# Role of Peroxisome Proliferator-Activated Receptor Gamma and Its Ligands in the Treatment of Hematological Malignancies

Authors: Tatiana M. Garcia-Bates; Geniece M. Lehmann; Patricia J. Simpson-Haidaris; Steven H. Bernstein; Patricia J. Sime; Richard P. Phipps
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) is a multifunctional transcription factor with important regulatory roles in inflammation, cellular growth, differentiation, and apoptosis. PPARγ is expressed in a variety of immune cells as well as in numerous leukemias and lymphomas. Here, we review recent studies that provide new insights into the mechanisms by which PPARγ ligands influence hematological malignant cell growth, differentiation, and survival. Understanding the diverse properties of PPARγ ligands is crucial for the development of new therapeutic approaches for hematological malignancies.


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## Body

## 1. Introduction

In order  to understand the
influence of PPARγ and its many ligands on hematological
malignancies and their normal cell counterparts, we first present background
material to orient the reader.Peroxisome
proliferator-activated receptors (PPARs)α
, β/δ
, and γ are members of the nuclear
hormone receptor superfamily of transcription factors that regulate several
metabolic pathways in a tissue-selective manner [1].  All PPARs form
heterodimers with members of the retinoid X receptor (RXR) subfamily of nuclear
hormone receptors and regulate initiation of transcription by binding to the
peroxisome proliferator response element (PPRE) in promoters of target genes.  Drug classes such as fibrates and
thiazolidinediones are used for lowering lipids and improving insulin
sensitivity, respectively, thus effectively reducing risk factors that lead to
cardiovascular disease [2, 3]  and diabetes [4, 5]. PPARγ agonists have both PPARγ-dependent and -independent effects on
coagulation, thrombosis, angiogenesis, and tumor growth and metastasis [6, 7].  PPARγ agonists also exert anti-inflammatory and antifibrotic
effects by negatively regulating the expression of proinflammatory genes and by
inhibiting myofibroblast differentiation [8–10]. Moreover,
PPARγ agonists modulate the activity of several transcription
factors (e.g., NF-κB, AP-1, and Stat3) [10–13] that regulate inflammation.


### 1.1. Structure of the Human PPARγ Gene

The
human PPARγ is located on chromosome 3, band 3p25 [14].  This gene gives rise to the two well-known
isoforms of PPARγ, PPARγ1, and PPARγ2, which function as transcriptional
activators or repressors in a context-dependent manner [15, 16].  Recent evidence suggests that the human PPARγ gene is composed of at least 11 exons
that give rise to 9 transcript variants due to the combination of differential
promoter usage, alternative RNA splicing, and polyadenylation site selection of
the primary transcript (Figure 1). To date, four promoters and three new exons
A′, 3′, and 4′ have been identified [14, 17–23]. Similar to exons A1 and A2,
exon A′ is noncoding and contributes to the 5′ UTR of several transcript
variants (Figure 1). Inclusion of exon 3′ in the processed transcript produces
a truncated PPARγ1 protein (γ1tr) [22], as does the read-through of
exon 4 to include intron 4 sequences (γORF4) [23]. Both truncated forms of PPARγ1 (γ1tr and γORF4) lack the coding regions for the
ligand binding domain and function in a dominant negative manner to wild type
PPARγ1. The truncated form of PPARγ (γ1tr) was discovered and cloned from chronic
myeloid leukemia K562 cells and enhanced cell proliferation [22].  Similarly, γORF4 protein was found to reside mainly
in the nucleus and enhanced cell growth [23]. The complexity in processing
the PPARγ primary transcript likely leads to specific
regulation of PPARγ functions in a context-dependent manner.  This may explain, at least in part, the
pleiotropic functions ascribed to PPARγ1 and PPARγ2 [23–29].Figure 1
Schematic
structure of the human PPARγ gene. The human PPARγ gene is located on chromosome 3, band
3p25, and is composed of at least 11 exons that give rise to 9 transcript
variants. Expression of PPARγ involves differential promoter usage in
combination with alternative splicing and polyadenylation site selection.  The relative positions of the four known PPARγ promoters are designated as Pγ1-Pγ4.
The noncoding exons A1, A′, and A2 are depicted by boxes in different
shades of gray or in black and white checked. These exons contribute to the 5′
UTR of transcripts γ1-γ1d, γ3 and γ1tr. The transcript variants 1γ-1γd, γ3, and γ4 encode the PPARγ1 isoform. Exon B (diagonal blue and
white hatched box) encodes the 28 additional amino acids found at the amino
terminus of human PPARγ2; the mouse PPARγ2 exon B encodes 30 amino acids. Exons 1–6 (light blue boxes)
are common in all PPARγ1 transcripts and when they are spliced
to exon B encode full-length PPARγ2.
Two additional exon regions have been recently identified, exon 3′
(horizontal light blue and white hatched box) and exon 4′ (small light blue
box).  Inclusion of either of these
coding regions in the processed mRNA transcript results in truncated PPARγ1 proteins lacking the ligand binding
domain (γ1tr and ORF4, resp.).  The sizes of the exon boxes approximate the
relative lengths of each exon; however, the introns (depicted as straight
lines) are not drawn to scale.  The
positions of the stop codons are depicted by the hexagonal red stop signs.


### 1.2. Posttranslational Modifications Regulate PPARγ Activity

Several reversible
posttranslational modifications occur that regulate the transactivation
potential of PPARγ (Figure 2). The phosphorylation status and activity of the PPARs
are regulated in both ligand-dependent and ligand-independent manners, the
details of which have been recently reviewed [30]. Whereas serine phosphorylation of PPARα
increases its transcriptional activity in hepatocytes,
MAPK/ERK-mediated phosphorylation of Ser84/112 on PPARγ1/2 leads to attenuation of PPARγ transcriptional activity and its possible relocalization
from the nucleus to the cytoplasm [30–33].  Furthermore, both
Ser84/112 phosphorylation [34] and ligand binding [35] contribute to the targeting
of PPARγ to ubiquitin-proteasome degradation. In
contrast, ERK5 activates PPARγ1 in a phosphorylation-independent
manner by directly interacting with the hinge-helix 1 region [36].Figure 2
Reversible
posttranslational modifications of PPARγ.
The superfamily of nuclear hormone receptors possesses conserved
structural and functional domains including PPARγ.  The A/B
domain is the hypervariable region containing the putative activation
function-1 (AF-1) domain. Human PPARγ2 contains a 28 amino acid amino terminal
region that arises from differential promoter use and splicing (see Figure 1).
The primary structure of the C-domain is the most conserved and contains the
DNA binding domain (DBD). The D-domain (Hinge) allows for conformational change
following ligand binding to promote coregulator (coactivator or corepressor)
docking; binding of ERK5 to the hinge helix 1 region potentiates
ligand-dependent PPARγ1 activity. The E/F region contains the ligand
binding domain (LBD) of PPARγ and the activation function-2 (AF-2)
domain that participates in ligand-dependent degradation mediated by the
ubiquitin-proteasome pathway. PPARγ heterodimerizes with its binding
partners, RXR family members, through the E/F domain as well.  Reversible posttranslational modifications of
PPARγ regulate its activation. In addition to
proteasome-mediated degradation, PPARγ can be phosphorylated by MAP kinases at
S84/112 (position of serine in PPARγ1/PPARγ2) or SUMO-1 modification.  Two SUMOylation consensus motifs have been
described. Whereas SUMOylation at a conserved ψKXEXXSP (where ψ is a
hydrophobic amino acid and X can be any residue) is linked to serine
phosphorylation events, SUMOylation at ψKXE/D motifs are not generally linked to MAPK
phosphorylation.  The lysine residues of
the three SUMOylation motifs identified on PPARγ1/2 are depicted in red.  The serine residue phosphorylated by MAPKs is
depicted in yellow.  Both serine
phosphorylation and SUMOylation negatively regulate PPARγ activity.In a recent
review, Straus and Glass [10] discuss various mechanisms
for nuclear hormone receptor-dependent transrepression of target genes by the
PPARs, Liver X Receptors (LXRs), and glucocorticoid receptor (GR).  Posttranslational modification with small
ubiquitin-like modifier (SUMO)-1 converts these nuclear hormones from
transactivators to transrepressors of gene expression [10, 37].  SUMOylated PPARγ1 binds to the corepressor complex
interfering with its clearance, thereby preventing transactivation of NF-κB target genes [10, 37].  To date, modifications of PPARγ with SUMO-1 occur on three lysine
residues (K79/107, K319/347, and K367/395) of
PPARγ1/2 [38–40]. Moreover, PPARγ’s dimerization partner, RXRα
, is also SUMOylated [41]. A summary of PPARγ posttranslational modifications is
shown in Figure 2.  SUMO competes with
ubiquitin for modification of lysines on some proteins, thereby rescuing the
protein from ubiquitin-proteasome mediated proteolysis [42].  In addition to increasing protein half-life,
SUMOylation plays a role in nuclear-cytoplasmic trafficking, cell-cycle
regulation, genome integrity, transcription, and cancer progression and
metastasis [43–47].


## 1.1. Structure of the Human PPARγ Gene

The
human PPARγ is located on chromosome 3, band 3p25 [14].  This gene gives rise to the two well-known
isoforms of PPARγ, PPARγ1, and PPARγ2, which function as transcriptional
activators or repressors in a context-dependent manner [15, 16].  Recent evidence suggests that the human PPARγ gene is composed of at least 11 exons
that give rise to 9 transcript variants due to the combination of differential
promoter usage, alternative RNA splicing, and polyadenylation site selection of
the primary transcript (Figure 1). To date, four promoters and three new exons
A′, 3′, and 4′ have been identified [14, 17–23]. Similar to exons A1 and A2,
exon A′ is noncoding and contributes to the 5′ UTR of several transcript
variants (Figure 1). Inclusion of exon 3′ in the processed transcript produces
a truncated PPARγ1 protein (γ1tr) [22], as does the read-through of
exon 4 to include intron 4 sequences (γORF4) [23]. Both truncated forms of PPARγ1 (γ1tr and γORF4) lack the coding regions for the
ligand binding domain and function in a dominant negative manner to wild type
PPARγ1. The truncated form of PPARγ (γ1tr) was discovered and cloned from chronic
myeloid leukemia K562 cells and enhanced cell proliferation [22].  Similarly, γORF4 protein was found to reside mainly
in the nucleus and enhanced cell growth [23]. The complexity in processing
the PPARγ primary transcript likely leads to specific
regulation of PPARγ functions in a context-dependent manner.  This may explain, at least in part, the
pleiotropic functions ascribed to PPARγ1 and PPARγ2 [23–29].Figure 1
Schematic
structure of the human PPARγ gene. The human PPARγ gene is located on chromosome 3, band
3p25, and is composed of at least 11 exons that give rise to 9 transcript
variants. Expression of PPARγ involves differential promoter usage in
combination with alternative splicing and polyadenylation site selection.  The relative positions of the four known PPARγ promoters are designated as Pγ1-Pγ4.
The noncoding exons A1, A′, and A2 are depicted by boxes in different
shades of gray or in black and white checked. These exons contribute to the 5′
UTR of transcripts γ1-γ1d, γ3 and γ1tr. The transcript variants 1γ-1γd, γ3, and γ4 encode the PPARγ1 isoform. Exon B (diagonal blue and
white hatched box) encodes the 28 additional amino acids found at the amino
terminus of human PPARγ2; the mouse PPARγ2 exon B encodes 30 amino acids. Exons 1–6 (light blue boxes)
are common in all PPARγ1 transcripts and when they are spliced
to exon B encode full-length PPARγ2.
Two additional exon regions have been recently identified, exon 3′
(horizontal light blue and white hatched box) and exon 4′ (small light blue
box).  Inclusion of either of these
coding regions in the processed mRNA transcript results in truncated PPARγ1 proteins lacking the ligand binding
domain (γ1tr and ORF4, resp.).  The sizes of the exon boxes approximate the
relative lengths of each exon; however, the introns (depicted as straight
lines) are not drawn to scale.  The
positions of the stop codons are depicted by the hexagonal red stop signs.


## 1.2. Posttranslational Modifications Regulate PPARγ Activity

Several reversible
posttranslational modifications occur that regulate the transactivation
potential of PPARγ (Figure 2). The phosphorylation status and activity of the PPARs
are regulated in both ligand-dependent and ligand-independent manners, the
details of which have been recently reviewed [30]. Whereas serine phosphorylation of PPARα
increases its transcriptional activity in hepatocytes,
MAPK/ERK-mediated phosphorylation of Ser84/112 on PPARγ1/2 leads to attenuation of PPARγ transcriptional activity and its possible relocalization
from the nucleus to the cytoplasm [30–33].  Furthermore, both
Ser84/112 phosphorylation [34] and ligand binding [35] contribute to the targeting
of PPARγ to ubiquitin-proteasome degradation. In
contrast, ERK5 activates PPARγ1 in a phosphorylation-independent
manner by directly interacting with the hinge-helix 1 region [36].Figure 2
Reversible
posttranslational modifications of PPARγ.
The superfamily of nuclear hormone receptors possesses conserved
structural and functional domains including PPARγ.  The A/B
domain is the hypervariable region containing the putative activation
function-1 (AF-1) domain. Human PPARγ2 contains a 28 amino acid amino terminal
region that arises from differential promoter use and splicing (see Figure 1).
The primary structure of the C-domain is the most conserved and contains the
DNA binding domain (DBD). The D-domain (Hinge) allows for conformational change
following ligand binding to promote coregulator (coactivator or corepressor)
docking; binding of ERK5 to the hinge helix 1 region potentiates
ligand-dependent PPARγ1 activity. The E/F region contains the ligand
binding domain (LBD) of PPARγ and the activation function-2 (AF-2)
domain that participates in ligand-dependent degradation mediated by the
ubiquitin-proteasome pathway. PPARγ heterodimerizes with its binding
partners, RXR family members, through the E/F domain as well.  Reversible posttranslational modifications of
PPARγ regulate its activation. In addition to
proteasome-mediated degradation, PPARγ can be phosphorylated by MAP kinases at
S84/112 (position of serine in PPARγ1/PPARγ2) or SUMO-1 modification.  Two SUMOylation consensus motifs have been
described. Whereas SUMOylation at a conserved ψKXEXXSP (where ψ is a
hydrophobic amino acid and X can be any residue) is linked to serine
phosphorylation events, SUMOylation at ψKXE/D motifs are not generally linked to MAPK
phosphorylation.  The lysine residues of
the three SUMOylation motifs identified on PPARγ1/2 are depicted in red.  The serine residue phosphorylated by MAPKs is
depicted in yellow.  Both serine
phosphorylation and SUMOylation negatively regulate PPARγ activity.In a recent
review, Straus and Glass [10] discuss various mechanisms
for nuclear hormone receptor-dependent transrepression of target genes by the
PPARs, Liver X Receptors (LXRs), and glucocorticoid receptor (GR).  Posttranslational modification with small
ubiquitin-like modifier (SUMO)-1 converts these nuclear hormones from
transactivators to transrepressors of gene expression [10, 37].  SUMOylated PPARγ1 binds to the corepressor complex
interfering with its clearance, thereby preventing transactivation of NF-κB target genes [10, 37].  To date, modifications of PPARγ with SUMO-1 occur on three lysine
residues (K79/107, K319/347, and K367/395) of
PPARγ1/2 [38–40]. Moreover, PPARγ’s dimerization partner, RXRα
, is also SUMOylated [41]. A summary of PPARγ posttranslational modifications is
shown in Figure 2.  SUMO competes with
ubiquitin for modification of lysines on some proteins, thereby rescuing the
protein from ubiquitin-proteasome mediated proteolysis [42].  In addition to increasing protein half-life,
SUMOylation plays a role in nuclear-cytoplasmic trafficking, cell-cycle
regulation, genome integrity, transcription, and cancer progression and
metastasis [43–47].


## 2. PPARγ Ligands

Transcriptional
activity of PPARγ is controlled primarily by ligand binding [48]. PPARγ has a large ligand binding pocket, which
enables it to bind a variety of ligands [49]. PPARγ ligands include both synthetic and natural
molecules [48]. Many of the naturally
occurring ligands are fatty acids or fatty acid derivatives obtained through
the diet or from intracellular signaling pathways. These include lysophosphatidic acid [50], nitrolinoleic acid [51], 9- and 13-hydroxyoctadecadienoic
acids (9- and 13-HODE) [48, 52], 15-hydroxyeicosatetraenoic
acid (15-HETE) [25], prostaglandin D2 (PGD2), and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2)
[25, 48, 49, 53–55]. 15d-PGJ2 is
thought to be the most potent endogenous ligand for PPARγ, activating it at low micromolar
concentrations [25, 52, 53]. PGD2 and 15d-PGJ2 are derived from
arachidonic acid by the catalytic activities of the cyclooxygenase-2 (Cox-2)
and prostaglandin D synthase [53, 54, 56]. PGD2 spontaneously undergoes a series of dehydration reactions to form the PGJ
family of prostaglandins, including 15d-PGJ2, and 15d-PGD2,
which can also transactivate PPARγ [56–60].Synthetic PPARγ ligands, including drugs of the thiazolidinedione
(TZD) family (e.g., ciglitazone,
pioglitazone, rosiglitazone, and troglitazone), have potent insulin-sensitizing
properties [3, 25, 49, 56, 61, 62]. Because of this, some are
commonly used for the treatment of type 2 diabetes [48, 61]. There also exist TZDs, such as TZD18, that act as dual PPARα
/PPARγ agonists [63].There are also
many non-TZD synthetic compounds that can function as PPARγ agonists. Some of these are:  L-tyrosine-based GW-7845 and GW-1929 [48, 52], diindolymethane analogs [48, 64], certain nonsteroidal
anti-inflammatory drugs (NSAIDs) (i.e., indomethacin, ibuprofen, flufenamic
acid, and fenoprofen [25, 27, 65]), and the novel synthetic
triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its
derivatives [48, 66]. CDDO binds to PPARγ with nanomolar affinity [48, 66] and displays
antiproliferative and differentiating activities, making it useful as a
chemotherapeutic agent. Derivatives of CDDO have more useful pharmacodynamic
and pharmacokinetic properties than CDDO itself [67, 68]. Importantly, some CDDO
derivatives are orally active and are remarkably well-tolerated in humans [69].PPARγ ligands, including CDDO, can reduce cell proliferation,
migration, cytokine production, expression of costimulatory, and adhesion
molecules and can promote apoptosis [48]. These findings suggest that
PPARγ ligands may be efficacious in the treatment of
hematological malignancies [48]. However, numerous side
effects have been observed in patients treated with TZDs [49]. For example,  troglitazone has caused hepatotoxicity [49]. TZDs also induce weight
gain, edema [70], increased lipoprotein(a)
concentrations [3, 49], and probably enhance risk of
heart failure and cardiac hypertrophy [48, 71, 72].  Therefore, it is highly desirable to develop PPARγ ligands with improved therapeutic profiles [48].The identification
of “selective PPARγ modulators” (SPPARγMs) has become the object of intense recent
interest, with the idea that one might modulate the genes necessary to achieve
therapeutic potential, while not affecting the genes involved in producing side
effects [49]. This concept is plausible
because SPPARγMs take advantage of the large PPARγ
ligand binding pocket, which allows a variety of ligands to bind in different
orientations [15, 61, 73–76].  SPPARγMs then induce specific conformational
changes of the receptor which create different interaction surfaces, favoring
the recruitment of only a subset of coregulators [48, 49, 77, 78].
This subset of coregulators will allow the induction of some, but not all
target genes [15, 49, 61, 74, 79–83].
The SPPARγM concept has been shown to hold true
for some currently recognized PPARγ ligands. For example, CDDO is a more potent
inducer of apoptosis than are TZDs [48]. This may be because the PPARγ target genes activated by CDDO are different
from those activated by TZDs [48]. CDDO is less effective than
rosiglitazone in recruiting coactivators, but it can effectively promote the
release of corepressors from PPARγ target genes [48].
A greater understanding of the activities of the various PPARγ ligands will depend on the identification of
the specific coregulators recruited to PPARγ target genes in response to binding to
specific ligands [25].


## 3. PPARγ and the Immune System

One of the earliest
indications of an important role for PPARγ
in the immune system was the discovery of its expression in mouse spleen [84]. After this finding, our laboratory and others
began searching for PPARγ
expression and function in immune cells. To date, PPARγ
expression has been found in monocytes/macrophages, dendritic cells,
granulocytes (i.e., neutrophils, eosinophils, and basophils), mast cells, T
cells, and B cells, and most recently our laboratory found PPARγ in human platelets
[84–90].PPARγ ligands have been shown to have anti-inflammatory
effects on cells of the innate and adaptive immune system [91–94]. In
macrophages, PPARγ has an important
role in regulating lipid metabolism, as well as in the generation of
macrophage-derived foam cells in atherosclerotic lesions [95–98]. Upon phorbol myristyl acetate (PMA)
stimulation, PPARγ ligands can inhibit macrophage activation and production of inflammatory cytokines (e.g., TNFα
, IL-1β, and IL-6), inducible nitric oxide synthase
(iNOS), gelatinase B, and scavenger receptor A (SR-A) [89, 99, 100]. Moreover, PPARγ activation
can skew macrophage differentiation into a more anti-inflammatory phenotype [101]. In dendritic cells, PPARγ activation can inhibit the production of IL-12
and of chemokines involved in the recruitment of Th1 lymphocytes, therefore,
favoring a type 2 immune response [90]. PPARγ ligands
also enhanced the development of a dendritic cell phenotype that: (1) has
increased endocytic activity and (2) induces the expansion of invariant natural
killer T (NKT) cells [102].PPARγ also
plays a role in T lymphocyte
function, and its levels are upregulated following their activation [103, 104]. PPARγ expression
and activation can inhibit T lymphocyte proliferation and reduce the production
of IFNγ, TNFα
, and IL-2 [92, 105, 106]. These inhibitory effects result from the
direct interaction between PPARγ and
the transcription factor nuclear factor of activated T cells (NFAT) [107]. Our laboratory demonstrated that mouse and
human T cells express PPARγ, and treatment with PPARγ ligands induces apoptosis in malignant T cells [103, 104].  Recent
findings reported by Wohlfert et al.
could illuminate yet another regulatory role for PPARγ in the
immune system [108]. In their study, PPARγ activation enhanced the generation of CD4+ CD25+ regulatory T cells (Tregs).
Tregs have been
demonstrated to play a key role in negatively regulating autoimmunity and
immune responses [109]. There are two different
subtypes of Tregs: thymus-derived natural Tregs (nTregs) and inducible Tregs
(iTregs), which develop from CD4+ CD25- effector T cells
in the periphery. [109–111]. Wohlfert et al. showed that
ciglitazone enhanced the conversion of effector T lymphocytes into inducible Tregs
(iTregs). Moreover, PPARγ
expression in natural Tregs (nTregs) was required for the in vivo effects of ligand treatment
in a murine model of graft versus host disease [108]. These findings suggest that PPARγ ligands
enhance the activity of Tregs while dampening the activation of other T
lymphocyte subsets. PPARγ was also shown to have a physiological role in
regulating B lymphocyte function. In studies using PPARγ haploinsufficient
mice, B lymphocytes exhibited increased proliferation and survival, enhanced
antigen specific immune responses and spontaneous NF-κB activation [15, 112]. Our laboratory demonstrated that both normal
and malignant B lymphocytes express PPARγ, and
that exposure to certain PPARγ
ligands inhibits B cell proliferation and can induce apoptosis [85, 93, 113].In summary, PPARγ
activation has antiproliferative and proapoptotic effects and dampens cytokine
production in several immune cells. PPARγ ligands can also attenuate several
inflammatory diseases such as inflammatory bowel disease
[114–119], multiple sclerosis
[120–122], rheumatoid arthritis [112, 123], and psoriasis
[124–126]. These findings suggest that PPARγ ligands
may be useful for the treatment of immunological diseases, which include myelo
and lymphoproliferative disorders.


## 4. PPARγ and Its Controversial Role as a Tumor Suppressor Gene

As evidence
accumulated to support that PPARγ ligands are inhibitors of cell
proliferation and inducers of cell differentiation, attention turned to the
role of PPARγ in the onset and development of cancer.
The potential of PPARγ ligands as anticancer drug therapies
has been explored in cells from various malignant tissues, including those of
adipose, colon, breast, prostate, lung, pancreas, bladder, and stomach origin [26, 127]. There is emerging evidence for
a direct role of PPARγ functional mutations in the initiation of several common
human cancers, such as colon, prostate, and thyroid
[28, 128–130]. For example, in a study of
55 patients with sporadic colon cancers, four somatic PPARγ mutations were found. [129]. Also, a hemizygous deletion
of PPARγ was identified in 40% of prostate cancers
[128]. Furthermore, a fusion
protein derived from the paired box gene 8 (PAX8) and PPARγ genes (PPARγ-PAX-8) was detected in thyroid cancers,
which causes PPARγ not only to be functionally inactive
but also  to function as a dominant
negative form of PPARγ [28, 131]. As described earlier, the
PPARγ gene is mapped to human chromosome 3,
band 3p25 [14]. Interestingly, 3p deletions
have been identified in several hematological cancers, including acute myeloid
leukemias (AML), myelodysplastic syndromes (MDS), Philadelphia
chromosome-positive chronic myeloid leukemia (CML), acute lymphoblastic
leukemias (ALL), chronic lymphoproliferative disorder (CLD), and non-Hodgkin's
lymphomas (NHL) [132]. These observations suggest
that PPARγ plays a role as a tumor suppressor gene
and, as such, may be a therapeutic target for cancer. Studies in liposarcoma [133] and in xenograft models of
prostate [134] and colon cancer [135] support this hypothesis. However,
a study using a large number of human tumor samples and cell lines (n=397),
including those from leukemias, found no detectable abnormalities, either in
PPARγ exon 3 (DBD) or in exon 5 (LBD),
suggesting that PPARγ gene mutations may occur, but are rare [136].The expression
levels and/or the transactivation of PPARγ may be impaired in certain cancers. In
human lung cancer, decreased expression of PPARγ correlated with poor prognosis [29] and well-differentiated
adenocarcinomas had more PPARγ expression than poorly differentiated varieties
[137]. In addition, a study
performed by Jansen et al. demonstrated that the abnormal PML-RARα
(promyelocytic leukemia-retinoic acid receptor
alpha) fusion protein found in acute promyelocitic leukemia (APL) interferes
with PPAR function [138]. Similarly, Hamadani et al.
showed that different X-RARα
fusion proteins found in APL can
inhibit the transactivation of PPARγ, and that this repression can be
released by the addition of PPARγ ligands [139, 140]. These findings suggest that
(1) PPARγ may be inactive in APL, (2) this may
contribute to the undifferentiated phenotype, and (3) PPARγ ligands may help sensitize APL cells to
the differentiating effects of all-trans-retinoic
acid (ATRA).


## 5. PPARγ and PPARγ Ligands as Potential Therapy for Hematological Malignancies

### 5.1. Myeloid Malignancies

#### 5.1.1. Acute Myeloid Leukemia (AML)

Acute myelogenous leukemia (AML) constitutes about 25% of
all leukemias in adults in the Western World. It ranks as the second most
frequent type of leukemia in adults after chronic lymphocytic leukemia, with
more than 13000 new cases, and nearly 9000 deaths from AML in the U.S.
in 2007 [141]. Unfortunately, this type of
leukemia has one of the lowest survival rates, about 20% [142]. There are several subtypes
of AML, including acute promyelocytic leukemia (APL). The most common cause of
APL is a translocation between chromosome 15 and 17, t(15;17), that leads to
the generation of the PML/RARα
fusion gene. The resulting fusion
protein arrests the maturation of myeloid cells at the promyelocytic stage and
leads to the increased proliferation of promyelocytes [143]. The cell lines typically
used to study APL are NB4 and HL-60. NB4 has the t(15;17) translocation, while HL-60
does not [144]. In addition to chemotherapy
and stem cell transplantation, treatments for APL also include differentiation
therapy using all-trans-retinoic acid (ATRA)
which has led to long-term disease-free survival in 70–80% of cases of
this AML subtype [145].An early study performed by Fujimura et al.
demonstrated that treatment with troglitazone inhibited HL-60 cell growth by a
G1 cell cycle arrest and induced their differentiation to monocytes [146]. A similar, G1 arrest was
observed in all other hematopoietic cell lines examined. Furthermore,
differentiation into the monocytic lineage was observed not only in the
myelogenous and promonocytic cell lines, but also in an erythroleukemia cell
line [146]. Data shown by
Yamakawa-Karakida et al. demonstrated that PPARγ activation by both troglitazone and
15d-PGJ2 inhibits proliferation and induces apoptosis in
promyelocytic leukemia cells under serum-free conditions [147]. The induction of apoptosis
was caspase-3 dependent, as treatment with a caspase-3 inhibitor completely
abolished cell death. Although there were no changes in antiapoptotic or
proapoptotic proteins, the expression levels of the proto-oncogene product c-myc were drastically reduced after 24
hours of troglitazone treatment while DNA binding by Tcf-4, a transcription
factor responsible for c-myc expression, was completely inhibited [147]. Troglitazone and 15d-PGJ2 were found by Liu et al. to significantly induce apoptosis in K562 and HL-60
cells by upregulating the levels of the proapoptotic protein Bax and
downregulating antiapoptotic proteins such as survivin and Bcl-2 [148]. Furthermore, these PPARγ ligands downregulated the expression of
cyclooxygenase-2 (COX-2), antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1,
upregulated Bax and activated caspase 3 in human monocytic leukemia cells [149]. Recent observations reported
by Han et al. revealed that 15d-PGJ2 was able to sensitize tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-resistant
leukemic HL-60 cells to TRAIL-induced apoptosis [150]. This effect of 15d-PGJ2 was PPARγ-independent, as treatment with a PPARγ antagonist did not rescue the cells
from apoptosis. These results were consistent with studies performed in other
cancer cells where 15d-PGJ2 enhanced TRAIL-induced apoptosis [151]. In a human eosinophilic
leukemia cell line, EoL-1, treatment with troglitazone caused a G0/G1 cell cycle arrest that correlated with increased mRNA levels of the
cyclin-dependent kinase (cdk) inhibitor, p21WAF1/CIP1. Troglitazone exerted a
similar induction of p21 mRNA accompanied by inhibition of cell proliferation
in U937 cells and in the KPB-M15 human myelomonoblastic cell line [152]. These findings suggest that
this PPARγ ligand inhibits myeloid leukemia cell
proliferation at least in part by upregulating p21 [152]. Aside from its growth
inhibitory and apoptosis-inducing properties, 15d-PGJ2 has also been
shown to decrease the expression of metalloproteinases in AML, therefore,
inhibiting leukemic cell adhesion and invasion of the extracellular matrix
(ECM) [153].A recent study investigated the antileukemia effects
and the molecular mechanism of action of a novel PPARγ ligand, DIM#34, in AML. DIM#34 can inhibit
cell growth and induce apoptosis through PPARγ-dependent and -independent mechanisms.
Cell death was associated with defective extracellular signal-regulated kinase
(ERK) activity, and inhibition of Bcl-2 phosphorylation [154].Konopleva et al. demonstrated growth inhibitory,
differentiative, and apoptotic effects of PPARγ ligands in cells from a variety of
leukemias, including AML [155]. Addition of RXR or RAR
ligands (i.e., LG100268 and ATRA, resp.) in combination with PPARγ ligands enhanced the differentiative
and growth-suppressive effects. Hirase et al. reported similar findings that
the antiproliferative, proapoptotic, and/or differentiating effects of TZDs on
HL-60 cells were further enhanced by the addition of the RXR-selective ligand, LG100268
[156]. PPARγ ligands have also been shown to inhibit
the clonal proliferation of U937 myelomonocytic leukemia cells by a G1 cell
cycle arrest, and that treatment with both PPARγ ligand (troglitazone) and LG100268 had
synergistic inhibitory effects on clonal growth [157]. Finally, recent work by
Yasugi et al. reported that both pioglitazone and 15d-PGJ2 inhibited
cell proliferation in NB4 cells and that combined with ATRA, these PPARγ ligands also induced myeloid differentiation
and lipogenesis [158].The PPARγ-ligand CDDO and its C-28 methyl ester
derivative (CDDO-Me) have also shown prodifferentiative properties in myeloid
leukemia cells [159–161]. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and
monocytic differentiation in primary AML cells. Interestingly, while
colony formation of AML progenitors was significantly inhibited, normal CD34+ progenitor cells were less affected. The more potent effect of CDDO-Me on leukemic
cells compared to normal progenitor cells suggests that CDDO-Me has potential as a new therapeutic
agent for the treatment of hematological malignancies [159]. Another group found that low
doses of CDDO promoted phagocytosis and granulocytic differentiation in HL-60
cells and primary blasts from AML patients through the regulation of CCAAT
enhancer-binding protein (CEBPA) [162]. CEBPA is an important
transcription factor for granulocytic differentiation. CDDO upregulated the
transcriptionally active p42 CEBPA, while downregulating the inactive p30
CEBPA, thereby enhancing CEBPA-regulated gene transcription. These findings
suggest the potential use of CDDO in the treatment of CEBPA-defective AML
subtypes.As proposed earlier, PPARγ transactivation may be impaired in AML,
and PPARγ ligands may be able to sensitize AML
cells to the prodifferentiation effects of ATRA [138, 139]. In light of this, a recent
study revealed that CDDO enhanced ATRA-induced differentiation and apoptosis both
in the ATRA-sensitive APL cell line, NB4, and an ATRA-resistant cell line, MR2 [163]. These effects were partially
dependent on PPARγ, as inhibition of PPARγ either by a specific inhibitor (T007)
or by siRNA diminished CDDO-induced APL differentiation [163].CDDO induces apoptosis in human myeloid leukemia
cells by promoting loss of mitochondrial membrane potential, leading to
cytochrome c release and activation of caspases [155, 160, 162, 164]. However, Bcl-xL
overexpression only partially inhibited cytochrome c release and caspase
activation, indicating that CDDO can activate caspases 3 and 8 in a cytochrome
c-independent manner [160]. Similar findings were shown
by Konopleva et al. where CDDO activated both caspase-dependent and -independent
cell death [164]. CDDO also promotes tumor
necrosis factor (TNF)-induced apoptosis in human leukemia cells. CDDO exposure
did not inhibit NF-κB translocation into the nucleus, but
rather inhibited a step after translocation, such as the NF-κB-dependent resynthesis of the inhibitor
of NF-κB, IκBα
[165]. Similarly, Shishodia et al.
demonstrated that CDDO-Me inhibited both constitutive and inducible NF-κB activity in human leukemic cells. In
contrast to the previous study [165], CDDO-Me-induced NF-κB inhibition occurred through
suppression of IκBα
kinase activation, IκBα
phosphorylation, IκBα
degradation, p65 nuclear translocation,
and NF-κB-mediated reporter gene transcription [166]. These results lead to a downregulation
of NF-κB target genes and enhanced apoptosis
induced by TNF and other chemotherapeutic agents.Another CDDO derivative, C-28 imidazole (CDDO-Im),
appears to be more potent than CDDO in inhibiting the growth of human leukemia
cells in vitro, as well as
murine melanoma and leukemia cells in
vivo [167]. The mechanism of CDDO and
CDDO-Im-induced apoptosis has been attributed to a disruption of intracellular
redox balance by increasing reactive oxygen species (ROS) and decreasing
intracellular glutathione (GSH) concentrations [168].Another subtype of AML is the acute myelomonocytic
leukemia (AMML). A well established cell line derived from a child with AMML,
THP-1, is often used to study this disease [169]. Several studies have shown
that macrophages and myelomonocytic leukemias express PPARγ and that PPARγ agonists can induce differentiation of
THP-1 cells into macrophages, as shown by the expression of CD36 scavenger
receptors, as well as CD11b, CD14, and CD18 [97]. Another study showed that PPARγ1 expression levels were upregulated by
9-cis retinoic acid (9-cis RA) in THP-1 cells coincident with suppression
of cell growth [170]. Moreover, addition of a
specific PPARγ ligand enhanced 9-cis RA-induced growth inhibition [170]. A reduction in THP-1 cell
migration also occurred in response to PPARγ ligands and was due to downregulation
of metalloproteinase-9 expression [171]. These findings suggest that
PPARγ ligands may be beneficial in preventing
metastasis of monocytic leukemia cells. Indeed, PPARγ ligands also have angiostatic
properties because of their inhibitory effects on endothelial differentiation and
on vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo [172]. Recently, Ho et al. reported
that the pigment epithelium derived factor (PEDF), a potent antiangiogenic
factor, can induce THP-1 apoptosis and necrosis by inducing PPARγ protein expression. In their study,
PEDF-induced apoptosis was shown to be PPARγ-induction-dependent. Treatment with
PPARγ antagonist and PPARγ siRNA attenuated PEDF-induced apoptosis.
Transient expression of PPARγ using a PPARγ expression plasmid reproduced the PEDF-effects.
Importantly, the PPARγ induced by PEDF was transcriptionally
active. These results suggest a PPARγ-dependent induction of apoptosis in
THP-1 cells [173].


#### 5.1.2. Chronic Myeloid Leukemia (CML)

Chronic
myelogenous leukemia (CML) is a myeloproliferative disorder that affects
all hematopoietic cell types. It constitutes 15 to 20% of adult leukemias [174]. The American Cancer Society
anticipated diagnosis of about 4570 new cases of CML in 2007 [174]. CML is characterized by a genetic abnormality known as Philadelphia
(Ph)
chromosome, resulting from a translocation between chromosomes 9 and 22,
t(9;22)(q34;q11). This translocation generates a fusion protein called BCR-ABL which
is a constitutively active tyrosine kinase responsible for uncontrolled cell
proliferation and enhanced cell survival [175]. Treatments for this disease include splenic
irradiation, stem cell transplantation, and interferon alpha (IFNα
) administration with combination chemotherapy.
A specific tyrosine kinase inhibitor, Imatinib, was introduced in the
late 1990s and is a standard treatment for CML. However, clinical resistance to
imatinib has been described in CML patients, where BCR-ABL gene mutations or amplifications
have occurred [176, 177].
Therefore, development of new therapeutic strategies to overcome imatinib
resistance is needed. Dual PPARα
and γ ligands have been tested, either alone or in
combination with Imatinib, to overcome drug resistance. A characteristic cell
line used to study CML is K562, which was established from a patient with CML
in the acute phase [178].  Recently, a study was performed using a
synthetic dual PPARα
/PPARγ agonist, TZD18, in human CML myeloid
blast crisis cell lines [63]. In this study, treatment
with TZD18, both alone and in combination with Imatinib, inhibited CML
proliferation and induced apoptosis. These effects were PPARα
and PPARγ- independent, as neither PPARα
nor PPARγ antagonists were able to rescue cell
proliferation and survival. These results were reported previously by the same
group in Ph-positive lymphocytic leukemia cell lines, where TZD18 promoted cell
death and acted synergistically to enhance the effect of Imatinib [179]. Hirase et al. tested the
effects of TZDs in K562 cells, which have an erythroid nature and the potential
to differentiate into megakaryocytes [180]. TZD inhibited both cell proliferation
and the erythroid phenotype of K562 cells. These results were accompanied by a
reduction in erythroid lineage-transcription factor, GATA-1, levels [180]. Therefore, PPARγ ligands may serve a therapeutic use for
the treatment of other types of myeloproliferative disorders where there is an
overproduction of erythrocytes, such as polycythemia vera (PV).


### 5.2. Lymphoid Malignancies

#### 5.2.1. Acute Lymphoblastic Leukemia (ALL) and Non-Hodgkin’s Lymphomas

Acute
lymphoblastic leukemia (ALL) is a malignant disorder that arises from uncontrolled
proliferation of lymphocytic progenitors. The disease is most commonly
diagnosed in children, but can also occur in adults. About 80–90% of ALL
patients can achieve complete remission with currently available therapy. Yet, many
patients eventually relapse, and only 35% of individuals have a long-term
leukemia-free survival (LFS) [181, 182]. Therefore, development of new treatment
approaches to improve both the cure rate and the quality of life of patients
with ALL is greatly needed. ALL involving hyperproliferation of B
lymphocyte progenitors (B-ALL) is frequently associated with a translocation
between the c-myc gene on chromosome
8q24 and any of the three immunoglobulin genes located on chromosomes 14q32,
2p11, or 22q11. This translocation results in c-myc overexpression and correlates with poor prognosis [183, 184]. The members of the Myc
family, including c-myc, are involved
in regulation of proliferation and development of normal and malignant cells [185].An investigation by Zang et al. revealed that the PPARγ ligands pioglitazone and 15d-PGJ2 suppressed cell growth in G1 phase and induced apoptosis in a dose-dependent
manner in B-ALL cell lines. Apoptosis was found to be partly caspase-dependent,
as treatment with a pan-caspase inhibitor partially reversed this effect [186]. Similar findings were shown
in B-ALL with t(14;18), in which troglitazone not only induced G1 phase growth
arrest and apoptosis, but also downregulated the expression of c-myc mRNA and protein [187].Our group has demonstrated that: (1) both normal and
malignant B lineage cells express PPARγ mRNA and protein, and (2) exposure to
certain small molecule PPARγ ligands, including 15d-PGJ2, inhibits proliferation and induces apoptosis in these cells [85, 113]. Subsequently, we reported
that PPARγ ligand-induced apoptosis was mainly PPARγ-independent, since it was not prevented
either by a PPARγ antagonist nor a dominant negative form
of PPARγ (PPARγ-DN) [94].  We reported that the apoptotic mechanism
regulated by 15d-PGJ2, but not by ciglitazone, was related to the
production of ROS and the reduction in intracellular GSH [94].
CD40 signaling through
CD40-ligand (CD40L) enhances B cell survival and prevents BCR-induced apoptosis
by activating the transcription factor NF-κB [188]. Therefore, we tested whether
CD40 ligation could protect normal and malignant B cells from PPARγ ligand-induced apoptosis. CD40L was
able to partially rescue normal and malignant B cells from PPARγ ligand-induced apoptosis by activating NF-κB.
Similarly, Piva et al. reported 15d-PGJ2-induced apoptosis in human
Burkitt’s lymphomas and multiple myeloma cell lines through inhibition of NF-κB
activity. These effects lead to the downregulation of NF-κB-dependent
antiapoptotic protein production and therefore decreased cell survival. The
apoptotic effects could also be mimicked by NF-κB
p65 subunit knockdown by siRNA [189]. These results suggest a possible mechanism
for the proapoptotic action of PPARγ
agonists.We have also demonstrated that PPARγ ligands can induce apoptosis in cells
from human T cell leukemias (Jurkat), lymphomas (J-Jahn), and T-ALL cells
(CCRF-CEM) by a PPARγ-dependent mechanism [103]. Interestingly, normal T
cells were not adversely affected by PPARγ ligands, suggesting the use of PPARγ agonists as selective therapeutic drugs
for T-cell malignancies [103]. However, data from Yang et
al. raised questions on the antiproliferative effects of PPARγ-ligands in T-lymphoma cells [190]. They demonstrated that low
concentrations of PPARγ-ligands promoted T-lymphoma cell
survival, while high concentrations promoted cell death. These results suggest
that in T-lymphoma cells, PPARγ ligands can have contradictory effects when
used at different concentrations and require further examination.Cutaneous T cell
lymphoma (CTCL) is a group of T cell malignancies that accumulate in the skin.
The most common CTCLs are (1) the Mycosis fungoides (MF), which develops as
patches, plaques, or tumors containing apoptosis-resistant CD4+ CD45RO+ helper/memory T cells; and (2) the Sézary syndrome (SS), which is the leukemic
form of CTCL that develops with erythroderma and the appearance of atypical T
cells in the peripheral blood [191]. Current therapies for CTCL
include the use of bexarotene, an RXR ligand, with good efficacy in the late
stages of the disease [191]. Zhang et al. demonstrated
the expression of PPARγ in three CTCL lines (MJ, Hut78, and HH)
and freshly isolated peripheral  blood lymphocytes (PBL) from SS patients with
circulating atypical T cells (CD4+CD26-) [192]. CDDO exposure caused a dose-dependent
induction of apoptosis in MF/SS cell lines and SS patients’ PBL [192]. These findings suggest that
PPARγ ligands may be beneficial for the
treatment of CTCL and may have synergistic effects when used in combination
with bexarotene.Mantle cell
lymphoma (MCL) is a rare type of non-Hodgkin’s lymphoma (NHL), constituting
about 6% of NHL [193, 194].  In 85% of MCL cases, a translocation between
chromosome 11 and 14, t(11;14), is involved in the pathogenesis. This translocation
leads to the overexpression of cyclin D1, a protein that increases cell
survival and proliferation by positively regulating cell cycle entry into the
S-phase [193]. Despite the success of
current therapies, patients with mantle cell lymphoma have a shorter life span
compared to patients with other B cell lymphomas [193]. Recently, a study
demonstrated that treatment with pioglitazone and rosiglitazone, as well as
with 15d-PGJ2 induced MCL cell apoptosis and downregulated cyclin D1
expression without altering cell cycle progression [195].


#### 5.2.2. Chronic Lymphoblastic Leukemia (CLL) and Diffuse Large B Cell Lymphoma (DLBCL)

CLL is a clinically heterogeneous disease
originating from B lymphocytes that differ in activation, maturation state, or
cellular subtype [196]. CLL is one of the most common forms of
leukemia in adults [141]. In B-CLL, resistance to apoptosis has
been associated with increased Bcl-2 expression, due to either promoter
hypomethylation or to chromosomal deletion of the genes which encode two
natural Bcl-2 antisense RNAs [197, 198].To date, there are few studies that evaluate the use
of PPARγ-ligands against these malignancies.  The effects of the triterpenoid CDDO were
evaluated in refractory B-CLL cells. CDDO induced apoptosis in a dose-dependent
manner in both previously untreated and chemoresistant CLL samples [199]. In this study, CDDO induced
the activation of caspase-8, but not caspase-9, indicating the involvement of a
mitochondrial-independent pathway [199]. CDDO also negatively
affected the levels of an endogenous caspase-8 inhibitor, c-FLIP (caspase-8
homolog Fas-ligand interleukin-1-converting enzyme (FLICE)-inhibitory protein).
However, downregulation of c-FLIP expression was not the sole pathway activated
by CDDO, as c-FLIP antisense oligonucleotides did not induce CLL apoptosis [199]. Subsequently, Inoue et al.
further investigated the mechanism of CDDO-induced apoptosis in primary B-CLL
and Jurkat cell lines. In contrast to the studies discussed earlier [160, 164, 199], where CDDO activated both
the intrinsic and extrinsic apoptotic pathways, Inoue et al. proposed that CDDO
induces apoptosis exclusively through the intrinsic pathway [200]. In their study, CDDO
exposure induced an initial caspase-independent mitochondrial depolarization,
followed by caspase cleavage. Using caspase inhibitors, the authors were able
to define caspase 9 as the primary activated caspase. Moreover, CDDO induced
cell death in caspase-8 and FADD-deficient but not in Bcl-xL-overexpressing
Jurkat T cells. In CLL, CDDO induced an initial release of proapoptotic
intermediates, cytochrome c, and Smac/DIABLO from the mitochondria and led to
apoptosis [200]. According to these results, CDDO
mainly activates the intrinsic apoptotic pathway in both cell lines [200].Diffuse large
B-cell lymphomas (DLBCLs) are the most common lymphoid neoplasms, composing 30–40% of adult NHL [201]. The gene expression pattern (using DNA
microarrays) of DLBCL was compared with that of normal B cells, including those
from the germinal center (GC) and in vitro-activated
peripheral blood B cells [202]. Based on the results, DLBCL were
classified into two groups: those resembling B cells from the GC (GC-DLBCL) and
those resembling in vitro-activated
B cells (ABC-DLBCL). Patients with cancer of the GC-DLBCL-type have a more
favorable prognosis than those with the ABC-DLBCL-type [202]. Although some DLBCL patients are cured with
current therapies, most succumb to the disease. In addition, poor prognosis
correlates with Bcl-2 overexpression, which may be responsible for the impaired
apoptotic response of ABC-DLBCL to chemotherapy [203, 204].Recently, a study by Ray et al. showed that CDDO
induced growth inhibition and apoptosis in human DLBCL and that these effects
were PPARγ-independent [205]. Interestingly, CDDO induced
NF-κB activation and enhanced DLBCL
apoptosis when combined with NF-κB inhibitors. These findings suggest
that NF-κB may be activated as a survival pathway
to antagonize the apoptotic effects of CDDO [205]. A recent study by Brookes et
al. elucidated another mechanism for CDDO-induced cell death [206]. In this study, CDDO, CDDO-Im,
and the dinitril derivative of CDDO, Di-CDDO induced both normal and malignant
B cell apoptosis. The CDDO derivatives were more effective than CDDO itself. It
was demonstrated that CDDO directly interacted with and modified several
mitochondrial protein thiols, resulting in large molecular weight protein
aggregates. These aggregates led to a loss in mitochondrial thiol status by constitutively
opening cyclosporin A-insensitive permeability
transition (PT) pores [206], thereby reducing mitochondrial
transmembrane potential and resulting in cell death. These findings suggest a
novel mechanism for triterpenoid-induced cell death and predict the development of new therapeutic drugs that can elicit unregulated PT pore formation in
cancer cells.


### 5.3. Multiple Myeloma

Multiple myeloma (MM) is a neoplastic
disorder characterized by clonal proliferation of differentiated plasma cells
in the bone marrow, accompanied by accumulation of monoclonal paraprotein
levels in serum and urine. Common clinical symptoms include bone lesions,
anemia, immunodeficiency, and renal failure [207]. MM constitutes ∼10% of hematological
cancers and ranks as the second most frequent hematological malignancy in the
United States after NHL [208, 209]. Current therapies for the disease
include chemotherapy with or without stem cell transplantation,
glucocorticosteroids, thalidomide, and the proteasome inhibitor Bortezomib
(Velcade) and combinations of these agents.
However, most of these treatments are not curative, and newer approaches
are needed [209]. The therapeutic potential of
PPARγ ligands has also been evaluated in MM [13, 210, 211]. PPARγ agonists have been demonstrated to have
inhibitory effects in Waldenstrom’s macroglobulinemia (WM), a rare plasma cell
malignancy [212]. In addition, our laboratory demonstrated
that human multiple myeloma cells modestly express PPARγ. Treatment with PPARγ ligands induced MM apoptosis via
caspase activation and mitochondrial depolarization. These proapoptotic effects
were not reversed by the addition of the MM growth factor IL-6. Moreover, we
showed that these cells express RXR and that addition of an RXR ligand (9-cis-RA) enhanced PPARγ-ligand-induced apoptosis [210]. Farrar’s group found that
PPARγ ligands 15d-PGJ2 and
troglitazone completely abolished IL-6-dependent MM cell proliferation and
induced apoptosis. PPARγ agonists inhibited MM cell survival by
specifically blocking the IL-6-dependent transactivation of STAT3 (signal
transducer and activator of transcription)-activated genes, including c-myc and mcl-1 [13]. Recently, the same group has
revealed that PPARγ ligands inhibit (1) MM cell adhesion to
bone marrow stromal cells (BMSC), (2) MM cell expression levels of adhesion
molecules, and (3) BMSC secretion of IL-6, which is triggered by MM cell
adhesion. The inhibitory effects of PPARγ ligands correlated with PPARγ-dependent transrepression of the
transcription factors 5′-CCAAT/enhancer-binding protein β (C/EBP-β) and NF-κB [213]. The PPARγ ligands CDDO and CDDO-Im have also been
tested in MM cells, both alone and in combination with the proteasome inhibitor
PS-341 (Bortezomib) [214–216]. The mechanisms of
CDDO-induced apoptosis include loss of mitochondrial membrane potential, which
increases release of ROS and depletes glutathione, as well as activation of
caspases and reduction of c-FLIP protein levels [214]. These results correlated
with the studies described earlier,
using CDDO in CLL [199]. Combination treatments of
CDDO-Im with Bortezomib had synergistic apoptotic effects in MM cells [215], abolished NF-κB and Bcl-2-mediated cytoprotective
effects and overcame drug resistance to Bortezomib [215]. Overall, these findings
suggest the use of CDDO-Im, either alone or in combination with bortezomib, to
treat drug-resistant MM and improve patient prognosis.


## 5.1. Myeloid Malignancies

### 5.1.1. Acute Myeloid Leukemia (AML)

Acute myelogenous leukemia (AML) constitutes about 25% of
all leukemias in adults in the Western World. It ranks as the second most
frequent type of leukemia in adults after chronic lymphocytic leukemia, with
more than 13000 new cases, and nearly 9000 deaths from AML in the U.S.
in 2007 [141]. Unfortunately, this type of
leukemia has one of the lowest survival rates, about 20% [142]. There are several subtypes
of AML, including acute promyelocytic leukemia (APL). The most common cause of
APL is a translocation between chromosome 15 and 17, t(15;17), that leads to
the generation of the PML/RARα
fusion gene. The resulting fusion
protein arrests the maturation of myeloid cells at the promyelocytic stage and
leads to the increased proliferation of promyelocytes [143]. The cell lines typically
used to study APL are NB4 and HL-60. NB4 has the t(15;17) translocation, while HL-60
does not [144]. In addition to chemotherapy
and stem cell transplantation, treatments for APL also include differentiation
therapy using all-trans-retinoic acid (ATRA)
which has led to long-term disease-free survival in 70–80% of cases of
this AML subtype [145].An early study performed by Fujimura et al.
demonstrated that treatment with troglitazone inhibited HL-60 cell growth by a
G1 cell cycle arrest and induced their differentiation to monocytes [146]. A similar, G1 arrest was
observed in all other hematopoietic cell lines examined. Furthermore,
differentiation into the monocytic lineage was observed not only in the
myelogenous and promonocytic cell lines, but also in an erythroleukemia cell
line [146]. Data shown by
Yamakawa-Karakida et al. demonstrated that PPARγ activation by both troglitazone and
15d-PGJ2 inhibits proliferation and induces apoptosis in
promyelocytic leukemia cells under serum-free conditions [147]. The induction of apoptosis
was caspase-3 dependent, as treatment with a caspase-3 inhibitor completely
abolished cell death. Although there were no changes in antiapoptotic or
proapoptotic proteins, the expression levels of the proto-oncogene product c-myc were drastically reduced after 24
hours of troglitazone treatment while DNA binding by Tcf-4, a transcription
factor responsible for c-myc expression, was completely inhibited [147]. Troglitazone and 15d-PGJ2 were found by Liu et al. to significantly induce apoptosis in K562 and HL-60
cells by upregulating the levels of the proapoptotic protein Bax and
downregulating antiapoptotic proteins such as survivin and Bcl-2 [148]. Furthermore, these PPARγ ligands downregulated the expression of
cyclooxygenase-2 (COX-2), antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1,
upregulated Bax and activated caspase 3 in human monocytic leukemia cells [149]. Recent observations reported
by Han et al. revealed that 15d-PGJ2 was able to sensitize tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-resistant
leukemic HL-60 cells to TRAIL-induced apoptosis [150]. This effect of 15d-PGJ2 was PPARγ-independent, as treatment with a PPARγ antagonist did not rescue the cells
from apoptosis. These results were consistent with studies performed in other
cancer cells where 15d-PGJ2 enhanced TRAIL-induced apoptosis [151]. In a human eosinophilic
leukemia cell line, EoL-1, treatment with troglitazone caused a G0/G1 cell cycle arrest that correlated with increased mRNA levels of the
cyclin-dependent kinase (cdk) inhibitor, p21WAF1/CIP1. Troglitazone exerted a
similar induction of p21 mRNA accompanied by inhibition of cell proliferation
in U937 cells and in the KPB-M15 human myelomonoblastic cell line [152]. These findings suggest that
this PPARγ ligand inhibits myeloid leukemia cell
proliferation at least in part by upregulating p21 [152]. Aside from its growth
inhibitory and apoptosis-inducing properties, 15d-PGJ2 has also been
shown to decrease the expression of metalloproteinases in AML, therefore,
inhibiting leukemic cell adhesion and invasion of the extracellular matrix
(ECM) [153].A recent study investigated the antileukemia effects
and the molecular mechanism of action of a novel PPARγ ligand, DIM#34, in AML. DIM#34 can inhibit
cell growth and induce apoptosis through PPARγ-dependent and -independent mechanisms.
Cell death was associated with defective extracellular signal-regulated kinase
(ERK) activity, and inhibition of Bcl-2 phosphorylation [154].Konopleva et al. demonstrated growth inhibitory,
differentiative, and apoptotic effects of PPARγ ligands in cells from a variety of
leukemias, including AML [155]. Addition of RXR or RAR
ligands (i.e., LG100268 and ATRA, resp.) in combination with PPARγ ligands enhanced the differentiative
and growth-suppressive effects. Hirase et al. reported similar findings that
the antiproliferative, proapoptotic, and/or differentiating effects of TZDs on
HL-60 cells were further enhanced by the addition of the RXR-selective ligand, LG100268
[156]. PPARγ ligands have also been shown to inhibit
the clonal proliferation of U937 myelomonocytic leukemia cells by a G1 cell
cycle arrest, and that treatment with both PPARγ ligand (troglitazone) and LG100268 had
synergistic inhibitory effects on clonal growth [157]. Finally, recent work by
Yasugi et al. reported that both pioglitazone and 15d-PGJ2 inhibited
cell proliferation in NB4 cells and that combined with ATRA, these PPARγ ligands also induced myeloid differentiation
and lipogenesis [158].The PPARγ-ligand CDDO and its C-28 methyl ester
derivative (CDDO-Me) have also shown prodifferentiative properties in myeloid
leukemia cells [159–161]. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and
monocytic differentiation in primary AML cells. Interestingly, while
colony formation of AML progenitors was significantly inhibited, normal CD34+ progenitor cells were less affected. The more potent effect of CDDO-Me on leukemic
cells compared to normal progenitor cells suggests that CDDO-Me has potential as a new therapeutic
agent for the treatment of hematological malignancies [159]. Another group found that low
doses of CDDO promoted phagocytosis and granulocytic differentiation in HL-60
cells and primary blasts from AML patients through the regulation of CCAAT
enhancer-binding protein (CEBPA) [162]. CEBPA is an important
transcription factor for granulocytic differentiation. CDDO upregulated the
transcriptionally active p42 CEBPA, while downregulating the inactive p30
CEBPA, thereby enhancing CEBPA-regulated gene transcription. These findings
suggest the potential use of CDDO in the treatment of CEBPA-defective AML
subtypes.As proposed earlier, PPARγ transactivation may be impaired in AML,
and PPARγ ligands may be able to sensitize AML
cells to the prodifferentiation effects of ATRA [138, 139]. In light of this, a recent
study revealed that CDDO enhanced ATRA-induced differentiation and apoptosis both
in the ATRA-sensitive APL cell line, NB4, and an ATRA-resistant cell line, MR2 [163]. These effects were partially
dependent on PPARγ, as inhibition of PPARγ either by a specific inhibitor (T007)
or by siRNA diminished CDDO-induced APL differentiation [163].CDDO induces apoptosis in human myeloid leukemia
cells by promoting loss of mitochondrial membrane potential, leading to
cytochrome c release and activation of caspases [155, 160, 162, 164]. However, Bcl-xL
overexpression only partially inhibited cytochrome c release and caspase
activation, indicating that CDDO can activate caspases 3 and 8 in a cytochrome
c-independent manner [160]. Similar findings were shown
by Konopleva et al. where CDDO activated both caspase-dependent and -independent
cell death [164]. CDDO also promotes tumor
necrosis factor (TNF)-induced apoptosis in human leukemia cells. CDDO exposure
did not inhibit NF-κB translocation into the nucleus, but
rather inhibited a step after translocation, such as the NF-κB-dependent resynthesis of the inhibitor
of NF-κB, IκBα
[165]. Similarly, Shishodia et al.
demonstrated that CDDO-Me inhibited both constitutive and inducible NF-κB activity in human leukemic cells. In
contrast to the previous study [165], CDDO-Me-induced NF-κB inhibition occurred through
suppression of IκBα
kinase activation, IκBα
phosphorylation, IκBα
degradation, p65 nuclear translocation,
and NF-κB-mediated reporter gene transcription [166]. These results lead to a downregulation
of NF-κB target genes and enhanced apoptosis
induced by TNF and other chemotherapeutic agents.Another CDDO derivative, C-28 imidazole (CDDO-Im),
appears to be more potent than CDDO in inhibiting the growth of human leukemia
cells in vitro, as well as
murine melanoma and leukemia cells in
vivo [167]. The mechanism of CDDO and
CDDO-Im-induced apoptosis has been attributed to a disruption of intracellular
redox balance by increasing reactive oxygen species (ROS) and decreasing
intracellular glutathione (GSH) concentrations [168].Another subtype of AML is the acute myelomonocytic
leukemia (AMML). A well established cell line derived from a child with AMML,
THP-1, is often used to study this disease [169]. Several studies have shown
that macrophages and myelomonocytic leukemias express PPARγ and that PPARγ agonists can induce differentiation of
THP-1 cells into macrophages, as shown by the expression of CD36 scavenger
receptors, as well as CD11b, CD14, and CD18 [97]. Another study showed that PPARγ1 expression levels were upregulated by
9-cis retinoic acid (9-cis RA) in THP-1 cells coincident with suppression
of cell growth [170]. Moreover, addition of a
specific PPARγ ligand enhanced 9-cis RA-induced growth inhibition [170]. A reduction in THP-1 cell
migration also occurred in response to PPARγ ligands and was due to downregulation
of metalloproteinase-9 expression [171]. These findings suggest that
PPARγ ligands may be beneficial in preventing
metastasis of monocytic leukemia cells. Indeed, PPARγ ligands also have angiostatic
properties because of their inhibitory effects on endothelial differentiation and
on vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo [172]. Recently, Ho et al. reported
that the pigment epithelium derived factor (PEDF), a potent antiangiogenic
factor, can induce THP-1 apoptosis and necrosis by inducing PPARγ protein expression. In their study,
PEDF-induced apoptosis was shown to be PPARγ-induction-dependent. Treatment with
PPARγ antagonist and PPARγ siRNA attenuated PEDF-induced apoptosis.
Transient expression of PPARγ using a PPARγ expression plasmid reproduced the PEDF-effects.
Importantly, the PPARγ induced by PEDF was transcriptionally
active. These results suggest a PPARγ-dependent induction of apoptosis in
THP-1 cells [173].


### 5.1.2. Chronic Myeloid Leukemia (CML)

Chronic
myelogenous leukemia (CML) is a myeloproliferative disorder that affects
all hematopoietic cell types. It constitutes 15 to 20% of adult leukemias [174]. The American Cancer Society
anticipated diagnosis of about 4570 new cases of CML in 2007 [174]. CML is characterized by a genetic abnormality known as Philadelphia
(Ph)
chromosome, resulting from a translocation between chromosomes 9 and 22,
t(9;22)(q34;q11). This translocation generates a fusion protein called BCR-ABL which
is a constitutively active tyrosine kinase responsible for uncontrolled cell
proliferation and enhanced cell survival [175]. Treatments for this disease include splenic
irradiation, stem cell transplantation, and interferon alpha (IFNα
) administration with combination chemotherapy.
A specific tyrosine kinase inhibitor, Imatinib, was introduced in the
late 1990s and is a standard treatment for CML. However, clinical resistance to
imatinib has been described in CML patients, where BCR-ABL gene mutations or amplifications
have occurred [176, 177].
Therefore, development of new therapeutic strategies to overcome imatinib
resistance is needed. Dual PPARα
and γ ligands have been tested, either alone or in
combination with Imatinib, to overcome drug resistance. A characteristic cell
line used to study CML is K562, which was established from a patient with CML
in the acute phase [178].  Recently, a study was performed using a
synthetic dual PPARα
/PPARγ agonist, TZD18, in human CML myeloid
blast crisis cell lines [63]. In this study, treatment
with TZD18, both alone and in combination with Imatinib, inhibited CML
proliferation and induced apoptosis. These effects were PPARα
and PPARγ- independent, as neither PPARα
nor PPARγ antagonists were able to rescue cell
proliferation and survival. These results were reported previously by the same
group in Ph-positive lymphocytic leukemia cell lines, where TZD18 promoted cell
death and acted synergistically to enhance the effect of Imatinib [179]. Hirase et al. tested the
effects of TZDs in K562 cells, which have an erythroid nature and the potential
to differentiate into megakaryocytes [180]. TZD inhibited both cell proliferation
and the erythroid phenotype of K562 cells. These results were accompanied by a
reduction in erythroid lineage-transcription factor, GATA-1, levels [180]. Therefore, PPARγ ligands may serve a therapeutic use for
the treatment of other types of myeloproliferative disorders where there is an
overproduction of erythrocytes, such as polycythemia vera (PV).


## 5.1.1. Acute Myeloid Leukemia (AML)

Acute myelogenous leukemia (AML) constitutes about 25% of
all leukemias in adults in the Western World. It ranks as the second most
frequent type of leukemia in adults after chronic lymphocytic leukemia, with
more than 13000 new cases, and nearly 9000 deaths from AML in the U.S.
in 2007 [141]. Unfortunately, this type of
leukemia has one of the lowest survival rates, about 20% [142]. There are several subtypes
of AML, including acute promyelocytic leukemia (APL). The most common cause of
APL is a translocation between chromosome 15 and 17, t(15;17), that leads to
the generation of the PML/RARα
fusion gene. The resulting fusion
protein arrests the maturation of myeloid cells at the promyelocytic stage and
leads to the increased proliferation of promyelocytes [143]. The cell lines typically
used to study APL are NB4 and HL-60. NB4 has the t(15;17) translocation, while HL-60
does not [144]. In addition to chemotherapy
and stem cell transplantation, treatments for APL also include differentiation
therapy using all-trans-retinoic acid (ATRA)
which has led to long-term disease-free survival in 70–80% of cases of
this AML subtype [145].An early study performed by Fujimura et al.
demonstrated that treatment with troglitazone inhibited HL-60 cell growth by a
G1 cell cycle arrest and induced their differentiation to monocytes [146]. A similar, G1 arrest was
observed in all other hematopoietic cell lines examined. Furthermore,
differentiation into the monocytic lineage was observed not only in the
myelogenous and promonocytic cell lines, but also in an erythroleukemia cell
line [146]. Data shown by
Yamakawa-Karakida et al. demonstrated that PPARγ activation by both troglitazone and
15d-PGJ2 inhibits proliferation and induces apoptosis in
promyelocytic leukemia cells under serum-free conditions [147]. The induction of apoptosis
was caspase-3 dependent, as treatment with a caspase-3 inhibitor completely
abolished cell death. Although there were no changes in antiapoptotic or
proapoptotic proteins, the expression levels of the proto-oncogene product c-myc were drastically reduced after 24
hours of troglitazone treatment while DNA binding by Tcf-4, a transcription
factor responsible for c-myc expression, was completely inhibited [147]. Troglitazone and 15d-PGJ2 were found by Liu et al. to significantly induce apoptosis in K562 and HL-60
cells by upregulating the levels of the proapoptotic protein Bax and
downregulating antiapoptotic proteins such as survivin and Bcl-2 [148]. Furthermore, these PPARγ ligands downregulated the expression of
cyclooxygenase-2 (COX-2), antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1,
upregulated Bax and activated caspase 3 in human monocytic leukemia cells [149]. Recent observations reported
by Han et al. revealed that 15d-PGJ2 was able to sensitize tumor
necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-resistant
leukemic HL-60 cells to TRAIL-induced apoptosis [150]. This effect of 15d-PGJ2 was PPARγ-independent, as treatment with a PPARγ antagonist did not rescue the cells
from apoptosis. These results were consistent with studies performed in other
cancer cells where 15d-PGJ2 enhanced TRAIL-induced apoptosis [151]. In a human eosinophilic
leukemia cell line, EoL-1, treatment with troglitazone caused a G0/G1 cell cycle arrest that correlated with increased mRNA levels of the
cyclin-dependent kinase (cdk) inhibitor, p21WAF1/CIP1. Troglitazone exerted a
similar induction of p21 mRNA accompanied by inhibition of cell proliferation
in U937 cells and in the KPB-M15 human myelomonoblastic cell line [152]. These findings suggest that
this PPARγ ligand inhibits myeloid leukemia cell
proliferation at least in part by upregulating p21 [152]. Aside from its growth
inhibitory and apoptosis-inducing properties, 15d-PGJ2 has also been
shown to decrease the expression of metalloproteinases in AML, therefore,
inhibiting leukemic cell adhesion and invasion of the extracellular matrix
(ECM) [153].A recent study investigated the antileukemia effects
and the molecular mechanism of action of a novel PPARγ ligand, DIM#34, in AML. DIM#34 can inhibit
cell growth and induce apoptosis through PPARγ-dependent and -independent mechanisms.
Cell death was associated with defective extracellular signal-regulated kinase
(ERK) activity, and inhibition of Bcl-2 phosphorylation [154].Konopleva et al. demonstrated growth inhibitory,
differentiative, and apoptotic effects of PPARγ ligands in cells from a variety of
leukemias, including AML [155]. Addition of RXR or RAR
ligands (i.e., LG100268 and ATRA, resp.) in combination with PPARγ ligands enhanced the differentiative
and growth-suppressive effects. Hirase et al. reported similar findings that
the antiproliferative, proapoptotic, and/or differentiating effects of TZDs on
HL-60 cells were further enhanced by the addition of the RXR-selective ligand, LG100268
[156]. PPARγ ligands have also been shown to inhibit
the clonal proliferation of U937 myelomonocytic leukemia cells by a G1 cell
cycle arrest, and that treatment with both PPARγ ligand (troglitazone) and LG100268 had
synergistic inhibitory effects on clonal growth [157]. Finally, recent work by
Yasugi et al. reported that both pioglitazone and 15d-PGJ2 inhibited
cell proliferation in NB4 cells and that combined with ATRA, these PPARγ ligands also induced myeloid differentiation
and lipogenesis [158].The PPARγ-ligand CDDO and its C-28 methyl ester
derivative (CDDO-Me) have also shown prodifferentiative properties in myeloid
leukemia cells [159–161]. CDDO-Me induced granulo-monocytic differentiation in HL-60 cells and
monocytic differentiation in primary AML cells. Interestingly, while
colony formation of AML progenitors was significantly inhibited, normal CD34+ progenitor cells were less affected. The more potent effect of CDDO-Me on leukemic
cells compared to normal progenitor cells suggests that CDDO-Me has potential as a new therapeutic
agent for the treatment of hematological malignancies [159]. Another group found that low
doses of CDDO promoted phagocytosis and granulocytic differentiation in HL-60
cells and primary blasts from AML patients through the regulation of CCAAT
enhancer-binding protein (CEBPA) [162]. CEBPA is an important
transcription factor for granulocytic differentiation. CDDO upregulated the
transcriptionally active p42 CEBPA, while downregulating the inactive p30
CEBPA, thereby enhancing CEBPA-regulated gene transcription. These findings
suggest the potential use of CDDO in the treatment of CEBPA-defective AML
subtypes.As proposed earlier, PPARγ transactivation may be impaired in AML,
and PPARγ ligands may be able to sensitize AML
cells to the prodifferentiation effects of ATRA [138, 139]. In light of this, a recent
study revealed that CDDO enhanced ATRA-induced differentiation and apoptosis both
in the ATRA-sensitive APL cell line, NB4, and an ATRA-resistant cell line, MR2 [163]. These effects were partially
dependent on PPARγ, as inhibition of PPARγ either by a specific inhibitor (T007)
or by siRNA diminished CDDO-induced APL differentiation [163].CDDO induces apoptosis in human myeloid leukemia
cells by promoting loss of mitochondrial membrane potential, leading to
cytochrome c release and activation of caspases [155, 160, 162, 164]. However, Bcl-xL
overexpression only partially inhibited cytochrome c release and caspase
activation, indicating that CDDO can activate caspases 3 and 8 in a cytochrome
c-independent manner [160]. Similar findings were shown
by Konopleva et al. where CDDO activated both caspase-dependent and -independent
cell death [164]. CDDO also promotes tumor
necrosis factor (TNF)-induced apoptosis in human leukemia cells. CDDO exposure
did not inhibit NF-κB translocation into the nucleus, but
rather inhibited a step after translocation, such as the NF-κB-dependent resynthesis of the inhibitor
of NF-κB, IκBα
[165]. Similarly, Shishodia et al.
demonstrated that CDDO-Me inhibited both constitutive and inducible NF-κB activity in human leukemic cells. In
contrast to the previous study [165], CDDO-Me-induced NF-κB inhibition occurred through
suppression of IκBα
kinase activation, IκBα
phosphorylation, IκBα
degradation, p65 nuclear translocation,
and NF-κB-mediated reporter gene transcription [166]. These results lead to a downregulation
of NF-κB target genes and enhanced apoptosis
induced by TNF and other chemotherapeutic agents.Another CDDO derivative, C-28 imidazole (CDDO-Im),
appears to be more potent than CDDO in inhibiting the growth of human leukemia
cells in vitro, as well as
murine melanoma and leukemia cells in
vivo [167]. The mechanism of CDDO and
CDDO-Im-induced apoptosis has been attributed to a disruption of intracellular
redox balance by increasing reactive oxygen species (ROS) and decreasing
intracellular glutathione (GSH) concentrations [168].Another subtype of AML is the acute myelomonocytic
leukemia (AMML). A well established cell line derived from a child with AMML,
THP-1, is often used to study this disease [169]. Several studies have shown
that macrophages and myelomonocytic leukemias express PPARγ and that PPARγ agonists can induce differentiation of
THP-1 cells into macrophages, as shown by the expression of CD36 scavenger
receptors, as well as CD11b, CD14, and CD18 [97]. Another study showed that PPARγ1 expression levels were upregulated by
9-cis retinoic acid (9-cis RA) in THP-1 cells coincident with suppression
of cell growth [170]. Moreover, addition of a
specific PPARγ ligand enhanced 9-cis RA-induced growth inhibition [170]. A reduction in THP-1 cell
migration also occurred in response to PPARγ ligands and was due to downregulation
of metalloproteinase-9 expression [171]. These findings suggest that
PPARγ ligands may be beneficial in preventing
metastasis of monocytic leukemia cells. Indeed, PPARγ ligands also have angiostatic
properties because of their inhibitory effects on endothelial differentiation and
on vascular endothelial growth factor (VEGF)-induced angiogenesis in vivo [172]. Recently, Ho et al. reported
that the pigment epithelium derived factor (PEDF), a potent antiangiogenic
factor, can induce THP-1 apoptosis and necrosis by inducing PPARγ protein expression. In their study,
PEDF-induced apoptosis was shown to be PPARγ-induction-dependent. Treatment with
PPARγ antagonist and PPARγ siRNA attenuated PEDF-induced apoptosis.
Transient expression of PPARγ using a PPARγ expression plasmid reproduced the PEDF-effects.
Importantly, the PPARγ induced by PEDF was transcriptionally
active. These results suggest a PPARγ-dependent induction of apoptosis in
THP-1 cells [173].


## 5.1.2. Chronic Myeloid Leukemia (CML)

Chronic
myelogenous leukemia (CML) is a myeloproliferative disorder that affects
all hematopoietic cell types. It constitutes 15 to 20% of adult leukemias [174]. The American Cancer Society
anticipated diagnosis of about 4570 new cases of CML in 2007 [174]. CML is characterized by a genetic abnormality known as Philadelphia
(Ph)
chromosome, resulting from a translocation between chromosomes 9 and 22,
t(9;22)(q34;q11). This translocation generates a fusion protein called BCR-ABL which
is a constitutively active tyrosine kinase responsible for uncontrolled cell
proliferation and enhanced cell survival [175]. Treatments for this disease include splenic
irradiation, stem cell transplantation, and interferon alpha (IFNα
) administration with combination chemotherapy.
A specific tyrosine kinase inhibitor, Imatinib, was introduced in the
late 1990s and is a standard treatment for CML. However, clinical resistance to
imatinib has been described in CML patients, where BCR-ABL gene mutations or amplifications
have occurred [176, 177].
Therefore, development of new therapeutic strategies to overcome imatinib
resistance is needed. Dual PPARα
and γ ligands have been tested, either alone or in
combination with Imatinib, to overcome drug resistance. A characteristic cell
line used to study CML is K562, which was established from a patient with CML
in the acute phase [178].  Recently, a study was performed using a
synthetic dual PPARα
/PPARγ agonist, TZD18, in human CML myeloid
blast crisis cell lines [63]. In this study, treatment
with TZD18, both alone and in combination with Imatinib, inhibited CML
proliferation and induced apoptosis. These effects were PPARα
and PPARγ- independent, as neither PPARα
nor PPARγ antagonists were able to rescue cell
proliferation and survival. These results were reported previously by the same
group in Ph-positive lymphocytic leukemia cell lines, where TZD18 promoted cell
death and acted synergistically to enhance the effect of Imatinib [179]. Hirase et al. tested the
effects of TZDs in K562 cells, which have an erythroid nature and the potential
to differentiate into megakaryocytes [180]. TZD inhibited both cell proliferation
and the erythroid phenotype of K562 cells. These results were accompanied by a
reduction in erythroid lineage-transcription factor, GATA-1, levels [180]. Therefore, PPARγ ligands may serve a therapeutic use for
the treatment of other types of myeloproliferative disorders where there is an
overproduction of erythrocytes, such as polycythemia vera (PV).


## 5.2. Lymphoid Malignancies

### 5.2.1. Acute Lymphoblastic Leukemia (ALL) and Non-Hodgkin’s Lymphomas

Acute
lymphoblastic leukemia (ALL) is a malignant disorder that arises from uncontrolled
proliferation of lymphocytic progenitors. The disease is most commonly
diagnosed in children, but can also occur in adults. About 80–90% of ALL
patients can achieve complete remission with currently available therapy. Yet, many
patients eventually relapse, and only 35% of individuals have a long-term
leukemia-free survival (LFS) [181, 182]. Therefore, development of new treatment
approaches to improve both the cure rate and the quality of life of patients
with ALL is greatly needed. ALL involving hyperproliferation of B
lymphocyte progenitors (B-ALL) is frequently associated with a translocation
between the c-myc gene on chromosome
8q24 and any of the three immunoglobulin genes located on chromosomes 14q32,
2p11, or 22q11. This translocation results in c-myc overexpression and correlates with poor prognosis [183, 184]. The members of the Myc
family, including c-myc, are involved
in regulation of proliferation and development of normal and malignant cells [185].An investigation by Zang et al. revealed that the PPARγ ligands pioglitazone and 15d-PGJ2 suppressed cell growth in G1 phase and induced apoptosis in a dose-dependent
manner in B-ALL cell lines. Apoptosis was found to be partly caspase-dependent,
as treatment with a pan-caspase inhibitor partially reversed this effect [186]. Similar findings were shown
in B-ALL with t(14;18), in which troglitazone not only induced G1 phase growth
arrest and apoptosis, but also downregulated the expression of c-myc mRNA and protein [187].Our group has demonstrated that: (1) both normal and
malignant B lineage cells express PPARγ mRNA and protein, and (2) exposure to
certain small molecule PPARγ ligands, including 15d-PGJ2, inhibits proliferation and induces apoptosis in these cells [85, 113]. Subsequently, we reported
that PPARγ ligand-induced apoptosis was mainly PPARγ-independent, since it was not prevented
either by a PPARγ antagonist nor a dominant negative form
of PPARγ (PPARγ-DN) [94].  We reported that the apoptotic mechanism
regulated by 15d-PGJ2, but not by ciglitazone, was related to the
production of ROS and the reduction in intracellular GSH [94].
CD40 signaling through
CD40-ligand (CD40L) enhances B cell survival and prevents BCR-induced apoptosis
by activating the transcription factor NF-κB [188]. Therefore, we tested whether
CD40 ligation could protect normal and malignant B cells from PPARγ ligand-induced apoptosis. CD40L was
able to partially rescue normal and malignant B cells from PPARγ ligand-induced apoptosis by activating NF-κB.
Similarly, Piva et al. reported 15d-PGJ2-induced apoptosis in human
Burkitt’s lymphomas and multiple myeloma cell lines through inhibition of NF-κB
activity. These effects lead to the downregulation of NF-κB-dependent
antiapoptotic protein production and therefore decreased cell survival. The
apoptotic effects could also be mimicked by NF-κB
p65 subunit knockdown by siRNA [189]. These results suggest a possible mechanism
for the proapoptotic action of PPARγ
agonists.We have also demonstrated that PPARγ ligands can induce apoptosis in cells
from human T cell leukemias (Jurkat), lymphomas (J-Jahn), and T-ALL cells
(CCRF-CEM) by a PPARγ-dependent mechanism [103]. Interestingly, normal T
cells were not adversely affected by PPARγ ligands, suggesting the use of PPARγ agonists as selective therapeutic drugs
for T-cell malignancies [103]. However, data from Yang et
al. raised questions on the antiproliferative effects of PPARγ-ligands in T-lymphoma cells [190]. They demonstrated that low
concentrations of PPARγ-ligands promoted T-lymphoma cell
survival, while high concentrations promoted cell death. These results suggest
that in T-lymphoma cells, PPARγ ligands can have contradictory effects when
used at different concentrations and require further examination.Cutaneous T cell
lymphoma (CTCL) is a group of T cell malignancies that accumulate in the skin.
The most common CTCLs are (1) the Mycosis fungoides (MF), which develops as
patches, plaques, or tumors containing apoptosis-resistant CD4+ CD45RO+ helper/memory T cells; and (2) the Sézary syndrome (SS), which is the leukemic
form of CTCL that develops with erythroderma and the appearance of atypical T
cells in the peripheral blood [191]. Current therapies for CTCL
include the use of bexarotene, an RXR ligand, with good efficacy in the late
stages of the disease [191]. Zhang et al. demonstrated
the expression of PPARγ in three CTCL lines (MJ, Hut78, and HH)
and freshly isolated peripheral  blood lymphocytes (PBL) from SS patients with
circulating atypical T cells (CD4+CD26-) [192]. CDDO exposure caused a dose-dependent
induction of apoptosis in MF/SS cell lines and SS patients’ PBL [192]. These findings suggest that
PPARγ ligands may be beneficial for the
treatment of CTCL and may have synergistic effects when used in combination
with bexarotene.Mantle cell
lymphoma (MCL) is a rare type of non-Hodgkin’s lymphoma (NHL), constituting
about 6% of NHL [193, 194].  In 85% of MCL cases, a translocation between
chromosome 11 and 14, t(11;14), is involved in the pathogenesis. This translocation
leads to the overexpression of cyclin D1, a protein that increases cell
survival and proliferation by positively regulating cell cycle entry into the
S-phase [193]. Despite the success of
current therapies, patients with mantle cell lymphoma have a shorter life span
compared to patients with other B cell lymphomas [193]. Recently, a study
demonstrated that treatment with pioglitazone and rosiglitazone, as well as
with 15d-PGJ2 induced MCL cell apoptosis and downregulated cyclin D1
expression without altering cell cycle progression [195].


### 5.2.2. Chronic Lymphoblastic Leukemia (CLL) and Diffuse Large B Cell Lymphoma (DLBCL)

CLL is a clinically heterogeneous disease
originating from B lymphocytes that differ in activation, maturation state, or
cellular subtype [196]. CLL is one of the most common forms of
leukemia in adults [141]. In B-CLL, resistance to apoptosis has
been associated with increased Bcl-2 expression, due to either promoter
hypomethylation or to chromosomal deletion of the genes which encode two
natural Bcl-2 antisense RNAs [197, 198].To date, there are few studies that evaluate the use
of PPARγ-ligands against these malignancies.  The effects of the triterpenoid CDDO were
evaluated in refractory B-CLL cells. CDDO induced apoptosis in a dose-dependent
manner in both previously untreated and chemoresistant CLL samples [199]. In this study, CDDO induced
the activation of caspase-8, but not caspase-9, indicating the involvement of a
mitochondrial-independent pathway [199]. CDDO also negatively
affected the levels of an endogenous caspase-8 inhibitor, c-FLIP (caspase-8
homolog Fas-ligand interleukin-1-converting enzyme (FLICE)-inhibitory protein).
However, downregulation of c-FLIP expression was not the sole pathway activated
by CDDO, as c-FLIP antisense oligonucleotides did not induce CLL apoptosis [199]. Subsequently, Inoue et al.
further investigated the mechanism of CDDO-induced apoptosis in primary B-CLL
and Jurkat cell lines. In contrast to the studies discussed earlier [160, 164, 199], where CDDO activated both
the intrinsic and extrinsic apoptotic pathways, Inoue et al. proposed that CDDO
induces apoptosis exclusively through the intrinsic pathway [200]. In their study, CDDO
exposure induced an initial caspase-independent mitochondrial depolarization,
followed by caspase cleavage. Using caspase inhibitors, the authors were able
to define caspase 9 as the primary activated caspase. Moreover, CDDO induced
cell death in caspase-8 and FADD-deficient but not in Bcl-xL-overexpressing
Jurkat T cells. In CLL, CDDO induced an initial release of proapoptotic
intermediates, cytochrome c, and Smac/DIABLO from the mitochondria and led to
apoptosis [200]. According to these results, CDDO
mainly activates the intrinsic apoptotic pathway in both cell lines [200].Diffuse large
B-cell lymphomas (DLBCLs) are the most common lymphoid neoplasms, composing 30–40% of adult NHL [201]. The gene expression pattern (using DNA
microarrays) of DLBCL was compared with that of normal B cells, including those
from the germinal center (GC) and in vitro-activated
peripheral blood B cells [202]. Based on the results, DLBCL were
classified into two groups: those resembling B cells from the GC (GC-DLBCL) and
those resembling in vitro-activated
B cells (ABC-DLBCL). Patients with cancer of the GC-DLBCL-type have a more
favorable prognosis than those with the ABC-DLBCL-type [202]. Although some DLBCL patients are cured with
current therapies, most succumb to the disease. In addition, poor prognosis
correlates with Bcl-2 overexpression, which may be responsible for the impaired
apoptotic response of ABC-DLBCL to chemotherapy [203, 204].Recently, a study by Ray et al. showed that CDDO
induced growth inhibition and apoptosis in human DLBCL and that these effects
were PPARγ-independent [205]. Interestingly, CDDO induced
NF-κB activation and enhanced DLBCL
apoptosis when combined with NF-κB inhibitors. These findings suggest
that NF-κB may be activated as a survival pathway
to antagonize the apoptotic effects of CDDO [205]. A recent study by Brookes et
al. elucidated another mechanism for CDDO-induced cell death [206]. In this study, CDDO, CDDO-Im,
and the dinitril derivative of CDDO, Di-CDDO induced both normal and malignant
B cell apoptosis. The CDDO derivatives were more effective than CDDO itself. It
was demonstrated that CDDO directly interacted with and modified several
mitochondrial protein thiols, resulting in large molecular weight protein
aggregates. These aggregates led to a loss in mitochondrial thiol status by constitutively
opening cyclosporin A-insensitive permeability
transition (PT) pores [206], thereby reducing mitochondrial
transmembrane potential and resulting in cell death. These findings suggest a
novel mechanism for triterpenoid-induced cell death and predict the development of new therapeutic drugs that can elicit unregulated PT pore formation in
cancer cells.


## 5.2.1. Acute Lymphoblastic Leukemia (ALL) and Non-Hodgkin’s Lymphomas

Acute
lymphoblastic leukemia (ALL) is a malignant disorder that arises from uncontrolled
proliferation of lymphocytic progenitors. The disease is most commonly
diagnosed in children, but can also occur in adults. About 80–90% of ALL
patients can achieve complete remission with currently available therapy. Yet, many
patients eventually relapse, and only 35% of individuals have a long-term
leukemia-free survival (LFS) [181, 182]. Therefore, development of new treatment
approaches to improve both the cure rate and the quality of life of patients
with ALL is greatly needed. ALL involving hyperproliferation of B
lymphocyte progenitors (B-ALL) is frequently associated with a translocation
between the c-myc gene on chromosome
8q24 and any of the three immunoglobulin genes located on chromosomes 14q32,
2p11, or 22q11. This translocation results in c-myc overexpression and correlates with poor prognosis [183, 184]. The members of the Myc
family, including c-myc, are involved
in regulation of proliferation and development of normal and malignant cells [185].An investigation by Zang et al. revealed that the PPARγ ligands pioglitazone and 15d-PGJ2 suppressed cell growth in G1 phase and induced apoptosis in a dose-dependent
manner in B-ALL cell lines. Apoptosis was found to be partly caspase-dependent,
as treatment with a pan-caspase inhibitor partially reversed this effect [186]. Similar findings were shown
in B-ALL with t(14;18), in which troglitazone not only induced G1 phase growth
arrest and apoptosis, but also downregulated the expression of c-myc mRNA and protein [187].Our group has demonstrated that: (1) both normal and
malignant B lineage cells express PPARγ mRNA and protein, and (2) exposure to
certain small molecule PPARγ ligands, including 15d-PGJ2, inhibits proliferation and induces apoptosis in these cells [85, 113]. Subsequently, we reported
that PPARγ ligand-induced apoptosis was mainly PPARγ-independent, since it was not prevented
either by a PPARγ antagonist nor a dominant negative form
of PPARγ (PPARγ-DN) [94].  We reported that the apoptotic mechanism
regulated by 15d-PGJ2, but not by ciglitazone, was related to the
production of ROS and the reduction in intracellular GSH [94].
CD40 signaling through
CD40-ligand (CD40L) enhances B cell survival and prevents BCR-induced apoptosis
by activating the transcription factor NF-κB [188]. Therefore, we tested whether
CD40 ligation could protect normal and malignant B cells from PPARγ ligand-induced apoptosis. CD40L was
able to partially rescue normal and malignant B cells from PPARγ ligand-induced apoptosis by activating NF-κB.
Similarly, Piva et al. reported 15d-PGJ2-induced apoptosis in human
Burkitt’s lymphomas and multiple myeloma cell lines through inhibition of NF-κB
activity. These effects lead to the downregulation of NF-κB-dependent
antiapoptotic protein production and therefore decreased cell survival. The
apoptotic effects could also be mimicked by NF-κB
p65 subunit knockdown by siRNA [189]. These results suggest a possible mechanism
for the proapoptotic action of PPARγ
agonists.We have also demonstrated that PPARγ ligands can induce apoptosis in cells
from human T cell leukemias (Jurkat), lymphomas (J-Jahn), and T-ALL cells
(CCRF-CEM) by a PPARγ-dependent mechanism [103]. Interestingly, normal T
cells were not adversely affected by PPARγ ligands, suggesting the use of PPARγ agonists as selective therapeutic drugs
for T-cell malignancies [103]. However, data from Yang et
al. raised questions on the antiproliferative effects of PPARγ-ligands in T-lymphoma cells [190]. They demonstrated that low
concentrations of PPARγ-ligands promoted T-lymphoma cell
survival, while high concentrations promoted cell death. These results suggest
that in T-lymphoma cells, PPARγ ligands can have contradictory effects when
used at different concentrations and require further examination.Cutaneous T cell
lymphoma (CTCL) is a group of T cell malignancies that accumulate in the skin.
The most common CTCLs are (1) the Mycosis fungoides (MF), which develops as
patches, plaques, or tumors containing apoptosis-resistant CD4+ CD45RO+ helper/memory T cells; and (2) the Sézary syndrome (SS), which is the leukemic
form of CTCL that develops with erythroderma and the appearance of atypical T
cells in the peripheral blood [191]. Current therapies for CTCL
include the use of bexarotene, an RXR ligand, with good efficacy in the late
stages of the disease [191]. Zhang et al. demonstrated
the expression of PPARγ in three CTCL lines (MJ, Hut78, and HH)
and freshly isolated peripheral  blood lymphocytes (PBL) from SS patients with
circulating atypical T cells (CD4+CD26-) [192]. CDDO exposure caused a dose-dependent
induction of apoptosis in MF/SS cell lines and SS patients’ PBL [192]. These findings suggest that
PPARγ ligands may be beneficial for the
treatment of CTCL and may have synergistic effects when used in combination
with bexarotene.Mantle cell
lymphoma (MCL) is a rare type of non-Hodgkin’s lymphoma (NHL), constituting
about 6% of NHL [193, 194].  In 85% of MCL cases, a translocation between
chromosome 11 and 14, t(11;14), is involved in the pathogenesis. This translocation
leads to the overexpression of cyclin D1, a protein that increases cell
survival and proliferation by positively regulating cell cycle entry into the
S-phase [193]. Despite the success of
current therapies, patients with mantle cell lymphoma have a shorter life span
compared to patients with other B cell lymphomas [193]. Recently, a study
demonstrated that treatment with pioglitazone and rosiglitazone, as well as
with 15d-PGJ2 induced MCL cell apoptosis and downregulated cyclin D1
expression without altering cell cycle progression [195].


## 5.2.2. Chronic Lymphoblastic Leukemia (CLL) and Diffuse Large B Cell Lymphoma (DLBCL)

CLL is a clinically heterogeneous disease
originating from B lymphocytes that differ in activation, maturation state, or
cellular subtype [196]. CLL is one of the most common forms of
leukemia in adults [141]. In B-CLL, resistance to apoptosis has
been associated with increased Bcl-2 expression, due to either promoter
hypomethylation or to chromosomal deletion of the genes which encode two
natural Bcl-2 antisense RNAs [197, 198].To date, there are few studies that evaluate the use
of PPARγ-ligands against these malignancies.  The effects of the triterpenoid CDDO were
evaluated in refractory B-CLL cells. CDDO induced apoptosis in a dose-dependent
manner in both previously untreated and chemoresistant CLL samples [199]. In this study, CDDO induced
the activation of caspase-8, but not caspase-9, indicating the involvement of a
mitochondrial-independent pathway [199]. CDDO also negatively
affected the levels of an endogenous caspase-8 inhibitor, c-FLIP (caspase-8
homolog Fas-ligand interleukin-1-converting enzyme (FLICE)-inhibitory protein).
However, downregulation of c-FLIP expression was not the sole pathway activated
by CDDO, as c-FLIP antisense oligonucleotides did not induce CLL apoptosis [199]. Subsequently, Inoue et al.
further investigated the mechanism of CDDO-induced apoptosis in primary B-CLL
and Jurkat cell lines. In contrast to the studies discussed earlier [160, 164, 199], where CDDO activated both
the intrinsic and extrinsic apoptotic pathways, Inoue et al. proposed that CDDO
induces apoptosis exclusively through the intrinsic pathway [200]. In their study, CDDO
exposure induced an initial caspase-independent mitochondrial depolarization,
followed by caspase cleavage. Using caspase inhibitors, the authors were able
to define caspase 9 as the primary activated caspase. Moreover, CDDO induced
cell death in caspase-8 and FADD-deficient but not in Bcl-xL-overexpressing
Jurkat T cells. In CLL, CDDO induced an initial release of proapoptotic
intermediates, cytochrome c, and Smac/DIABLO from the mitochondria and led to
apoptosis [200]. According to these results, CDDO
mainly activates the intrinsic apoptotic pathway in both cell lines [200].Diffuse large
B-cell lymphomas (DLBCLs) are the most common lymphoid neoplasms, composing 30–40% of adult NHL [201]. The gene expression pattern (using DNA
microarrays) of DLBCL was compared with that of normal B cells, including those
from the germinal center (GC) and in vitro-activated
peripheral blood B cells [202]. Based on the results, DLBCL were
classified into two groups: those resembling B cells from the GC (GC-DLBCL) and
those resembling in vitro-activated
B cells (ABC-DLBCL). Patients with cancer of the GC-DLBCL-type have a more
favorable prognosis than those with the ABC-DLBCL-type [202]. Although some DLBCL patients are cured with
current therapies, most succumb to the disease. In addition, poor prognosis
correlates with Bcl-2 overexpression, which may be responsible for the impaired
apoptotic response of ABC-DLBCL to chemotherapy [203, 204].Recently, a study by Ray et al. showed that CDDO
induced growth inhibition and apoptosis in human DLBCL and that these effects
were PPARγ-independent [205]. Interestingly, CDDO induced
NF-κB activation and enhanced DLBCL
apoptosis when combined with NF-κB inhibitors. These findings suggest
that NF-κB may be activated as a survival pathway
to antagonize the apoptotic effects of CDDO [205]. A recent study by Brookes et
al. elucidated another mechanism for CDDO-induced cell death [206]. In this study, CDDO, CDDO-Im,
and the dinitril derivative of CDDO, Di-CDDO induced both normal and malignant
B cell apoptosis. The CDDO derivatives were more effective than CDDO itself. It
was demonstrated that CDDO directly interacted with and modified several
mitochondrial protein thiols, resulting in large molecular weight protein
aggregates. These aggregates led to a loss in mitochondrial thiol status by constitutively
opening cyclosporin A-insensitive permeability
transition (PT) pores [206], thereby reducing mitochondrial
transmembrane potential and resulting in cell death. These findings suggest a
novel mechanism for triterpenoid-induced cell death and predict the development of new therapeutic drugs that can elicit unregulated PT pore formation in
cancer cells.


## 5.3. Multiple Myeloma

Multiple myeloma (MM) is a neoplastic
disorder characterized by clonal proliferation of differentiated plasma cells
in the bone marrow, accompanied by accumulation of monoclonal paraprotein
levels in serum and urine. Common clinical symptoms include bone lesions,
anemia, immunodeficiency, and renal failure [207]. MM constitutes ∼10% of hematological
cancers and ranks as the second most frequent hematological malignancy in the
United States after NHL [208, 209]. Current therapies for the disease
include chemotherapy with or without stem cell transplantation,
glucocorticosteroids, thalidomide, and the proteasome inhibitor Bortezomib
(Velcade) and combinations of these agents.
However, most of these treatments are not curative, and newer approaches
are needed [209]. The therapeutic potential of
PPARγ ligands has also been evaluated in MM [13, 210, 211]. PPARγ agonists have been demonstrated to have
inhibitory effects in Waldenstrom’s macroglobulinemia (WM), a rare plasma cell
malignancy [212]. In addition, our laboratory demonstrated
that human multiple myeloma cells modestly express PPARγ. Treatment with PPARγ ligands induced MM apoptosis via
caspase activation and mitochondrial depolarization. These proapoptotic effects
were not reversed by the addition of the MM growth factor IL-6. Moreover, we
showed that these cells express RXR and that addition of an RXR ligand (9-cis-RA) enhanced PPARγ-ligand-induced apoptosis [210]. Farrar’s group found that
PPARγ ligands 15d-PGJ2 and
troglitazone completely abolished IL-6-dependent MM cell proliferation and
induced apoptosis. PPARγ agonists inhibited MM cell survival by
specifically blocking the IL-6-dependent transactivation of STAT3 (signal
transducer and activator of transcription)-activated genes, including c-myc and mcl-1 [13]. Recently, the same group has
revealed that PPARγ ligands inhibit (1) MM cell adhesion to
bone marrow stromal cells (BMSC), (2) MM cell expression levels of adhesion
molecules, and (3) BMSC secretion of IL-6, which is triggered by MM cell
adhesion. The inhibitory effects of PPARγ ligands correlated with PPARγ-dependent transrepression of the
transcription factors 5′-CCAAT/enhancer-binding protein β (C/EBP-β) and NF-κB [213]. The PPARγ ligands CDDO and CDDO-Im have also been
tested in MM cells, both alone and in combination with the proteasome inhibitor
PS-341 (Bortezomib) [214–216]. The mechanisms of
CDDO-induced apoptosis include loss of mitochondrial membrane potential, which
increases release of ROS and depletes glutathione, as well as activation of
caspases and reduction of c-FLIP protein levels [214]. These results correlated
with the studies described earlier,
using CDDO in CLL [199]. Combination treatments of
CDDO-Im with Bortezomib had synergistic apoptotic effects in MM cells [215], abolished NF-κB and Bcl-2-mediated cytoprotective
effects and overcame drug resistance to Bortezomib [215]. Overall, these findings
suggest the use of CDDO-Im, either alone or in combination with bortezomib, to
treat drug-resistant MM and improve patient prognosis.


## 6. Conclusions and Future Directions

In summary,
although the exact role of PPARγ in controlling malignant cell growth
and apoptosis remains unclear, PPARγ has been commonly implicated as a tumor
suppressor in hematological cancers (see Figure 3 for overview). Evidently, a
better understanding of the mechanism of action of PPARγ is needed. It is important that studies
be performed to carefully analyze PPARγ levels, as well as the activation
status of PPARγ in hematological cancers. In addition,
since many of the existing studies have demonstrated that the proapoptotic and
antiproliferative effects of PPARγ ligands are independent of the receptor;
additional studies are required to elucidate PPARγ-dependent from independent events by
using tissue specific knockouts, siRNA approaches, and overexpression studies.
Understanding the mechanisms of action of these agents has become a priority to
develop drugs that have beneficial effects on tumor suppression without having
major side effects. Certain advances may be possible through the discovery of
SPPARγMs that can activate only a subset of desired
genes. This will require the identification of PPARγ target genes that mediate the
antitumorigenic effects in hematological malignancies.Figure 3
Mechanisms of action of PPARγ ligands in hematological malignancies. PPARγ ligands can bind to and activate PPARγ
to regulate gene transcription or they can exert PPARγ-independent mechanisms.
PPARγ ligands have antiproliferative,
prodifferentiation, antimetastatic, and proapoptotic effects on several
hematological malignancies making them promising candidates for use in
therapeutic regimens.The fact that PPARγ can be modified by phosphorylation
through MAP kinases and that this modification decreases PPARγ transcriptional activity, and the fact
that PPARγ activation itself increases PPARγ degradation by the proteasome may be
exploited for therapeutic benefit. PPARγ ligands in combination with inhibitors
of MAP kinases and/or proteasome inhibitors (e.g., Bortezomib) may be useful in
the treatment of malignancy. Therefore, studies should be performed to assess
the effectiveness of these combination therapies as well as those combining
PPARγ ligands with drugs such as Imatinib or
RXR/RAR ligands. Our current knowledge of the anticancer potential of PPARγ ligands predicts that such therapies
may prove to be of great benefit for future treatments of hematological
cancers.



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*Source: 834612-2008-05-26.xml*