The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells.
ABSTRACT Diminished apoptosis, a critical event in tumorigenesis, is linked to down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer cells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primary product of 15-LOX-1 metabolism of linoleic acid, restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for linoleic and arachidonic acid metabolites. PPAR-delta promotes colonic tumorigenesis. NSAIDs suppress PPAR-delta activity in colon cancer cells. The mechanistic relationship between 15-LOX-1 and PPAR-delta was previously unknown. Our current study shows that (i) 13-S-HODE binds to PPAR-delta, decreases PPAR-delta activation, and down-regulates PPAR-delta expression in colorectal cancer cells; (ii) the induction of 15-LOX-1 expression is a critical step in NSAID down-regulation of PPAR-delta and the resultant induction of apoptosis; and (iii) PPAR-delta is an important signaling receptor for 13-S-HODE-induced apoptosis. The in vivo relevance of these mechanistic findings was demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our findings indicate that the down-regulation of PPAR-delta by 15-LOX-1 through 13-S-HODE is an apoptotic signaling pathway that is activated by NSAIDs.
Article: The role of 12/15-lipoxygenase in the expression of interleukin-6 and tumor necrosis factor-alpha in macrophages.[show abstract] [hide abstract]
ABSTRACT: 12/15-lipoxygenase (12/15-LO) enzyme and products have been associated with inflammation and atherosclerosis. However, the mechanism of effects of the 12/15-LO products has not been fully clarified. To study the role of 12/15-LO in cytokine expression, experiments with direct additions of the12/15-LO products, 12(S)-hydroxyeicosa tetraenoic acid or 12(S)-hydroperoxyeicosa-5Z, 8Z, 10E, or 14Z-tetraenoic acid to macrophages were first carried out, and results showed that the 12/15-LO products stimulated mRNA and protein expression of IL-6 and TNF-alpha in a dose-dependent manner. In contrast, an inactive analogue of 12(S)-hydroxyeicosa tetraenoic acid had no effect. To further explore the role of endogenous 12/15-LO in cytokine expression, we used an in vitro and in vivo model to test the effect of 12/15-LO overexpression. The models included Plox-86 cells, a J774A.1 cell line that stably overexpresses leukocyte-type 12/15-LO and primary mouse peritoneal macrophages (MPMs) from 12/15-LO transgenic mice. The results showed a clear increase in IL-6 and TNF-alpha expression in Plox-86 cells and MPMs from 12/15-LO transgenic mice, compared with mock-transfected J774A.1 cells and MPMs from control C57BL6 mice. IL-1beta, IL-12, and monocyte chemoattractant protein (MCP)-1 mRNA were also increased in Plox-86 cells. These data clearly suggest a clear role of 12/15-LO pathway in cytokine production. We also demonstrated that signaling pathways including protein kinase C, p38 MAPK (p38), c-jun NH(2)-terminal kinase as well as nicotinamide adenine dinucleotide phosphate oxidase are important for 12-(S)-hydroxyeicosatetraenoic acid-induced increases in IL-6 and TNF-alpha gene expression. These results suggest a potentially important mechanism linking 12/15-LO activation to chronic inflammation and atherosclerosis.Endocrinology 04/2007; 148(3):1313-22. · 4.46 Impact Factor
Article: Animal products, diseases and drugs: a plea for better integration between agricultural sciences, human nutrition and human pharmacology.[show abstract] [hide abstract]
ABSTRACT: Eicosanoids are major players in the pathogenesis of several common diseases, with either overproduction or imbalance (e.g. between thromboxanes and prostacyclins) often leading to worsening of disease symptoms. Both the total rate of eicosanoid production and the balance between eicosanoids with opposite effects are strongly dependent on dietary factors, such as the daily intakes of various eicosanoid precursor fatty acids, and also on the intakes of several antioxidant nutrients including selenium and sulphur amino acids. Even though the underlying biochemical mechanisms have been thoroughly studied for more than 30 years, neither the agricultural sector nor medical practitioners have shown much interest in making practical use of the abundant high-quality research data now available. In this article, we discuss some specific examples of the interactions between diet and drugs in the pathogenesis and therapy of various common diseases. We also discuss, using common pain conditions and cancer as specific examples, how a better integration between agricultural science, nutrition and pharmacology could lead to improved treatment for important diseases (with improved overall therapeutic effect at the same time as negative side effects and therapy costs can be strongly reduced). It is shown how an unnaturally high omega-6/omega-3 fatty acid concentration ratio in meat, offal and eggs (because the omega-6/omega-3 ratio of the animal diet is unnaturally high) directly leads to exacerbation of pain conditions, cardiovascular disease and probably most cancers. It should be technologically easy and fairly inexpensive to produce poultry and pork meat with much more long-chain omega-3 fatty acids and less arachidonic acid than now, at the same time as they could also have a similar selenium concentration as is common in marine fish. The health economic benefits of such products for society as a whole must be expected vastly to outweigh the direct costs for the farming sector.Lipids in Health and Disease 01/2011; 10:16. · 2.17 Impact Factor
Article: Effect of polyunsaturated fatty acids on drug-sensitive and resistant tumor cells in vitro.[show abstract] [hide abstract]
ABSTRACT: Previous studies showed that γ-linolenic acid (GLA, 18: 3 ω-6), arachidonic acid (AA, 20:4 ω-6), eicosapentaenoic acid (EPA, 20: 5 ω-3) and docosahexaenoic acid (DHA, 22:6 ω-3) have selective tumoricidal action. In the present study, it was observed that dihomo-gamma-linolenic acid (DGLA) and AA, EPA and DHA have cytotoxic action on both vincristine-sensitive (KB-3-1) and resistant (KB-Ch(R)-8-5) cancer cells in vitro that appeared to be a free-radical dependent process but not due to the formation of prostaglandins, leukotrienes and thromboxanes. Uptake of vincristine and fatty acids was higher while their efflux was lower in KB-3-1 cells compared with KB-Ch(R)-8-5 cells, suggesting that drug resistant cells have an effective efflux pump. GLA, DGLA, AA, EPA and DHA enhanced the uptake and decreased efflux in both drug-sensitive and drug-resistant cells and augmented the susceptibility of tumor cells especially, of drug-resistant cells to the cytotoxic action of vincristine. These results suggest that certain polyunsaturated fatty acids have tumoricidal action and are capable of enhancing the cytotoxic action of anti-cancer drugs specifically, on drug-resistant cells by enhancing drug uptake and reducing its efflux. Thus, polyunsaturated fatty acids either by themselves or in combination with chemotherapeutic drugs have the potential as anti-cancer molecules.Lipids in Health and Disease 09/2011; 10:159. · 2.17 Impact Factor
The 15-lipoxygenase-1 product 13-S-hydroxyocta-
decadienoic acid down-regulates PPAR-? to
induce apoptosis in colorectal cancer cells
Imad Shureiqi*†‡, Wei Jiang*, Xiangsheng Zuo*, Yuanqing Wu*, Julie B. Stimmel§, Lisa M. Leesnitzer§,
Jeffrey S. Morris¶, Hui-Zhen Fan*, Susan M. Fischer?, and Scott M. Lippman*
Departments of *Clinical Cancer Prevention,†Gastrointestinal Medical Oncology, and¶Biostatistics, University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Boulevard, Houston, TX 77030;§Nuclear Receptor System Research, GlaxoSmithKline, Inc., Research Triangle Park, NC 27709; and
?Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, P.O. Box 789, Smithville, TX 78957
Edited by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, and approved June 11, 2003 (received for
review February 24, 2003)
Diminished apoptosis, a critical event in tumorigenesis, is linked to
down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorec-
is the primary product of 15-LOX-1 metabolism of linoleic acid,
restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs)
transcriptionally up-regulate 15-LOX-1 expression to induce apopto-
sis. Peroxisome proliferator-activated receptors (PPARs) are nuclear
receptors for linoleic and arachidonic acid metabolites. PPAR-? pro-
motes colonic tumorigenesis. NSAIDs suppress PPAR-? activity in
colon cancer cells. The mechanistic relationship between 15-LOX-1
and PPAR-? was previously unknown. Our current study shows that
(i) 13-S-HODE binds to PPAR-?, decreases PPAR-? activation, and
down-regulates PPAR-? expression in colorectal cancer cells; (ii) the
induction of 15-LOX-1 expression is a critical step in NSAID down-
regulation of PPAR-? and the resultant induction of apoptosis; and
(iii) PPAR-? is an important signaling receptor for 13-S-HODE-induced
apoptosis. The in vivo relevance of these mechanistic findings was
demonstrated in our tumorigenesis studies in nude mouse xenograft
models. Our findings indicate that the down-regulation of PPAR-? by
15-LOX-1 through 13-S-HODE is an apoptotic signaling pathway that
is activated by NSAIDs.
human cancers (2–4). Restoring apoptosis is an important
anticarcinogenic mechanism (2, 4, 5). Loss of apoptosis in
colorectal cancer cells is linked to down-regulation in 15-
lipoxygenase-1 (15-LOX-1) expression, and the primary product
of 15-LOX-1 acting on linoleic acid, 13-S-hydroxyoctadecadi-
enoic acid (13-S-HODE) (6), restores apoptosis in colorectal
cancer cells (7). Nonsteroidal antiinflammatory drugs
found previously that NSAIDs transcriptionally up-regulate the
expression of 15-LOX-1 to induce apoptosis in colorectal cancer
cells (11–13). These findings led us to search for downstream
receptors involved in 15-LOX-1-induced apoptosis, none of
which were identified previously. Peroxisome proliferator-
activated receptors (PPARs) can act as nuclear receptors for
polyunsaturated fatty acids (arachidonic and linoleic acids) and
their metabolites (14). PPAR-? expression promotes colonic
tumorigenesis (15). He et al. (16) found that NSAIDs suppress
PPAR-? activity in colon cancer cells (by interfering with
PPAR-? binding to DNA) as early as 10 h after treatment. These
investigators speculated that the effects of NSAIDs on PPAR-?
and fatty acid metabolism may be linked (16). Because 13-S-
HODE is a critical mediator of NSAID-induced apoptosis in
colorectal cancer cells (11, 12), and linoleic acid can bind
PPAR-? (17), the present study investigated whether 13-S-
HODE is the link between these two NSAID effects.
poptosis, an important regulatory event that maintains
homeostasis in normal epithelial cells (1), is diminished in
Materials and Methods
Materials. We obtained DLD-1 (colon cancer) and RKO (rectal
cancer) cells, sulindac, NS-398, sulindac sulfone, and indomethacin
as described (11, 12). Celecoxib was obtained from LKT Labora-
tories (St. Paul, MN), and rabbit anti-human PPAR-? antibody was
obtained from Santa Cruz Biotechnology. The HCT-116 parental
PPAR-? WT (PPAR-? ???) colon cancer cell line, HCT-116
PPAR-?-null cell line (PPAR-? ???) (15), and PPAR-? DNA-
response element (DRE) reporter vector were gifts from Bert
Vogelstein (The Johns Hopkins University School of Medicine,
Baltimore). pMH-100-TK-luc reporter and Gal4-PPAR-? ligand-
binding domain (LBD) plasmids were provided by Ronald M.
Evans (The Salk Institute, La Jolla, CA). Rabbit polyclonal anti-
serum to recombinant human 15-LOX-1 was a gift from Mary
Mulkins and Elliot Sigal (Roche Bioscience) and was also gener-
ated by Lampire Biological Laboratories (Pipersville, PA) by
injecting the recombinant human 15-LOX-1 protein (provided by
Mary Mulkins and Elliot Sigal) into rabbits. We obtained 13-S-
HODE and linoleic acid (formulated in DMSO) from Cayman
Chemical (Ann Arbor, MI). Other reagents, molecular-grade sol-
vents, and chemicals were obtained either from commercial man-
ufacturers or as specified below.
Cell Culture. HCT-116 cells were grown in McCoy’s 5A medium
(modified) containing 10% FBS and 1% penicillin?streptomycin
12). Cells were treated with: sulindac, indomethacin, and sulindac
1.35–13.5 ?M 13-S-HODE or linoleic acid as described (12).
PPAR-? Ligand-Binding Reporter Assays. Cells were transfected in
24-well plates with 0.4 ?g of pMH-100-TK-luc per well, 0.4 ?g of
GAL4-PPAR-? LBD per well (18), 0.2 ?g of pSV-?gal [which
encodes ?-galactosidase (?-gal)] per well, and 3 ?l of Lipo-
fectamine 2000 (Invitrogen) per well and incubated overnight.
13-S-HODE or linoleic acid was added at 13.5 ?M, and cells were
cultured for 6 h. Control experiments used DMSO (the vehicle for
harvested and lysed, and luciferase activity was measured by using
a luciferase assay kit (Promega). Luciferase activity levels were
normalized to the relative ?-gal activity as measured by using a
commercial kit (Invitrogen).
Transfection with a 15-LOX-1 Expression Vector. 15-LOX-1 cDNA
was subcloned from a 15-LOX-1 cDNA-carrying pIND vector
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: 13-S-HODE, 13-S-hydroxyoctadecadienoic acid; 15-LOX-1, 15-lipoxygen-
vector-transfected RKO cells; LBD, ligand-binding domain; NSAIDs, nonsteroidal antiin-
flammatory drugs; PPARs, peroxisome proliferator-activated receptors; TUNEL, terminal
deoxynucleotidyltransferase-mediated dUTP nick end labeling; W-RKO, WT RKO cells.
‡To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
August 19, 2003 ?
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(Invitrogen) [provided by Joseph Cornicelli (Parke-Davis)] into a
entry segment)-GFP vector (Qbiogene) and used for transient
transfections (with Lipofectamine 2000) into RKO and DLD-1
PPAR-? Activation Assays. To measure PPAR-? activation levels, we
used a luciferase reporter construct containing PPAR-? DRE in
pBV-luc vector (16), which was transfected into cells with Lipo-
(13.5 ?M), and luciferase activity was measured as described above
for the PPAR-? ligand-binding reporter assays.
Scintillation Proximity Assays of 13-S-HODE Binding to PPAR-?. The
PPAR-? LBDs, expressed in Escherichia coli as polyhistidine-
on streptavidin-modified scintillation proximity assay beads (19).
The radioligand for PPAR-? was [3H]GW362433X. The assay
buffer was 50 mM Hepes (pH 7)?50 mM KCl?5 mM 3-[(3-
mg/ml BSA?10 mM DTT. Increasing concentrations of 13-S-
HODE were incubated in samples for at least 1 h at room
temperature before the bound reactivity for each well was deter-
Northern Blot Analysis of PPAR-? RNA Expression. Total RNA was
isolated, and Northern blot analyses were performed as described
(13). The PPAR-? probe was a 471-bp PPAR-? cDNA fragment
generated by RT-PCR using human liver mRNA (Clontech) with
primers 5?-AGC-AGC-CTC-TTC-CTC-AAC-GAC-CAG-3? and
Western Blot Analysis of PPAR-? and 15-LOX-1. Protein samples were
prepared and subjected to SDS?PAGE under reducing conditions
(12). After transfer, blots were probed with a solution of rabbit
antibody to human 15-LOX-1 (1:2,000 dilution) or PPAR-? (1:500
(Amersham Biosciences), as described (12).
Supporting Information. For further details, see Supporting Mate-
rials and Methods, which is published as supporting information
on the PNAS web site, www.pnas.org.
13-S-HODE Binds to PPAR-? in Colorectal Cancer Cells. We used the
Gal4-PPAR-? LBD reporter assay to measure the ability of 13-S-
express cyclooxygenase (COX)-1 and -2, and in DLD-1 cells, which
do not express COX-1 or -2. 13-S-HODE was 3.6- to 6.1-fold
stronger than was the identical concentration of linoleic acid in
activating the PPAR-? LBD (Fig. 1 A and B) (13-S-HODE vs.
linoleic acid and control, P ? 0.0001 for RKO and P ? 0.0012 for
DLD-1). By using scintillation proximity assays, we found that
activity for the Gal4-PPAR-? LBD and pMH-100-TK-luc reporter plasmid system was measured and normalized to ?-gal activity (relative to 100,000 ?-gal units). Values
to 100,000 ?-gal units). Values shown are the fold activation relative to control (DMSO-treated cells) and represent the means of triplicate experiments. Error bars
represent SEM. (F and G) Effects of 13-S-HODE on PPAR-? expression. Western blot analyses for PPAR-? expression in cells treated with 13-S-HODE or linoleic acid,
cultured for 24 h, and then harvested; repeated experiments showed similar results. Control cells (0) were treated with DMSO only. Equal loading was assessed by
probing for actin. (H) Effects of 15-LOX-1 expression on PPAR-? expression. RKO and DLD-1 cells were transfected with 15-LOX-1 expression vector, empty vector, or
transfection media alone (mock transfection). Western blots show the expression of 15-LOX-1 and PPAR-? in cells harvested at 24 h. Similar results were observed at
48 h and with repeated experiments. Lanes: S, standard 15-LOX-1 recombinant protein; 1 and 5, 15-LOX-1 expression vector transfected cells; 2 and 6, empty
vector-transfected cells; 3 and 7, mock transfections; 4 and 8, cells without transfections. (I and J) Effects of 15-LOX-1 expression on PPAR-? activity. DLD-1 (I) and RKO
(J) cells were transfected with 15-LOX-1 expression vector or the empty vector and with DRE-luciferase reporter and pSV-?gal vectors. Luciferase activity values were
reduced PPAR-? activation significantly compared (t test) with empty-vector-transfected RKO (E-RKO) (P ? 0.0125) and empty-vector-transfected DLD-1 (P ? 0.0052)
Shureiqi et al. PNAS ?
August 19, 2003 ?
vol. 100 ?
no. 17 ?
13-S-HODE binds directly to PPAR-? with a Ki of 10.8 ?M
13-S-HODE Down-Regulates PPAR-? Expression and Activation in
Colorectal Cancer Cells. Compared with linoleic acid (control),
cells (10 h, P ? 0.0001) (Fig. 1D), 44% in RKO cells (18 h, P ?
0.0001) (Fig. 1E), and in a concentration-dependent manner in
both cell lines (24 h). In DLD-1 cells, 13-S-HODE reduced the
activation of PPAR-? by 6.6% at 1.35 ?M, 37% at 6.75 ?M, 40%
at 13.5 ?M, 66% at 27 ?M, and 74% at 54 ?M; in RKO cells,
34% at 6.75 ?M, 45% at 13.5 ?M, 63% at 27 ?M, and 74% at 54
?M. We also found that 13-S-HODE down-regulated PPAR-?
and RKO cells (Fig. 1 F and G). In contrast, linoleic acid failed to
decrease PPAR-? expression (Fig. 1 F and G).
15-LOX-1 Expression Down-Regulates PPAR-? Expression and Tran-
scriptional Activity in Colorectal Cancer Cells. RKO and DLD-1 cells
transfected with pAdenoVator-CMV5-IRES-GFP vector carrying
15-LOX-1 cDNA expressed 15-LOX-1, whereas cells transfected
with empty pAdenoVator-CMV5-IRES-GFP vector or transfec-
tion medium alone (mock transfection) did not (Fig. 1H). 13-S-
HODE levels in 15-LOX-1-transfected DLD-1 cells (6.14 ? 0.43
ng??g of protein) were higher than in empty-vector-transfected
or nontransfected (2.05 ? 0.08, P ? 0.001) DLD-1 cells at 24 h
(data not shown). PPAR-? expression was lower in 15-LOX-1-
transfected cells than in empty-vector- or mock-transfected cells
(Fig. 1H). PPAR-? activity was significantly lower in 15-LOX-1-
transfected than in empty-vector-transfected cells (Fig. 1 I and J).
NSAIDs Down-Regulate PPAR-? Expression in Colorectal Cancer Cells.
We examined several NSAIDs for their ability to alter PPAR-?
expression; we began with sulindac (a nonselective COX inhibitor)
and celecoxib (a selective COX-2 inhibitor), which exhibit clinical
chemopreventive activity in colonic tumorigenesis (21, 22). Sulin-
dac and celecoxib reduced PPAR-? RNA expression in RKO and
DLD-1 cells at 48 h (Fig. 2 Top). Celecoxib reduced PPAR-?
protein expression in a time-dependent manner starting at 48 h
posttreatment (Fig. 2 Middle). Similarly, other NSAIDs (NS-398,
indomethacin, sulindac, and sulindac sulfone) down-regulated
PPAR-? protein expression (Fig. 2 Bottom).
15-LOX-1 Up-Regulation, PPAR-? Down-Regulation, and NSAID-
Induced Apoptosis. Given the temporal relationship between
NSAID up-regulation of 15-LOX-1 (at 24 h) (11, 12) and down-
regulation of PPAR-? (at 48 h), we examined the possible mech-
anistic link between these cellular events by creating an in vitro
system of RKO cells in which NSAID-induced 15-LOX-1 expres-
sion is blocked by 15-LOX-1-antisense (AS). Stable transfection of
an AS construct for 15-LOX-1 blocked the ability of celecoxib to
induce 15-LOX-1 expression (Fig. 3A) and suppressed the growth-
inhibitory effects of celecoxib (data not shown) in four selected
clones. Furthermore, celecoxib failed to reduce PPAR-? activity in
the two 15-LOX-1-AS clones (clones 3 and 4) tested for this effect.
SEM) of PPAR-? activity with DMSO (control) treatment in clone
4. In contrast, celecoxib reduced PPAR-? activity in pcDNA3.1
empty-vector-transfected RKO cells (E-RKO); PPAR-? activity in
celecoxib-treated cells was 66 ? 5.3% of the levels in DMSO-
treated cells at 48 h. The differences between the effects of
celecoxib on PPAR-? activity in E-RKO versus 15-LOX-1-AS-
transfected clones were statistically significant (P ? 0.0125). In
contrast, the effects of celecoxib on PPAR-? activity were not
statistically different between E-RKO and W-RKO. We selected
to further characterize the effects that blocking 15-LOX-1 expres-
sion would have on the response of RKO cells to celecoxib in vitro.
Celecoxib failed to up-regulate 15-LOX-1 or down-regulate
PPAR-? expression in 15-LOX-1-AS4-RKO cells cultured as long
as 240 h after celecoxib treatment (Fig. 3B). In contrast, celecoxib
up-regulated 15-LOX-1 and down-regulated PPAR-? in W-RKO
and E-RKO (Fig. 3B). Celecoxib increased 13-S-HODE levels (by
a mean of 2.57-fold in nine experiments) in W-RKO (vs. controls)
48 h after treatment (P ? 0.0001). The level of celecoxib-increased
13-S-HODE was inhibited by ?50% in 15-LOX-1-AS4-RKO cells
(P ? 0.0006 in nine experiments), compared with levels in cele-
coxib-treated E-RKO and W-RKO. 13-S-HODE levels in cele-
coxib-treated E-RKO and W-RKO were similar (P ? 0.14 in nine
To further study the link between 15-LOX-1 up-regulation,
sured the effects of celecoxib on apoptosis in W-RKO, 15-LOX-
1-AS4-RKO, and E-RKO. Celecoxib’s ability to inhibit growth and
induce apoptosis (measured by TUNEL, sub-G1, and DNA lad-
dering assays) was markedly lower in 15-LOX-1-AS4-RKO than in
W-RKO or E-RKO (Fig. 3 C–F).
PPAR-?-Knock-Out Suppresses Apoptosis Induction by Celecoxib and
13-S-HODE. We next evaluated the role of PPAR-? in apoptosis
induction by celecoxib or 13-S-HODE in an experimental PPAR-
?-knock-out model in HCT-116 colon cancer cells. Celecoxib
inhibited cell growth and induced apoptosis in WT HCT-116 cells,
which express PPAR-?, whereas these effects were markedly sup-
pressed in PPAR-?-null HCT-116 cells (Fig. 4). Parallel to these
findings, 13-S-HODE inhibition of cell growth and induction of
apoptosis were markedly diminished in PPAR-?-null vs. WT HCT-
of celecoxib on PPAR-? protein expression in DLD-1 cells. Similar results were
protein expression in RKO cells. Cells were treated with NSAIDs, harvested 72 h
later, processed for Western blotting, and probed with PPAR-? antibody. Lanes:
1, control; 2, NS-398; 3, indomethacin; 4, sulindac; 5, sulindac sulfone. Similar
results were observed with DLD-1 cells (data not shown) and with repeated
NSAID effects on PPAR-? expression in colorectal cancer cells. (Top)
www.pnas.org?cgi?doi?10.1073?pnas.1631086100Shureiqi et al.
116 cells (Fig. 4). Linoleic acid failed to show significant growth-
inhibitory or apoptotic effects in either cell line.
Celecoxib Effects on 15-LOX-1, PPAR-?, and Tumorigenesis in Vivo. To
examine the in vivo relevance of our in vitro findings, we tested the
mechanistic link between celecoxib’s induction of 15-LOX-1 ex-
pression, down-regulation of PPAR-?, and inhibition of tumori-
genesis in a nude mouse model with xenografts of WT-RKO and
RKO cells transfected with 15-LOX-1-AS or empty vector. Cele-
with pcDNA3.1 empty vector (Fig. 5A) and in W-RKO (data not
shown). In contrast, the antitumorigenic effects of celecoxib were
blocked in RKO cells that were stably transfected with 15-LOX-
1-AS clones 3 (Fig. 5B) and 4 (Fig. 5C). The tumor growth rates in
the non-celecoxib-treated animal groups (Fig. 5 A–C, solid lines)
were variable and inconsistent with specific 15-LOX-1-AS effects.
Celecoxib up-regulated 15-LOX-1 and down-regulated PPAR-?
protein expression in W-RKO (Fig. 5D) and empty-vector-
transfected cells (data not shown) but not in cells stably transfected
with 15-LOX-1-AS clone 3 or 4 (Fig. 5D).
and apoptosis. (A) Effects of stable transfection of 15-LOX-1-AS on celecoxib-
induced 15-LOX-1 in RKO cells. S, standard positive control of human 15-LOX-1
recombinant protein; ?, control cells that were treated with DMSO (celecoxib
1–4, AS-transfected clones. (B) Effects of celecoxib on 15-LOX-1 and PPAR-? in
stably transfected RKO cells with 15-LOX-1-AS clone 4 in protracted cell-culture
celecoxib on growth inhibition by celecoxib. Growth ratios of celecoxib (cele)-
treated WT RKO (W-RKO), E-RKO, and 15-LOX-1-AS clone 4 stably transfected
cells (15-LOX-1-AS) to control cells (DMSO-treated only) at 72 h. Mean ? SEM of
triplicate experiments. (cele, 15-LOX-1-AS vs. cele, W-RKO, P ? 0.0008; cele,
Effects of blocking 15-LOX-1 expression on celecoxib-induced apoptosis mea-
and 15-LOX-1-AS cells are means ? SEM of triplicate experiments at 72 h after
celecoxib or DMSO (control) treatment. (cele, 15-LOX-1-AS vs. cele, W-RKO, P ?
0.0001; cele, 15-LOX-1-AS vs. cele, E-RKO, P ? 0.0001). (E) Effects of blocking
15-LOX-1 expression on celecoxib-induced apoptosis as measured by sub-G1
fractions in RKO cells. Sub-G1 fractions in W-RKO, E-RKO, and 15-LOX-1-AS
treated with either celecoxib or DMSO (control) and harvested 72 h later are
means ? SEM of triplicate experiments. (cele, 15-LOX-1-AS vs. cele, W-RKO, P ?
0.0015; cele, 15-LOX-1-AS vs. cele, E-RKO, P ? 0.0001). (F) Effects of blocking
15-LOX-1 expression on celecoxib-induced apoptosis as measured by DNA lad-
dering assay. Lanes: 1, standard DNA ladder; 2, W-RKO cells; 3, W-RKO cells
treated with celecoxib; 4, E-RKO cells; 5, E-RKO cells treated with celecoxib; 6,
15-LOX-1-AS (control); 7, 15-LOX-1-AS cells treated with celecoxib.
Effects of celecoxib-induced 15-LOX-1 expression on PPAR-? expression
divided by the number of viable cells in the control experiment (DMSO-treated
are PPAR-? null. 13-S-HODE and linoleic acid (LA) concentrations were 13.5 ?M.
Means ? SEMs of triplicate experiments are shown. (13-S-HODE, PD?, vs. 13-S-
experiments showed similar results. (B) Effects of silencing PPAR-? expression on
apoptosis induction by 13-S-HODE and celecoxib, measured by TUNEL assay.
Means ? SEMs of triplicate experiments are shown. (13-S-HODE, PD? vs. 13-S-
PD? vs. linoleic acid, PD?, P ? 0.672.) Similar results were found with sub-G1
fraction assays and in triplicate experiments (data not shown). (C) Effects (mea-
celecoxib and 13-S-HODE. Lanes: 1, standard DNA ladder; 2, PD? cells treated
cells treated with 13-S-HODE; 5, PD? cells treated with 13-S-HODE; 6, PD? cells
treated with linoleic acid; 7, PD? cells treated with linoleic acid; 8, PD? cells
treated with celecoxib; 9, PD? cells treated with celecoxib.
PPAR-? effects on celecoxib and 13-S-HODE-induced apoptosis in colon
Shureiqi et al.PNAS ?
August 19, 2003 ?
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no. 17 ?
We found that (i) 13-S-HODE, the main product of 15-LOX-1,
bound to PPAR-?, suppressed PPAR-? activation, and down-
regulated PPAR-? expression; (ii) NSAIDs induced 15-LOX-1
expression to down-regulate PPAR-? expression and induce
apoptosis; and (iii) PPAR-? is an important signaling receptor
for both NSAIDs and 13-S-HODE for inducing apoptosis. These
results demonstrate the binding of 13-S-HODE to PPAR-? and
the biological effects of this binding on PPAR-? expression and
activation. The role of 13-S-HODE in suppressing PPAR-?
expression is important because of the recently recognized role
of PPAR-? in promoting colonic tumorigenesis (15, 16, 23). Both
exogenous and endogenous 13-S-HODE (the latter formed after
15-LOX-1 transfection) down-regulated PPAR-? expression and
activity. 13-S-HODE bound to PPAR-?, decreased PPAR-?
activation, and down-regulated PPAR-? expression at the same
concentration that we previously showed to induce apoptosis in
colon cancer cells (12); these results suggest that these biological
effects of 13-S-HODE are linked. This study found that these
effects are mechanistically linked in PPAR-?-null cancer-cell
experiments, showing that the loss of PPAR-? expression mark-
edly suppressed apoptosis induction by 13-S-HODE. Linoleic
effects, which indicates that these effects are specific to 13-S-
HODE. Our results, therefore, define a previously unknown
signaling pathway for inducing apoptosis in colorectal cancer
cells: the metabolism of linoleic acid by 15-LOX-1 leads to
13-S-HODE production, which down-regulates PPAR-? expres-
sion and activity to induce apoptosis (Fig. 5E).
be therapeutically modulated (by NSAIDs) to induce apoptosis. A
link during apoptosis induction between LOX metabolism and
NSAID–PPAR-? signaling was postulated (16) but never con-
firmed. Our prior studies, which showed that 15-LOX-1-induced
expression by NSAIDs is crucial to NSAID-induced apoptosis, led
us to investigate the existence of a mechanistic link to PPAR-?. We
hypothesized, based on our previous findings that NSAIDs up-
regulate 15-LOX-1 starting at 24 h, that if NSAIDs affect PPAR-?
expression through a 15-LOX-1 signaling pathway, this effect on
PPAR-? would occur after 24 h posttreatment (11, 12). Other prior
findings that NSAIDs had no effect on PPAR-? RNA expression
for as long as 36 h after treatment (16) supported this prediction.
Indeed NSAIDs down-regulated PPAR-? expression at 48 h post-
treatment in the present study, which was consistent with our
hypothesis. These findings indicated the temporal relationship
between NSAID up-regulation of 15-LOX-1 and down-regulation
We used a 15-LOX-1-AS model to investigate whether the
temporally associated NSAID effects of up-regulated 15-LOX-1,
down-regulated PPAR-? (by 13-S-HODE), and apoptosis induc-
tion are linked mechanistically. The 15-LOX-1-AS construct
blocked celecoxib-induced 15-LOX-1 expression, which in turn
inhibited celecoxib from forming 13-S-HODE, inducing apoptosis,
and inhibiting cell growth. Furthermore, blocking 15-LOX-1 pre-
vented PPAR-? down-regulation by celecoxib, thus indicating the
mechanistic link between 15-LOX-1 up-regulation, PPAR-? down-
in vitro findings to the in vivo setting of a nude mouse xenograft
model. Our findings in this model established the physiological
relevance of the mechanistic link between celecoxib induction of
15-LOX-1 expression, down-regulation of PPAR-?, and inhibition
of tumorigenesis. In WT or empty vector-transfected xenografts,
celecoxib up-regulated 15-LOX-1 expression, down-regulated
blocked in xenografts in which the expression of 15-LOX-1 was
15-LOX-1 expression is crucial to celecoxib’s ability to down-
regulate PPAR-? and inhibit tumorigenesis.
Based on findings in a colon-cancer cell system with overex-
pressed PPAR-?, He et al. (16) concluded that reducing PPAR-?
activity is an important mechanism of NSAID-induced apoptosis.
Park et al. (15) (of the same group) subsequently found that
found, however, that high concentrations of sulindac sulfide (?80
LOX-1 expression on PPAR-? expression and
tumorigenesis in vivo. Wild type RKO (W-
1-AS clone 3 (15-LOX AS-3), 15-LOX-1-AS
clone 4 (15-LOX AS-4), and E-RKO were
grown as xenografts in nude mice. Animals
control diet. Celecoxib inhibited the growth
of E-RKO (A) but not 15-LOX AS-3 (B) or 15-
LOX AS-4 (C) cells. (D) 15-LOX-1 and PPAR-?
Western blot analyses of W-RKO, 15-LOX
AS-3, and 15-LOX AS-4 tissue xenografts.
Lanes: S, standard 15-LOX-1 recombinant
protein; 1, W-RKO (WT); 2, W-RKO ? cele-
celecoxib; 5, 15-LOX AS-4 (AS-4); 6, 15-LOX
AS-4 ? celecoxib. Actin expression was used
to assess equal loading. (E) Proposed model
naling pathway that can be modulated by
NSAIDs to induce apoptosis and inhibit colo-
Effects of celecoxib-induced 15-
www.pnas.org?cgi?doi?10.1073?pnas.1631086100Shureiqi et al.