# The Role of PPARγ Receptors and Leukotriene B4 Receptors in Mediating the Effects of LY293111 in Pancreatic Cancer

Authors: Thomas E. Adrian; Rene Hennig; Helmut Friess; Xianzhong Ding
Journal: PPAR Research
 (2008)

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## Abstract

Pancreatic cancer is a devastating disease in which current therapies are inadequate. Separate lines of research have identified the 5-lipoxygenase/leukotriene B4 receptor pathway and the PPARγ pathway as potential targets for prevention or treatment of this disease. LY293111 was originally designed as a potent leukotriene B4 receptor antagonist for treatment of inflammatory conditions. LY293111 was also known to have inhibitory effects on 5-lipoxygenase, which is upstream of the production of leukotrienes. LY293111 was shown to have potent anticancer effects in pancreatic cancer and several other solid malignancies, where it caused cell cycle arrest and marked apoptosis. Subsequently, it came to light that LY293111 exhibited PPARγ agonist activity in addition to its effects on the 5-lipoxygenase pathway. This raises the question of which of the two targets is of greatest importance with regard to the anticancer effects of this agent. The evidence to date is not conclusive, but suggests that the effects of LY293111 may be mediated by both LTB4 receptors and PPARγ.


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## Body

## 1. Introduction

Pancreatic Cancer is a devastating disease with more than 80% of all patients presenting with surgically inoperable tumors. It remains the fourth leading cause of cancer death in both men and women in the USA. The median survival is usually less than six months even with the addition of chemotherapy [1–3]. Surgical resection is the only effective
treatment option, but there are few long-term survivors even after apparent
curative resection [1–3]. Alternative
effective treatment strategies are desperately needed for this disease.


### 1.1. Fatty Acids and Human Cancer

Epidemiological and animal studies show that
a high fat consumption is associated with a higher incidence and growth of
tumors at several specific organ sites including breast, pancreas, and prostate
[4–11]. Recent
studies indicate that diets containing a high proportion of polyunsaturated
omega-6 fatty acids (n−6 FA), such as linoleic acid (the precursor of
arachidonic acid) are associated with a more advanced disease stage at the time
of diagnosis of several kinds of cancer [4–6, 8, 10, 11]. In contrast,
long-chain n−3 fatty acids, such as docosahexaenoic acid and eicosapentaenoic
acid (EPA) inhibit the growth and metastasis of several cancers including
pancreatic cancer [7, 9]. Omega 3 fatty acids inhibit tumor growth by a number
of mechanisms including suppression of COX-2 expression and, for EPA at least,
the alternative substrate produces different cyclooxygenase (PGE3)
and lipoxygenase (LTB5) products that have anti-inflammatory and anticancer
effects.


### 1.2. Eicosanoid Pathways

Arachidonic acid is
a substrate for three distinctively different enzymatic pathways. Among them,
prostaglandin endoperoxide synthases (cyclooxygenases) catalyze the committed
step that leads to prostaglandin biosynthesis [12–14]. The second
pathway is the epoxygenase pathway that appears to have no role in cancer. The
third pathway for metabolizing arachidonic acid, the lipoxygenase pathway
catalyzes the incorporation of one oxygen molecule into polyunsaturated fatty
acids to yield a 1-hydroperoxy-2, 4-trans, cis-pentadiene product [14–16]. Mammalian lipoxygenases possess
regiospecificity during interaction with substrate, and on this basis have been
designated as arachidonate 5-, 12-, and 15-lipoxygenase (5-LOX, 12-LOX, and
15-LOX) [14–16]. The three distinct enzymes insert oxygen at
carbon 5, 12 or, 15 of arachidonic acid, and the primary product is 5S-, 12S-,
or 15S-hydroperoxyeicosatetraenoic acid (5-, 12-, or 15-HPETE), which can be
further reduced by glutathione peroxidase to hydroxy forms (5-, 12-, 15-HETE),
respectively [14–16]. 5-LOX is
noteworthy because it is the only pathway that can turn arachidonic acid into
leukotrienes [15, 17]. The activity of 5-LOX is dependent upon a second factor
termed 5-LOX-activating protein (FLAP) [15, 17]. Considerable effort has been
expended by the pharmaceutical industry to produce inhibitors of FLAP, 5-LOX as
well as leukotriene antagonists, because the 5-LOX products, leukotrienes (LTB4,
LTC4, LTD4, and LTE4) have been implicated as
mediators of inflammation and immediate hypersensitivity reactions, in
particular, human bronchial asthma [18, 19].


### 1.3. Leukotriene Receptor Antagonists and the Development of LY293111

The pharmaceutical industry has focused on several targets to suppress
leukotriene activity in inflammatory conditions such as bronchial asthma [18, 19].
One approach is to directly inhibit 5-lipoxygenase activity, thereby blocking
secretion of all leukotrienes. The most
widely studied clinical inhibitor of 5-lipoxygenase is zileuton, which inhibits
the active site of 5-lipoxygenase atconcentrations that do not
inhibit cyclooxygenase, 12-lipoxygenase, or 15-lipoxygenase [18–22]. Another avenue to inhibit leukotriene
formation is via blocking FLAP activity, thus preventing cytoplasmic to
membrane translocation and activation of 5-lipoxygenase [16–18]. MK-0591 is a
widely used 5-lipoxygenase-activating protein inhibitor for
biomedical research [16–18]. Even though it strongly inhibits
5-lipoxygenase activity and blocks leukotriene generation, its use in clinic is
limited by marked side effects. The final pharmacological approach to block
leukotriene activity is to selectively block the actionsof LTB4 or the sulfidopeptide leukotrienes using specific receptor antagonists.Several synthetic LTB4 receptor
antagonists have been developed. Early compounds included SC-41930;
ONO-4057, which was orally
active; LY223982, a benzophenone dicarboxylic acid; and LY255283 a hydroxyacetophenone
[23–27]. These latter
two compounds from the Lilly Research Laboratories potently block LTB4 binding to its receptors within the nM range and inhibit the biological
functions of LTB4 in vitro [28, 29]. Unfortunately, they
showed poor oral bioavailability [28]. In 1995, investigators at the Lilly
Research Laboratories reported a new LTB4 antagonist, LY293111. This
compound is a novel derivative of LY255283, but is orally stable and more potent
as an LTB4 receptor antagonist [28, 29]. Compared with other LTB4 receptor
antagonists, LY293111 is superior at blocking the cellular functions induced by
LTB4 [28, 30].


### 1.4. Inflammation, Cyclooxygenases, Lipoxygenases, and Cancer

The
epidemiological data show a clear and strong association between chronic
inflammatory conditions and cancer development, even though the conditions
causing inflammation may vary [31–36]. It can be due to chronic infection caused by
a virus, bacteria, or parasite or it may be due to noninfective, physical, or
chemical irritant [31–36]. For example, chronic infection with the bacterium Helicobacter pylori causes atrophic gastritis, which can lead to
dysplasia and adenocarcinoma [37].
Hepatitis B and C viruses account for more than 80% of cases of
hepatocellular carcinoma worldwide [34].
The inflammatory bowel diseases, ulcerative colitis and Crohn's disease,
predispose to the development of cancers of the large bowel and/or terminal
ileum, although a causative infectious agent has never been conclusively
identified [38]. For noninfectious
inflammation, chronic reflux of gastric acid and bile into the distal esophagus
causes chemical injury and on the long-term can lead to Barrett's esophagus and
eventually to esophageal adenocarcinoma [35].
Thus it is apparent that chronic inflammation is a common underlying
theme in the development of many different malignancies.Although the mechanisms for the association between inflammation and
cancer are not fully understood, growth factors, cytokines, and chemokines
released into inflammatory environment are associated with tumor development
and progression [32, 36]. High
concentrations of free radicals and nitric oxide can induce DNA damage and
promote cancer development [32, 36].
Over the past decade, much attention has been paid on the role of
cyclooxygenases in cancer development, specifically its inducible isoform, the
cyclooxygenase 2 (COX-2) [39–41]. COX-2 is active within both inflamed and
malignant tissues [37–39]. The expression of COX-2 and COX-2 metabolites
increases during the multistage progression of tumors [39–41]. By metabolizing
arachidonic acid to prostaglandins, COX-2 induces cellular resistance to
apoptosis, modulation of cellular adhesion and motility, promotion of
angiogenesis, and immunosuppression [42–47]. Epidemiological data has implicated COX-2 in
the pathogenesis of a number of epithelial malignancies, especially colorectal
cancer [48, 49]. Inhibition of the enzyme with COX inhibitors is associated
with a dramatic reduction in the incidence, morbidity and mortality of
colorectal cancer [48–51]. Recent
attention has also been focused on the role of 5-LOX, 12-LOX, and 15-LOX in
cancer [52–60]. In
pancreatic cancer, activation of the 5-LOX and 12-LOX pathways enhances cancer
cell proliferation, while the 15-LOX pathway is protective against cancer
development [61–64].


### 1.5. The 5-Lipoxygenase/LeukotrieneB4 Pathway and Cancer

Accumulating
evidence suggests that the 5-LOX pathway has profound influence on the
development and progression of human cancers [61–64]. 5-LOX is overexpressed in pancreatic cancer
tissues but is not expressed in normal pancreatic ductal cells [65]. Furthermore,
this pathway is already up-regulated in pancreatic intraepithelial neoplasias
(PanINs), which are the precursor lesions of pancreatic adenocarcinoma [66]. Blockade of 5-LOX activity inhibits
proliferation and induces apoptosis in pancreatic cancer cells both in vitro
and in vivo [67–69]. Pancreatic cancer cells secrete LTB4 and LTB4 induces proliferation in these cells [62]. Two
G-protein-coupled LTB4 receptors (BLT1 and BLT2) have been cloned
and characterized. BLT1 and BLT2 are high- and low-affinity LTB4 receptors, respectively, and form a gene cluster in humans. Both BLT1 and BLT2
are up-regulated in pancreatic cancer tissues, and expression was seen in all
of the tested pancreatic cancer cell lines [65, 70]. As with other proteins in
the 5-LOX/LTB4 pathway, BLT1 and BLT2 are already up-regulated in
pancreatic intraepithelial neoplasias (PanIN lesions) which are the precursors
of pancreatic adenocarcinomas [70]. This suggests that they may be valuable
targets for chemoprevention.


### 1.6. PPARγ and Pancreatic Cancer

Peroxisome proliferator activated receptor-γ
(PPARγ) is a member of the nuclear receptor superfamily of ligand-activated
transcription factors. PPARγ is expressed at high levels in adipose tissue and
plays a central role in adipocyte differentiation and energy homeostasis.
Recent studies have implicated PPARγ in the pathogenesis of several human
malignancies [71–74]. Previous
studies have suggested that PPARγ is up-regulated in pancreatic cancer [75]. Our
own studies, employing two separate commercially available antibodies, show
that PPARγ is expressed in pancreatic cancer, but that expression in the cancer
cells does not appear to be different from that in normal pancreatic ductal cells (Figure 1). In contrast, PPARγ staining was seen in the islets surrounding cancers, but
not in islet cells from normal pancreatic tissues obtained from multiorgan
donors (Figure 1). In animal models,
PPARγ
ligands have preventive effects against chemical carcinogenesis [76]. Several studies
have shown that PPARγ agonists, including the natural ligand 15-deoxy-Δ12,14-prostaglandin J2, and thiazolidinedione antidiabetic agents, such as citiglitizone
and rosiglitizone, inhibit growth and induce apoptosis in pancreatic cancer [75, 77–81]. In contrast,
one paper suggests induction of differentiation without apoptosis [82]. The
apoptosis appears to be preceded by a morphological change to a more differentiated
cell type which perhaps undergoes apoptosis when DNA repair turns out to be not
possible [80]. In some studies, PPARγ agonists also block invasion and
angiogenesis [83, 84]. However, this is controversial since PPARγ agonists
induce secretion of vascular endothelial growth factor, which would have a
promoting effect on metastatic tumor growth [85].Immunocytochemistry of PPARγ receptor in normal human pancreas and in a
PanIN lesion and a pancreatic cancer. The PPARγ receptor antibody shows a
similar intensity of nuclear staining in normal ducts and in pancreatic cancer
cells as well as cells in the PanIN lesion. In contrast, no staining is seen in
normal islets, but nuclear staining is seen in islets from tissue adjacent to a
cancer. These pictures are representative of eight samples of each tissue type.
Staining was similar using antibodies from two different commercial sources.


### 1.7. LY293111 and Cancer

As might be expected from the growth-stimulatory effects of LTB4 in pancreatic cancer, the LTB4 receptor antagonist, LY293111
inhibits cancer growth and induces apoptosis both in vitro and in vivo [86–89]. LY293111
inhibits proliferation and induces apoptosis in a wide range of pancreatic
cancer cell lines as well as cells of other tumor types, such as breast,
prostate, and colon cancer cells [86–89]. These effects on growth and apoptosis
are both time and concentration dependent, with effects seen at 100–500 nM in vitro
[86, 87]. To confirm the involvementof LTB4 receptors in mediating the effect of LY293111 on humanpancreatic cancer cell proliferation, another selective LTB4 receptor antagonist, U75302 was used in comparison with a selective LTD4 antagonist, LY171883 [86]. U75302 inhibits the proliferation of pancreatic
cancercells; but it is less potent than LY293111 as expected from the lower receptor affinity of this drug [28, 29, 90]. In contrast, the selective LTD4 antagonist, LY171883 had no significant effect on pancreatic cancer
cell growth. LY293111 causes cell
cycle arrest in the S phase of the cell cycle with suppression of expression of
cyclin A, cyclin E, and cdk2. In
parallel with growth inhibition, LY293111 induced apoptosis in all cancer cell
lines tested [86, 87]. LY293111 induced
dramatic morphological changes inhuman pancreatic cancer cells
following a short period of treatment [86, 87]. The treated cells became
rounded and exhibited membrane blebbing, chromatin condensation, and
nuclear fragmentation,
finally they were detached from the microplate.
Induction of DNA fragmentation by LY293111 was confirmed by TUNEL assay
(terminal deoxynucleotidyl transferase-mediated nick end labeling)
and apoptosis was also established by annexin V binding [86, 87]. Apoptosis
is triggered through the mitochondrial pathway, with a change in the ratio of
proapoptotic proteins, such as Bax to antiapoptotic proteins, such as Bcl-2 and
Mcl-1, release of cytochrome C, activation of caspase (but not caspase 8), and subsequent
activation of the downstream caspase cascade with activation of caspase 3 and
caspase 7 and cleavage of the caspase 3 substrate, poly ADP-ribose polymerase
(PARP) [87].LY293111 markedly slows down the growth of
subcutaneous xenografts of human pancreatic cancer in athymic mice at a dose of
250 mg/kg/day [86]. To confirm the antipancreatic cancer effect of LY293111,
pancreatic cancer cells with stable expression of enhanced green
fluorescent protein (GFP) were
orthotopically implanted into the duodenal lobe of the pancreas of
athymic mice. Our data show that LY293111 significantly inhibits the growth of
the orthotopically implanted pancreatic cancer cells in concert with blocking metastatic
spread to the liver and other organs [91]. LY293111 also dramatically increased the number of TUNEL positive cells
in pancreatic tumors harvested from the subcutaneous transplant experiments in
athymic mice [86, 91].


### 1.8. LY293111 as a PPARγ Agonist

Following
the disclosure of anticancer effects of LY293111, researchers at the Lilly
Research Laboratories found that LY293111 is also a PPARγ agonist [92, 93]. This finding was initially
based on structural analysis and was supported by functional studies. The PPARγ
agonist activity of LY293111 is evidenced by its ability to induce adipogenic
differentiation in vitro [92].
Normalization of circulating glucose levels by LY293111
in the ZDF rat diabetes model further suggests that LY293111 is an
antidiabetic, PPARγ
agonist [92]. Further studies suggested that the anticancer effect of
LY293111 might be mediated, at least in part, by PPARγ [92, 93]. More extensive studies have
subsequently shown that LY293111 is also an inhibitor of 5-lipoxygenase,
although this effect is less potent than the LTB4 and PPARγ targets.


### 1.9. Mechanisms by Which LY293111 Functions in Cancer

Since our findings suggest that all
pancreatic cancer cells express both PPARγ
and BLT1, it is possible that the anticancer effects of LY293111 could be
mediated by either receptor or both receptors. It has been reported that PPARγ
negative-expressing cancer cells are less responsive to LY293111-induced growth
inhibition [92, 93]. However, there is also evidence in favor of BLT1 being the
major target. Firstly, the effects of LY293111 on proliferation and apoptosis
are extremely potent. A comparison between the effects of LY293111 and the PPARγ
agonist, ciglitazone is shown in Figure 2. As this figure shows, LY293111 is
approximately 50 times more potent than ciglitazone in inhibiting the
proliferation of Panc-1 and S2-013 human pancreatic cancer cells. LY293111 was
also more potent than another PPARγ
agonist, rosiglitazone and the PPARα agonist, WY-14643. The antiproliferative
effects and induction of apoptosis are seen at 250 nM LY293111, which is much
lower than the IC50 of the drug for PPARγ receptors (~4 μM) [92, 93]. Indeed, its effects
on cancer cells are more potent than several PPARγ agonists, including ciglitazone and
rosiglitazone. Secondly, LY293111 is able to completely inhibit the effects of
LTB4 on proliferation and MAP kinase activation in pancreatic cancer
cells [62]. However, preliminary studies have shown that the antiproliferative
effects of LY293111 in pancreatic cancer are not inhibited by the PPARγ
antagonist, GW9662 in vitro (Figure 3). Finally, data from our own
studies and those of others show that PPARγ agonists induce cell cycle arrest in the
G0/G1 phase, whereas LY293111 induces S phase cell cycle arrest [82, 87]. It is even possible that anticancer effects of
LY293111 might also be partially mediated by other unknown mechanisms. However, based on the current data, both the
leukotriene B4 receptor and PPARγ are likely to be involved in the antitumor activity
of LY293111.Comparison of the effects of different concentrations of LY293111 and
ciglitazone on proliferation of two human pancreatic cancer cell lines (Panc-1
and S2-013) after 24 and 96 hours of treatment, measured by thymidine
incorporation. LY293111 was approximately 50 times more potent than ciglitazone
at inhibiting proliferation of both cell lines. Data shown is mean±SEM from
four separate experiments.Figure 3
Effect of a PPARγ receptor antagonist, GW9662 on the inhibition of
proliferation induced by LY293111 in AsPC-1 human pancreatic cancer cells after
24 hours of treatment. These cells express both the PPARγ receptor and LTB4
(BLT1 and BLT2) receptors. While GW9662 alone was able to significantly
increase thymidine incorporation, it was not able to block the inhibitory
effect of different concentrations of LY293111.


## 1.1. Fatty Acids and Human Cancer

Epidemiological and animal studies show that
a high fat consumption is associated with a higher incidence and growth of
tumors at several specific organ sites including breast, pancreas, and prostate
[4–11]. Recent
studies indicate that diets containing a high proportion of polyunsaturated
omega-6 fatty acids (n−6 FA), such as linoleic acid (the precursor of
arachidonic acid) are associated with a more advanced disease stage at the time
of diagnosis of several kinds of cancer [4–6, 8, 10, 11]. In contrast,
long-chain n−3 fatty acids, such as docosahexaenoic acid and eicosapentaenoic
acid (EPA) inhibit the growth and metastasis of several cancers including
pancreatic cancer [7, 9]. Omega 3 fatty acids inhibit tumor growth by a number
of mechanisms including suppression of COX-2 expression and, for EPA at least,
the alternative substrate produces different cyclooxygenase (PGE3)
and lipoxygenase (LTB5) products that have anti-inflammatory and anticancer
effects.


## 1.2. Eicosanoid Pathways

Arachidonic acid is
a substrate for three distinctively different enzymatic pathways. Among them,
prostaglandin endoperoxide synthases (cyclooxygenases) catalyze the committed
step that leads to prostaglandin biosynthesis [12–14]. The second
pathway is the epoxygenase pathway that appears to have no role in cancer. The
third pathway for metabolizing arachidonic acid, the lipoxygenase pathway
catalyzes the incorporation of one oxygen molecule into polyunsaturated fatty
acids to yield a 1-hydroperoxy-2, 4-trans, cis-pentadiene product [14–16]. Mammalian lipoxygenases possess
regiospecificity during interaction with substrate, and on this basis have been
designated as arachidonate 5-, 12-, and 15-lipoxygenase (5-LOX, 12-LOX, and
15-LOX) [14–16]. The three distinct enzymes insert oxygen at
carbon 5, 12 or, 15 of arachidonic acid, and the primary product is 5S-, 12S-,
or 15S-hydroperoxyeicosatetraenoic acid (5-, 12-, or 15-HPETE), which can be
further reduced by glutathione peroxidase to hydroxy forms (5-, 12-, 15-HETE),
respectively [14–16]. 5-LOX is
noteworthy because it is the only pathway that can turn arachidonic acid into
leukotrienes [15, 17]. The activity of 5-LOX is dependent upon a second factor
termed 5-LOX-activating protein (FLAP) [15, 17]. Considerable effort has been
expended by the pharmaceutical industry to produce inhibitors of FLAP, 5-LOX as
well as leukotriene antagonists, because the 5-LOX products, leukotrienes (LTB4,
LTC4, LTD4, and LTE4) have been implicated as
mediators of inflammation and immediate hypersensitivity reactions, in
particular, human bronchial asthma [18, 19].


## 1.3. Leukotriene Receptor Antagonists and the Development of LY293111

The pharmaceutical industry has focused on several targets to suppress
leukotriene activity in inflammatory conditions such as bronchial asthma [18, 19].
One approach is to directly inhibit 5-lipoxygenase activity, thereby blocking
secretion of all leukotrienes. The most
widely studied clinical inhibitor of 5-lipoxygenase is zileuton, which inhibits
the active site of 5-lipoxygenase atconcentrations that do not
inhibit cyclooxygenase, 12-lipoxygenase, or 15-lipoxygenase [18–22]. Another avenue to inhibit leukotriene
formation is via blocking FLAP activity, thus preventing cytoplasmic to
membrane translocation and activation of 5-lipoxygenase [16–18]. MK-0591 is a
widely used 5-lipoxygenase-activating protein inhibitor for
biomedical research [16–18]. Even though it strongly inhibits
5-lipoxygenase activity and blocks leukotriene generation, its use in clinic is
limited by marked side effects. The final pharmacological approach to block
leukotriene activity is to selectively block the actionsof LTB4 or the sulfidopeptide leukotrienes using specific receptor antagonists.Several synthetic LTB4 receptor
antagonists have been developed. Early compounds included SC-41930;
ONO-4057, which was orally
active; LY223982, a benzophenone dicarboxylic acid; and LY255283 a hydroxyacetophenone
[23–27]. These latter
two compounds from the Lilly Research Laboratories potently block LTB4 binding to its receptors within the nM range and inhibit the biological
functions of LTB4 in vitro [28, 29]. Unfortunately, they
showed poor oral bioavailability [28]. In 1995, investigators at the Lilly
Research Laboratories reported a new LTB4 antagonist, LY293111. This
compound is a novel derivative of LY255283, but is orally stable and more potent
as an LTB4 receptor antagonist [28, 29]. Compared with other LTB4 receptor
antagonists, LY293111 is superior at blocking the cellular functions induced by
LTB4 [28, 30].


## 1.4. Inflammation, Cyclooxygenases, Lipoxygenases, and Cancer

The
epidemiological data show a clear and strong association between chronic
inflammatory conditions and cancer development, even though the conditions
causing inflammation may vary [31–36]. It can be due to chronic infection caused by
a virus, bacteria, or parasite or it may be due to noninfective, physical, or
chemical irritant [31–36]. For example, chronic infection with the bacterium Helicobacter pylori causes atrophic gastritis, which can lead to
dysplasia and adenocarcinoma [37].
Hepatitis B and C viruses account for more than 80% of cases of
hepatocellular carcinoma worldwide [34].
The inflammatory bowel diseases, ulcerative colitis and Crohn's disease,
predispose to the development of cancers of the large bowel and/or terminal
ileum, although a causative infectious agent has never been conclusively
identified [38]. For noninfectious
inflammation, chronic reflux of gastric acid and bile into the distal esophagus
causes chemical injury and on the long-term can lead to Barrett's esophagus and
eventually to esophageal adenocarcinoma [35].
Thus it is apparent that chronic inflammation is a common underlying
theme in the development of many different malignancies.Although the mechanisms for the association between inflammation and
cancer are not fully understood, growth factors, cytokines, and chemokines
released into inflammatory environment are associated with tumor development
and progression [32, 36]. High
concentrations of free radicals and nitric oxide can induce DNA damage and
promote cancer development [32, 36].
Over the past decade, much attention has been paid on the role of
cyclooxygenases in cancer development, specifically its inducible isoform, the
cyclooxygenase 2 (COX-2) [39–41]. COX-2 is active within both inflamed and
malignant tissues [37–39]. The expression of COX-2 and COX-2 metabolites
increases during the multistage progression of tumors [39–41]. By metabolizing
arachidonic acid to prostaglandins, COX-2 induces cellular resistance to
apoptosis, modulation of cellular adhesion and motility, promotion of
angiogenesis, and immunosuppression [42–47]. Epidemiological data has implicated COX-2 in
the pathogenesis of a number of epithelial malignancies, especially colorectal
cancer [48, 49]. Inhibition of the enzyme with COX inhibitors is associated
with a dramatic reduction in the incidence, morbidity and mortality of
colorectal cancer [48–51]. Recent
attention has also been focused on the role of 5-LOX, 12-LOX, and 15-LOX in
cancer [52–60]. In
pancreatic cancer, activation of the 5-LOX and 12-LOX pathways enhances cancer
cell proliferation, while the 15-LOX pathway is protective against cancer
development [61–64].


## 1.5. The 5-Lipoxygenase/LeukotrieneB4 Pathway and Cancer

Accumulating
evidence suggests that the 5-LOX pathway has profound influence on the
development and progression of human cancers [61–64]. 5-LOX is overexpressed in pancreatic cancer
tissues but is not expressed in normal pancreatic ductal cells [65]. Furthermore,
this pathway is already up-regulated in pancreatic intraepithelial neoplasias
(PanINs), which are the precursor lesions of pancreatic adenocarcinoma [66]. Blockade of 5-LOX activity inhibits
proliferation and induces apoptosis in pancreatic cancer cells both in vitro
and in vivo [67–69]. Pancreatic cancer cells secrete LTB4 and LTB4 induces proliferation in these cells [62]. Two
G-protein-coupled LTB4 receptors (BLT1 and BLT2) have been cloned
and characterized. BLT1 and BLT2 are high- and low-affinity LTB4 receptors, respectively, and form a gene cluster in humans. Both BLT1 and BLT2
are up-regulated in pancreatic cancer tissues, and expression was seen in all
of the tested pancreatic cancer cell lines [65, 70]. As with other proteins in
the 5-LOX/LTB4 pathway, BLT1 and BLT2 are already up-regulated in
pancreatic intraepithelial neoplasias (PanIN lesions) which are the precursors
of pancreatic adenocarcinomas [70]. This suggests that they may be valuable
targets for chemoprevention.


## 1.6. PPARγ and Pancreatic Cancer

Peroxisome proliferator activated receptor-γ
(PPARγ) is a member of the nuclear receptor superfamily of ligand-activated
transcription factors. PPARγ is expressed at high levels in adipose tissue and
plays a central role in adipocyte differentiation and energy homeostasis.
Recent studies have implicated PPARγ in the pathogenesis of several human
malignancies [71–74]. Previous
studies have suggested that PPARγ is up-regulated in pancreatic cancer [75]. Our
own studies, employing two separate commercially available antibodies, show
that PPARγ is expressed in pancreatic cancer, but that expression in the cancer
cells does not appear to be different from that in normal pancreatic ductal cells (Figure 1). In contrast, PPARγ staining was seen in the islets surrounding cancers, but
not in islet cells from normal pancreatic tissues obtained from multiorgan
donors (Figure 1). In animal models,
PPARγ
ligands have preventive effects against chemical carcinogenesis [76]. Several studies
have shown that PPARγ agonists, including the natural ligand 15-deoxy-Δ12,14-prostaglandin J2, and thiazolidinedione antidiabetic agents, such as citiglitizone
and rosiglitizone, inhibit growth and induce apoptosis in pancreatic cancer [75, 77–81]. In contrast,
one paper suggests induction of differentiation without apoptosis [82]. The
apoptosis appears to be preceded by a morphological change to a more differentiated
cell type which perhaps undergoes apoptosis when DNA repair turns out to be not
possible [80]. In some studies, PPARγ agonists also block invasion and
angiogenesis [83, 84]. However, this is controversial since PPARγ agonists
induce secretion of vascular endothelial growth factor, which would have a
promoting effect on metastatic tumor growth [85].Immunocytochemistry of PPARγ receptor in normal human pancreas and in a
PanIN lesion and a pancreatic cancer. The PPARγ receptor antibody shows a
similar intensity of nuclear staining in normal ducts and in pancreatic cancer
cells as well as cells in the PanIN lesion. In contrast, no staining is seen in
normal islets, but nuclear staining is seen in islets from tissue adjacent to a
cancer. These pictures are representative of eight samples of each tissue type.
Staining was similar using antibodies from two different commercial sources.


## 1.7. LY293111 and Cancer

As might be expected from the growth-stimulatory effects of LTB4 in pancreatic cancer, the LTB4 receptor antagonist, LY293111
inhibits cancer growth and induces apoptosis both in vitro and in vivo [86–89]. LY293111
inhibits proliferation and induces apoptosis in a wide range of pancreatic
cancer cell lines as well as cells of other tumor types, such as breast,
prostate, and colon cancer cells [86–89]. These effects on growth and apoptosis
are both time and concentration dependent, with effects seen at 100–500 nM in vitro
[86, 87]. To confirm the involvementof LTB4 receptors in mediating the effect of LY293111 on humanpancreatic cancer cell proliferation, another selective LTB4 receptor antagonist, U75302 was used in comparison with a selective LTD4 antagonist, LY171883 [86]. U75302 inhibits the proliferation of pancreatic
cancercells; but it is less potent than LY293111 as expected from the lower receptor affinity of this drug [28, 29, 90]. In contrast, the selective LTD4 antagonist, LY171883 had no significant effect on pancreatic cancer
cell growth. LY293111 causes cell
cycle arrest in the S phase of the cell cycle with suppression of expression of
cyclin A, cyclin E, and cdk2. In
parallel with growth inhibition, LY293111 induced apoptosis in all cancer cell
lines tested [86, 87]. LY293111 induced
dramatic morphological changes inhuman pancreatic cancer cells
following a short period of treatment [86, 87]. The treated cells became
rounded and exhibited membrane blebbing, chromatin condensation, and
nuclear fragmentation,
finally they were detached from the microplate.
Induction of DNA fragmentation by LY293111 was confirmed by TUNEL assay
(terminal deoxynucleotidyl transferase-mediated nick end labeling)
and apoptosis was also established by annexin V binding [86, 87]. Apoptosis
is triggered through the mitochondrial pathway, with a change in the ratio of
proapoptotic proteins, such as Bax to antiapoptotic proteins, such as Bcl-2 and
Mcl-1, release of cytochrome C, activation of caspase (but not caspase 8), and subsequent
activation of the downstream caspase cascade with activation of caspase 3 and
caspase 7 and cleavage of the caspase 3 substrate, poly ADP-ribose polymerase
(PARP) [87].LY293111 markedly slows down the growth of
subcutaneous xenografts of human pancreatic cancer in athymic mice at a dose of
250 mg/kg/day [86]. To confirm the antipancreatic cancer effect of LY293111,
pancreatic cancer cells with stable expression of enhanced green
fluorescent protein (GFP) were
orthotopically implanted into the duodenal lobe of the pancreas of
athymic mice. Our data show that LY293111 significantly inhibits the growth of
the orthotopically implanted pancreatic cancer cells in concert with blocking metastatic
spread to the liver and other organs [91]. LY293111 also dramatically increased the number of TUNEL positive cells
in pancreatic tumors harvested from the subcutaneous transplant experiments in
athymic mice [86, 91].


## 1.8. LY293111 as a PPARγ Agonist

Following
the disclosure of anticancer effects of LY293111, researchers at the Lilly
Research Laboratories found that LY293111 is also a PPARγ agonist [92, 93]. This finding was initially
based on structural analysis and was supported by functional studies. The PPARγ
agonist activity of LY293111 is evidenced by its ability to induce adipogenic
differentiation in vitro [92].
Normalization of circulating glucose levels by LY293111
in the ZDF rat diabetes model further suggests that LY293111 is an
antidiabetic, PPARγ
agonist [92]. Further studies suggested that the anticancer effect of
LY293111 might be mediated, at least in part, by PPARγ [92, 93]. More extensive studies have
subsequently shown that LY293111 is also an inhibitor of 5-lipoxygenase,
although this effect is less potent than the LTB4 and PPARγ targets.


## 1.9. Mechanisms by Which LY293111 Functions in Cancer

Since our findings suggest that all
pancreatic cancer cells express both PPARγ
and BLT1, it is possible that the anticancer effects of LY293111 could be
mediated by either receptor or both receptors. It has been reported that PPARγ
negative-expressing cancer cells are less responsive to LY293111-induced growth
inhibition [92, 93]. However, there is also evidence in favor of BLT1 being the
major target. Firstly, the effects of LY293111 on proliferation and apoptosis
are extremely potent. A comparison between the effects of LY293111 and the PPARγ
agonist, ciglitazone is shown in Figure 2. As this figure shows, LY293111 is
approximately 50 times more potent than ciglitazone in inhibiting the
proliferation of Panc-1 and S2-013 human pancreatic cancer cells. LY293111 was
also more potent than another PPARγ
agonist, rosiglitazone and the PPARα agonist, WY-14643. The antiproliferative
effects and induction of apoptosis are seen at 250 nM LY293111, which is much
lower than the IC50 of the drug for PPARγ receptors (~4 μM) [92, 93]. Indeed, its effects
on cancer cells are more potent than several PPARγ agonists, including ciglitazone and
rosiglitazone. Secondly, LY293111 is able to completely inhibit the effects of
LTB4 on proliferation and MAP kinase activation in pancreatic cancer
cells [62]. However, preliminary studies have shown that the antiproliferative
effects of LY293111 in pancreatic cancer are not inhibited by the PPARγ
antagonist, GW9662 in vitro (Figure 3). Finally, data from our own
studies and those of others show that PPARγ agonists induce cell cycle arrest in the
G0/G1 phase, whereas LY293111 induces S phase cell cycle arrest [82, 87]. It is even possible that anticancer effects of
LY293111 might also be partially mediated by other unknown mechanisms. However, based on the current data, both the
leukotriene B4 receptor and PPARγ are likely to be involved in the antitumor activity
of LY293111.Comparison of the effects of different concentrations of LY293111 and
ciglitazone on proliferation of two human pancreatic cancer cell lines (Panc-1
and S2-013) after 24 and 96 hours of treatment, measured by thymidine
incorporation. LY293111 was approximately 50 times more potent than ciglitazone
at inhibiting proliferation of both cell lines. Data shown is mean±SEM from
four separate experiments.Figure 3
Effect of a PPARγ receptor antagonist, GW9662 on the inhibition of
proliferation induced by LY293111 in AsPC-1 human pancreatic cancer cells after
24 hours of treatment. These cells express both the PPARγ receptor and LTB4
(BLT1 and BLT2) receptors. While GW9662 alone was able to significantly
increase thymidine incorporation, it was not able to block the inhibitory
effect of different concentrations of LY293111.


## 2. Effect of LY293111 in Combination with Other Agents in Cancer

Several studies have demonstrated that
LY293111 enhances anticancer effects of gemcitabine, which is widely
used as the standard therapy in pancreatic cancer patients in adjuvant and
palliative treatment settings [89, 91, 94]. Gemcitabine only improves survival by
a few weeks, but clinical data show improvement in the quality of life for
pancreatic cancer patients. The effects of LY293111 in combination with gemcitabine
were investigated in an orthotopic model of pancreatic cancer in athymic mice [91]. This model is superior to subcutaneous
transplantation since it is less likely to modify the biological characteristic
of pancreatic cancer cells, providing a favourable growth environment for them.
It also allows easy monitoring of hepatic and lymph node metastasis with GFP
stable expressing cells. In this model,
animals without any treatment following implantation of GFP-expressing, S2-O13
pancreatic cancer cells developed end-stage disease with invasive cancer
obstructing the duodenum and bile duct [91]. The animals develop liver, lung,
and lymph node metastases and eventually peritoneal carcinomatosis with
malignant ascites and cachexia [91]. Either gemcitabine or LY293111 alone
significantly inhibited tumor growth and reduced the incidence of liver
metastasis. However the combination of LY293111 and gemcitabine was
significantly more effective than either treatment alone in blocking tumor
growth [91]. Combined treatment also
significantly relieved tumor-induced cachexia and maintained stable body
weights compared with either drug alone, and also significantly decreased the
incidence of biliary obstruction and metastasis [91]. These experimental
results show that combined therapy of gemcitabine and LY293111 potently
inhibits the growth and metastases of the very rapidly growing and aggressive
pancreatic adenocarcinoma and suggest that it might be a valuable way for
treatment of pancreatic cancer patients. LY293111 has also been shown to
increase the effectiveness of gemcitabine in a colon cancer model [89]. The in
vitro effects of LY293111 have been tested with other classical
chemotherapeutic agents. The effects of the active metabolite of irinotecan,
SN-38 or the active metabolite of capecitabine, 5′-DFUR were enhanced by
LY293111 in multiple cell lines, including breast, bladder, and sarcoma cells [94].


## 3. Clinical Trials with LY293111

Three phase I clinical trials with
LY293111 alone or in combination with gemcitabine or irinotecan have been
reported. LY293111 was generally well tolerated [95–97]. The side
effects were mild to moderate; the major ones gastrointestinal with diarrhea
and pain. These initial phase I trials looked promising and LY293111 could be
safely administered orally [95–97]. For example,
in combination with gemcitabine, three patients had partial responses [96]. One
had pancreatic cancer previously treated with gemcitabine, one with pancreatic
cancer previously treated with 5-flurouracil and radiation, and one with non-small-cell
lung cancer treated with one prior regimen. Two phase II trials have been
completed and preliminary data reported in abstract form [98, 99]. One of these
compared the combination of LY293111 with gemcitabine compared with gemcitabine
with placebo in pancreatic cancer [98]. The second compared LY293111 with
cisplatin and gemcitabine versus the placebo combined with the latter two drugs
in patients with non-small-cell lung cancer [99]. Unfortunately, LY293111 did
not improve progression-free survival in either of these two trials [98, 99].


## 4. Conclusions

LY293111 is an interesting compound that has biological effects on several different
targets. It acts as an antagonist on LTB4 receptors, as a PPARγ agonist
and as a 5-lipoxygenase inhibitor. Indeed, some investigators have referred to
LY293111 as a multiple eicosanoid pathway inhibitor [94, 99]. Since LY293111
has anticancer effects on multiple tumor types, the target involved in
mediating the effects of the drug is clearly of interest. The evidence to date
is not conclusive, but suggests that effects may be mediated by both LTB4 receptors and PPARγ.
Preliminary reports regarding the phase II clinical trials have unfortunately
been disappointing. It remains unknown whether this compound will eventually
find a use in the clinic for cancer therapy; however this recent clinical
experience perhaps makes this unlikely now.



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*Source: 827096-2009-01-27.xml*