# The Roles of Dietary PPARγ Ligands for Metastasis in Colorectal Cancer

Authors: Hiroki Kuniyasu
Journal: PPAR Research
 (2008)

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## Abstract

Dietary peroxisome proliferator-activated receptor (PPAR)γ ligands, linoleic acid (LA) and conjugated linoleic acid (CLA), showed anticancer effects in colorectal carcinoma cells. LA is metabolized by two pathways. Cyclooxygenase (COX)-2 produces procarcinogenic prostaglandin E2, whereas 15-lipoxygenase (LOX)-1 produces PPARγ ligands. The 15LOX-1 pathway, which is dominant in colorectal adenomas, was downregulated and inversely COX-2 was upregulated in colorectal cancer. LA and CLA inhibited peritoneal metastasis of colorectal cancer cells in nude mice. The inhibitory effect was abrogated by PPARγ antisense treatment. A continuous LA treatment provided cancer cells quiescence. These quiescent cells formed dormant nests in nude mice administrated LA. The quiescent and dormant cells showed downregulated PPARγ and upregulated nucleostemin. Thus, short-term exposure to dietary PPARγ ligands inhibits cancer metastasis, whereas consistent exposure to LA provides quiescent/dormant status with possible induction of cancer stem and/or progenitor phenotype. The complicated roles of dietary PPARγ ligands are needed to examine further.


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## Body

## 1. Introduction

Colorectal cancer is the third most
common malignant neoplasm worldwide and the third leading cause of cancer
deaths in Japan [1]. The frequency of
colorectal cancer in Japan doubled
in the last three decades according to the alteration of life style from
Japanese to Western [2]. Especially, the increase in fat intake and decrease in
fiber intake have been regarded as the most important nutritional influence on
colon cancer development [3, 4]. In this review,
we focused on the fatty acids, which possess ligand activity for peroxisome
proliferator-activated receptor (PPAR)γ in foods. PPARγ is activated by endogenous secreted
prostaglandins and fatty acids. 15-deoxy-δ(12,14)-prostaglandin J2 is a strong
endogenous ligand of PPARγ [5]. Linoleic acid
(LA) is one of the essential fatty acids, which we must intake from food.
Metabolic products of LA, such as 9-hydroxyoctadecadienoic
acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), and
13-oxooctadecadienoic acid (13-OXO), are known as
PPARγ ligands [6].
Conjugated linoleic acid is a stereoisomer of LA [7]. CLA
is contained in beef, lamb, and also in vegetable oils [8]. Roles of
these dietary PPARγ ligands on PPARγ activation are not still unclear.
Colorectal cancer is a good model for influence of nutritional factors to
cancer development and progression [9, 10]. In this review,
roles of LA and CLA for colorectal cancer progression and therapeutic
possibility are discussed as dietary PPARγ ligands.


## 2. Metabolic Pathways of Linoleic Acid

Prostaglandins (PGs) are bioactive
lipids derived from the metabolites of membrane polyunsaturated fatty acids
(PUFAs), and play important roles in a number of biological processes [11]. Cyclooxygenases-2 (COX-2)-dependent overproduction of PGE2 is hypothesized
to be an important part of sustained proliferative and chronic inflammatory
conditions [12, 13]. Several in vivostudies
hypothesize that a high amount of ω-6 PUFA such as LA might
enhance colorectal carcinogenesis via stimulation of colonic epithelial cell
proliferation [14–17]. In fact, rats treated with a genotoxic agent, azoxymethane (AOM), and fed a diet supplemented with LA
develop more tumors than those treated with AOM alone [18].The oxidative metabolites of LA, in particular, 9-HODE,
13-HODE, and 13-OXO, have biological
effects as a PPARγ ligand [6]. CLA, a
strong ligand for PPARγ, has a substantial
anticarcinogenic effect [8, 19]. Synthesized
PPARγ ligands including
troglitazon have been shown to be effective
chemopreventive agents in a rat model of carcinogenesis and in AOM-induced
colon cancer in mice [20]. If LA provides PPARγ
ligands, LA might act as an anticarcinogenic agent.Arachidonic
acid is a fatty acid and a component of lipid membranes, and also a major
substrate for lipoxygenase enzymes. 15lipooxygenase-1 (15LOX-1) is known for
its anti-inflammatory properties and has a profound influence on the
development and progression of cancers [21, 22]. Recent studies show that
ligand activation of PPARγ
in colorectal cancer cells attenuates colonic inflammation and causes a
reduction growth via the induction of apoptosis [23, 24]. Furthermore, it has
been reported that a number of metabolites generated by 15LOX-1 can function as
endogenous activators and ligands for PPARγ
[25, 26]. We confirmed the anticarcinogenic effect of LA by in vitro
transformation assay using a rat intestinal cell line, IEC6, which expressed
15LOX-1, but not COX2. LA treatment inhibited AOM-induced transformation in
IEC6 cells [27]. Many literatures reported that PPARγ possesses an anticarcinogenic effect in
colorectal cancer [20]. Moreover, a decrease in PPARγ expression is associated with cancer
metastasis [28, 29]. Inhibitory effect of PPARγ to cancer metastasis is reported in several
cancers, such as nonsmall cell lung cancer, colon cancer, thyroid cancer, and
breast cancer [30–33].


## 3. Switching of LA Metabolic Pathways from 15lox-1 to Cox-2 in Colorectal Cancer Development

Inhibitory
effect of LA on intestinal epithelial cell transformation elucidated above
suggests that 15LOX-1 LA metabolism suppresses colon carcinogensis [27]. We
next focused on the dual roles of LA in human colon cancer development.
Expression of 15LOX-1 and COX-2 was examined in human colon adenoma and
carcinoma to elucidate the balance of the two LA metabolic pathways in
malignant transformation of human colon epithelium.We
examined the expressions of COX-2 and 15LOX-1 in 54 adenomas, 21
carcinoma-in-adenoma lesions, and 36 serosa-invading advanced carcinomas in the
colon [34]. We examined 15LOX-1 mRNA and COX-2 protein by in situ hybridization
and immunohistochemistry, respectively. In the nonpathological
colon mucosa, which expressed 15LOX-1 but not COX-2, proliferation of colon
epithelial cells was controlled at constitutive levels. 15LOX-1 mRNA was found in 96% of
adenomas, 43% of adenoma in carcinoma-in-adenoma lesions, and 10% of carcinoma
in carcinoma-in-adenoma lesions, but not in advanced carcinoma (P<.0001).
In contrast, COX-2 production was found in 11% of adenomas, 52% of adenoma in
carcinoma-in-adenoma lesions, 71% of carcinoma in carcinoma-in-adenoma lesions,
and 92% of advanced carcinoma (P<.0001). Concurrence of COX-2 induction with 15LOX-1 downregulation
was found in 6% adenomas, in 33% adenoma components, and 71% carcinoma
components of carcinoma-in-adenoma
lesions (all mucosal cancer), in 89% cases in
nonmetastatic serosa-invading carcinomas, and in 100% cases of nodal
metastasized carcinomas. Our
data showed that induction of COX-2 expression and downregulation of 15LOX-1
were sequentially increased from adenomas, adenoma components, and carcinoma
components in carcinoma-in-adenoma lesions, to invasive carcinomas. Interestingly, low grade-adenoma components with in carcinoma-in-adenoma lesions showed COX-2 expression and 15LOX-1 downregulation at
more frequency than low grade-adenomas, which suggests that the biological
property is different in the same histological atypia. In contrast, high
grade-adenoma components showed no difference from high grade-adenomas in expressions of COX-2 and
15LOX-1, which suggests that high grade-adenomas might already possess
malignant potential as high as adenoma components in carcinoma-in-adenoma lesions. This sequential alteration of concurrence of COX-2
induction with 15LOX-1 downregulation possibly shows close association of the
switching of LA-metabolizing pathways with colon cancer development and
progression.15LOX-1 is revealed as an apoptosis inducer in human
cancers and inhibits cancer progression. The
reduction of 15LOX-1 and the isoform 15LOX-2 is correlated with the disease
progression of breast cancer and the poor clinical outcome [35]. Induction of
15LOX-1 provides apoptosis in oral cancer [36]. 15LOX-1 expression is downregulated
in colon adenomas, and ectopic expression of 15LOX-1 induces apoptosis in
Caco-2 colon cancer cells [37].In expression of 15LOX-1, inflammatory cytokines play an
important role. Interleukin (IL)-4 and IL-13 induce 15LOX-1 expression via
Jak2/Tyk2/Stats pathway [36, 38–40]. In prostate cancer, ratio of ω-3/ω-6 fatty acids is associated with expressions of 15LOX-1 and COX-2 [41].
15LOX-1 induction by nonsteroidal anti-inflammatory drugs (NSAIDs), such as
sulindac sulfone, provides apoptosis in SW480 colon cancer cells. Sulindac
sulfone inhibits GMP (cGMP)-phosphodiesterases and activates protein kinase G,
which enhances 15LOX-1 expression transcriptionally [42]. GATA-6
transcriptional factor is involved in NSAID-induced 15LOX-1 induction in RKO
and DLD-1 colon cancer cells [43]. GATA-6 activates 15LOX-1 promoter in Caco-2
colon cancer cells but not in the cells induced differentiation by
sodium-butyrate [44]. These are supported by conventional NSAIDs activates PPARγ
[45]. COX-2 expression also involves Jak2/Stats pathway and nuclear factor (NFκB) [46–48]. Activation of PPARγ
downregulates COX-2 expression by inhibition of NFκB and activator protein
(AP)-1 [49]. This negative regulation of COX-2 expression by PPARγ
activation might be one of the
mechanisms of reversal expressions between 15LOX-1 and COX-2. Furthermore, promoter DNA
methylation is responsible for silencing 15LOX-1 expression, which is reversed
by 5-aza-2-deoxycytidine treatment [50, 51]. The epigenetic alteration might be
a trigger to switch 15LOX-1 repression and COX-2 upregulation along with
malignant transformation and disease progression in colorectal cancer. Thus,
switching of LA metabolism from 15LOX-1 to COX-2 is thought to be a common
mechanism to escape from antitumoral effect of LA.


## 4. Antitumor Effect of CLA

We
next argue the effect of CLA, which is an isomer of linoleic acid and is
established as a PPARγ ligand without metabolization by 15LOX-1.
CLA is a natural content of some foods, such as beef, lamb, and also vegetable
oils [8]. CLA is one of essential fatty acids, which possesses characteristic
bioactive properties [8]. CLA is composed of positional- and stereoisomers
of octadecadienoate (18 : 2). Chemoprotective properties of CLA are reported in
experimental cancer models and in vitro examinations [8, 19]. Differently from
LA, CLA is not a precursor of prostaglandines. CLA activates PPARγ as a ligand [7, 52]. Through activation of PPARγ, CLA might act as an antimetastatic agent as
well as an anticarcinogenic agent. We examined the antimetastatic effect of CLA
on peritoneal dissemination [53]. Cell growth of MKN28 human gastric cancer
cells and Colo320 human colon cancer cells was suppressed by CLA in a
dose-dependent manner with an increment in apoptosis. CLA significantly
inhibited invasion into type IV collagen-coated membrane of MKN28 and Colo320
cells. CLA-induced growth inhibition was recovered by the exposure to antisense
S-oligodeoxynucleotides (ODN) for PPARγ in both cell lines. BALB/c nu-nu mice were
inoculated with MKN28 and Colo320 cells into their peritoneal cavity, and
administrated with CLA intraperitoneally (weekly, 4 times). CLA treatment did
not affect food intake or weight gain of mice. CLA treatment significantly
decreased metastatic foci of both cells in the peritoneal cavity. Survival rate
in mice inoculated with MKN28 or Colo320 cells was significantly recovered by
CLA treatment. PPARγ initiates transcription of genes associated
with energy homeostasis, cell growth, and anti-/proinflammatory
effect [24, 54–57]. Protein production in MKN28 and Colo320 cells
treated with CLA showed a decrease in epidermal growth factor receptor (EGFR)
and transforming growth factor (TGF)-α and an increase in Bax. Our results showed
that CLA inhibits cell growth and invasion, and induces apoptosis in cancer
cells. Our data are supported with the reports, which indicate that PPARγ downregulates EGFR, and upregulates Bax,
p21Waf-1, and E-cadherin, which are associated with antiproliferative, proapoptotic,
and prodifferentiation effects [33, 58–60].We also reported the
tumor suppressive effect of CLA on established peritoneal tumors using a
syngeneic mouse peritoneal metastasis model [61]. C57BL6
mice were inoculated with LL2 cells into their peritoneal cavity. Two weeks
after the inoculation, colonized peritoneal cancer foci (2.2±0.4 mm in
diameter) were treated with CLA administrated intraperitoneally (600 pmol/mouse, weekly, twice). CLA
treatment decreased the number of peritoneal tumors to 26% of that in untreated
mice (P<.0001). CLA treatment also decreased size of peritoneal
tumors to 27% of that in untreated mice (P<.0001). In CLA-treated
tumors, proliferating cells were decreased (P<.0001), whereas
apoptotic cells were increased (P<.0010). CLA-treated LL2 tumors
showed decrease in PPARγ and EGFR proteins, and increase in Bax
protein in comparison with untreated tumors.


## 5. Antitumor Effect of Linoleic Acid

We confirmed antimetastatic
effect of PPARγ by CLA in gastric and colon cancer cells. We
next examined antimetastatic effect of LA.The effect of LA on peritoneal metastasis was examined using in vitro
treatment of cancer cells and mouse peritoneal metastasis models as well as CLA
examination. Firstly, cell growth of MKN28 human gastric cancer cells and
Colo320 human colon cancer cells were suppressed by LA in a dose-dependent
manner with increment of apoptosis. LA significantly inhibited invasion into
type IV collagen-coated membrane of MKN28 and Colo320 cells (P<.05).
The inhibition of growth and invasion and the induction of apoptosis by LA were
abrogated by exposure to antisense S-ODN for PPARγ.
Moreover, the decrease in 15LOX-1 expression by exposure to antisense S-ODN for
15LOX-1 suppressed the inhibition of growth and invasion and the induction of
apoptosis by LA. LA-induced growth inhibition was recovered by the exposure to
antisense S-ODN for PPARγ
or 15LOX-1. BALB/c nu-nu mice inoculated with MKN28 and Colo320 cells into
their peritoneal cavities were administrated IP with LA (weekly, 4 times). The
LA treatment significantly diminished the number of metastatic foci of both
cells in the peritoneal cavity (P<.05). Protein production in MKN28
and Colo320 cells treated with LA showed a decrease in EGFR and an increase in
Bax. PPARγ
activation is reported to decrease EGFR expression and increase Bax expression
[58, 60]. Our data suggest
that LA possesses the same mechanism to CLA of PPARγ
ligand; however, its efficacy
was 103 times weaker than CLA. LA-metabolites thought to be weaker
agonists of PPARγ. MKN28 and Colo320 cells expressed both
COX-2 and 15LOX-1. At least, relative high concentration of LA might be
metabolized dominantly by 15LOX-1, which consequently provides antimetastatic
effect in these cells.Thus,
LA and CLA suppress occurrence of cancer metastasis and
reduce existing metastatic tumors in animal models. These findings encourage
clinical usage of LA and CLA for treatment of cancer metastasis.


## 6. Effect of LA on Cancer Dormancy

A short-term
treatment with LA or CLA induced apoptosis in a dose-dependent manner through
PPARγ
activation as described above. On the contrary, in a long-term continuous
treatment with adequate concentrations, LA induced reversible cell growth-arrest in cancer
cells that escaped
from apoptosis [62].Cancer cell tumorigenicity in nude mice depends on
several factors in cancer cells themselves and their host. To form
macroscopical tumors, cancer cells must survive in their host tissue against
host immunity, and proliferate with utilization of the host endothelial cells
to make tumor vasculature. These steps are similar to those in metastasized
cancer cells on the metastasis targets [63]. If cancer cells do not endure the
attacks by host immunity, they cannot form tumor. When they survive but not
proliferate, the condition resembles quiescent or static dormancy. When they
survive and proliferate but do not generate tumor vasculature, the cancer cells
stay microscopical cell aggregation; the condition is to be an equivalent
condition to tumor dormancy [64].In
vitro cell growth was suppressed by 48-hour treatment with LA in a dose-dependent manner in MKN28 and Colo320 cells.
Continuous treatment with LA induced quiescence in both cells at 5 to 7 weeks
after the LA treatment. The finding was also observed in Ku-7 bladder cancer
cells and DU145 prostate cancer cells (Figure1). In LA-induced quiescent
cancer cells, protein production of Bcl-2 was increased, whereas Bak, EGFR, and
vascular endothelial growth factor (VEGF) levels were decreased [62]. These alterations might be associated
with inhibition of cell growth, angiogenesis, and apoptosis [65, 66], which explained
well the characteristics of LA-treated cancer cell aggregation. These
alterations of protein levels were the same as those in cells treated with PPARγ
ligands, toroglitazon, and CLA [62]. The PPARγ expression was also
decreased in quiescent MKN28 and Colo320 cells by continuous treatment with LA.
The LA metabolites by 15LOX-1 activate mitogen-activated protein kinase (MAPK)
and increase PPARγ phosphorylation,
but downregulate PPARγ activity [67].
Continuous PPARγ phosphorylation
might decrease PPARγ expression by long-term
LA treatment.Effect of LA on growth inhibition in Ku-1 bladder cancer cells
and DU145 prostate cancer cells by long-term treatment. Ku-7 cells and DU145
cells were continuously treated with LA (100μg/mL) for the indicated period with weekly reseeding by 1×105 cells per well. If cells were less than 1×105 cells per well, all
cells were reseeded. Standard deviation of each cell number was less than 10%
of the value.
(a)(b)When MKN28 and Colo320
cells were inoculated to nude mice subcutaneous tissue, LA-induced quiescent
MKN28 and Colo320 cells showed higher tumorigenicity than nontreated cells in
nude mice. In the contrary to the tumorigenicity, LA-induced quiescent cancer
cells showed 1/10 slower tumor growth than nontreated cells. LA withdrawal after the inoculation
provided regrowth in the cancer cells, which subsequently grew into macroscopic
tumors. LA-induced quiescent cells were different from growth-arrest cells by
aging or senescence, which are irreversible, and refractory to growth factors
[68].In mice treated with
LA weekly administration after the inoculation, inoculated quiescent cancer
cells did not form macroscopical tumors. Histological examination revealed less
than 500μm-sized cancer-cell
aggregations in the inoculation site. These cancer cell nests showed no
proliferative activity, no vascularity, and no immune cell infiltration [62]. These features of the cancer cell nests
were similar to those in tumor dormancy status [64]. The dormant cells
expressed undetectable levels of PPARγ
protein, which suggests that inactivation of PPARγ
might be associated with tumor dormancy formation. In contrast, withdrawal of
LA might break the dormancy status in cancer cells. Moreover, PPARγ-negative
dormant cells expressed increased levels of nucleostemin. Nucleostemin possesses a role for maintaining
stemness [69, 70]. PPARγ
inhibits Wnt and LIF (leukemia inhibitory factor) signaling pathways, which
maintain pluripotency and self-renewal of stem cells [5, 71]. Downregulation of
PPARγ
might induce dedifferentiation and stem cell/progenitor phenotype in cancer
cells, which might be associated with cancer dormancy and metastasis.In
this story, several possibilities are considered. Firstly, LA-induced PPARγ
downregulation provides stemness in cancer cells. Secondly, LA-induced
apoptosis abolishes PPARγ-positive
cancer cells and PPARγ-negative
cancer stem cell/progenitor cells remain. Thirdly, LA possesses direct effect on cancer stem
niche. To confirm the mechanism underlying LA-induced cancer dormancy, further
examination is requested to focus on the relation of PPARγ
with cancer stem cells.


## 7. Conclusion

In this article, we described that the
dietary PPARγ ligands, LA and CLA,
are deeply involved in colorectal cancer development and progression through
PPARγ activation. LA and
CLA provide remarkable anticancer effects by short-term treatment. In contrast,
long-term continuous treatment with LA induces quiescent and dormancy in cancer
cells with PPARγ downregulation.
These conditions might be associated with phenotypes of cancer stem
cells/progenitor cells. LA is a dietary factor to be taken from food everyday.
Cancer cells might be continuously exposed to various concentrations of LA in human body. Taken
together, short-term administration of LA and CLA is an effective therapeutic
tool for cancer metastasis. We are requested to evaluate the effect of dietary
LA on cancer metastasis for prevention of cancer metastasis and cancer dormancy.



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