<title>1. INTRODUCTION</title> <p>PPAR<italic>γ</italic> can regulate inflammatory responses to
prevent chronic inflammation [##REF##16905700##1##], but more importantly, it plays an important
role in the sensing and regulation of metabolism [##REF##16360030##2##]. These functions, especially the regulation of
metabolism, may be involved in the documented ability of PPAR<italic>γ</italic> to modulate the malignant phenotype
[##REF##17896990##3##]. This aspect of PPAR<italic>γ</italic> articulates with the resurgence of
interest in metabolism and cancer [##REF##10098401##4##, ##UREF##0##5##] which has underscored the 50-year old
findings of Warburg that the metabolism of tumor cells is deranged; aerobic
glycolysis rather than oxidative phosphorylation is the mode of tumor
metabolism [##UREF##1##6##]. Recent findings suggest
that many oncogenes and suppressor proteins target metabolic pathways, and in
the context of Warburg's early discovery, they form a new, revealing paradigm
[##UREF##2##7##]. The survival and malignant
potential of a tumor are critically dependent on its adaptation to a variety of
stress situations and nutrient limitations. 
To generate adequate energy from the relatively inefficient glycolytic
pathway, the flux from glucose to lactate must be maintained at a high rate
[##REF##16640561##8##]. Thus, vascularity and
neoangiogenesis as a response not only to hypoxia but also to the depletion of
nutrients play a critical role in tumor progression [##REF##1375931##9##]. In this context, the mobilization of proline
from the degradation of extracellular matrix in the tumor microenvironment has
come to our attention. The use of
proline as alternative stress substrate and the regulation of this response by
stress signals has been a focus of our research effort.</p>
<p>Recommended by Dipak Panigrahy</p> <p>Proline is metabolized by its own specialized enzymes with their own tissue and subcellular localizations and mechanisms of regulation. The central enzyme in this metabolic system is proline oxidase, a flavin adenine dinucleotide-containing enzyme which is tightly bound to mitochondrial inner membranes. The electrons from proline can be used to generate ATP or can directly reduce oxygen to form superoxide. Although proline may be derived from the diet and biosynthesized endogenously, an important source in the microenvironment is from degradation of extracellular matrix by matrix metalloproteinases. Previous studies showed that proline oxidase is a p53-induced gene and its overexpression can initiate proline-dependent apoptosis by both intrinsic and extrinsic pathways. Another important factor regulating proline oxidase is peroxisome proliferator activated receptor gamma (PPAR<italic>γ</italic>). Importantly, in several cancer cells, proline oxidase may be an important mediator of the PPAR<italic>γ</italic>-stimulated generation of ROS and induction of apoptosis. Knockdown of proline oxidase expression by antisense RNA markedly decreased these PPAR<italic>γ</italic>-stimulated effects. These findings suggest an important role in the proposed antitumor effects of PPAR<italic>γ</italic>. Moreover, it is possible that proline oxidase may contribute to the other metabolic effects of PPAR<italic>γ</italic>.</p>
<title>2. PROLINE METABOLISM</title> <p>Proline
is the only secondary amino acid incorporated into protein. Because the alpha nitrogen is contained
within a pyrrolidine ring, proline cannot be metabolized by generic amino acid
enzymes, that is, aminotransferases, decarboxylases, and racemases [##REF##5500436##10##, ##REF##2410198##11##]. Instead, a special family of
enzymes evolved with their own subcellular localizations and mechanisms of
regulation. There is a little overlap
between the activity of these enzymes and that for generic amino acids. Thus, the metabolic system is distinct and
can be responsive to special metabolic requirements. The enzymes for the proline metabolic scheme
had been characterized by the 1960s and the general system is shown
schematically in ##FIG##0##Figure 1##. 
Pyrroline-5-carboxylate, in tautomeric equilibrium with glutamic-<italic>γ</italic>-semialdehyde, is a central
intermediate. It is not only the
committed precursor of proline but also the immediate product of proline
degradation. Importantly, it is an
obligate intermediate bridging the urea cycle and the tricarboxylic acid cycle
and can play an anaplerotic role for both metabolic cycles [##REF##5500436##10##, ##REF##2410198##11##]. The complete metabolic system is not present
in all tissues.</p> <p>The
role of proline in proteins has been characterized and reviewed by others [##REF##12539244##12##], and the topic is outside the
scope of this review. However, functions
beyond its contribution to proteins have also been recognized in a variety of
animal and plant species. In
prokaryotes, proline is thought to have antioxidant and osmoprotective
functions [##REF##2410198##11##]. Regulatory roles have
been proposed for parasitic trematodes although the mechanisms are not
understood [##REF##6854470##13##]. In a variety of higher
plants, proline is thought to be an osmoprotectant and the metabolism of
proline has been linked to the synthesis of polyphenolic compounds [##UREF##3##14##]. Proline has been identified as a critical
metabolic substrate in the initiation of flight in insects. In addition, insects can detect and are
attracted to proline. The finding that proline
is at high concentrations in plant floral nectar has led to the proposal that
proline is the basis of a coevolution to optimize insect-mediated pollination
[##REF##16365739##15##]. During the molecular biological
explosion of the 1990s, the genes for proline metabolism were cloned from a
variety of sources, making possible studies defining functions for this special
metabolic system.</p> <p>An
interesting feature of proline metabolism is that the interconversions of
proline and pyrroline-5-carboxylate form a <italic>proline
cycle</italic>. Proline oxidase (POX), a.k.a.
proline dehydrogenase (PRODH), is tightly bound to mitochondrial inner
membranes (the enzyme will be designated POX, but the gene will be referred to
as <italic>PRODH</italic>). The enzyme is a flavoprotein and electrons
from proline are passed into the electron transport chain at site II with
cytochrome c as the electron acceptor [##REF##5500436##10##, ##REF##2410198##11##]. 
Pyrroline-5-carboxylate, the product of proline degradation, can be
converted to glutamate and <italic>α</italic>-ketoglutarate to contribute
anaplerotically to the TCA cycle [##REF##2410198##11##]. 
However, it is also converted back to proline by pyrroline-5-carboxylate
reductase in the cytosol to form a metabolic cycle. Coupled by pyridine nucleotides (NADP/NADPH
preferentially over NAD/NADH), the proline cycle forms a metabolic interlock
with glucose-6-phosphate dehydrogenase and the pentose phosphate pathway and
serves as a redox shuttle to convert reducing potential from the pentose
phosphate pathway into an ATP-generating system in mitochondria [##REF##6892988##16##–##REF##3729412##18##]. The magnitude of ATP generation, however, is
small compared to the TCA cycle and oxidative phosphorylation. The glycolytic pathway, with optimized flux,
also can generate ATP more efficiently. 
Thus, the contribution of the proline cycle to redox and energetics was
considered trivial previously. However,
as the mechanisms for upregulating POX were elucidated, it became clear that
the system serves as an important accessory source for energy under stress
conditions.</p> <p>Proline is available
from dietary proteins and can be biosynthesized from either glutamate or
ornithine [##REF##5500436##10##, ##REF##2410198##11##]. However, an abundant
source is from degradation of collagen in the extracellular matrix, connective
tissue, and bone [##REF##598383##19##]. Since 25% of the
residues in collagen is either proline or hydroxyproline and collagen is the
most abundant (by mass) protein in the body, it serves as an ample reservoir of
proline. Additionally, matrix
metalloproteinases (MMPs), the family of enzymes which degrade collagen and
other proteins in the extracellular matrix, are markedly upregulated under a
variety of conditions. Importantly,
upregulation of MMPs occurs during
tumor progression and invasion [##REF##17587815##20##, ##REF##16680569##21##] as well as during inflammation and
wound healing [##UREF##4##22##, ##REF##16914544##23##]. MMP upregulation
has been considered an important physical component of invasion, that is
allowing for tumor cells to escape from their basement membrane site and
migrate through tissue. Recently, it has
been shown that a variety of biologically active factors are released from
binding sites on ECM with activation of the MMPs [##REF##11704682##24##]. However, the utilization of proline or
hydroxyproline as a source of metabolic substrate has not been considered. That degradation of collagen occurs during
carcinogenesis in the skin tumor model has been convincingly demonstrated
[##REF##3986956##25##]. Recently, using breast and
prostate cancer xenografts and novel imaging methodology, investigators have
shown that hypoxia mediates collagen fiber breakdown and restructuring [##UREF##5##26##].</p> <title>3. POX AND APOPTOSIS</title> <p>P53
is considered the most important cancer suppressor protein [##REF##9039259##27##]. It is mutated in 85% of all human tumors and
germ-line mutations in p53 result in the Li-Fraumeni syndrome, a familial
syndrome with predisposition to early cancers in a variety of tissues [##UREF##6##28##]. To screen for p53 target genes, Polyak et al.
[##REF##9305847##29##] used an adenoviral-p53 expression construct and serial analysis of gene
expression. Only 14 out of 7202 genes
monitored were induced more than 7-fold, and POX was one of these and
designated as p53-induced gene-6 (PIG6). 
Using a construct where POX expression was under the control of
tetracycline, the overexpression of POX produced proline-dependent ROS [##REF##11280728##30##] and induced proline-dependent
apoptosis [##UREF##7##31##–##REF##16619034##35##]. Subsequently, it was
shown that POX overexpression produced its effects through generation of
proline-dependent mitochondrial superoxide (##FIG##1##Figure 2##)
[##REF##15817612##34##]. It is this superoxide which plays
a critical role in signaling to produce not only the release of cytochrome c
from mitochondria and the activation of the caspases in the intrinsic
(mitochondrial) limb of programmed cell death, but also it activated the
extrinsic (death receptor) limb by increasing the production of TRAIL
[##REF##16619034##35##]. A number of other signaling
systems respond to POX-mediated signaling including downregulation of MEK/ERK
phosphorylation [##REF##16619034##35##], downregulation of COX-2 with decreased PGE2 production,
and blockade of the progression through the cell cycle [##UREF##8##36##].</p> <p>The findings from
the tissue culture system have been translated into an animal model. In studies using DLD-POX cells to form
xenografts in athymic mice, the expression of POX markedly inhibited tumor
formation [##UREF##9##37##]. In mice given
doxycycline to suppress POX expression in DLD-POX cells, or in animals injected
with DLD-vector cells, tumors formed rapidly. 
By week 2 all these animals developed palpable tumors and by week 3, the
animals had to be sacrificed due to the size of the tumors. By contrast, in mice without doxycycline in
which POX was overexpressed, few tumors were detected. By week 2, only 1 out of 16 animals had
palpable tumors. Thus, the expression of
POX markedly inhibited the formation of xenografts.</p> <p>The
relevance of these changes in POX was pursued by immunohistochemical studies in
human tissues. Ninety-two paired normal
and cancer tissues from a variety of tumors were examined using
immunohistochemistry. The findings were
striking from gastrointestinal tumors (stomach, colon, pancreas) in which the
level of POX expression was markedly decreased or undetectable in 79% of the
tumors [##UREF##8##36##]. We are currently
investigating the genetic or epigenetic mechanism for the decrease in POX
expression, but based on these findings, we propose that POX is a potential
cancer suppressor protein.</p> <p>The
mechanism for the POX-mediated, proline-dependent generation of superoxide may
be due to leakage of electrons from the electron transport chain, a mechanism
proposed for other sources of mitochondrial superoxide. However, recent studies from structural
biology suggest that the generation of superoxide is an intrinsic property of
the enzyme. White et al. [##REF##17344208##38##] described
interesting findings using recombinant <italic>Thermus
thermophilus</italic> POX/PRODH. Unlike the
POX/PRODH from certain prokaryotic species, for example <italic>Escherichia coli</italic>, which have a bifunctional enzyme, embodying the
activities of POX and pyrroline-5-carboxylate dehydrogenase in a single
protein, the enzyme from <italic>T. thermophilus</italic> is monofunctional and produces pyrroline-5-carboxylate in a manner similar to
the enzyme in animal tissues, and thus may serve as a good model for human POX
[##REF##17344208##38##]. These workers found that the
flavin adenine dinucleotide is located in a domain exposed to solvent
oxygen. Thus, the electrons from proline
can be used to reduce oxygen to superoxide (##FIG##2##Figure 3##). In addition, they found an adjacent <italic>α</italic>-helix which can shield the FAD and
block its access to solvent oxygen. The
interpretation of these findings includes the intriguing possibility that POX
can be switched from an ATP-generating function to a superoxide-producing
function. Although a number of enzymes
have been proposed as generators of superoxide, these enzymes are cytosolic
(xanthine oxidase) or are associated with cell membranes (NADPH oxidase) with
their own specified functions.</p> <p>These
aforementioned functions of POX have been emphasized for their relevance to
cancer, but another function deserves mention. 
Proline functions as a neurotransmitter, inhibiting glutamatergic
neurons [##REF##1350201##39##]. Additionally, a
high-affinity transporter has been discovered and cloned from the brain
[##REF##1350201##39##]. The relevance to neurological
systems extends to lower species. 
Mutations in POX/PRODH result in “sluggishness” in <italic>Drosophila melanogaster</italic> [##REF##8096642##40##] and the PRO/Re mice, defective in
POX/PRODH, exhibit “gating” defects, a functional neurologic defect [##REF##10192398##41##]. In humans, mutations in <italic>PRODH</italic> have been associated with risk for early schizophrenia
[##REF##11891283##42##]. Although there has been a number
of studies supporting or contradicting this conclusion, evidence supports the
relevance of POX mutations. It has been
shown that the mutations in PRODH associated with the neuropsychiatric syndrome
have a biochemical phenotype with markedly decreased activity in the enzyme
[##REF##15662599##43##].</p> <title>4. REGULATION OF POX</title> <p>The induction of
POX by p53 suggested that it served special functions and was not simply a
“housekeeping enzyme.” To screen for
potential regulators, Pandhare et al. [##REF##16303758##44##] made a POX-promoter,
luciferase-reporter construct, and cotransfected a variety of transcriptional
factors corresponding to binding sites identified in the <italic>PRODH</italic> promoter. Although
Jun, Fos, and p65 of NF-<italic>κ</italic>B produced modest stimulatory effects (&lt;2-fold), a
marked activation of the <italic>PRODH</italic> promoter was observed with cotransfection of PPAR<italic>γ</italic>. This
finding was interesting, indeed, since this pleiotropic factor not only plays
an important role in metabolism [##REF##16360030##2##], especially of adipocytes, but also it is
an important modulator of inflammatory responses [##REF##16905700##1##]. The wide use of the thiazolidinediones (TZDs)
in the management of hyperglycemia in type 2 diabetes mellitus is an example of
the former [##REF##16511590##45##]. For the latter, some
investigators have suggested that PPAR<italic>γ</italic> provides a mechanism to downregulate
inflammatory stress responses and avoid the pathologic consequences of chronic
inflammation [##REF##17418635##46##]. Attracting
considerable attention recently is the finding in a variety of cultured cancer
cells that TZDs will block cell proliferation and induce apoptosis
[##REF##17582802##47##–##REF##16397256##49##]. Epidemiologic data from
patients with type 2 DM treated with TZDs suggest that these ligands of PPAR<italic>γ</italic> are protective against lung cancer but
not against colon or prostate cancer [##REF##17442990##50##]. 
With the impressive in vitro data and suggestive findings from
epidemiology, oncologists have proposed that PPAR<italic>γ</italic> is an attractive target for cancer
treatment.</p> <title>5. MECHANISM OF TZDs IN INDUCING <italic>PRODH</italic>
</title> <p>Pandhare
et al. [##REF##16303758##44##] showed that cotransfection of PPAR<italic>γ</italic> activated the <italic>PRODH</italic> promoter 8-fold, and troglitazone, a widely used TZD before
it was taken off the market because of side effects, further increased the
magnitude of this activation. The
combination of PPAR<italic>γ</italic> expression and troglitazone treatment
activated the <italic>PRODH</italic> promoter more
than 10-fold (##FIG##3##Figure 4##). 
The effect could be generalized to a variety of colorectal cancer cells
and could be elicited by four different TZDs. 
That troglitazone induced POX through a PPAR<italic>γ</italic> mediated binding to the peroxisomal
proliferator response element was shown using several methods. First, an electrophoretic shift mobility
assay showed a troglitazone-stimulated formation of a nuclear complex with the
labeled PPRE sequence from the <italic>PRODH</italic> promoter. That PPAR<italic>γ</italic> was present in this complex was shown
with chromatin immunoprecipitation assays. 
In this assay, formaldehyde was used to cross-link DNA-protein complexes
and then the DNA was sheared by sonication. 
After immunoprecipitation with specific anti-PPAR<italic>γ</italic> antibody, the PPRE sequences of the <italic>PRODH</italic> promoter were amplified using polymerase chain reaction.</p> <p>Although
these studies showed that PPAR<italic>γ</italic> and its pharmacologic ligands are directly
involved in the activation of the <italic>PRODH</italic> promoter, the integration of signaling by the PPAR<italic>γ</italic> assembly to physiologically regulate <italic>PRODH</italic> expression may be more
complex. The interaction with retinoid-X
receptors (RXR) is a requisite for PPAR<italic>γ</italic> function [##UREF##10##51##]. Moreover, a number of coactivators interact
with liganded PPAR<italic>γ</italic> and RXR to form an active
transcriptional complex. These include
steroid receptor PPAR<italic>γ</italic>-coactivator-1 (PGC-1) and steroid
receptor coactivator-1 (SRC-1) [##REF##9744270##52##]. The
specific coactivator may depend on the cell type and stimuli. In the context of metabolism, PGC-1 may be
especially relevant since it responds to signaling from other
metabolism-regulating hormones and cytokines [##REF##17018837##53##]. The specific effect of these coactivators on <italic>PRODH</italic> expression, however, has not been
elucidated, but it is an area of emphasis of our current work.</p> <title>6. CONTRIBUTION OF POX TO THE PPAR<italic>γ</italic>
EFFECTS ON ROS AND APOPTOSIS</title> <p>The
discovery that PPAR<bold><italic>γ</italic></bold> has a marked inhibitory effect on
cultured cancer cells stimulated a large number of studies using a variety of
cancer cells. The TZDs augmented
differentiation, slowed proliferation, and induced apoptosis. Although this effect was generally observed,
there were a few reports of TZDs actually stimulating the growth of certain
cultured cancer cells [##REF##17453334##54##]. 
Nevertheless, the preponderance of studies showed that TZDs inhibited
growth [##REF##17582802##47##–##REF##16397256##49##]. Although the mechanism
of this effect was not well understood, several investigators found that TZDs
induced the generation of ROS, and they concluded that ROS was the mechanism
for inducing apoptosis as has been reported for many experimental models. The actual mechanism by which ROS production
was induced by TZDs, however, remained unknown.</p> <p>Since
POX is a p53-induced gene and has been established as a mechanism for
generating superoxide that initiates apoptosis, the PPAR<bold><italic>γ</italic></bold> induction of POX raised the
attractive hypothesis that POX may be involved in the apoptotic mechanism
observed with the TZDs. To answer this
question, Pandhare et al. [##REF##16303758##44##] showed that in colorectal cancer cells,
troglitazone not only induced POX, but also markedly increased the production
of ROS as has been shown by others in other cultured cells. More importantly, the knockdown of POX with
antisense RNA markedly decreased the generation of troglitazone-stimulated
ROS. These studies strongly suggested
that the ROS presumed to be the mechanism for TZD-stimualted apoptosis was due,
at least in part, to its induction of POX. 
Thus, POX plays an important role in the apoptotic effect of TZDs, at
least in tissue culture. This finding was soon confirmed by others. Working with nonsmall cell lung cancer cells,
Kim et al. [##REF##17535976##55##] showed that rosiglitazone induced apoptosis through an
ROS-dependent mechanism, and that the induction of POX by rosiglitazone played
a critical role in the production of apoptosis. 
These are exciting findings but require further corroboration and
extension to other cultured cancer cells.</p> <p>The
effects of TZDs in cultured cells have been extended to several tumor models in
animals and the results are encouraging. 
In athymic mice, the growth rates of xenografts of ovarian, thyroid, and
bladder cancer are markedly affected by a variety of PPAR<italic>γ</italic>-stimulating agents [##REF##17582802##47##–##REF##16397256##49##]. Not only is
tumor growth inhibited but survival of the host animal is prolonged. Although
the mechanism underlying these effects remains unclear, it appears that the
cells in the tumors are apoptotic perhaps due to decreased expression of COX-2
[##UREF##11##56##]. Recent work in our laboratory
links POX expression to downregulation of COX-2 [##UREF##8##36##]. There are direct effects on the tumor as well
as effects on angiogenesis. There are no
studies of the effects of PPAR<italic>γ</italic> on POX expression in animals or on the
role of POX in mediating the PPAR<italic>γ</italic>-mediated antitumor effects.</p> <title>7. PARADOXES AND POSSIBLE SOLUTIONS</title> <p>The
enthusiasm generated by these antitumor effects of PPAR<italic>γ</italic> and the TZDs was somewhat blunted by
the finding that in C57Bl/6J-APC<sup>Min/+</sup> mice, activation of PPAR<italic>γ</italic>-mediated signaling promotes rather than
inhibits the development of colon tumors [##UREF##12##57##]. 
APC is the tumor suppressor protein in adenomatous polyposis coli and is
an integral part of the Wnt/<italic>β</italic>-catenin signaling system. The Min mutation blocks the formation of the
tetrameric complex (APC, axin, GSK-3<italic>β</italic>, <italic>β</italic>-catenin) which allows for
phosphorylation of <italic>β</italic>-catenin leading to its proteasomal
degradation. Accumulated <italic>β</italic>-catenin translocates into the nucleus
to form transcriptional complexes with TCF/LEF to induce target genes involved
in proliferation [##REF##11463572##58##]. However, in
keeping with the earlier reports that activation of PPAR<italic>γ</italic> or its ligands had antitumor effects,
recent studies have shown marked reduction in tumor growth or survival of
animals with peritoneal carcinomatosis with various PPAR<italic>γ</italic> ligands. These recent studies include ovarian cancers
[##REF##17582802##47##], anaplastic thyroid carcinomas [##REF##16331265##48##], and bladder tumors [##REF##16397256##49##]. Thus, the debate continues: “. . . the action of PPAR<italic>γ</italic> on cell cycle, proliferation, differentiation,
and apoptosis seems to depend on the cell type and/or the mutational events
that predispose tissue to cancer development” [##REF##11463572##58##]. The importance of coactivators or
corepressors cannot be overemphasized. 
Interactions with and contributions of the microenvironment must also be
considered in understanding these different effects.</p> <p>A
common target of these signaling pathways is the matrix metalloproteinases
(MMP) [##REF##16510589##59##, ##REF##17597617##60##]. Differential effects on
these enzymes may explain, in part, the variability in the aforementioned
effects of PPAR<italic>γ</italic> activation. Increased PPAR<italic>γ</italic> signaling will downregulate MMP whereas
certain MMP are target genes of <italic>β</italic>-catenin/TCF-LEF. The transcriptional system constitutively
upregulated by the APC<sup>Min</sup> mutation increases the expression of
MMP-7. Just how these mechanisms
articulate for regulating MMP remains unclear. 
However, in the context of the aforementioned induction of POX by PPAR<italic>γ</italic>, the differential effects on MMP may be
relevant. In a given experimental model,
the availability of ECM and the effects on MMP may determine the relative
availability of proline as a stress substrate for POX. Furthermore, the consequences of POX
induction may also be two-edged. Under
stimulation of p53, POX can use proline to generate mitochondrial superoxide to
initiate apoptosis by both intrinsic and extrinsic pathways [##REF##15817612##34##]. Recent work has shown that POX overexpression
will also blockade the cell cycle [##UREF##13##61##]. 
Thus, upregulation of POX in the presence of MMP to generate free proline
will activate antitumor mechanisms. On
the other hand, POX also can generate ATP and it is upregulated by
downregulation of mTOR signaling under nutrient stress. With the availability of proline,
upregulation of POX can support cell survival [##UREF##14##62##]. Like several mediators of metabolic
regulators, for example, p53 and PPAR<italic>γ</italic>, POX also can play a two-edged
regulatory role.</p> <title>8. THE ROLE OF POX IN ANTITUMOR
EFFECTS OF PPAR<italic>γ</italic>
</title> <p>Additional
work is needed to translate these findings in cultured cancer cells to animal
models and eventually to clinical trials. 
As a first step, studies are being undertaken to monitor the expression
of POX in mice administered TZDs. 
Assuming that certain tissues in intact animals will respond as in
cultured cells, the effect of POX upregulation on spontaneous tumors in that
tissue can be investigated. The inhibition of POX by proline analogues or the
blockade of MMPs, specifically prolidase, may limit the availability of proline
in that tissue. Also, control of dietary
proline could be important. With the
insights gained by these animal studies, it may be possible to design clinical
trials in which perturbations of the POX-mediated effects can be
pharmacologically attacked as an adjunct to the use of TZDs or other PPAR<italic>γ</italic> activators. Furthermore, PPAR<italic>γ</italic> activation with or without POX can be
used in combination with other chemotherapeutic modalities.</p> <title>9. CONTRIBUTION OF POX TO OTHER
PPAR<italic>γ</italic>-MEDIATED EFFECTS</title> <p>The
consequences of POX induction and its role in PPAR<italic>γ</italic>-mediated
metabolic effects other than that on cancer have not been explored. However, it is intriguing that the
well-established metabolic effects of PPAR<italic>γ</italic> could be mediated in part by induction
of POX. Nevertheless, the known effects
of POX and PPAR<italic>γ</italic> invite speculation, but these specific
questions have not been experimentally addressed. Thus, these questions remain in the realm of
future plans. Of special consideration
are the following effects of PPAR<italic>γ</italic>: (1) increased
insulin sensitivity, (2) decreased inflammation, and (3) increased osteopenia.</p> <p>There
are potential links between degradation of proline and insulin-related
metabolic effects. Certainly, POX uses
proline to generate intermediates for anaplerosis of the TCA cycle which could
make oxidative metabolism more efficient. 
Investigators have cited the importance of these intermediates as building
blocks rather than as energy substrates. 
Furthermore, the metabolic interlock of the proline cycle and glucose
metabolism through the pentose phosphate pathway could affect insulin
sensitivity since it opens an alternative pathway for glucose metabolism. Thus, glucose would not only be metabolized
by oxidative phosphorylation in the TCA cycle and converted to lactate by glycolysis, but also would be
converted to CO<sub>2</sub> by interconversions and cycling through the pentose phosphate
shunt.</p> <p>The
PPAR<italic>γ</italic> signaling pathway is frequently considered as a response to inflammatory stress, that is,
to prevent chronic inflammation. 
Inflammatory cells such as macrophages will respond to inflammatory
signals such as prostaglandins and this will induce POX in macrophages and
induce apoptosis. Furthermore, COX-2 may
be regulated by the expression of POX and the generation of proline-mediated
ROS [##UREF##8##36##].</p> <p> The final metabolic consideration is the demonstrated effects
in animals and in humans that TZDs will result in osteopenia [##REF##17332064##63##]. From histologic and metabolic studies, PPAR<italic>γ</italic> appears to decrease osteogenesis and
increase osteoloysis. There are
decreased numbers of osteoblasts and increased numbers of osteoclasts
[##REF##17669705##64##]. Since bone is primarily made up of
calcified collagen, it is not surprising that collagen synthesis is decreased
and collagen degradation is increased. 
Since collagen synthesis requires the incorporation of proline, the
degradation of proline by increased POX would be a biochemical process
consistent with osteoclastic function.</p> <p>Another
interesting area involves a physiologic/pathophysiologic source of natural
ligands for PPAR<italic>γ</italic>, that is, oxidized low-density
lipoproteins (oxLDL). Their precursor,
low-density lipoproteins (LDL) are synthesized in the liver and are the
carriers for 60% of total serum cholesterol, and they are widely known as the
“bad cholesterol.” Recent studies
suggest that LDL is oxidized in human blood and tissues under various
pathological conditions. OxLDL may be an
important player in the development of atherosclerosis, promoting apoptosis in
endothelial cells, increasing proliferation of smooth muscle cells, and
upregulating inflammatory signaling in macrophages. The result is the formation of atheromatous
plaques. Mechanisms of oxLDL-induced
effects are being intensively investigated, but there is a considerable
evidence supporting a role for PPAR<italic>γ</italic> activation [##REF##9568715##65##]. Additionally, oxidized LDL activates p53 [##REF##11027537##66##, ##REF##17289842##67##] and stimulates the formation of mitochondrial ROS [##REF##15805232##68##] to induce cell
death. Since all these mechanisms are linked
to POX activity, it is tempting to speculate that POX may be involved.</p> <p>Although
oxLDL is mainly associated with atherosclerosis, several studies point to the
correlation between serum oxLDL levels and cancer risk in humans [##UREF##15##69##, ##REF##17673194##70##]. This prompted us to study the possible role
of POX in the oxLDL-mediated effects on carcinogenic pathways. First, we transfected breast, prostate,
colon, cervical, ovarian, and lung cancer cell lines with the POX
promoter-luciferase reporter and found that oxLDL treatment activated the POX
promoter in a dose- and time-dependent manner. 
This effect was further augmented by the addition of 2.5 mM
proline. We also found that oxLDL
treatment increased POX gene expression as compared to nonoxidized LDL, or a
solvent control [Zabirnyk O and Phang JM, unpublished results]. These preliminary studies suggest a role of
proline oxidase in the oxLDL-mediated effects on PPAR<italic>γ</italic> activation and initiation of apoptotic
cell death.</p> <p>In
summary, POX, a p53-induced gene, is markedly upregulated by overexpression of
PPAR<italic>γ</italic> or by the addition of TZDs. The effect is generalizable to a variety of
cells and to all the TZDs. The mechanism
of this effect appears to be by transcriptional activation by activating the
POX promoter at the PPRE site. The PPAR<italic>γ</italic> effect on apoptosis is mediated by the
generation of ROS, and knockdown of POX by siRNA markedly decreases or blocks
the effects of PPAR<italic>γ</italic> on ROS formation and apoptosis in
colorectal cancer cell or nonsmall cell lung cancer cell, respectively. These findings suggest that POX may play a
critical role in PPAR<italic>γ</italic>-mediated
antitumor effects. Furthermore, it may
offer an explanation for the inconsistent findings observed in different animal
systems. It also may offer an adjunctive
therapeutic approach to optimize the PPAR<italic>γ</italic>-mediated
antitumor effects. Finally, a
speculative proposal for the articulation of POX-dependent metabolic effects on
the metabolic syndrome with PPAR<italic>γ</italic> activation is presented.</p>
<title>ACKNOWLEDGMENTS</title> <p>This
research is supported by the Intramural Research Program of the NIH, National
Cancer Institute, Center for Cancer Research. 
This project also has been funded in part with Federal funds from the
National Cancer Institute, National Institutes of Health under Contract no.
N01-CO-12400. The content of this
publication does not necessarily reflect the views or policies of the Department
of Health and Human Services, nor does the mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Government.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Proline metabolic pathway. Abbreviations: 
PRO, proline; P5C, Δ<sup>1</sup>-pyrroline-5-carboxylate;
GLU, glutamate; GSA, glutamic-gamma-semialdehyde; ORN, ornithine. Enzyme names not shown: A, P5C Synthase; B, P5C dehydrogenase;
C, spontaneous; D, ornithine aminotransferase.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Proline oxidase-induced apoptosis. 
Abbreviations: ROS, reactive oxygen species; TRAIL, tumor necrosis
factor related apoptosis-inducing ligand; DR5, death receptor
5 NFAT, nuclear factor of activated T cells; MEK, MAP
kinase; ERK, extracellular-signal regulated kinase.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>Structure of proline dehydrogenase (proline
oxidase) from <italic>Thermus thermophilus</italic>. The flavine
adenine dinucleotide at the active site is shown in yellow. The
flexible alpha helix adjacent to the FAD is shown in violet and
blue. Access of the FAD to solvent O<sub>2</sub> allows direct
reduction of O<sub>2</sub> to form superoxide radicals. The figure
is used with permission from Dr. Jack Tanner, University of
Missouri-Columbia, and the Journal of Biological Chemistry.</p></caption></fig> <fig id="fig4" position="float"><label>Figure 4</label><caption><p>Induction of proline oxidase by PPAR<italic>γ</italic>
and its pharmacologic ligand, troglitazone. (a) Activation of the
POX promoter using a luciferase reporter assay. HEK 293
colorectal cancer cells were transfected with equivalent amounts
of cDNA of PPAR<italic>γ</italic> or vector plasmid as control. The cells
were also transfected with <italic>POX</italic>-Luc and
pRL-null. Troglitazone (25 <italic>μ</italic>M) or Me<sub>2</sub>SO in
control was added after 10 hours as indicated. At 24–36
hours after transfection, the cell lysates were harvested, and the
<italic>POX</italic> promoter luciferase activity was determined
using the Dual Luciferase Assay kit. (b) Troglitazone increases
the binding of PPAR<italic>γ</italic> to the PPRE in the <italic>POX</italic>
promoter. HCT 116 colorectal cancer cells were treated with or
without 25 <italic>μ</italic>M troglitazone for 36 hours and nuclear extracts
were prepared. The binding of PPAR<italic>γ</italic> to the PPRE was
evaluated by an electrophoretic mobility shift analysis assay
using the double-stranded POX-PPRE oligonucleotide probe. Unlabeled POX-PPRE probe (100x) was used as a competitor (lane 4).
(c) Chromatin immunoprecipitation assay of the
<italic>POX</italic> promoter in troglitazone-treated HCT 116
cells. 
HCT 116 cells were incubated with 1% formaldehyde to fix protein-DNA
complexes. DNA was sheared by sonication. Soluble chromatin-DNA
complexes were immunoprecipitated using PPAR<italic>γ</italic> antibody and
immunoprecipitates were analyzed by PCR with specific primers for
the <italic>POX</italic> promoter region containing the
POX-PPRE.</p></caption></fig>
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PPAR Res. 2008 Jul 28; 2008:542694