<p>Recommended by Dipak Panigrahy</p> <p>Today, there is increasing evidence that PPAR<italic>γ</italic> agonists, including thiazolidinediones (TDZs) and nonthiazolidinediones, block the motility and invasiveness of glioma cells and other highly migratory tumor entities. However, the mechanism(s) by which PPAR<italic>γ</italic> activators mediate their antimigratory and anti-invasive properties remains elusive. This letter gives a short review on the debate and adds to the current knowledge by applying a PPAR<italic>γ</italic> inactive derivative of the TDZ troglitazone (Rezulin) which potently counteracts experimental glioma progression in a PPAR<italic>γ</italic> independent manner.</p>
<p>Gliomas are the most common primary tumors in the central nervous system, with
glioblastomas as the most malignant entity [##REF##11150363##1##]. Despite multimodal therapy regimens incl uding
neurosurgical resection, radio- and polychemotherapy, the prognosis of glioma
patients remains poor. Less than 3% of affected patients survive more than five
years after diagnosis [##REF##15685439##2##]. Rapid proliferation, tumor-induced neurodegeneration,
and brain edema [##UREF##0##3##] as well as diffuse brain invasion are
pathological hallmarks of these tumors and are likely to determine unfavorable
prognosis. Because local invasion of neoplastic cells into the surrounding
brain is perhaps the most important aspect in the biology of gliomas that preclude successful
treatment, pharmacological inhibition of glioma cell migration and brain
invasion is considered as a highly promising strategy for adjuvant glioma
therapy.</p> <p>Today, there is increasing evidence that PPAR<italic>γ</italic>
agonists, including thiazolidinediones (TDZs) and nonthiazolidinediones, block the
motility and invasiveness of glioma cells and other highly migratory tumor
entities. GW7845, an investigational non-TDZ PPAR<italic>γ</italic> ligand, binds and activates human PPAR<italic>γ</italic>
at low nanomolar concentrations and thus possesses a higher potency than TDZs such
as pioglitazone (Actos), troglitazone (Rezulin), rosiglitazone (Avandia), and the
experimental PPAR<italic>γ</italic> agonist ciglitazone, respectively, which require submicromolar doses [##REF##9836622##4##]. Grommes et al. [##REF##15665144##5##] demonstrated that 30 <italic>μ</italic>M concentrations
of GW7845 reduced the viability of rat (C6) and human glioma cells (U-87 MG,
A172), which could be attributed to a G<sub>1</sub> cell cycle arrest and
increased cell death. Besides its antiproliferative and cytotoxic properties, the authors demonstrated for
the first time that GW7845 counteracts migration and invasion of C6 rat glioma
cells in vitro (spheroid outgrowth, Boyden chamber assay). A subsequent study revealed that the FDA-approved
TDZ pioglitazone exhibits antiglioma properties similar to GW7845 [##REF##16887936##6##]. Alike GW7845, micromolar doses of
pioglitazone (30 <italic>μ</italic>M) counteract C6 rat glioma cell invasiveness in vitro (Boyden chamber assay). In
this study, Grommes et al. [##REF##16887936##6##] demonstrated profound in vivo antiglioma properties of pioglitazone. Following C6
glioma cell implantation into the striata of adult rats, oral or intracerebral
drug application effectively decelerated glioma progression, resulting in an
improved clinical outcome and 80% reduction of tumor volume at 3 weeks after
tumor implantation. Immunohistochemical analyses of pioglitazone-treated
animals revealed that protein levels of MMP-9 (<italic>matrix metalloproteinase 9</italic>), which has shown to be intimately
involved in glioma migration and invasion [##REF##12439751##7##], were substantially reduced in the bulk tumor
and the tumor margins. However, the data regarding the antiglioma properties of
pioglitazone are somewhat contradictory in a mouse glioma model (GL261 glioma
cells, C57Bl/6 mice). Grommes et al. demonstrated that oral application of
pioglitazone increased the number of surviving animals after 30 days of
treatment. By employing the same model, Spagnolo and coworkers observed no
effect on survival following oral drug application, while intracerebral
injection of pioglitazone increased the mean survival time [##REF##17410715##8##]. We have recently shown that the TDZ troglitazone
reduces the viability and proliferation of rat (F98), mouse (SMA-560), and
human (U-87 MG) glioma cells slightly but significantly more potent than the remaining TZDs tested 
(troglitazone &gt; pioglitazone &gt; rosiglitazone &gt; ciglitazone) [##REF##17541035##9##]. By employing an ex vivo glioma invasion 
model [##REF##15871520##10##], troglitazone effectively blocked glioma progression and brain invasion and consistent with the
in vitro data presented by Grommes and coworkers, we confirmed that troglitazone (30 <italic>μ</italic>M) antagonized rat
F98 glioma cell migration (scratch wound healing, Boyden chamber assay).</p> <p>Inhibition of cell motility and invasiveness by PPAR<italic>γ</italic>
activators has also been described for other neoplastic cells and thus appears not
to be restricted to glioma. Liu et
al. [##REF##12779083##14##] showed that GW7845 (5 <italic>μ</italic>M) as well as the FDA-approved
TDZs pioglitazone and rosiglitazone (both 25 <italic>μ</italic>M) inhibits the invasive properties of human MDA-MB-231
breast cancer cells. In this study, treatment with PPAR<italic>γ</italic>
agonists was associated with increased <italic>tissue inhibitor of matrix metalloproteinase 1</italic> (TIMP-1) mRNA and
protein levels, which are likely to contribute to the anti-invasive effects observed. Recently, Yang et al. [##REF##18021457##15##] 
demonstrated that troglitazone (10–30 <italic>μ</italic>M)
inhibits migration and invasiveness of a human ovarian carcinoma ES-2 cells.
Anti-invasive properties were also shown for the TDZ ciglitazone, although with
a lower potency. Extended analyses by Yang et al. revealed that troglitazone
(20 <italic>μ</italic>M) inhibits focal adhesion formation associated with reduced focal adhesion
kinase (FAK) activity. FAK, an ubiquitously expressed
nonreceptor tyrosine kinase, has shown to be a vitally important regulator of
cancer cell migration and invasion. FAK is highly expressed in many tumor
entities and activated by autophosphorylation [##REF##16997283##16##], which has shown to be reduced by more than 80% in troglitazone-treated
ES-2 cells [##REF##18021457##15##]. Based on these data, the authors concluded that troglitazone may
inhibit ES-2 cell migration and invasion by preventing FAK activation. Concordantly,
inhibition of FAK kinase activity by the investigational small molecule TAE226 reduced the invasive properties of human U-87 MG, U251, and LN18 glioma cells by more than 50% [##REF##17431114##17##], suggesting that FAK activation decisively
promotes migration and invasion also of glioma cells. In all, these data demonstrate that PPAR<italic>γ</italic>
activators belonging to different chemical classes effectively diminish glioma
progression in vitro, ex vivo, and in vivo [##REF##15665144##5##, ##REF##16887936##6##, ##REF##17541035##9##], which occurs at least in part by the inhibition of glioma 
cell migration and invasiveness.</p> <p>Given the fact that cancer cell migration and invasion are highly complex processes [##REF##15674479##18##], the mechanism(s) by which PPAR<italic>γ</italic> agonists exert their
antimigratory and anti-invasive properties requires further investigation. Besides MMP-9,
TIMP-1, and FAK, which have been shown to be involved in the antimigratory
activities of PPAR<italic>γ</italic> agonists, we recently demonstrated that already low doses of troglitazone
block <italic>transforming growth factor beta</italic> (TGF-<italic>β</italic>)
release [##REF##17541035##9##], a cytokine which plays a pivotal role in glioma
cell motility [##REF##11716069##19##]. Several in vitro studies revealed that exogenously added TGF-<italic>β</italic>
<sub>1</sub> and TGF-<italic>β</italic>
<sub>2</sub> elicit a strong stimulation of migration in a variety of
glioma cells [##REF##8054266##20##–##REF##15520202##23##], while TGF-<italic>β</italic> gene silencing has shown to reduce glioma cell motility and invasiveness [##REF##15492287##24##]. In agreement with these findings, inhibition
of TGF-<italic>β</italic> signaling by the investigational type I TGF-<italic>β</italic> receptor antagonist,
SB-431542 reduced the invasive properties of human D-54 MG and rat F98 glioma
cells by approximately 70% [##REF##17541035##9##, ##REF##15210860##25##]. The role of TGF-<italic>β</italic> as molecular target for
glioma therapy has been facilitated by studies using surgically resected glioma
tissues, which revealed an intriguing correlation between tumor grade and the
expression of TGF-<italic>β</italic> ligands and their corresponding receptors I and
II. High-grade gliomas express high levels of TGF-<italic>β</italic>RI, TGF-<italic>β</italic>RII, and TGF-<italic>β</italic>
ligands, while the expression levels of these molecules have been shown to be
weak in low-grade gliomas and normal brain tissue [##REF##10861501##26##–##REF##7635563##28##]. A comprehensive transcriptome-wide study by
Demuth et al. [##UREF##1##29##] using 111 glial tumor samples and 24 normal
brain specimens identified the TGF-<italic>β</italic> signaling pathway to be predominantly enriched
in glial tumors compared to normal brain. In all, these data implicate that
glioma cells release TGF-<italic>β</italic> ligands at high doses and fortify their promigratory
and proinvasive properties in an autocrine manner, thus promoting glioma
progression. Given the fact that 10 <italic>μ</italic>M doses of troglitazone allay TGF-<italic>β</italic>
release of glioma cells (F98, SMA-560, U-87 MG) by more than 50% [##REF##17541035##9##], we hypothesized that the abrogation of glioma
cell motility and invasiveness by troglitazone and other PPAR<italic>γ</italic>
activators is primarily
driven by the inhibition of TGF-<italic>β</italic> signaling and thus, troglitazone and related
compounds may be considered for adjuvant glioma therapy to counteract TGF-<italic>β</italic>-mediated
brain invasion.</p> <p>However, the mechanism(s) by which PPAR<italic>γ</italic> activators mediate their antimigratory and anti-invasive properties remains 
elusive. We have shown that PPAR<italic>γ</italic> inhibition by the investigational antagonist GW9662, either alone or in combination
with troglitazone, does not affect rat F98 glioma cell invasiveness in a Boyden
chamber assay, suggesting that the effects observed are not mediated by PPAR<italic>γ</italic> [##REF##17541035##9##]. Simultaneously, Yang and coworkers [##REF##18021457##15##] have shown that PPAR<italic>γ</italic> knockdown by siRNA did not counteract the
anti-invasive features of troglitazone using human ovarian carcinoma ES-2
cells, underscoring the idea that the PPAR<italic>γ</italic>
agonists counteract cancer cell migration by a yet unknown off-target activity.
To validate these preliminary findings, we analyzed the effects of a troglitazone
derivative, Δ2-troglitazone, which has been shown to be PPAR<italic>γ</italic>-inactive [##REF##15109648##11##, ##REF##15735046##31##, ##REF##16728570##32##]. In case the antiglioma properties of
troglitazone are solely or predominantly due to PPAR<italic>γ</italic> activation, Δ2-troglitazone
should display no or a considerably lower inhibitory potency on glioma cell
viability than troglitazone. Initially, concentration-dependent
inhibition of glioma cell viability by troglitazone and Δ2-troglitazone was investigated using glioma cell lines derived from mouse
(SMA-560), rat (F98), and human (U-87 MG, U-373 MG). As shown by MTT assay, both
compounds inhibited glioma cell growth in a concentration-dependent manner with
similar potencies (Figures ##FIG##0##1(a)##, ##FIG##0##1(c)##). Even though numerous PPAR<italic>γ</italic>-dependent mechanisms
have been identified (for review see Tatenhorst et al., this issue), these data
suggest that PPAR<italic>γ</italic> activation is not an imperative prerequisite for the inhibition of glioma cell viability in vitro, which is in line with
previous studies using human PC-3 and LNCaP prostate cancer and human A549 lung
carcinoma cells [##REF##15109648##11##, ##REF##15735046##31##]. Next, we analyzed the reduction of glioma cell viability using IC<sub>90</sub> doses of Δ2-troglitazone and
equimolar doses of troglitazone. In all four cell lines tested, Δ2-troglitazone displays
a slightly but significantly higher potency compared with troglitazone.
However, with IC<sub>90</sub> doses ranging from 93 <italic>μ</italic>M (SMA-560) to 132 <italic>μ</italic>M (U-87
MG), the antiproliferative properties of Δ2-troglitazone can be
regarded as moderate.</p> <p>Next, we analyzed the effects of troglitazone and Δ2-troglitazone
on TGF-<italic>β</italic> release by glioma cells. Hjelmeland et al. [##REF##15210860##25##] have shown that secretion of activated TGF-<italic>β</italic>
<sub>1</sub> is a common attribute of glioma cells (U-87 MG, U-373 MG, D-54 MG, D-270 MG, D-423
MG, D-538 MG), while simultaneous release of TGF-<italic>β</italic>
<sub>2</sub> was found only
sporadically (D-54 MG, U-373 MG, D-423 MG). In accordance with these findings, quantification of TGF-<italic>β</italic>
<sub>1</sub> and TGF-<italic>β</italic>
<sub>2</sub> transcript
levels by real-time PCR revealed that U-373 MG and SMA-560 glioma cells express
both TGF-<italic>β</italic>
<sub>1</sub> and TGF-<italic>β</italic>
<sub>2</sub>, respectively, 
while TGF-<italic>β</italic>
<sub>1</sub> is clearly
the predominant isoform in F98 and U-87 MG glioma cells (data not shown). Repeated
quantification (<italic>n</italic> ≥ 7) of absolute TGF-<italic>β</italic>
<sub>1</sub> levels following cultivation of glioma cells for 48 hours in serum-free medium
revealed that all cell lines investigated secrete TGF-<italic>β</italic>
<sub>1</sub> (F98: 8.45 ± 1.59 ng/mL; SMA-560: 2.7 ± 0.54 ng/mL; U-87 MG: 2.55 ± 0.68 ng/mL, U-373 MG: 0.43 ± 0.08 ng/mL), while both troglitazone and Δ2-troglitazone inhibit
TGF-<italic>β</italic>
<sub>1</sub> release in a dose-dependent manner (Figures ##FIG##0##1(b)##, ##FIG##0##1(d)##). The
finding that Δ2-troglitazone
counteracts TGF-<italic>β</italic>
<sub>1</sub> release indicates that this effect is not PPAR<italic>γ</italic> dependent. Again, Δ2-troglitazone displays
a significantly higher potency as compared with troglitazone (##FIG##0##Figure 1(f)##). In
case of Δ2-troglitazone, 90% inhibition
of TGF-<italic>β</italic>
<sub>1</sub> release was found at concentrations ranging from 5 <italic>μ</italic>M
(F98) to 14 <italic>μ</italic>M (U-87 MG, U-373 MG), whereas troglitazone required 11 <italic>μ</italic>M (F98)
to 30 <italic>μ</italic>M (U-373 MG) to achieve the same effects. Strikingly, troglitazone as
well as Δ2-troglitazone is approximately 10 fold more potent inhibitors of TGF-<italic>β</italic>
<sub>1</sub> release than
of glioma cell proliferation, suggesting that both effects may not be essentially
interlinked.</p> <p>In agreement with the finding that TGF-<italic>β</italic>
<sub>1</sub> promotes glioma cell migration and brain
invasion, treatment of glioma cells with micromolar doses of Δ2-troglitazone
effectively blocks their migrative properties (##FIG##1##Figure 2##). Already 10 <italic>μ</italic>M doses
of Δ2-troglitazone inhibit F98 glioma cell migration in a Boyden chamber assay, while migration was completely
suppressed at 20 <italic>μ</italic>M. An intriguing question is whether inhibition of glioma
cell migration alone is sufficient to counteract glioma progression. To address
this issue we employed rat organotypic hippocampal brain slice cultures (OHSCs)
to monitor glioma progression and brain invasion in the organotypic brain
environment [##REF##15857402##12##]. Here, eGFP-labelled F98 glioma cells were implanted into the
entorhinal cortex of OHSCs (##FIG##2##Figure 3(a)##). The tumor infiltration area was
quantified up to 12 days by fluorescence microscopy. A continuous increase of
the bulk tumor mass was observed in solvent-matched control experiments
at all time periods. 12 days after glioma cell implantation, the tumor
infiltration area increased approximately 4.5 fold compared to the initial
tumor size at day 1 after implantation (Figures ##FIG##2##3(b)##, ##FIG##2##3(c)##). In contrast, the
tumor infiltration size remained stable over the period of 12 days after
treatment with 10 <italic>μ</italic>M Δ2-troglitazone. This
finding indicates that Δ2-troglitazone is not
able to reduce existing tumor masses, but effectively inhibits tumor
progression and brain invasion in an organotypic environment. Given the fact
that 10 <italic>μ</italic>M doses of Δ2-troglitazone
significantly affect TGF-<italic>β</italic>
<sub>1</sub> release (##FIG##0##Figure 1(d)##) and glioma cell
motility (##FIG##1##Figure 2##) but not glioma cell viability (##FIG##0##Figure 1(c)##), these data suggest that glioma cell
migration is an essential requirement for glioma progression in a system closely resembling extracellular matrix environment present
in the brain.</p> <p>TGF-<italic>β</italic> antagonism is considered as a therapeutic strategy
including the development of antisense regimens, inhibition of pro-TGF-<italic>β</italic> processing, scavenging of TGF-<italic>β</italic> by the TGF-<italic>β</italic>-binding proteoglycan decorin, and blocking of
TGF-<italic>β</italic> receptor I kinase activity [##REF##16454748##33##]. The finding that troglitazone and its derivative Δ2-troglitazone effectively
inhibit TGF-<italic>β</italic> release suggests readily available PPAR<italic>γ</italic> activators and structurally related PPAR<italic>γ</italic> inactive compounds as
candidate drugs for adjuvant glioma therapy. Besides its promigratory and proinvasive activities, TGF-<italic>β</italic> is considered
as one of the most potent immunosuppressive factors released by gliomas allowing
glioma cells to escape from immune surveillance [##REF##10795886##34##, ##REF##17414317##35##]. Friese et al. [##REF##15492287##24##] demonstrated that combined TGF-<italic>β</italic>
<sub>1</sub> 
and TGF-<italic>β</italic>
<sub>2</sub> knock down in human LNT-229 glioma cells results in a
loss of tumorigenicity when xenografted into CD1 nude mice, and natural killer
cells isolated from these animals show an activated phenotype. More than 10
years ago, Ständer et al. [##REF##9930319##36##] have shown that inhibition of TGF-<italic>β</italic> signaling
by decorin increases the number of B and T cells (CD45+), T helper cells
(CD4+), cytotoxic T cells (CD8+), and, most prominently, of activated T cells
(CD25+) infiltrating the tumor in an intracerebral C6 rat glioma model. By employing
an SMA-560 mouse glioma model, Tran et al. [##REF##17522330##37##] have shown that inhibition of TGF-<italic>β</italic> signaling by the TGF-<italic>β</italic> RI
kinase inhibitor SX-007 increased T-cell (CD3+) infiltration into the tumor. Due
to the fact that inhibition of TGF-<italic>β</italic> signaling has been shown to enhance
antiglioma immune responses in vivo [##REF##15492287##24##, ##REF##9930319##36##, ##REF##17522330##37##] it appears likely that troglitazone, inhibiting
TGF-<italic>β</italic>
<sub>1</sub> release at clinically achievable doses [##REF##17541035##9##, ##REF##10496299##38##], restores immune
surveillance. However, the yet-unknown protein/proteins mediating the inhibition of glioma progression by troglitazone
and Δ2-troglitazone remain(s) to be identified and may represent
future targets for structure-relationship studies. Moreover, PPAR<italic>γ</italic> inactive derivatives of
known PPAR<italic>γ</italic> agonists which retain their propensity to counteract glioma progression might be
further developed to minimize potential PPAR<italic>γ</italic>
mediated side effects in glioma patients.</p>
<title>ACKNOWLEDGMENTS</title> <p>SMA-560 mouse glioma cells [##REF##9402588##39##] were kindly provided by D. D. Bigner (Durham, USA). Thanks are due to Anita Betz, 
University of Würzburg for her skilful assistance in synthesizing Δ2-troglitazone. This study was
supported by the Köln Fortune Program (Faculty of Medicine, University of Cologne)
and the Wilhelm Sander-Stiftung (2008.010.1) to E. H.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>
<italic>Troglitazone (TRO) and the PPAR<italic>γ</italic> inactive </italic>Δ<italic>2-troglitazone (</italic>Δ<italic>2-TRO) reduce 
glioma cell viability and TGF-<italic>β</italic><sub>1</sub> release</italic>. Δ2-TRO was synthesized as previously described in [##REF##15109648##11##]. (a), (c) Concentration-dependent
inhibition of glioma cell viability by TRO (a) or Δ2-TRO (c) in the
indicated cell lines are given as mean ± SEM percentage relative to time- and
solvent-matched controls. Cell viability assays (MTT
assay, 96 hours) were performed as described earlier [##REF##15857402##12##, ##REF##6606682##13##]. Inhibitory
concentrations IC<sub>50</sub> and IC<sub>90</sub>, defined as concentrations
shown to inhibit tumor cell viability by 50% or 90%,
respectively, were determined by nonlinear regression data analysis: TRO: F98 (62 <italic>μ</italic>M, 166 <italic>μ</italic>M), SMA-560 (26 <italic>μ</italic>M, 407 <italic>μ</italic>M), U-87 MG (120 <italic>μ</italic>M, 324 <italic>μ</italic>M), and U-373 MG (123 <italic>μ</italic>M,
331 <italic>μ</italic>M); Δ2-TRO: F98 (46 <italic>μ</italic>M, 95 <italic>μ</italic>M), SMA-560 (23 <italic>μ</italic>M, 93 <italic>μ</italic>M), U-87 MG (78 <italic>μ</italic>M, 132 <italic>μ</italic>M), and U-373 MG (71 <italic>μ</italic>M, 126 <italic>μ</italic>M). <italic>Troglitazone and the PPAR<italic>γ</italic> inactive </italic>Δ<italic>2-troglitazone
reduce TGF-<italic>β</italic><sub>1</sub> release at low micromolar doses</italic>: (b), (d) quantification
of TGF-<italic>β</italic>
<sub>1</sub> release by F98, SMA-560, U87-MG, and U-373 MG glioma cell
culture supernatants following TRO (b)
or Δ2-TRO (d) treatment for 48 hours. TGF-<italic>β</italic>
<sub>1</sub> protein levels in glioma cell culture supernatants were determined as described
in [##REF##17541035##9##] using the mouse/rat/porcine/canine or the human quantikine
TGF-<italic>β</italic>
<sub>1</sub> ELISA Kit (R&amp;D Systems, Minneapolis, Minn, USA), respectively. Each
experiment was repeated at least 3 times (<italic>n</italic> ≥ 3). Drug concentrations shown
to inhibit TGF-<italic>β</italic>
<sub>1</sub> release by 50% or 90%, respectively, were
determined by nonlinear regression data analysis: TRO: F98 (7 <italic>μ</italic>M, 11 <italic>μ</italic>M),
SMA-560 (8 <italic>μ</italic>M, 15 <italic>μ</italic>M), U-87 MG (8 <italic>μ</italic>M, 28 <italic>μ</italic>M), and U-373 MG (10 <italic>μ</italic>M, 30 <italic>μ</italic>M); Δ2-TRO:F98 (3 <italic>μ</italic>M, 5 <italic>μ</italic>M), SMA-560
(3 <italic>μ</italic>M, 8 <italic>μ</italic>M), U-87 MG (4 <italic>μ</italic>M, 14 <italic>μ</italic>M), and U-373 MG (4 <italic>μ</italic>M, 14 <italic>μ</italic>M). Δ<italic>2-Troglitazone
displays higher potencies than troglitazone</italic>. Using IC<sub>90</sub> concentrations
of Δ2-TRO and equimolar concentrations of TRO,
the PPAR<italic>γ</italic> inactive Δ2-TRO displays a significantly stronger
effect in both experimental paradigms (*** = <italic>P</italic> &lt; .001, <italic>t</italic>-test) (e), (f).</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>
<italic>The PPAR<italic>γ</italic> inactive</italic> Δ <italic>2-troglitazone (</italic>Δ<italic>2-TRO) inhibits glioma cell 
migration</italic>. The glioma cell
migration assay (Boyden chamber; QCM-FN Migration Assay, Chemicon, Temecula, Calif, USA) was performed as described recently [##REF##17541035##9##]. Briefly, F98 rat
glioma cells, pretreated with the test compound or solvent for 24 hours, were
transferred into each Boyden chamber. After 24 hours of incubation, cells which
migrated through the fibronectin-coated
chamber membranes (8 micron pore diameter) were quantified according to
the manufacturer’s protocol. Experiments were repeated 3 times (<italic>n</italic> = 3). (** = <italic>P</italic> &lt; .01; *** = <italic>P</italic> &lt; .001; <italic>t</italic>-test). Right panel: representative
microphotographs of F98 glioma cells which migrated though the
fibronectin-coated chamber membranes after treatment with Δ2-TRO (20 <italic>μ</italic>M) or solvent only.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>
Δ<italic>2-Troglitazone inhibits
glioma progression in an organotypic glioma transplantation model</italic>. (a) Organotypic hippocampal glioma
invasion assay was performed as described earlier [##REF##15871520##10##, ##REF##15857402##12##, ##REF##16731757##30##]. In brief, enhanced green
fluorescent protein (eGFP) positive F98 rat glioma cells were transplanted into
the entorhinal cortex of organotypic rat brain slice cultures one day after preparation. DAI
= days after implantation. DG = dentate gyrus. EC = entorhinal cortex. (b) Tumor progression was monitored by fluorescent
microscopy over the time course of 12 days. Quantification of the tumor
infiltration area at day 1 to day 12 after transplantation derived from 3
independent experiments is shown. For each experiment, the tumor infiltration
area at DAI 1 was defined as 100%. Data are given as mean ± SD percentage. At
DAI 12, the tumor infiltration area significantly increased to 448 ± 71 % (<italic>P</italic> = .002, <italic>t</italic>-test) in solvent-matched controls but
remained unchanged following Δ2-TRO treatment (75 ± 22 %; <italic>P</italic> = .18, <italic>t</italic>-test). Starting from DAI 2,
differences in tumor progression (TRO versus Δ2-TRO) reached
statistical significance (<italic>P</italic> &lt; .01, <italic>t</italic>-test) <bold>(c)</bold> A
continuous increase of the bulk tumor masses was observed in solvent-matched
controls while 10 <italic>μ</italic>M concentrations of Δ2-TRO effectively
blocked tumor progression. Right column: magnification of the indicated border
area between bulk tumor mass and rat brain tissue. In controls, F98 glioma
cells have diffusely migrated into the adjacent brain parenchyma, while a sharp
tumor border was observed following Δ2-TRO treatment (scale bar: 200 <italic>μ</italic>m).</p></caption></fig>
<graphic xlink:href="PPAR2008-513943.001"/> <graphic xlink:href="PPAR2008-513943.002"/> <graphic xlink:href="PPAR2008-513943.003"/>
[{"label": ["3"], "surname": ["Savaskan", "Heckel", "Hahnen"], "given-names": ["NE", "A", "E"], "article-title": ["Small interfering RNA-mediated xCT silencing in gliomas inhibits neurodegeneration \nand alleviates brain edema"], "italic": ["Nature Medicine"], "year": ["2008"], "volume": ["14"], "issue": ["6"], "fpage": ["629 "], "lpage": ["632"]}, {"label": ["29"], "surname": ["Demuth", "Rennert", "Hoelzinger"], "given-names": ["T", "JL", "DB"], "article-title": ["Glioma cells on the run-the migratory transcriptome of 10 human glioma cell lines"], "italic": ["BMC Genomics"], "year": ["2008"], "volume": ["9, article 54"]}]
PPAR Res. 2008 Sep 14; 2008:513943