# PPARγ and Apoptosis in Cancer

Authors: Heath A. Elrod; Shi-Yong Sun
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptors (PPARs) are ligand binding transcription factors which function in many physiological roles including lipid metabolism, cell growth, differentiation, and apoptosis. PPARs and their ligands have been shown to play a role in cancer.  In particular, PPARγ ligands including endogenous prostaglandins and the synthetic thiazolidinediones (TZDs) can induce apoptosis of cancer cells with antitumor activity. Thus, PPARγ ligands have a potential in both chemoprevention and therapy of several types of cancer either as single agents or in combination with other antitumor agents. Accordingly, the involvement of PPARγ and its ligands in regulation of apoptosis of cancer cells have been extensively studied. Depending on cell types or ligands, induction of apoptosis in cancer cells by PPARγ ligands can be either PPARγ-dependent or -independent. Through increasing our understanding of the mechanisms of PPARγ ligand-induced apoptosis, we can develop better strategies which may include combining other antitumor agents for PPARγ-targeted cancer chemoprevention and therapy. This review will highlight recent research advances on PPARγ and apoptosis in cancer.


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## Body

## 1. Introduction

Peroxisome proliferator-activated receptors (PPARs)
are ligand binding transcription factors belonging to the nuclear receptor
superfamily which includes receptors for steroids, thyroid hormone, and
retinoids [1, 2].
PPARs function in a variety of roles including regulation of lipid metabolism,
immune function, cell growth, differentiation, and apoptosis [2].  PPARs are involved in several diseases
including obesity, diabetes, cardiovascular disease, and cancer [3]. Three different subtypes of
PPARs have been identified, PPARα, PPARβ/δ, and PPARγ, each encoded by separate genes. The three
isoforms share functions as well as have distinct activities [2].PPARs function by regulating gene transcription via
binding to DNA sequences known as peroxisome proliferator response elements
(PPREs) located in the promoter regions of target genes. PPREs are direct
repeats of the consensus sequence with a spacing of one nucleotide (AGGTCA N
AGGTCA) [4].
PPARs bind to PPREs as heterodimers with retinoid X receptors (RXR). The
heterodimer PPAR/RXR can bind other transcriptional coactivators or
corepressors to influence gene transcription [1]. Ligand binding to PPARs
induces conformational changes that release corepressors from the heterodimer
and recruit coactivators to allow for target gene transcription [5].Synthetic and endogenous PPAR ligands have been used
to elucidate the role of PPARs.
Specifically, thiazolidinediones (TZDs) including pioglitazone,
ciglitazone, troglitazone, and rosiglitazone are synthetic PPARγ ligands which are insulin-sensitizing agents
developed to treat diabetes mellitus [2]. The naturally occurring
prostaglandin, 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2),
is generally considered to be an endogenous PPARγ ligand [6, 7].
The promiscuous nature of PPARs may lead to the binding of multiple ligands
resulting in the activation of many cellular pathways. These ligands have been
extensively studied and shown to exert antineoplastic properties including
induction of apoptosis.Apoptosis or programed cell death is a highly
regulated process critical for normal development and tissue homeostasis.
Aberrant regulation of apoptosis can lead to cancer.  Apoptosis is induced from signals inside or
outside the cell including radiation, viral infection, growth factors, and
hormones [23]. Apoptosis involves signature
morphological changes induced by caspases, which are activated upon induction
of apoptotic signaling and cleave downstream molecules to facilitate the
apoptotic cascade [24]. The induction of apoptosis
can occur through two pathways: the intrinsic apoptotic pathway which involves
signaling through the mitochondria and the extrinsic apoptotic pathway which is
initiated through activation of cell surface death receptors [25]. Apoptotic signaling through
the intrinsic pathway primarily involves activation of the proapoptotic Bcl-2
family members Bax and Bak, which facilitate release of cytochome C from the
mitochondria and subsequent caspase-9 cleavage or activation. The activated
caspase-9 will finally cleave or activate the downstream effector caspases such
as caspase-3 and -7, leading to apoptosis. This pathway is negatively regulated
by several antiapoptotic Bcl-2 family members such as Bcl-2 and Bcl-XL [26]. Apoptotic signaling through
the extrinsic pathway is initiated by ligand binding to death receptors or by
induction of trimerization of the receptors [27]. The death receptors belong
to the tumor necrosis factor (TNF) receptor superfamily, which includes Fas,
TNFR1, DR3, DR4 (TRAIL-R1), DR5 (TRAIL-R2), and DR6. Upon ligand binding and
trimerization of death receptors, the intracellular death domain of the death
receptors recruits adapter proteins such as Fas-associated death domain (FADD),
forming a death-inducing signaling complex (DISC) which helps recruit
procaspase-8 to the DISC. Caspase-8 is then activated, leading to activation of
the downstream effector caspases such as caspase-3 and -7. The effector
caspases can also be activated by death receptors indirectly through
caspase-8-mediated cleavage of Bid, which facilitates Bax activation and
subsequent release of cytochome C from the mitochondria. Thus, the Bid cleavage
links the two apoptotic pathways [28]. Cellular FLICE inhibitory
protein (c-FLIP), an inactive homolog of caspase-8, primarily functions as an
inhibitor of the extrinsic apoptotic pathway by preventing caspase-8
activation, whereas inhibitors of apoptosis protein (IAPs) such as survivin
mainly suppress the intrinsic apoptotic pathway by inhibiting caspase-9 as well
as caspase-3 activation (Figure 1).Figure 1
Schema for basic apoptotic signaling
pathways and possible mechanisms underlying PPARγ ligand-induced apoptosis. Ligation
of death ligands (e.g., TRAIL) with their receptors (e.g., DR5) results in
formation of the death-inducing signaling complex (DISC), in which
pro-caspase-8 will be recruited through the death adaptor protein FADD and
cleaved to generate activated caspase-8. This process is inhibited by c-FLIP.
Certain stress signals (e.g., DNA damage) can target mitochondria and induce
cytochrome C release from the mitochondria into the cytosol leading to caspase-9
activation by forming an apoptosome via binding to Apaf-1.  Both caspase-8 and caspase-9 activate
downstream procaspase-3, -6, and -7, leading to cleavages of their target death
proteins such as PARP. In addition, truncated Bid (tBid), activated by caspase-8
via cleavage, facilitates insertion of Bax into the mitochondrial membrane
leading to cytochrome C release. Therefore, tBid may serve as a link between
the extrinsic and intrinsic apoptotic pathways. Inhibitors of apoptosis
proteins (IAPs) such as survivin can bind to activated caspase-9 and prevent
its action on effector caspases, whereas SMAC/DIABLO binds to IAPs, leaving
caspase-9 free to activate the effector caspases. PPARγ ligands may induce apoptosis through
induction of DR5 and/or downregulation of c-FLIP and/or survivin.PPARs, particularly PPARγ, and their ligands play a role in
regulation of both apoptotic pathways. Thus, this review will specifically
focus on the role of PPARγ and its ligands in regulation of tumor cell
apoptosis. Some of the underlying mechanisms resulting in apoptosis of tumor
cells in PPARγ-dependent and -independent manners will be
highlighted.


## 2. PPARγ Agonists Induce Apoptosis of Cancer Cells

PPARγ agonists (e.g., TZDs) have been shown
to induce apoptosis in a variety of cancer cells including lymphoma, multiple
myeloma, bladder, gastric, esophageal, pancreatic, hepatoma, colon, breast,
brain, and lung cancer cells [8, 12, 29–39].
However, many of the underlying mechanisms of the apoptotic properties of TZDs
remain unknown. In general, this induction of apoptosis is PPARγ-dependent and/or -independent depending on
cell types or ligands (Table 1).Table 1
PPARγ agonists induce apoptosis in cancer.
PPARγ agonistPPARγTumor typeMolecular mediator(s) of  apoptosisReference15d-PGJ2IndependentBreastUnknown[8]Troglitazone and 15d-PGJ2DependentThyroidc-myc[9]CiglitazoneDependentThyroidPPARγ[10]RosiglitazoneDependentThyroidNF-κB, cyclinD1, caspase-3[11]Troglitazone and 15d-PGJ2UnknownColonc-myc, c-jun, GADD153[12]TroglitazoneDependentLungGADD153[13]TroglitazoneIndependentColonEGR-1, NAG-1[14, 15]15d-PGJ2DependentColonEGR-1, NAG-1[15]TroglitazoneDependentLungERK1/2[16]TroglitazoneDependent and independentColonp53, POX[17]TroglitazoneIndependentProstateBcl-2, Bcl-XL[18]15d-PGJ2IndependentOralStat3[19]15d-PGJ2IndependentProstate, bladderCaspase-3, -7[20]15d-PGJ2IndependentMultiple myeloma, burkitt
lymphomaNF-kappa-B, cIAP-1, XIAP, c-FLIP[21]RosiglitazoneDependentBreastPPARγ, p53[22]


### 2.1. PPARγ-Dependent Apoptosis

In thyroid
cancer cell lines, it has been shown that the expression of PPARγ correlates with the sensitivity of
troglitazone and 15d-PGJ2 to cell death. Thyroid cancer cells that
did not express PPARγ showed no growth inhibition after treatment
with troglitazone and 15d-PGJ2 compared with thyroid cancer cells
that did express PPARγ and were sensitive to growth inhibition by
troglitazone and 15d-PGJ2, suggesting PPARγ-dependent growth inhibition. Growth
inhibition by troglitazone was due to apoptosis as was seen by DNA laddering [9]. Another study in thyroid
cancer cell lines also implicates PPARγ as an important target. In this study,
ciglitazone was effective in reducing the growth of thyroid cancer cells that
expressed PPARγ, but had no effect in reducing growth in a
thyroid cancer cell line that do not express PPARγ [10].
After introduction of wild-type PPARγ into the PPARγ-deficient cells, these cells became responsive
to ciglitazone. Moreover, overexpression of PPARγ in thyroid cancer cells significantly
increased apoptosis compared to cells transfected with empty vector or with a
vector carrying a mutated nonfunctional PPARγ cDNA [10].
Collectively, it appears that the presence of PPARγ at least partly contributes to the induction
of apoptosis by PPARγ ligands in thyroid cancer. The recent findings
from a transgenic mouse study [11] may provide an explanation
for why thyroid cancer is susceptible to treatment with PPARγ agonists. Mice harboring a knockin
dominant negative mutant thyroid hormone receptor β (TRβPV/PV mouse) spontaneously develop
follicular thyroid carcinoma similar to human thyroid cancer. Using the
offspring from the cross of TRβPV/+ and PPARγ+/− mice, Kato et al. [11] found that thyroid
carcinogenesis progressed significantly faster in TRβPV/PV mice with PPARγ insufficiency from increased cell
proliferation and reduced apoptosis. Reduced PPARγ protein activated the NF-κB signaling pathway, resulting in the
activation of cyclin D1 and repression of critical genes involved in apoptosis.
Treatment of TRβPV/PV mice with a PPARγ agonist, rosiglitazone, delayed the
progression of thyroid carcinogenesis by decreasing cell proliferation and
activating apoptosis. These results suggest that PPARγ is a critical modifier in thyroid
carcinogenesis.Other molecular mediators of apoptosis have been
examined in PPARγ-dependent models.  In thyroid cancer cells, troglitazone
increased c-Myc expression
without changing the expression of Bcl-2
or Bax  [9]. In contrast, in colon cancer
cells, both troglitazone and 15d-PGJ2 have been shown to
downregulate c-Myc expression [12].
Thus, whether c-Myc is involved in mediating PPARγ agonist-induced apoptosis needs further
investigation.In colon and lung cancer cells, troglitazone was
reported to increase the expression of growth arrest and DNA-damage inducible
153 (GADD153) [12, 13],
a key apoptosis-regulated gene particularly involved in endoplasmic reticulum
(ER) stress-induced apoptosis [40]. Further analysis revealed
that troglitazone did not stimulate GADD153 mRNA levels in undifferentiated
3T3-L1 cells lacking PPARγ expression, whereas its induction was
clearly observed in differentiated adipocytes expressing PPARγ, suggesting the importance of PPARγ in troglitazone-induced GADD153
expression. In lung cancer cells, inhibition of GADD153 gene expression by an
antisense phosphorothionate oligonucleotide attenuated the troglitazone-induced
growth inhibition [13].
These findings collectively suggest that GADD153 might be a candidate factor
implicated in TZD-induced growth inhibition and apoptosis.Several studies have demonstrated the importance of
ERK and its regulated genes in PPARγ agonist-induced apoptosis [14–16, 41].
In human lung cancer cells, troglitazone induced apoptosis as well as PPARγ and ERK1/2 accumulation in the nucleus.
Both PPARγ siRNA and U0126, a specific inhibitor
of ERK1/2, blocked these effects of troglitazone, suggesting that
troglitazone-induced apoptosis is PPARγ- and ERK1/2-dependent. Moreover,
inhibition of ERK1/2 by U0126 also significantly decreased the levels of PPARγ, suggesting a positive crosstalk
between PPARγ and ERK1/2 or an autoregulatory
feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and
apoptosis [16].Proline oxidase (POX) is a redox enzyme localized in
the mitochondrial inner membrane and functions as a p53-induced gene that can
mediate apoptosis through generation of reactive oxygen species (ROS) [17].
A recent study in colon cancer cells showed that troglitazone enhanced the
binding of PPARγ to PPRE in the POX promoter, activated
the POX promoter, and increased endogenous POX expression. Blocking of PPARγ activation either by the antagonist
GW9662 or deletion of the PPAR-responsive element in the POX promoter only
partially decreased the POX promoter activation in response to troglitazone,
suggesting also the involvement of PPARγ-independent mechanisms. Further,
troglitazone induced p53 protein expression in HCT116 cells, which may be the
possible mechanism for PPARγ-independent POX activation, since POX
has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15
cells, with both mutant p53 and mutant PPARγ, troglitazone did not activate POX,
whereas it did in HT29 cells, with a mutant p53 and wild type PPARγ, indicating that both PPARγ-dependent and -independent mechanisms
are involved in the troglitazone-induced POX expression [17].
Thus, this study suggests that troglitazone-induced apoptosis involves
targeting POX gene expression for generation of ROS.


### 2.2. PPARγ-Independent Apoptosis

To help
discern the PPARγ-dependent and -independent properties of TZDs,
TZD derivatives lacking PPARγ activity were developed. These derivatives
have a double bond adjoining the terminal thiazolidine-2,4-dione ring which
abolishes ligand binding to PPARγ [42]. Shiau et al. [18] showed that the pioglitazone,
troglitazone, and ciglitazone derivatives (Δ2-PG, Δ2-TG, Δ2-CG) were unable to activate PPARγ compared to pioglitazone, troglitazone, and
ciglitazone which showed significant activation of PPARγ. When troglitazone and Δ2-TG were tested for growth inhibition in two
prostate cancer cell lines: one cell line expressing high levels of PPARγ (PC-3) and one deficient of PPARγ expression (LNCaP), the LNCaP cells were more
sensitive to troglitazone compared to PC-3 cells despite being deficient in
PPARγ. As well, Δ2-TG which cannot activate PPARγ was more effective than troglitazone in
suppressing growth in both PC-3 and LNCaP cells. Both troglitazone and Δ2-TG induced cytochome C release and DNA
fragmentation in these cells, attributing the growth inhibition to apoptosis.
These results suggest that TZDs can induce apoptosis independent of PPARγ activation. The induction of apoptosis in this
study appears to be partly due to the inhibition of the antiapoptotic function
of Bcl-2 and Bcl-XL. It is thought that Bcl-2 and Bcl-xL sequester
proapoptotic molecules such as Bax and Bak through heterodimerization through
BH3 domain binding which inhibits the proapoptotic function of Bax and Bak [43, 44].
Both troglitazone and Δ2-TG reduced the association of Bak with Bcl-2
and Bcl-XL causing the cells to undergo apoptosis as shown by
cytochrome C release and caspase-9 activation. Moreover, Bcl-XL overexpression protected LNCaP cells from troglitazone- and Δ2-TG-induced apoptosis [18]. Collectively, these results
show that PPARγ ligands trigger apoptosis independent of PPARγ and primarily target activation of the
intrinsic apoptotic pathway, at least in the tested prostate cancer cells.It was shown that 15d-PGJ2, but
not rosiglitazone and ciglitazone, induced apoptosis in oral squamous cell
carcinoma cells, suggesting that 15d-PGJ2 is acting through pathways
other than activation of PPARγ. In this study, the apoptotic effect of
15d-PGJ2 was associated with downregulation of the oncogene Stat3
which was not seen with rosiglitazone or ciglitazone [19].  Similarly, in bladder and prostate cancer
cells, 15d-PGJ2 and troglitazone inhibited cell growth but
rosiglitazone and pioglitazone had no effect on growth inhibition. 15d-PGJ2 inhibited cell growth by induction of apoptosis, while troglitazone
induced cell cycle arrest [20].
Thus, the induction of apoptosis can also be selective for certain PPARγ ligands.Other mediators of apoptosis in PPARγ ligand-induced cell death include the
early growth response-1 (EGR-1) transcription factor. EGR-1 has been linked to
apoptosis and shown to be activated by ERK. In colon cancer cells, EGR-1 was
induced dramatically by troglitazone but not by other PPARγ ligands [14].
Inhibition of ERK phosphorylation abolished EGR-1 induction by troglitazone,
suggesting an ERK-dependent induction of EGR-1. Given that troglitazone-induced
apoptosis is accompanied by the biosynthesis of EGR-1, these results suggest
that PPARγ-independent EGR-1 induction is a unique
property of troglitazone compared with other PPARγ ligands and may play an important role
in troglitazone-induced apoptosis [14].
One of the EGR-1-regulated genes is proapoptotic nonsteroidal anti-inflammatory
drug (NSAID)-activated gene (NAG-1) [15].
A recent study has demonstrated that the novel TZD derivative MCC-555 exerts a
PPARγ-independent upregulation of NAG-1.
Moreover, NAG-1 induction contributes to MCC-555-induced apoptosis as
downregulation of NAG-1 by siRNA suppressed MCC-555-induced apoptosis [41]. As well, NAG-1 induction was
also observed in colon cancer cells treated with troglitazone or 15d-PGJ2 [15].
Importantly, both agents induce NAG-1 expression through an EGR-1-dependent
mechanism. However, troglitazone, but not 15d-PGJ2, increases EGR-1
binding to the EGR-1 binding site located within region -73 to -51 of the NAG-1
promoter; this effect has an important role in the transactivation of
TGZ-induced NAG-1 expression. The effect of 15d-PGJ2 is probably
PPARγ-dependent because a PPARγ antagonist inhibited the 15d-PGJ2-induced
expression of NAG-1, whereas TGZ-induced NAG-1 expression was not inhibited by
the PPARγ antagonist [15].Multiple myeloma and Burkitt lymphoma cells express
constitutively active NF-κB. 15d-PGJ2 was reported to
suppress constitutive NF-κB activity and potently induce apoptosis
in both types of B-cell malignancies. NF-κB inhibition is accompanied by rapid
downregulation of NF-κB-dependent antiapoptotic gene products,
including cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2,
X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), and c-FLIP. These
effects were mimicked by the proteasome inhibitor MG-132, but not by
troglitazone, suggesting that 15d-PGJ2-induced apoptosis is
independent of PPARγ [21].
Thus, the inhibition of NF-κB may play a major role in the
proapoptotic activity of 15d-PGJ2 in aggressive B-cell malignancies
characterized by aberrant regulation of NF-κB. Another study in MCF7 breast cancer
cells has shown that both PPARγ and p53 are involved in rosiglitazone-induced
apoptosis. However, the NF-κB sequence in the p53 promoter region is
required for rosiglitazone to increase p53 transcription in this study [22].


## 2.1. PPARγ-Dependent Apoptosis

In thyroid
cancer cell lines, it has been shown that the expression of PPARγ correlates with the sensitivity of
troglitazone and 15d-PGJ2 to cell death. Thyroid cancer cells that
did not express PPARγ showed no growth inhibition after treatment
with troglitazone and 15d-PGJ2 compared with thyroid cancer cells
that did express PPARγ and were sensitive to growth inhibition by
troglitazone and 15d-PGJ2, suggesting PPARγ-dependent growth inhibition. Growth
inhibition by troglitazone was due to apoptosis as was seen by DNA laddering [9]. Another study in thyroid
cancer cell lines also implicates PPARγ as an important target. In this study,
ciglitazone was effective in reducing the growth of thyroid cancer cells that
expressed PPARγ, but had no effect in reducing growth in a
thyroid cancer cell line that do not express PPARγ [10].
After introduction of wild-type PPARγ into the PPARγ-deficient cells, these cells became responsive
to ciglitazone. Moreover, overexpression of PPARγ in thyroid cancer cells significantly
increased apoptosis compared to cells transfected with empty vector or with a
vector carrying a mutated nonfunctional PPARγ cDNA [10].
Collectively, it appears that the presence of PPARγ at least partly contributes to the induction
of apoptosis by PPARγ ligands in thyroid cancer. The recent findings
from a transgenic mouse study [11] may provide an explanation
for why thyroid cancer is susceptible to treatment with PPARγ agonists. Mice harboring a knockin
dominant negative mutant thyroid hormone receptor β (TRβPV/PV mouse) spontaneously develop
follicular thyroid carcinoma similar to human thyroid cancer. Using the
offspring from the cross of TRβPV/+ and PPARγ+/− mice, Kato et al. [11] found that thyroid
carcinogenesis progressed significantly faster in TRβPV/PV mice with PPARγ insufficiency from increased cell
proliferation and reduced apoptosis. Reduced PPARγ protein activated the NF-κB signaling pathway, resulting in the
activation of cyclin D1 and repression of critical genes involved in apoptosis.
Treatment of TRβPV/PV mice with a PPARγ agonist, rosiglitazone, delayed the
progression of thyroid carcinogenesis by decreasing cell proliferation and
activating apoptosis. These results suggest that PPARγ is a critical modifier in thyroid
carcinogenesis.Other molecular mediators of apoptosis have been
examined in PPARγ-dependent models.  In thyroid cancer cells, troglitazone
increased c-Myc expression
without changing the expression of Bcl-2
or Bax  [9]. In contrast, in colon cancer
cells, both troglitazone and 15d-PGJ2 have been shown to
downregulate c-Myc expression [12].
Thus, whether c-Myc is involved in mediating PPARγ agonist-induced apoptosis needs further
investigation.In colon and lung cancer cells, troglitazone was
reported to increase the expression of growth arrest and DNA-damage inducible
153 (GADD153) [12, 13],
a key apoptosis-regulated gene particularly involved in endoplasmic reticulum
(ER) stress-induced apoptosis [40]. Further analysis revealed
that troglitazone did not stimulate GADD153 mRNA levels in undifferentiated
3T3-L1 cells lacking PPARγ expression, whereas its induction was
clearly observed in differentiated adipocytes expressing PPARγ, suggesting the importance of PPARγ in troglitazone-induced GADD153
expression. In lung cancer cells, inhibition of GADD153 gene expression by an
antisense phosphorothionate oligonucleotide attenuated the troglitazone-induced
growth inhibition [13].
These findings collectively suggest that GADD153 might be a candidate factor
implicated in TZD-induced growth inhibition and apoptosis.Several studies have demonstrated the importance of
ERK and its regulated genes in PPARγ agonist-induced apoptosis [14–16, 41].
In human lung cancer cells, troglitazone induced apoptosis as well as PPARγ and ERK1/2 accumulation in the nucleus.
Both PPARγ siRNA and U0126, a specific inhibitor
of ERK1/2, blocked these effects of troglitazone, suggesting that
troglitazone-induced apoptosis is PPARγ- and ERK1/2-dependent. Moreover,
inhibition of ERK1/2 by U0126 also significantly decreased the levels of PPARγ, suggesting a positive crosstalk
between PPARγ and ERK1/2 or an autoregulatory
feedback mechanism to amplify the effect of ERK1/2 on cell growth arrest and
apoptosis [16].Proline oxidase (POX) is a redox enzyme localized in
the mitochondrial inner membrane and functions as a p53-induced gene that can
mediate apoptosis through generation of reactive oxygen species (ROS) [17].
A recent study in colon cancer cells showed that troglitazone enhanced the
binding of PPARγ to PPRE in the POX promoter, activated
the POX promoter, and increased endogenous POX expression. Blocking of PPARγ activation either by the antagonist
GW9662 or deletion of the PPAR-responsive element in the POX promoter only
partially decreased the POX promoter activation in response to troglitazone,
suggesting also the involvement of PPARγ-independent mechanisms. Further,
troglitazone induced p53 protein expression in HCT116 cells, which may be the
possible mechanism for PPARγ-independent POX activation, since POX
has been shown to be a downstream mediator in p53-induced apoptosis. In HCT15
cells, with both mutant p53 and mutant PPARγ, troglitazone did not activate POX,
whereas it did in HT29 cells, with a mutant p53 and wild type PPARγ, indicating that both PPARγ-dependent and -independent mechanisms
are involved in the troglitazone-induced POX expression [17].
Thus, this study suggests that troglitazone-induced apoptosis involves
targeting POX gene expression for generation of ROS.


## 2.2. PPARγ-Independent Apoptosis

To help
discern the PPARγ-dependent and -independent properties of TZDs,
TZD derivatives lacking PPARγ activity were developed. These derivatives
have a double bond adjoining the terminal thiazolidine-2,4-dione ring which
abolishes ligand binding to PPARγ [42]. Shiau et al. [18] showed that the pioglitazone,
troglitazone, and ciglitazone derivatives (Δ2-PG, Δ2-TG, Δ2-CG) were unable to activate PPARγ compared to pioglitazone, troglitazone, and
ciglitazone which showed significant activation of PPARγ. When troglitazone and Δ2-TG were tested for growth inhibition in two
prostate cancer cell lines: one cell line expressing high levels of PPARγ (PC-3) and one deficient of PPARγ expression (LNCaP), the LNCaP cells were more
sensitive to troglitazone compared to PC-3 cells despite being deficient in
PPARγ. As well, Δ2-TG which cannot activate PPARγ was more effective than troglitazone in
suppressing growth in both PC-3 and LNCaP cells. Both troglitazone and Δ2-TG induced cytochome C release and DNA
fragmentation in these cells, attributing the growth inhibition to apoptosis.
These results suggest that TZDs can induce apoptosis independent of PPARγ activation. The induction of apoptosis in this
study appears to be partly due to the inhibition of the antiapoptotic function
of Bcl-2 and Bcl-XL. It is thought that Bcl-2 and Bcl-xL sequester
proapoptotic molecules such as Bax and Bak through heterodimerization through
BH3 domain binding which inhibits the proapoptotic function of Bax and Bak [43, 44].
Both troglitazone and Δ2-TG reduced the association of Bak with Bcl-2
and Bcl-XL causing the cells to undergo apoptosis as shown by
cytochrome C release and caspase-9 activation. Moreover, Bcl-XL overexpression protected LNCaP cells from troglitazone- and Δ2-TG-induced apoptosis [18]. Collectively, these results
show that PPARγ ligands trigger apoptosis independent of PPARγ and primarily target activation of the
intrinsic apoptotic pathway, at least in the tested prostate cancer cells.It was shown that 15d-PGJ2, but
not rosiglitazone and ciglitazone, induced apoptosis in oral squamous cell
carcinoma cells, suggesting that 15d-PGJ2 is acting through pathways
other than activation of PPARγ. In this study, the apoptotic effect of
15d-PGJ2 was associated with downregulation of the oncogene Stat3
which was not seen with rosiglitazone or ciglitazone [19].  Similarly, in bladder and prostate cancer
cells, 15d-PGJ2 and troglitazone inhibited cell growth but
rosiglitazone and pioglitazone had no effect on growth inhibition. 15d-PGJ2 inhibited cell growth by induction of apoptosis, while troglitazone
induced cell cycle arrest [20].
Thus, the induction of apoptosis can also be selective for certain PPARγ ligands.Other mediators of apoptosis in PPARγ ligand-induced cell death include the
early growth response-1 (EGR-1) transcription factor. EGR-1 has been linked to
apoptosis and shown to be activated by ERK. In colon cancer cells, EGR-1 was
induced dramatically by troglitazone but not by other PPARγ ligands [14].
Inhibition of ERK phosphorylation abolished EGR-1 induction by troglitazone,
suggesting an ERK-dependent induction of EGR-1. Given that troglitazone-induced
apoptosis is accompanied by the biosynthesis of EGR-1, these results suggest
that PPARγ-independent EGR-1 induction is a unique
property of troglitazone compared with other PPARγ ligands and may play an important role
in troglitazone-induced apoptosis [14].
One of the EGR-1-regulated genes is proapoptotic nonsteroidal anti-inflammatory
drug (NSAID)-activated gene (NAG-1) [15].
A recent study has demonstrated that the novel TZD derivative MCC-555 exerts a
PPARγ-independent upregulation of NAG-1.
Moreover, NAG-1 induction contributes to MCC-555-induced apoptosis as
downregulation of NAG-1 by siRNA suppressed MCC-555-induced apoptosis [41]. As well, NAG-1 induction was
also observed in colon cancer cells treated with troglitazone or 15d-PGJ2 [15].
Importantly, both agents induce NAG-1 expression through an EGR-1-dependent
mechanism. However, troglitazone, but not 15d-PGJ2, increases EGR-1
binding to the EGR-1 binding site located within region -73 to -51 of the NAG-1
promoter; this effect has an important role in the transactivation of
TGZ-induced NAG-1 expression. The effect of 15d-PGJ2 is probably
PPARγ-dependent because a PPARγ antagonist inhibited the 15d-PGJ2-induced
expression of NAG-1, whereas TGZ-induced NAG-1 expression was not inhibited by
the PPARγ antagonist [15].Multiple myeloma and Burkitt lymphoma cells express
constitutively active NF-κB. 15d-PGJ2 was reported to
suppress constitutive NF-κB activity and potently induce apoptosis
in both types of B-cell malignancies. NF-κB inhibition is accompanied by rapid
downregulation of NF-κB-dependent antiapoptotic gene products,
including cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2,
X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), and c-FLIP. These
effects were mimicked by the proteasome inhibitor MG-132, but not by
troglitazone, suggesting that 15d-PGJ2-induced apoptosis is
independent of PPARγ [21].
Thus, the inhibition of NF-κB may play a major role in the
proapoptotic activity of 15d-PGJ2 in aggressive B-cell malignancies
characterized by aberrant regulation of NF-κB. Another study in MCF7 breast cancer
cells has shown that both PPARγ and p53 are involved in rosiglitazone-induced
apoptosis. However, the NF-κB sequence in the p53 promoter region is
required for rosiglitazone to increase p53 transcription in this study [22].


## 3. PPAR Agonists Augment Death Receptor-Induced Apoptosis

Apoptosis induced by death receptors can be initiated
through binding of death receptor ligands such as TRAIL or Fas ligand. PPARγ ligands can increase death receptor expression
and augment death receptor-induced apoptosis. The linkage between PPARγ and TRAIL/death receptor-induced
apoptosis came from the early work showing that the PPARγ ligand pioglitazone enhances
TRAIL-induced apoptosis through induction of p21 (WAF1) [45, 46].
Subsequently, there are multiple studies demonstrating that different PPARγ ligands have the ability to enhance
TRAIL-induced apoptosis in various types of cancer cells both in vitro and in vivo [34, 47–51].
The majority of the studies using various approaches including PPARγ antagonists, PPARγ siRNA or dominant-negative PPARγ mutants conclude that PPARγ ligands enhance TRAIL/death
receptor-induced apoptosis through PPARγ-independent mechanisms [34, 47–49]
(Table 2).Table 2
Combination of anticancer drugs with PPARγ ligands enhances tumor cell death.
PPARγ agonist + antitumor agentPPARγTumor typeMolecular mediator(s) of apoptosisReferenceTroglitazone or ciglitazone or
GW1929 + TRAILIndependentLungDR5, c-FLIP[34]15d-PGJ2 + TRAILIndependentLeukemia, prostateDR5[47]Troglitazone + TRAILIndependentGlioblastoma, neuroblastomac-FLIP, survivin, DR5[52]Rosiglitazone + TRAILIndependentRenal, glioma, breast, prostateROS, DR5, c-FLIP[49]15d-PGJ2 or ciglitazone
or troglitazone or CDDO or CDDO-Me + TRAILIndependentProstate, ovarian, colonc-FLIP[50]Troglitazone + TRAIL or troglitazone
+ etoposide or paclitaxelIndependentGliomaPTP1B, STAT3, c-FLIP, Bcl-2[53]15d-PGJ2 + MK886DependentLungPPARγ, RXRα[54]15d-PGJ2 + IndomethacinCiglitazone + MK886 +
13-cis-retinoic acidRosiglitazone + LG100268 or all
transretinoic acidDependent and independentLeukemia, lymphoma, myelomaBcl-2, caspase-9[55]15d-PGJ2 + LG100268 or
all trans-retinoic acidCDDO + LG100268 or all transretinoic
acidRosiglitazone + carboplatinDependentLung, ovarian, colonMT1H, MT1X, MTIIA[56]TZD18 + imatinibIndependentLeukemiaBax, NF-κB[57, 58]RS5444 + paclitaxelDependentThyroidp21WAF1/CIP1[59]15dPGJ2 + docetaxelIndependentLungBcl-2, BAD, cyclin D1, p53[60]Among these studies, Kim et al. [50]
first reported their important findings that a variety of natural and synthetic
ligands of PPARγ including 15d-PGJ2, ciglitazone,
troglitazone, and the triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic
acid (CDDO) selectively reduce the levels of c-FLIP, and hence sensitize tumor
but not normal cells to apoptosis induction by TRAIL. Both PPARγ agonists and antagonists displayed
these effects, regardless of the levels of PPARγ expression and even in the presence of
a PPARγ dominant-negative mutant, indicating a
PPARγ-independent mechanism. Importantly,
PPARγ agonists induced ubiquitination and
proteasome-dependent degradation of c-FLIP, without concomitant reductions in
c-FLIP mRNA, thus demonstrating a mechanism by which PPARγ agonists decrease c-FLIP through
facilitating ubiquitin/proteasome-dependent c-FLIP degradation.Our group has shown that PPARγ agonists including ciglitazone, troglitazone,
and GW1929 induce the expression of death receptor 5 (DR5) including increasing
the cell surface distribution of DR5, reducing the levels of c-FLIP, and
enhancing TRAIL-induced apoptosis in human lung cancer cells [34]. When c-FLIP was
overexpressed or DR5 was silenced, PPARγ ligands showed diminished ability to enhance
TRAIL-induced apoptosis, indicating that both DR5 induction and c-FLIP
downregulation are two critical events accounting for enhancement of
TRAIL-induced apoptosis by PPARγ ligands. Moreover, we have shown that
the modulation of DR5 and c-FLIP expression is independent of PPARγ because the use of a PPARγ antagonist and silencing of PPARγ did not effect the ability of the PPARγ ligands to induce DR5 or downregulate c-FLIP [34].Consistently, 15d-PGJ2 has also been shown
to induce DR5 expression and augment TRAIL-induced apoptosis in Jurkat leukemia
cells and PC-3 prostate cancer cells. This induction of DR5 by 15d-PGJ2 occurs
posttranscriptionally through increasing DR5 mRNA stability, which is
independent of PPARγ activation because two other PPARγ agonists pioglitazone and rosiglitazone did
not upregulate DR5 expression and pretreatment with GW9662, a PPARγ inhibitor did not block the induction of DR5
by 15d-PGJ2 [47].
As well, DR5 upregulation contributes to the sensitization of TRAIL-induced
apoptosis by 15d-PGJ2 since the knockdown of DR5 using DR5 siRNA
decreased apoptosis induced by the combination of TRAIL and 15d-PGJ2 compared to control cells [47].In renal cancer
cells, rosiglitazone, in addition to downregulating c-FLIP expression, also
increases DR5 expression at the mRNA level [49]. The use of a DR5/Fc chimeric
protein or DR5 siRNA attenuated rosiglitazone and TRAIL-induced apoptosis,
indicating a critical role of DR5 induction in this death process.
Interestingly, rosiglitazone induced the generation of ROS, whereas cotreatment
with glutathione, which can scavenge ROS, prevented ROS generation, DR5
upregulation, and enhancement of TRAIL-induced apoptosis by rosiglitazone,
suggesting that ROS-mediated transcriptional activation of DR5 is important for
sensitization of renal cancer cells to TRAIL-induced apoptosis [49].As well, glioblastoma and neuroblastoma cells can be
sensitized to TRAIL-induced apoptosis by troglitazone [51].
In addition to upregulation of DR5 and reduction of c-FLIP, troglitazone
downregulated survivin levels in these cells, all of which may explain the
synergy observed with troglitazone and TRAIL treatment. Importantly, normal
astrocytes did not become sensitive to TRAIL-induced apoptosis when treated
with troglitazone, suggesting limited toxicity to normal tissues with this
treatment [51].
Downregulation of survivin by PPARγ ligands was also observed in breast
cancer cells; this event contributes to enhancement of TRAIL-induced apoptosis
because enforced expression of ectopic survivin partially protected cells from
troglitazone/TRAIL-induced apoptosis. Different from other studies, this study
did not find that troglitazone altered the levels of the key proteins in the
death receptor-mediate apoptotic pathway including DR4, DR5, and c-FLIP.
Instead, they found that troglitazone decreased cyclin D3 levels through
inducing ubiqutin/proteasome-mediated protein degradation. Importantly, cyclin
D3 downregulation is associated with troglitazone-induced survivin reduction
and enhancement of TRAIL-induced apoptosis in human breast cancer cells as
silencing of cyclin D3 reduced the levels of survivin and promoted
TRAIL-induced apoptosis [48].
Currently, it is unclear how cyclin D3 regulates survivin expression.In human glioma cells, it has been shown that
troglitazone activates protein-tyrosine phosphatase-1B (PTP-1B), which
subsequently reduces phosphotyrosine 705 STAT3 (pY705-STAT3) via a PPARγ-independent pathway [53].
Reduction of pY705-STAT3 in glioma cells caused downregulation of c-FLIP and
Bcl-2. When given in combination with TRAIL or caspase-dependent
chemotherapeutic agents, such as etoposide and paclitaxel, troglitazone
exhibited a synergistic effect by facilitating caspase-8 and -9 activities.
Thus, it appears that PTP-1B plays a critical role in the downregulation of
activated STAT3, as well as c-FLIP and Bcl-2 [53].
However, it is also not clear how PTP-1B regulates the expression of c-FLIP and
Bcl-2.Although PPARγ agonists downregulate c-FLIP through
promoting its degradation, the detailed mechanisms by which PPARγ agonists trigger
ubiquitin/proteasome-dependent degradation of c-FLIP are unknown. Moreover, the
mechanisms underlying PPARγ agonist-induced upregulation of DR5
expression have not been addressed as well. Nonetheless, the sensitization of
TRAIL-induced death by PPARγ agonists may have relevant clinical
implications as TRAIL is currently being tested in clinical trials for cancer.
Identification of tumors that can overcome TRAIL resistance by treatment with
PPARγ agonists will enhance tumor specific targeting
by TRAIL and reduce toxicity of normal tissues as TRAIL has been shown to
induce death in tumor cells while sparing normal cells.PPARγ agonists have also been shown to effect
Fas-mediated apoptosis. In HT-29 colon cancer cells, there was a synergistic
effect on induction of apoptosis when the anti-Fas agonistic antibody, CH11,
was combined with 15d-PGJ2 or ciglitazone [61]. As well, rosiglitazone
sensitized the breast cancer cell line, MDA-MB-231, to the antitumor effects of
CH11 as well as TNF-α [62].
In uterine leiomyoma cells, ciglitazone downregulated the antiapoptotic protein
Bcl-2 and upregulated Bax and Fas while enhancing PARP cleavage and caspase-8
activation, suggesting that ciglitazone induces apoptosis in a Fas- and
caspase-dependent mechanism [63].


## 4. PPARγ Agonists in Combination with Other Anticancer Agents Enhance Apoptosis

Combination therapy regimens are effective in the
clinical treatment of various cancers. Many studies have shown that combining
PPARγ agonists with anticancer agents can
further sensitize tumor cells to apoptosis (Table 2). As we continue to elucidate
the molecular mechanisms of PPARγ in the regulation of cancer formation
and development, combining PPARγ agonists with other targeted anticancer
agents may be an effective strategy for chemoprevention and treatment of
various cancers.One such combination involves the 5-lipoxygenase
inhibitor MK866. MK866 blocks the 5-lipoxygenase pathway of arachidonic acid
metabolism, increases the expression of PPARα and PPARγ in breast cancer cells, and induces apoptosis [64].
As well, in lung cancer cells, MK886 increased PPARγ reporter activity. The combination of MK886
with 15d-PGJ2 generated greater growth-inhibitory effects including
apoptosis than each single agent alone in A549 lung cancer cells [54].
Moreover, MK866 increased the expression of RXRα whose heterodimerization with PPARγ is thought to be necessary for the
proapoptotic effect of PPARγ. When MK866 was combined with ciglitazone and
the RXR agonist, 13-cis-retinoic acid, there was a superadditive growth
inhibitory effect compared to each drug alone [54].
These results suggest that the induction of PPARγ and RXRα by MK866 sensitizes tumor cells to apoptosis
by PPARγ ligands or retinoids. In leukemia, lymphoma,
and myeloma cells, exposure to rosiglitazone, 15d-PGJ2, or
CDDO in combination with the RXR agonist, LG100268, or the retinoic acid
receptor (RAR) agonist, all transretinoic acid, augmented the growth-inhibitory
effects in these cells [55].
In agreement, treatment of breast cancer cells with another RXR selective
ligand, AGN194204, and the PPARγ ligand γ-linolenic
acid showed an additive growth inhibitory response [65].
Thus, combining retinoids with PPAR ligands may prove to be a successful
treatment in some cancers. This approach may be useful in the clinic as lower
doses of each drug can be used to inhibit growth and a more optimal therapeutic
index can be achieved.Other drug combinations that show synergy with PPARγ ligands include the platinum-based drugs. The
combination of rosiglitazone with carboplatin in lung, ovarian, and colon
cancer models showed a synergistic inhibition of growth. These results are PPARγ-dependent as a non-TZD PPARγ ligand was also able to enhance growth
inhibition when combined with carboplatin, and the PPARγ antagonist GW9662 significantly reduced the
synergistic effect of rosiglitazone and carboplatin [56]. This synergy is related to
the metallothioneins which are heavy metal binding proteins that play a role in
platinum drug resistance. Rosiglitazone reduces metallothionein gene expression
through a PPARγ-dependent mechanism as treatment with the PPARγ antagonist GW9662 abrogated metallothionein
reduction by rosiglitazone and the non-TZD PPARγ agonist GW1929 was also able to reduce
metallothionein expression. Moreover, overexpression of the metallothionein
MT1H reduced the synergistic effect of rosiglitazone and carboplatin.
Therefore, it appears that the downregulation of metallothioneins contributes
to the synergism of PPARγ ligands and carboplatin. This synergistic
effect of the combination of rosiglitazone and carboplatin was also observed in
vivo in xenograft models of lung and ovarian cancer as well as a
carcinogen-induced model of colon cancer [56]. Platinum-based drugs are
currently being used in the clinic to treat lung and ovarian cancer, therefore,
the use of PPARγ ligands to enhance the efficacy of platinum
drugs in the treatment of these cancers would be a great advancement in
treating these two deadly diseases.A dual PPARα/γ ligand, TZD18, can induce apoptosis in adult lymphocytic leukemia and chronic
myeloid leukemia cell lines [57, 58].
When TZD18 was combined with the bcr-abl tyrosine kinase inhibitor, imatinib,
in these cell lines, there was enhanced growth inhibition. Treatment of
leukemia patients with imatinib has been a successful therapy, however
resistance to imatinib is a problem. These data suggest that combining TZD18
with imatinib is a potential therapy for treating imatinib resistant disease.
In these studies, the growth inhibitory effects of TZD18 appeared to be
independent of PPARα or PPARγ [57, 58].A common mutation found in anaplastic thyroid cancer
is a PAX8/PPARγ rearrangement which results in downregulation
of PPARγ, suggesting that PPARγ may be a tumor suppressor gene in this type of
cancer [59].
In addition, the fusion protein, PAX8-PPARγ, resulting from this rearrangement can act as
a dominant negative inhibitor of wild-type PPARγ [66].
Treatment with the novel PPARγ agonist RS5444 in anaplastic thyroid cancer
cells lines resulted in growth inhibition and PPARγ activation. When RS5444 was combined with
paclitaxel, a standard treatment for anaplastic thyroid cancer, enhanced
apoptosis-inducing effects were observed [59].
Similarly, the combination of a PPARγ agonist and docetaxel also exerted
enhanced apoptosis-inducing and antitumor effects in human lung cancer cells.
In addition, 15d-PGJ2 combined with docetaxel significantly reduced
tumor volume compared with control, 15d-PGJ2, or docetaxel alone in
both A549 and H460 xenografts. This combination showed a significant increase
in apoptosis associated with inhibition of Bcl-2 and cyclin D1 expression and
overexpression of caspase-3 and p53 pathway genes. However, enhanced expression
of caspase 3 and inhibition of cyclin D1 by the combination was not reversed by
GW9662, thus suggesting a possible PPARγ-independent mechanism underlying
enhanced apoptosis-inducing and antitumor effects by the combination of 15d-PGJ2 and docetaxel [60].


## 5. PPARγ Antagonists Exert Apoptosis-Inducing Effects

Although
most of the antitumor effects of PPARγ ligands are attributed to PPARγ agonists, there is evidence that PPARγ antagonists can have antiproliferative and
apoptotic effects on tumor cells. In one study, two PPARγ antagonists, T0070907 and GW9662, were tested in a panel of cancer
cell lines and were able to inhibit cell growth and induce apoptosis. Combining
the PPARγ agonist, pioglitazone, with the PPARγ antagonists T0070907 or GW9662 actually increased growth
inhibition in a colon cancer cell line compared to each agent alone [67]. In breast cancer cells, the PPARγ antagonist GW9662 inhibited growth and also surprisingly
enhanced rosiglitazone-induced growth inhibition [68]. The effects of this enhanced growth inhibition appeared to
be independent of PPARγ activity as the combination of GW9662 and rosiglitazone did
not result in activation of PPARγ as compared to rosiglitazone alone which did
activate PPARγ [68]. How the combination of PPARγ agonists and antagonists can enhance tumor
growth inhibition needs further investigation.Human
primary squamous cellcarcinoma, lymph node metastasis, and squamous
cell carcinoma cell lines express high levels of PPARγ [69].
The specific PPARγ antagonists T0070907, GW9662, and
BADGE, but not agonists (i.e., pioglitazone and rosiglitazone) induced apoptosis
in squamous cell carcinoma cell lines by interfering with adhesion
to the extracellular matrix and disrupting survival signals, and
thus inducing anoikis. Furthermore, the PPARγ antagonists strongly
inhibited the invasion of squamous cell carcinomas.These results
imply a potentially important and novel role for the inhibition of
PPARγ function via the use of specific
antagonists in the treatment of oral squamous cell carcinoma and the
prevention of tumor invasion and metastasis [69].
Similarly, these antagonists also induced apoptosis in colorectal cancer cells
as well as altered cell morphology which was linked to alterations in
microtubules. The PPARγ antagonists reduced the levels of α and β tubulin
which prevented microtubule formation. This mechanism is unique from the known
antimicrotubule drugs for the treatment of cancer such as the taxanes which
alter microtubule polymerization. These data suggest that PPARγ antagonists may be used as cancer therapy
particularly in cancers that are not responsive to antimicrotubule therapy [70].In
contradiction to data suggesting that activation of PPARγ can reduce tumor growth, treatment with PPARγ ligands increased the number of colon tumors
in the Min mouse model of
familial
adenomatous polyposis [71, 72].
In this model, PPARγ may be playing a role in tumor promotion.
Thus, it appears that
activation or inhibition of PPARγ can have dual roles in tumorigenesis depending
on the type of cancer models examined. Determining the mechanisms of PPARγ ligands in cancer either dependent or
independent of PPARγ action will be critical to understanding how
to best target tumor cells for effective therapy.


## 6. Activation of PPARγ as a Mechanism for Certain Anticancer Agents to Induce Apoptosis

In addition to PPARγ ligands, certain antitumor agents
induce apoptosis through activation of endogenous PPARγ. It was reported that the NSAID,
sulindac, induced apoptosis, and upregulated PPARγ expression in oral squamous carcinoma cells [73].
When PPARγ was silenced with PPARγ antisense oligonucleotides, sulindac lost its
growth-inhibitory effects compared to control cells transfected with PPARγ sense oligonucleotides in which significant
growth inhibition was observed. Therefore, PPARγ is an important mediator of cell growth
induced by sulindac [73].
Similarly, β-carotene was shown to induce apoptosis
and increase PPARγ expression at both mRNA and protein
levels in MCF-7 breast cancer cells. The presence of the PPARγ antagonist GW9662 partially attenuated β-carotene-induced cell death, thus
suggesting that PPARγ is involved in β-carotene-induced apoptosis in this cell
line [74].Butyrate is a histone deacetylase inhibitor with the
capacity to induce apoptosis of cancer cells. Its growth-inhibitory effects
were suggested previously to be dependent on PPARγ activation [75]. A recent study has shown
that stimulation of cells with butyrate increased PPARγ expression and activity as well as
phospho-p38 MAPK protein levels and caspase-3 activity. Butyrate-induced
upregulation of PPARγ was abrogated by coincubation with the
p38 MAPK inhibitor SB203580. Treatment of cells with butyrate resulted in both
increased caspase-8 and -9 activity and reduced expression of XIAP and
survivin. Moreover, these effects were almost completely abolished in cells
expressing a dominant-negative PPARγ mutant [76]. These results collectively
suggest PPARγ as a key target in the butyrate-induced
signaling cascade leading to apoptosis.Capsaicin (N-vanillyl-8-methyl-alpha-nonenamide), a
spicy component of hot pepper, is a homovanillic acid derivative that
preferentially induces certain cancer cells to undergo apoptosis and has a
putative role in cancer chemoprevention. In colon cancer cells, capsaicin
induced apoptotic cell death; this effect was completely blocked by bisphenol A
diglycidyl ether, a specific PPARγ antagonist, but not by capsazepine, a
specific antagonist for vanilloid receptor [77]. Thus, it seems that
capsaicin-induced apoptotic cell death in colon cancer cells is associated with
the PPARγ pathway without the involvement of the
vanilloid receptor.Abnormally elevated expression or activation of cyclooxygenase-2
(COX-2) is often associated with cell proliferation and transformation.
However, increased numbers of studies have suggested that induction of COX-2
can be proapoptotic [78–81].
In COX-2-mediated apoptosis, production of prostaglandin D2(PGD2)
and 15d-PGJ2 and activation of PPARγ have been considered important
mechanisms. For example, the alkylphospholipid type antitumor agent
ET-18-O-CH3, at the same concentration ranges that induce apoptosis, induced
COX-2 expression in H-ras transformed human breast epithelial cells (MCF10A-ras).
The addition of a selective COX-2 inhibitor SC-58635 and COX-2 gene knockdown
blocked ET-18-O-CH3-induced apoptosis, suggesting that COX-2 induction by this
drug is causally linked to its apoptosis-inducing activity. ET-18-O-CH3
treatment resulted in elevated release of 15d-PGJ2 and DNA binding
and transcriptional activity of PPARγ. These data suggest that ET-18-O-CH3
likely induces COX-2 expression and production of 15d-PGJ2, leading to
induction of apoptosis in MCF10A-ras cells [79].In agreement, several chemotherapeutics including
paclitaxel, cisplatin, and 5-fluorouracil induced COX-2 expression and
prostaglandin (PG) synthesis, accompanied by a substantial decrease of
viability and enhanced apoptosis [80]. Cells were significantly
less sensitive to apoptotic death when either COX-2 expression or its activity
was suppressed by siRNA or by the selective COX-2 inhibitor NS-398. Experiments
performed to clarify how COX-2 leads to apoptosis revealed a profound proapoptotic
action of PGD2 and its dehydration product, 15d-PGJ2, because chemotherapeutic-induced apoptosis was prevented by siRNA targeting
lipocalin-type PGD synthase (L-PGDS), which catalyzes the isomerization of PGH2 to PGD2. Moreover, apoptosis by chemotherapeutics, PGD2 and
15d-PGJ2, was suppressed by the PPARγ antagonist, GW-9662 or PPARγ siRNA. Collectively, this study
suggests that COX-2 induction and synthesis of
L-PGDS-derived PPARγ-activating PGs are a decisive target by
which several chemotherapeutics induce apoptosis [80]. As well, the novel natural
compound, a cycloanthranilylproline derivative (Fuligocandin B) was recently
reported to sensitize leukemia cells to TRAIL-induced apoptosis through
COX-2-dependent 15d-PGJ2 production. However, the synergy mediated
by 15d-PGJ2 works in a PPARγ-independent manner as PPARγ siRNA failed to block the synergy [82].These findings have important clinical impact on the
treatment of cancer patients if this mechanism, particularly to
chemotherapeutic agents, is common in different types of cancer cells. Because
NSAIDs possess COX-2-inhibitory activity and are commonly used by many people
including cancer patients, caution should be taken during cancer chemotherapy
to avoid potentially diminished therapeutic efficacy due to COX-2 inhibition.


## 7. Conclusions

The role of
PPARs, particularly PPARγ, in cancer is an evolving field.
Understanding of the molecular mechanisms underlying PPAR-mediated regulation
of apoptosis of tumor cells will continue to expand. Accordingly, targeting
PPARs, especially PPARγ for cancer chemoprevention and therapy may prove
to be very effective and will remain an interesting research topic. As we
continue to address specific signaling pathways that lead to cancer, we can
further elucidate how PPARs and their ligands contribute to these pathways and
design effective combinations of therapy that target multiple steps in the
oncogenic process. PPARγ ligands have the potential to sensitize
cancer cells to or overcome resistance to chemotherapy or other anticancer
drug-based therapies. Thus, exploring mechanism-driven PPARγ ligand-based combination regimens for
both cancer chemoprevention and therapy should be the focus of this future
study. Specific types of tumors and unique tumor microenvironments also behave
differently to PPAR activation or inhibition. Therefore, a close examination of
individual tumor types and their response to PPAR stimulation will be critical
for successful cancer therapy targeting PPARs, particularly PPARγ. Many studies have revealed that TZDs
exert PPARγ-independent effects on induction of
apoptosis in various cancer cells. Although some of the TZDs are clinically
used drugs for treatment of diabetes with acceptable or manageable side effects
or toxicity, they were not originally developed as anticancer drugs and hence
are not optimal for cancer treatment. Therefore, it is necessary to use them as
lead compounds for synthesizing analogs as anticancer drugs that possess better
or optimized cancer chemopreventive or therapeutic efficacy.



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*Source: 704165-2008-07-02.xml*