because 15-LOX-2, similar to other
lipoxygenases, seems to undergo translocation to cell membranes on
activation (31)
, it is unlikely that adding exogenous
15S-HETE completely mimics the intracellular sites of formation under
more physiological conditions. 15S-HETE made at the nuclear membrane
could function as a PPAR ligand within the same cell in which it is
formed. We have recently identified several cell strains established
from prostatectomy specimens, which in contrast to commercially
available tumor cell lines, express high levels of 15-LOX-2 mRNA and
form 15S-HETE from exogenous
AA.5
Future studies selectively eliminating expression of 15-LOX-2 and
PPAR in 15-LOX-2-positive cells may shed more insight on other
aspects of this signaling pathway in prostate.
The limited tissue distribution of 15-LOX-2 strongly suggests that its
biology is crucial to normal prostate function and that its reduced
expression is critical to Pca development. The mechanisms whereby
15-LOX-2 and PPAR contribute to prostate cell function and how
reductions in these pathways contribute to the malignant phenotype need
clarification. 15S-HETE increased expression of the PPAR-regulated
a-FABP, an effect that was not previously observed in
PPAR-agonist-treated breast cancer or
PGJ2-treated Pca cell lines (17
, 27)
. The activation of PPAR-dependent transcription was not
demonstrated in these latter Pca cell studies (17)
. In
contrast, we readily demonstrated up-regulation of a-FABP by both a
synthetic PPAR-agonist and 15S-HETE in PC3 Pca cells by Northern
analysis and real-time RT-PCR. Although a-FABP expression is associated
with adipogenesis, it is clearly not limited in its expression to
adipocytes. Its expression includes epithelial cells such as urothelium
(15
, 34) . The function of this gene in prostate remains to
be established, but it may contribute to secretory cell
differentiation. As FABP overexpression inhibits proliferation in
breast cancer cell lines (35)
, it is tempting to speculate
that reduced expression in prostate because of reduced
15-LOX-2/15S-HETE activation of PPAR could directly contribute to
altered tumor cell proliferation or differentiation. Whether this or
changes in expression of other PPAR-regulated genes are more crucial
in Pca development and progression remains to be established. In the
present studies, increase in a-FABP expression was not attributed to
increase in PPAR, because BRL 49653 and 15S-HETE caused
down-regulation of PPAR under our experimental conditions.
Concordant with these observations and further supporting a connection
between these pathways in vivo, in a subset of eight
snap-frozen benign and tumor pairs in which 15-LOX-2 mRNA was reduced
in tumor versus benign, PPAR mRNA quantitated by
real-time RT-PCR was higher in tumor versus benign in seven
of eight.4
These results indicate that 15S-HETE activation
of PPAR in benign prostate may result in down-regulation of PPAR
(or alternatively, that loss of the endogenous ligand results in
"compensatory" up-regulation of this nuclear receptor in at least
some tumors).
PPAR is expressed in other tissues that express 15-LOX-2, further
supporting a role for 15S-HETE formation in modulating expression of
PPAR-regulated genes. In addition to prostate, 15-LOX-2 mRNA is
detected in skin, lung, and cornea (7
, 31)
. 15-LOX-2 was
cloned originally from human hair rootlets (7)
, and in the
skin, 15-LOX-2 is expressed strongly in sebaceous glands.4
Interestingly, PPAR is also expressed in skin sebaceous glands, and
synthetic PPAR agonists are additive with androgens in inducing
sebocyte differentiation (36)
. Hence, a 15-LOX-2-PPAR
pathway may contribute to differentiation in other tissues besides
prostate. That 15-LOX-2 is reduced in Pca and 15S-HETE may be an
endogenous ligand for PPAR in prostate, as demonstrated herein,
further supports the rationale for evaluation of PPAR-agonists in
the treatment and/or prevention of Pca. During the preparation of this
manuscript, Mueller et al. reported encouraging results from
a Phase II clinical study in which use of the synthetic PPAR-agonist
troglitazone resulted in serum prostate-specific antigen
decreases in 33% of patients with androgen-dependent Pca and 14% of
patients with androgen-independent Pca (28)
. Also,
understanding the relationship between androgens, 15-LOX-2, and PPAR
in the prostate may help identify patient subsets that are particularly
likely to benefit from such novel therapies.
|
ACKNOWLEDGMENTS |
|---|
and a-FABP probes and Jean McClure
and Brent Weedman for help with figures. |
FOOTNOTES |
|---|
1 Supported in part by Department of Defense Grant
PC970260 (to S. B. S.), a Vanderbilt University Intramural
Interdisciplinary Discovery Grant (to S. B. S.), a Discovery Grant
from the Vanderbilt Ingram Cancer Center (to S. B. S., A. R. B.),
NIH Grant GM-53638 (to A. R. B.), and Specialized Program of Research
Excellence (SPORE) Grant CA58204 from the National Cancer Institute
(T. M. W.). 
2 To whom requests for reprints should be
addressed, at Department of Pathology, C-3321 Medical Center North,
Vanderbilt University Medical Center, Nashville, TN 37232-2561. Phone:
(615) 343-2338; Fax: (615) 343-7023; E-mail: scott.shappell{at}mcmail.vanderbilt.edu
3 The abbreviations used are: AA, arachidonic
acid; Pca, prostate carcinoma; 15-LOX, 15-lipoxygenase;
15-HETE, 15-hydroxyeicosatetraenoic acid; PPAR, peroxisome
proliferator-activated receptor ; PPRE, PPAR response element;
HPLC, high-performance liquid chromatography; RP, radical
prostatectomy; RT, reverse transcription; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; a-FABP, adipocyte type fatty
acid binding protein; PG, prostaglandin.
4 S. B. Shappell, unpublished observations.
5 S. B. Shappell and D. M. Peehl,
unpublished observations.
Received 8/11/00. Accepted 11/29/00.
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