Download Tetradecylthioacetic acid inhibits growth of rat

Carcinogenesis vol.22 no.11 pp.1747–1755, 2001
Tetradecylthioacetic acid inhibits growth of rat glioma cells
ex vivo and in vivo via PPAR-dependent and PPAR-independent
pathways
Kjetil Berge3,4, Karl J.Tronstad3, Esben N.Flindt1,3,
Thomas H.Rasmussen2, Lise Madsen, Karsten Kristiansen1
and Rolf K.Berge
Department of Clinical Biochemistry, Haukeland Hospital, University of
Bergen, N-5021 Bergen, Norway, 1Department of Biochemistry and Molecular
Biology, University of Southern Denmark, DK-5230 Odense M and
2Department of Environmental Medicine, Institute of Community Health,
University of Southern Denmark, DK-5230 Odense M, Denmark
3These
authors contributed equally to this work
4To
whom correspondence should be addressed
Email: [email protected]
The peroxisome proliferator-activated receptors (PPARs)
are transcription factors involved in fatty acid metabolism
and energy homeostasis. The PPARs also play crucial roles in
the control of cellular growth and differentiation. Especially,
the recently emerged concept of ligand-dependent PPARγmediated inhibition of cancer cell proliferation through
induction of G1-phase arrest and differentiation is of clinical
interest to cancer therapy. Tetradecylthioacetic acid (TTA)
is a sulphur-substituted saturated fatty acid analog with
unique biochemical properties. In this study, we investigated
the effects of TTA-administration on cell proliferation in
glioma cancer models. The rat glioma cell line BT4Cn,
whether grown in culture or implanted in rats, expressed
significant levels of PPARγ and PPARδ, with PPARγ being
the predominant PPAR subtype. In BT4Cn cells, TTA
activated all PPAR subtypes in a dose-dependent manner. In
cell culture experiments, the PPARγ-selective ligand
BRL49653 moderately inhibited growth of BT4Cn cells,
whereas administration of TTA resulted in a marked growth
inhibition. Administration of the PPARγ-selective antagonist
GW9662 abolished BRL49653-induced growth inhibition,
but only marginally reduced the effect of TTA. TTA reduced
tumor growth and increased the survival time of rats with
implanted BT4Cn tumor. TTA-induced apoptosis in BT4Cn
cells, and the administration of TTA led to cytochrome c
release from mitochondria and increased the glutathione
content in glioma cells. In conclusion, our results indicate
that TTA inhibits proliferation of glioma cancer cells through
both PPARγ-dependent and PPARγ-independent pathways,
of which the latter appears to predominate.
Introduction
Development of cancer is associated with dysregulation of cellcycle control, apoptosis and/or differentiation. Several members
of the family of nuclear hormone receptors (NHR) play crucial
roles in the control of cellular homeostasis, and administration
of their cognate ligands has successfully been used in cancer
Abbreviations: ANT, adenine nucleotide translocase; ∆ψ, mitochondrial
membrane potential; NHR, nuclear hormone receptor; PPAR, peroxisome
proliferator-activated receptor; PPRE, peroxisome proliferator response
element; PTP, permeability transition pore; RXR, retinoid-x receptor; TTA,
tetradecylthioacetic acid.
© Oxford University Press
treatment. Thus, retinoids have been used in the management of
leukemia (1), head and neck cancer (2) and skin cancer (3).
Recently, members of the peroxisome proliferator-activated
receptor (PPAR) subfamily of the NHRs, have been demonstrated to be intimately involved in the control of cellular
proliferation and differentiation, and accordingly, PPAR agonists
have been employed in the treatment of certain cancers (4,5).
The PPAR family comprises PPARα, PPARγ and PPARδ
(also called PPARβ, FAAR or NUC1). The PPARs bind as
heterodimers with retinoic-x acid receptor (RXR) to a subset
of DR-1 elements, peroxisome proliferator response elements
(PPREs), and have been shown to regulate expression of genes
involved in the transport, metabolism and storage of fatty
acids. Transcriptional activation of the PPAR–RXR heterodimers is enhanced upon binding of a large variety of ligands
including saturated and unsaturated fatty acids, arachidonic acid
derivatives and a wide range of synthetic drugs with different
subtype specificities [reviewed by (6–9)].
For the last 10 years, administration of peroxisome proliferators, PPARα agonists, has been known to induce hepatocarcinogenesis in rodents (10). However, cancer development is
probably induced by mechanisms secondary to PPARα transcriptional activation (9). A role in growth regulation for the two
remaining PPAR subtypes has also been suggested. Whereas upregulation of PPARδ expression has been associated with colon
cancer (11) and activation of PPARδ stimulates post-confluent
proliferation of pre-adipocytes (12,13), opposite effects on cell
proliferation are mediated by activation of PPARγ. This PPAR
subtype is well known for its role in adipocyte differentiation
(14). Furthermore, studies of different cancer cell lines have
demonstrated that administration of PPARγ ligands causes G1phase cell cycle arrest (15–18) and induces expression of
differentiation markers (19–22). Furthermore, PPARγ agonists
have been reported to induce apoptosis in certain settings
(23–25), supporting the idea that activation of PPARγ could be
of therapeutic value in cancer treatment.
For the last decade, our group has focused on the hypolipidemic properties of the synthetic fatty acid analog tetradecylthioacetic acid (TTA) (26). The hypolipidemic effect of TTA is
probably associated with the ability of TTA to activate PPARα
in the liver (27,28), as several genes containing PPREs are
activated upon TTA treatment of rats and hepatocytes in culture.
Recently, it has been shown that TTA is a ligand for all PPAR
subtypes (28–30) (L.Madsen, et al., submitted for publication).
It is well documented that the PPARα subtype plays a central role
in hepatic lipid metabolism, but the tissue-specific expression
patterns of the PPARs and the fact that PPARγ and PPARδ
probably play important roles in fatty acid metabolism in most
of these tissues, makes it probable that TTA could also act
through PPARs outside the liver. The new data concerning
PPARδ- and PPARγ-mediated regulation of proliferation make
an analysis of the effects of TTA even more interesting.
Glioma cancers are among the most aggressive cancers and
are difficult to treat with chemotherapeutic agents. The BT4Cn
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K.Berge et al.
cell line is a permanent rat glioma cell line, originally induced
in fetal rat brain cells after transplacental exposure to ethylnitrosourea (31). The BT4Cn cell line has been used to study the
potentials of anticancer drugs and gene therapy in the treatment
of glioma cancer (32,33). Whereas the stages of glioma growth
with the concomitant changes in morphological structure are
well characterized, relatively little is known about the underlying molecular events or the potential of intervention through
transcription factors involved in growth regulation. In this study,
we investigated the ability of TTA to affect glioma cell proliferation ex vivo and in vivo. Our results indicate that TTA inhibits
BT4Cn growth by PPARγ-dependent and PPARγ-independent
pathways, and that TTA induces apoptosis, possibly via release
of cytochrome c from the mitochondria.
Materials and methods
Chemicals
Chemicals used were: TTA (prepared at the Department of Chemistry, University
of Bergen, Norway), as described previously (34), Wy14,643 (Calbiochem, San
Diego, CA), BRL49653 (kindly provided by Novo-Nordisk A/S, Bagsvaerd,
Denmark), L-165041 (kindly provided by Merck Research Laboratories,
Darmstadt, Germany) (35) and GW9662 (kindly provided by Glaxo Wellcome
Research and Development, Uxbridge, UK) (36). Chemicals were dissolved in
dimethyl sulfoxide (DMSO) and added to a final concentration of 0.1% DMSO
in the culture medium. All other chemicals and solvents were of reagent grade
from common commercial sources.
Cell culture
The continuous neoplastic cell line BT4Cn were obtained from fetal rat brain
cells (31). The cells were grown at 37°C, 5% CO2 and 95% air in Dulbecco’s
modified Eagle medium [DMEM, high glucose (4.5 g/l)] (Gibco) supplemented
with 10% fetal calf serum (Sigma, St. Louis, MO), streptomycin (100 µg/ml)
and penicillin (62.5 µg/ml). In apoptosis and cytochrome c release experiments,
cells were grown in DMEM supplemented with 10% new born calf serum,
streptomycin (100 µg/ml), penicillin (62.5 µg/ml) and three times the prescribed
concentration of non-essential amino acids (all from Sigma, St. Louis, MO).
Media was changed routinely every 2 days and cells were passaged by
trypsinization before confluence. When carrying out growth experiments, cells
were plated at low density (~5% confluence) and allowed to settle for 4 h
before the medium was changed to medium containing relevant chemicals.
Animal studies
The experiments described have been carried out according to the rules of
the Norwegian Animal Research Authority (NARA). Male Norwegian brown
rats, BD-IX, were obtained from the Gades Institute, Haukeland Hospital,
Bergen, Norway. They were housed in cages in pairs, and maintained on a
12 h cycle of light and dark at a temperature of 20 ⫾ 3°C. During the
experiments they weighed 250–400 g. They were fed a commercial standard
pelleted food and provided tap water ad libitum. Fatty acids were administered
by oro-gastric intubation. TTA and palmitic acid were dissolved in 0.5%
(w/v) sodium carboxymethyl cellulose at a final stock solution of 75 mg/ml.
The animals were administered once a day at a dose of 300 mg/kg body wt,
and the feeding was started the day after tumor implantation.
The tumors were implanted subcutaneously or intracranially using stereotactical techniques (37). Before surgery the rats were anaesthetized with
0.2 ml Hypnorm-Dormicum/100 g body wt. For subcutaneous implantation,
a tumor was established in vivo by subcutaneous injection of 5⫻106 tumor
cells in the rat’s neck. After ~2 weeks, the tumor was removed and cut into
2⫻2 mm tissue pieces and subcutaneously implanted in the leg. The tissue
piece was entered and established 1 cm below the skin incision. The tumor
sizes were measured daily and tumor tissue was collected and frozen in liquid
nitrogen after ~2 weeks. The intracranially implanted rats were killed when
neurological signs were evident.
Growth assays
DNA amounts were determined by fluorometry. Cell samples were harvested
in triplicate by trypsinization and dissolved in 1 ml high salt harvest buffer
(10 mM Tris, 10 mM EDTA, 2 M NaCl, pH 7.4). Samples were sonicated
and properly diluted in high salt buffer (2 mM Tris, 2 mM EDTA, 2 M NaCl,
pH 7.4) and the DNA amount was measured on a Hoefer DyNA Quant 200
in high salt buffer containing 0.1 µg/ml Hoechst 33258.
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Plasmids
The PPREx3-tk-luc reporter construct containing three copies of the PPRE
from the acyl-CoA promoter (38) was kindly provided by R.Evans. Expression
vectors encoding murine PPARα (pSG5–PPARα) (kindly provided by
J.D.Tugwood) (6), murine PPARδ (pSG5–PPARδ) (kindly provided by
by P.Grimaldi) (39), murine PPARγ2 (pSPORT–PPARγ2) (kindly provided
B.Spiegelman) (40), and murine RXRα (pCMXRXRα) (kindly provided by
R.Evans) (41) have been described previously.
Transient transfection
BT4Cn cells were plated in 6-well plates 24 h before transfection. One hour
prior to the transfection the medium was changed. Each well was transfected
with 3 µg reporter plasmid DNA, 1 µg PPAR expression vector or pSG5 and
1 µg of β-galactosidase control plasmid by the calcium–phosphate method.
For transfections with PPARδ, 1 µg RXRα-vector was included. A total
amount of 6 mg plasmid DNA/well was used. Five hours later the cells were
shocked with 12.5% glycerol in phosphate-buffered saline (PBS) pH 7.3 at
room temperature, and fresh media containing 10% (v/v) resin-charcoalstripped calf serum (AG-1X-8 resin, Bio-Rad, Richmond, CA; Activated
Powder Charcoal, Bie & Berntsen, Roedovre, Denmark) was added together
with the indicated compound or vehicle (DMSO) alone. After 24 h, cells were
harvested and the luciferase and β-galactosidase activities measured in a
Berthold MicroLumat LB96P luminometer using the commercial kit (GalactoLight, Applied Biosystems, Foster City, CA). Luciferase values were
normalized to the β-galactosidase values.
RT–PCR
Total RNA was purified, reverse transcribed (M-MLV Reverse Transcriptase
kit, Life Technologies, Carlsbad, CA) and selected cDNA amplified by 25 or
28 cycles of PCR as described previously by Hansen et al. (42). TBP served
as internal standard. The following primers were used: TBP: 5⬘-ACCCTTCACCAATGACTCCTATG-3⬘ and 5⬘-ATGATGACTGCAGCAAATCGC–
3⬘; PPARα: 5⬘-CCCTGCCTTCCCTGTGAACTGAC-3⬘ and 5⬘-GGGACTCATCTGTACTGGTGGGGAC-3⬘; PPARδ: 5⬘-GCTATGACCAGGCCTGCAGG-3⬘ and 5⬘-CCCTTGCACCCCTCACACGCGTC-3⬘; PPARγ: 5⬘-GAGCTGA- CCCAATGGTTGCTG-3⬘ and 5⬘-GCTTCAATCGGATGGTTCTTC-3⬘.
Fragments were separated on 6% polyacrylamide gels and bands were
quantified by phosphoimager (Molecular Dynamics, Sunnyvale, CA) analysis.
Western blotting
One hundred micrograms of whole cell protein in SDS sample buffer
(50 mM Tris–HCl pH 6.8, 10% glycerol, 2.5% SDS, 10 mM DTE, 10 mM
β-glycerophosphate, 10 mM NaF, 0.1 mM Na orthovanadate, 1 mM PMSF,
1⫻ Complete™) were separated by SDS–PAGE (10% gels) and blotted onto
a PVDF membrane (MSI). Equal loading and transfer were confirmed by
Amido black staining. PPARγ was detected by enhanced chemiluminescence
using mouse anti-PPARγ antibody (E-8, Santa Cruz Biotechnology, Santa Cruz,
CA) and goat horseradish peroxidase-conjugated antimouse antibody (DAKO).
Glutathione assay
Cells suspended in distilled H2O were extracted with equal volumes of cold
5% sulfosalicylic acid with 50 µM dithioerythriol before they were placed
at –80°C. Sample preparation and HPLC-analysis was performed according
to the method described by Svardal et al. (43).
Determination of cytochrome c
The cells were harvested by scraping and washed in PBS before they were
homogenized in H-buffer (0.25 M sucrose, 2 mM HEPES, 0.2 mM EGTA,
pH 7.40) using a ball-bearing homogenizer (44) (EMBL, Heidelberg, Germany).
Cell debris was removed by centrifugation at 1930 g for 15 min. The supernatant
(total homogenate) was centrifuged at 80 000 g for 90 min for preparation of
cytosolic (supernatant) fraction. Cytochrome c was determined using a rat/mouse
cytochrome c immunoassay provided by R&D Systems, Minneapolis, MN.
Fluorescence microscopy
For microscopic analysis of apoptosis, cells were grown in 8-well chamber slides
for 6 days. The cells were fixed in 4% formaldehyde over night and incubated
for 30 min with 3 µg/ml Hoechst 33342 in the dark. The cells were analysed in
a Leica Orthoplan fluorescence microscope.
Statistics
All fluorometry measurements were performed at least in triplicate and repeated
a minimum of two times. Data were then normalized to the control of each
experiment. To obtain a measure of dispersion for the overall mean of the control
group, the weighed average of the sample standard deviation (SD) from all
control experiments were used. A Dunnet two-tailed multiple comparison test
TTA inhibits growth of rat glioma cells
Figure 1. Effect of TTA (A) and palmitic acid (B) on DNA accumulation in
BT4Cn cells. Cells were exposed to fatty acids (10, 30 or 100 µM) or
vehicle (DMSO) for 8 days. DNA was determined by fluorometry, and the
results represent measurement on 10 (TTA) or 2 (palmitic acid) independent
experiments. DNA [means ⫾ SD (n ⫽ 2–10)] is presented as percent of
DMSO-control; *P ⬍ 0.01.
were applied to test for equality of means of each sample compared with the
control at the significance level α ⫽ 0.05 and α ⫽ 0.01.
Results
Inhibition of glioma cell growth
TTA has chemical and physical properties almost identical to
normal saturated fatty acids, but TTA cannot undergo βoxidation (45). Figure 1 shows that the presence of 100 µm TTA
reduced the accumulation of DNA by ~45% in rat BT4Cn glioma
cells after 8 days of treatment, whereas the control (palmitic
acid) had no effect on DNA accumulation. These data are in
agreement with previous findings showing that TTA reduced
[3H]thymidine incorporation in and reduced proliferation of both
rat and human glioma cells (46).
Expression of PPARs in rat glioma BT4Cn cells
We have recently shown that TTA activates human PPARα and
PPARγ in a concentration-dependent manner (28). Activation of
PPARγ has been associated with growth inhibition in several
experimental systems and hence, the decreased BT4Cn cell
proliferation could be mediated via PPAR activation. Using
semi-quantitative RT–PCR analysis, RNA expression of the
different PPARs in BT4Cn cells was assayed. Expression of
PPARα mRNA was at the detection limit, whereas a significant
expression of PPARδ and especially PPARγ mRNA was
observed (Figure 2A). We observed a marked 3-fold increase in
the level of PPARγ mRNA at confluence. By 4 days of postconfluent growth, the level of PPARγ mRNA had declined to a
level ~2-fold above that observed in exponentially growing cells.
Expression of PPARδ followed a similar pattern. However, the
magnitude of alterations varied from experiment to experiment,
and the differences in expression levels were not statistically
significant. Expression of PPARδ and PPARγ mRNA in
confluent cells was approximately one-third to one-fourth of that
observed in white adipose tissue, a tissue with known high
expression of PPARγ, and a lower but still significant level of
PPARδ expression (47). Western blotting was used to examine
PPARγ expression at the protein level. As shown in Figure
2C, the PPARγ protein level paralleled the PPARγ mRNA
expression profile (Figure 2A and B).
Transactivation of PPARs in rat glioma cells
Several recent reports have focused on the involvement of
PPARγ in the inhibition of cancer cell growth (20,48,49). In
contrast, overexpression of PPARδ has been linked to the
development of colorectal cancers (11), and in addition,
activation of PPARδ induces clonal expansion of pre-adipocytes
(12). It is known that ligand-dependent PPAR-mediated
transactivation exhibits cell type specificity. To examine how
PPAR subtype-selective ligands and TTA-induced PPARdependent transactivation, BT4Cn cells were cotransfected with
the PPAR responsive PPREx3-TK-luc reporter construct and
PPARα-, PPARγ- or PPARδ-expression vectors and then treated
with PPAR-selective agonists or TTA. Figure 3A shows that
cotransfection with vectors expressing either PPAR subtype led
to a significant transactivation of the reporter even in the absence
of added ligand. Addition of Wy14,643 only very modestly
enhanced PPARα-mediated transactivation. More pronounced
and dose-dependent induction of PPARδ- and PPARγ-mediated
transactivation was observed with the PPARδ-selective ligand
L165041 and the PPARγ-selective ligand BRL 49653,
respectively. Interestingly, TTA activated all PPAR subtypes in
a dose-dependent manner. We have shown previously that TTA
most potently activates human PPARδ- and human PPARαmediated transactivation, and that higher concentrations of TTA
were required to achieve significant activation of human PPARγ
(28,30). In the present study using rodent (mouse) PPARs, a
similar trend is apparent in that 100 µM TTA was needed to
enhance PPARγ-mediated transactivation, whereas 30 µM TTA
slightly enhanced PPARα- and PPARδ-dependent transactivation suggesting that TTA activates rodent PPARs in BT4Cn
glioma cells in the ranking order PPARα 艌 PPARδ ⬎ PPARγ
(Figure 3A).
To examine the transactivation potential of endogenously
expressed PPAR subtypes, BT4Cn cells were transiently transfected with the PPAR responsive PPREx3-TK-luc reporter
plasmid, and treated with Wy14,643, L-165041, BRL 49653 or
TTA using the concentrations shown to significantly enhance
PPAR-mediated transactivation. In all cases, addition of ligands
only slightly enhanced transactivation possibly reflecting the
low levels of endogenous PPARs present in the BT4Cn glioma
cells (Figure 3B). Of the PPAR subtype-selective ligands, we
found that BRL49653 most efficiently enhanced transactivation
in keeping with PPARγ being the predominant PPAR subtype in
BT4Cn cells.
Effect of PPAR ligands on BT4Cn growth
To investigate how PPAR activation affected BT4Cn proliferation, we carried out a series of experiments to determine the effect
of PPAR-selective ligands. As a measure of cell proliferation we
determined the accumulation of DNA in the cultures. Figure 4A
and B show that Wy14,643 and L-165041, at concentrations
sufficient to activate PPARα and PPARδ, respectively, did not
affect the accumulation of DNA after 8 days of treatment. However, BRL49653 (Figure 4C) reduced the DNA accumulation to
~85% of the control. Low concentrations of L-165041 were
found to increase growth, supporting the notion that activation
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K.Berge et al.
Figure 2. Expression of PPAR mRNA and protein in BT4Cn cells ex vivo. (A) Expression of PPAR subtype mRNAs was determined by RT–PCR using TBP
as internal standard (PPARα and PPARδ: 28 cycles; PPARγ: 25 cycles). (B) Phosphoimager quantitated RT–PCR profiles from two independent experiments
of PPAR mRNA expression normalized to TBP. (C) Western blot analysis of PPARγ expression. Equal loading and transfer were assessed by amido black
staining and probing for TBP. Cells were harvested during exponential growth 2 days after seeding (pre-confluence), at confluence 4 days after seeding
(confluence) and at 8 days after seeding (post-confluence). exp, Exponential phase (pre-confluence); confl, confluent phase; post, post-confluent phase;
rliver, rat liver; mbrain, mouse brain; mWAT, mouse white adipose tissue; 3T3-L1, 3T3-L1 adipocytes.
of PPARδ induces proliferation. The diminished effect observed
by 1 µM L-165041 may relate to the ability of L-165041 to
activate PPARγ (35). In conclusion, of the PPAR-selective
ligands only BRL49653 was able to inhibit the growth of rat
BT4Cn cells, but notably TTA was a much more potent inhibitor
of growth than BRL49653.
The transfection analysis showed that 100 µM TTA activated
all three PPAR subtypes (Figure 3). Consequently, we analysed
whether combinations of PPAR-selective ligands led to a more
pronounced inhibition of proliferation than either ligand alone.
None of the employed combinations of PPAR-selective ligands
reduced growth beyond that observed with BRL49653 alone
(Figure 4D). Similarly, simultaneous addition of TTA and the
PPAR-selective ligands did not result in an inhibition of growth
exceeding that observed with TTA alone (Figure 4E).
The results described above showed that BRL49653 and TTA
both reduced growth with TTA being by far the most potent
inhibitor. As TTA at 100 µM induced PPARγ-mediated transactivation, we decided to investigate to what extent PPARγmediated processes contributed to TTA-induced growth arrest.
To this end, we took advantage of the PPARγ antagonist
GW9662. In separate experiments we have shown that transactivation by 1 µM BRL49653 is completely abolished by the
addition of 10 µM GW9662 (data not shown). Administration
of 1 mM or 10 µM GW9662 did not affect the growth of BT4Cn
cells. As described above, 1 µM BRL49653 modestly inhibited
growth, whereas the BRL49653-mediated inhibition was
neutralized by the addition of 10 µM GW9662. Administration
of TTA reduced DNA accumulation to 55% of the control, and
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the simultaneous addition of the PPARγ antagonist only restored
the DNA accumulation to 65% of the control (Figure 4F). Thus,
PPARγ-dependent processes contribute to TTA-mediated
growth inhibition. However, the contribution of PPARγdependent pathways is clearly of minor importance in TTAmediated arrest of BT4Cn cell proliferation.
TTA-induced apoptosis in BT4Cn cells
We then investigated whether apoptosis contributed to the
reduction in DNA accumulation observed upon treatment of
rat BT4Cn cells with TTA. Figure 5 shows that 200 µM TTA
induced massive apoptosis in BT4Cn cells, as determined by
chromatin condensation. When exponentially growing BT4Cn
cells were treated with increasing concentrations of TTA we
observed a dose-dependent increase in cells exhibiting morphological changes, including chromatin condensation, associated
with apoptosis. Thus, following treatment for 24 h with 50, 100
and 200 µM TTA, 11%, 14% and 83% of the cells, respectively,
appeared apoptotic. Less than 5% of the control cells showed
morphological apoptotic characteristics.
Cytochrome c release from mitochondria, and subsequent
activation of caspases are closely associated with induction and
execution of the apoptotic process. Interestingly, TTA-induced
apoptosis and increased considerably the release of cytochrome
c from mitochondria in BT4Cn cells (Figure 6).
Activation of apoptosis is associated with generation of reactive oxygen species (ROS), and TTA may activate mitochondrial
ROS production as it affects the mitochondrial function (50).
Furthermore, mitochondrial loss of cytochrome c stimulates
TTA inhibits growth of rat glioma cells
Figure 3. The effect of TTA and PPAR-selective ligands on PPAR-mediated
transactivation in BT4Cn cells. (A) BT4Cn cells were transiently transfected
with the PPREx3-TK-luc reporter plasmid and the indicated PPAR
expression vector and grown in the presence of PPAR-selective ligands,
TTA or vehicle (DMSO) for 24 h. Ligands used were: Wy14,643 (10, 30
and 100 µM), BRL49653 (0.1, 0.5 and 1 µM), L-165041 (0.1, 0.5 and
1 µM) and TTA (10, 30 and 100 µM). Background activity from cells
transfected with PPREx3-TK-luc reporter only and treated with vehicle is
indicated by ‘-’. (B) BT4Cn cells were transiently transfected with the
PPREx3-TK-luc reporter plasmid, and incubated in the presence or absence
of PPAR-selective ligands and TTA for 24 h. Ligands used were: Wy
(Wy14,643) (100 µM), BRL (BRL49653) (1 µM), L (L-165041) (1 µM)
and TTA (100 µM). Results are presented as β-galactosidase-normalized
luciferase activity ⫾ SD. Similar results were obtained in at least three
separate experiments, each performed in duplicate.
Figure 5. Apoptotic effect of TTA on BT4Cn cells. Cells were treated with
vehicle (medium) (A) and 200 µM TTA (B) for 3 days. The cells were
incubated with Hoechst 33342 and condensed chromatin was detected by
fluorescence microscopy.
Figure 6. Cytochrome c release from mitochondria. Cells were grown in the
presence or absence of TTA (200 µM) for 18 h. Cytochrome c [means ⫾
SD (n ⫽ 2)] in the cytosolic fraction was measured as described in
‘Materials and methods’, and is presented as ng/mg protein.
Figure 4. The effect of PPAR ligands on BT4Cn cell growth. Cells were
treated with the PPAR ligands Wy14,643 (10, 30 and 100 µM) (A),
L-165041 (0.1, 0.5, 1 µM) (B), BRL49653 (0.1, 0.5, 1 µM) (C), BRL49653
(1 µM), Wy14,643 (100 µM), L-165041 (1 µM) (D), Wy14,643 (100 µM),
L-165041 (1 µM), BRL49653 (1 µM) (E) or TTA (100 µM), BRL49653
(1.0 µM) and/or the PPARγ-selective antagonist GW9662 (1 and 10 µM)
(F), as indicated for 8 days. DNA [means ⫾ SD (n ⫽ 3)] was determined
by fluorometry and is presented as percent of DMSO-control. *P ⬍ 0.01.
mitochondrial superoxide production (51). Thus, ROS and the
resulting cellular redox change can be a part of the signal transduction pathway leading to apoptosis. Interestingly, treatment
with 100 µM TTA increased the cellular content of glutathione
(Table I). However, when compared with 100 µM TTA, treatment
with 200 µM TTA gave a higher degree of apoptosis and resulted
in a reduced cellular content of glutathione. These data indicate
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K.Berge et al.
Table I. Effect of TTA on the glutathione content in BT4Cn cells
Treatment
Glutathione nmol/mg protein
Control
50 µM TTA
100 µM TTA
150 µM TTA
200 µM TTA
0.99
1.83
3.54
5.68
2.77
⫾
⫾
⫾
⫾
⫾
0.74
0.44
0.99
0.77
1.85
Cells were grown in the presence or absence of TTA (50, 100, 150 and
200 µM) for 6 days. Glutathione contents [means ⫾ SD (n ⫽ 3)] in total
cell homogenate were measured by HPLC analysis, and are presented as
nmol/mg protein.
that TTA treatment may elicit compensatory adjustments in order
to counteract changes in the cellular redox state associated with
cytochrome c release and apoptosis.
The effect of TTA on tumor growth in vivo
To test whether TTA would also affect growth of glioma cells in
a setting resembling the conditions for an in vivo glioma, rats
were intracranially implanted with BT4Cn tumors. First, we
examined the expression of PPARs in implanted tumors. Figure
8 shows that PPAR subtype expression in implanted tumors
paralleled that observed for BT4Cn cells grown in culture
(Figure 2). PPARα expression was at the detection limit and
PPARγ was clearly the predominant PPAR subtype expressed in
the tumors. In rats with intracranially implanted BT4Cn tumors,
TTA increased the survival time compared with an equal dose
of palmitic acid (Figure 7A). Average survival time was
16.9 ⫾ 1.8 and 14.6 ⫾ 1.2 days for TTA and palmitic acid fed
animals, respectively. In another set of in vivo experiments, TTA
was shown to inhibit the growth of subcutaneously implanted
BT4Cn tumors (Figure 7B).
Discussion
In the present study we show that TTA is able to inhibit rat
glioma cell growth ex vivo and in vivo. PPARγ is the predominant
PPAR subtype expressed in cultured BT4Cn cells and in intracranially implanted BT4Cn tumor cells (Figures 2A and 8). Of
the PPAR subtype-selective ligands, only BRL49653 inhibited
the growth of BT4Cn cells ex vivo, an effect that was abolished
by the PPARγ antagonist GW9662 (Figure 4F). These data
indicate that only PPARγ mediated ligand-induced inhibition
of growth in BT4Cn cells. TTA was a far more potent inhibitor
of growth than BRL49653 (Figure 4D and E). The potential of
TTA as a PPARγ ligand was confirmed in transfection experiments (Figure 3A and B). Treatment with TTA in combination
with BRL49653 did not lead to additional inhibition of growth
(Figure 4E), indicating that TTA could fully exploit the
potential of PPARγ activation in growth inhibition. However,
the irreversible PPARγ antagonist GW9662 did only partly
reduce the TTA-mediated growth arrest, clearly demonstrating
the existence of PPARγ-independent pathways in the antiproliferative activity of TTA. Moreover, our data show that
PPARγ-independent pathways predominate in TTA-mediated
growth inhibition.
BRL49653 reduced the accumulation of DNA in glioma
cells and modestly induced apoptosis (data not shown). TTA
increased the level of apoptosis in confluent BT4Cn cultures
(Figure 5). Thus, our results indicate that ligand-mediated
activation of PPARγ elicited an apoptotic response, which
contributed to the observed reduction in DNA accumulation
1752
Figure 7. (A) Effect of TTA and palmitic acid on the survival of rats with
intracranially implanted BT4Cn tumors. Rats were fed TTA (- - -) or
palmitic acid (–) at a dose of 300 mg/day/kg body weight for 17 ⫾ 4 days.
Fatty acids were administered by oro-gastric intubation. Treatment was
started the day after implantation. The values are presented as percent
survival of n ⫽ 8–9 rats in each group. (B) Effect of TTA and palmitic acid
on the growth of subcutaneously implanted BT4Cn tumors. Rats were fed
TTA (j) or palmitic acid (m) at a dose of 300 mg/day/kg body weight for
14 days. Fatty acids were administered by oro-gastric intubation. The values
are presented as mean tumor volume (mm3) ⫾ SD (n ⫽ 10). *P ⬍ 0.05
compared with PA control.
in treated BT4Cn cells. The PPARγ-mediated induction of
apoptosis may relate to the well-established negative cross talk
between PPARγ and NF-kB. Administration of PPARγ ligands
have been shown to inhibit NF-κB activity thus quenching
anti-apoptotic signals (52–55). However, our results also suggest that administration of TTA activates additional PPARγindependent apoptotic pathways.
The induction of apoptosis observed in response to administration of TTA was paralleled by a substantial mitochondrial
release of cytochrome c into the cytosol (Figure 6). Cytosolic
cytochrome c is a pro-apoptotic factor that induces caspasedriven apoptotic cascades (56,57). The exact mechanism behind
the TTA-mediated release of cytochrome c is unknown.
However, millimolar concentrations of long-chain 3-thia fatty
acids have been shown to uncouple oxidative phosphorylation
by a fatty acid cycle mechanism resulting in dissipation of the
mitochondrial membrane potential (∆ψ) (50). Furthermore, it
has been shown that long-chain fatty acids interact directly
with and induces the opening of the mitochondrial permeability
transition pore (PTP) in a Ca2⫹-dependent manner (58,59).
The mitochondrial adenine nucleotide translocase (ANT) seems
to be central to the protonophoric activity of long-chain fatty
acids (60). ANT is assumed to be part of the PTP (61,62),
TTA inhibits growth of rat glioma cells
brain barrier. As TTA may provide non-toxic therapy against
human glioma cancers, TTA becomes a candidate for a new
class of potent drugs in the treatment of glioma cancers.
Acknowledgements
We thank Bjorn Netteland for technical help. This work was supported by the
University of Bergen, the Norwegian Research Council, the Norwegian Cancer
Society, the Danish Biotechnology Program, the Danish Natural Science
Research Council, the Danish Cancer Society and the Novo Nordisk Foundation. We are grateful to Novo Nordisk, Merck Research laboratories and
Glaxo Welcome for providing ligands.
References
Figure 8. Expression of PPAR mRNA in intracranial BT4Cn implants.
RT–PCR profiles of PPAR subtype mRNA using TBP as internal standard
(25/28 cycles). PA, palmitic acid treatment; TTA, TTA treatment (n ⫽ 2).
and it has been reported that ANT is inhibited by long-chain
3-thia fatty acids (50). Mitochondrial permeability transition
is considered to be a critical and rate-limiting event in apoptosis
and can be triggered by oxidative stress, ADP deficiency and
dissipation of ∆ψ. It is conceivable that interactions with ANT,
dissipation of ∆ψ and triggered generation of reactive oxygen
species are associated with increased release of cytochrome c,
and thus play important roles in TTA-induced apoptosis.
Inhibition of mitochondrial ANT and release of cytochrome
c into cytosol is thought to stimulate production of reactive
oxygen species (51,63). Therefore, the increased level of
glutathione detected in BT4Cn cells treated with low doses of
TTA (Table I) may represent an adaptation to the altered redox
conditions in order to escape from cell death. In this notion,
increasing the TTA dose to 200 µM apparently led to changes
that were beyond the cellular adaptive capacity as a higher
level of apoptosis was observed in these cultures. Furthermore,
the concomitant reduction in glutathione content in these
cells is in accordance with previous reports showing loss in
glutathione during apoptosis (51,64). We suggest that the
mitochondrial release of cytochrome c and altered cellular
redox conditions following TTA treatment may well be a PPAR
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The limited therapeutic possibilities for the treatment of
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of new and selective drugs and the search for molecular targets
of relevance to cancer therapy. Studies in our laboratory have
shown that 1-14C-labeled TTA is found in rat brain after
intravenous administration, and thus is able to cross the blood–
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Received February 1, 2001; revised May 22, 2001; accepted June 22, 2001
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