Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
Hepatocellular carcinoma (HCC) affects more than 500
000 people worldwide annually, and the 5-year mortality
exceeds 95%. More than half of those people are in
China[1]. Present measures, including mainly surgical resection, embolization
therapy, ethanol injection, and microwave coagulation
therapy, delay or temporarily control progress, but do not
eradicate HCC. Therefore, a new molecular target for
anticancer therapy in HCC is needed.
Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear hormone receptor that mediates the
transcriptional regulation of genes. The PPARγ gene is
located in human chromosome 3p25; PPARγ includes 4
isoforms: PPARγ1, PPARγ2, PPARγ3, and PPARγ4, which are
widely distributed[2]. The PPARγ protein forms a heterodimer
with retinoid X receptor a, and the complex subsequently
binds to a specific DNA sequence designated as the
peroxisome proliferating response element (PPRE), which is located
in the promoter region of PPARγ targeted genes and
modulates their transcription[3]. Recently, the role of
PPARγ in tumors has been extensively studied, and
PPARγ agonists have been shown to have direct effects on tumor cells,
including breast, colon, lung, stomach, and liver
cancer[4,5]. Thiazolidinedione (TZD) groups are synthetic ligands of
PPARγ, including troglitazone, rosiglitazone, pioglitazone,
and ciglitazone, which have been used widely in patients
with insulin-resistant diabetes mellitus. Of note, the highest
affinity for PPARγ in the TZD is
rosiglitazone[6]. It has been reported that rosiglitazone inhibits the cell proliferation and
colony formation via inducing apoptosis in MCF-7 breast
cancer cells[7]. However, those effects of rosiglitazone on
hepatoma cells have been poorly understood until now.
The phosphatase and tensin homologue deleted on
chromosome 10 gene (PTEN) is a major tumor suppressor gene
located on human chromosome 10q23.3. The PTEN protein dephosphorylates inositol phospholipid intermediates
of the phosphatidylinositol 3-kinase (PI3K) pathway and
inhibits the activation of downstream targets including Akt.
It has been suggested that the loss of PTEN could
stimulate cell proliferation, reduce apoptosis, and induce tumor
angiogenesis[8,9]. Previous studies have shown that
inactivated PTEN was detected in HCC for its loss, mutation, or
low expression[10], and PPARγ activation by rosiglitazone
could upregulate the transcription of PTEN in colorectal
and breast cancer cells[11]. However, how rosiglitazone
affects PTEN in HCC has not been elucidated.
Thus, we hypothesized that the upregulation of PTEN
by rosiglitazone could inhibit tumor cell growth, which might
be one of the anticancer mechanisms. To verify this
hypothesis, the present study investigated the effects of
rosiglitazone on the PTEN expression and cell growth in HCC,
as well as the underlying mechanisms.
Materials and methods
Reagents Rosiglitazone was purchased from Cayman
Chemical Company (Ann Arbor, MI, USA). GW9662, LY294002, and Z-VAD-FMK were obtained from Sigma
Chemical Co (St Louis, MO, USA) and were dissolved in 0.1%
DMSO. Mouse monoclonal anti-human PTEN, rabbit polyclonal anti-human phosphorylated-Akt (Ser473) and Akt,
and horseradish peroxidase-conjugated goat
anti-mouse/rabbit IgG secondary antibody were provided by Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
Cell culture The human HCC cell line Hep3B was kindly
donated by Prof Li WEN (Surgical Laboratory Department
of Sun-Yat-Sen University, Guangzhou, China). The cells
were maintained in the Dulbecco's modified Eagle's medium
(DMEM, Gibco BRL, Grand Island, NY, USA) containing 10%
(v/v) fetal bovine serum (Bio-Whittaker, Walkersville, MD,
USA), penicillin (100 U/mL), and streptomycin (100 mg/L).
The cells were maintained at 37 °C in an incubator with a
humidified atmosphere of 5% CO2.
RNA isolation and RT-PCR The total cellular RNA was
extracted using TRIzol reagent (Sigma-Aldrich, USA)
according to the manufacturer's instructions. Reverse
transcription with oligo (dT) primer was used to generate cDNA from
the total RNA extracts. The PTEN and β-actin genes were
amplified using specific sets of primers. The primers for the
human PTEN gene were: sense 5'-AGT TTG TGG TCT GCC
AGC TA-3', and antisense 5'- TCA GAG TCA GTG GTG TCA GA-3' (470 bp). The primers for the human
b-actin gene as an internal control were: sense 5'-GTG GAC ATC
CGC AAA GAC-3', antisense 5'-GAA AGG GTG TAA CGC AACT-3' (303 bp). The PCR cycle was as follows: 94 °C
for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C
for 1 min using the TaKaRa Bio Kit (Osaka, Japan)
according to the manufacturer's recommendations with some
modifications, and was repeated for 28 cycles. The
amplified product was loaded onto 15 g/L agarose gel containing
ethidium bromide (0.5 g/L) and quantified by
densitometry using the Image Master VDS system and associated
software (Pharmacia, Pfizer, NY, USA).
Western blotting analysis The Hep3B cells were rinsed
twice with cold PBS buffer, and lysed in an ice-cold lysis
buffer including 0.1% SDS, 150 mmol/L NaCl, 50 mmol/L
Tris-HCl (pH 7.5), 1% Nonidet P-40, and a protease inhibitor
cocktail (Boehringer Mannheim, Lewes, UK) for 30 min at 4 °C.
The supernatant was collected by centrifugation at 15 000×
g for 20 min. The protein concentration was determined with
Coomassie brilliant blue G-250 (Aldrich Chemical, Milwaukee,
WI, USA). The cell extracts (50 µg/lane) were separated via
SDS-PAGE and electrotransferred to polyvinylidene fluoride
membranes (Immobilon, Bedford, MA, USA). After
blocking in the blocking buffer (20 mmol/L Tris-HCl, pH 7.6, 150
mmol/L NaCl, 0.1 % Tween-20, and 5% non-fat dry milk), the
membranes were incubated with primary antibodies
overnight at 4 °C and then incubated with horseradish
peroxidase-conjugated secondary antibody for 1 h. The blots were
developed using an enhanced chemiluminescence detection
system (Amersham Pharmacia Biotech, Piscataway, NJ, USA)
according to the manufacturer's instructions.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay for cell viability The status of cell
growth was determined by MTT (Amresco, Solon, OH, USA)
assay. Briefly, exponentially growing cells were diluted to a
concentration of 2.5×104 cells/mL with DMEM, planted in
96-well plates (Corning Inc, Corning, NY, USA) with 200
μL/well, and then routinely incubated for 12 h. After being
treated with drugs and incubated for 48 h (triplicate wells
for each sample), the cells were exposed to 20 µL/well MTT
(5 g/L) The medium was then removed after being
incubated for 4 h, and DMSO (200 µL/well) was added to
dissolve the formazan product. Finally, the plate was read in
enzyme-linked immunity implement (Bio-Rad 2550, Hercules, CA, USA) at 570 nm.
Analysis of DNA fragmentation DNA fragmentation was
assessed using a modification of a previously described
method[12]. Briefly, the Hep3B cells were treated
with rosiglitazone at the indicated concentrations and time points,
washed with PBS twice, collected, and lysed for 30 min on
ice in 400 μL lysis buffer (10 mmol/L Tris-HCl, 10 mmol/L
EDTA, and 10 mmol/L NaCl, 1% SDS, pH 8.0). Then an
aliquot of the cell lysate was removed for the determination of
protein and DNA content. The lysate was treated with 100
µL of proteinase K (500 mg/L) and incubated on ice for 15
min. RNase10 μL (200 mg/L) was added to react for 1 h at
50 °C. DNA was extracted using phenol:chloroform (1:1,
v/v) and then loaded onto 1.5% agarose gel containing ethidium
bromide (500 mg/L) for electrophoresis. The DNA band was
visualized by UV light and photographed.
Flow cytometry analysis 1×106
Hep3B cells treated with rosiglitazone were collected, washed twice with ice-cold PBS,
and fixed in 70% ethanol at 4 °C for 12 h. The cells were
centrifuged and stained for 30 min with RNase A (50 mg/L)
supplemented with propidium iodide (50 mg/L) at 4 °C in the
dark. The apoptotic rate was assayed by flow cytometry
(EPICS-Elite, Beckman Coulter, Miami, FL USA), and the data
were analyzed using CellQuest software (Becton Dickinson,
Mountain View, CA, USA). For each sample, 10 000 cells
were measured. Hypodiploid cells with a sub-G1 peak
(apoptotic peak) were defined as apoptotic cells.
Hoechst 33258 staining assay Apoptotic
morphological changes in the Hep3B cells were detected by Hoechst
33258 staining. 1×104 Hep3B cells were washed with ice-cold
PBS and fixed with 4% paraformaldehyde in PBS for 10 min at
4 °C. The cells were washed again with PBS and stained with
Hoechst 33258 (5 mg/L) for 10 min. The washed cells were
then observed under an Advanced Fluorescence Microscope
(Nikon 80i, Tokyo, Japan) by an observer blind to the cell
treatment.
Detection of caspases activity After the rosiglitazone
treatment, the activation of caspases-3, -8, and -9 was
detected with 200 µg of the cell lysate using the colorimetric
protease assay kit (KeyGEN Biotech, Nanjing, China)
according to the manufacturer's protocol. The activity of
caspases-3, -8, and -9 was converted through the
measurement of absorbance [optical density (OD) 405 values] using
a microplate reader at a wavelength of 405 nm. Non-specific
reactions were corrected by subtracting the background
absorbance readings from the combination of the cell lysate
and buffer.
Gene silencing by small interfering RNA The PTEN
small interfering RNA and non-specific control siRNA were
purchased from Ribobio (Guangzhou, China). The cells were
plated onto 6-well plates, maintained in antibiotic medium
for 24 h, and grown to about 50% confluence. PTEN siRNA
or control siRNA were transfected with Lipofectamine 2000
reagent (Invitrogen, Madison, WI, USA). Briefly,
oligomer-fectamine was diluted at 1:50 in OptiMEMI reduced serum
medium (GIBCO, Palo Alto, Calif, USA), mixed gently, and
incubated for 5 min at room temperature. Subsequently, a
mixture of siRNA was added and incubated for 20 min. The
mixture was diluted by adding medium to each well, and the
final concentration of siRNA in each well was set as 100
nmol/L. The cells were then incubated for 48 h until
processed.
Statistical analysis Data were expressed as mean±SD
of at least 3 separate experiments. Differences between
the groups were assessed with one-way ANOVA and
Student-Newman-Keuls q test using SPSS 11.0 for Windows
(SPSS Inc, Chicago, IL, USA). P values less than 0.05 were
considered statistically significant.
Results
Rosiglitazone stimulated PTEN expression of Hep3B
cells in a dose- and time-dependent manner To determine
the effect of rosiglitazone on the expression of PTEN protein
in HCC cells, the Hep3B cells were treated with various
concentrations of rosiglitazone and harvested at different time
points. Western blotting showed that rosiglitazone increased
the expression level of PTEN in a dose- (Figure 1A) and
time-dependent manner (Figure 1B). The most effective dose was
20 µmol/L treated for 24 h. However, the PTEN expression in
the Hep3B cells declined slightly after being treated with
rosiglitazone for a longer time period (eg 48 h) or at higher
concentrations (40 and 80 µmol/L), since the cells were
already greatly damaged (data not shown).
Rosiglitazone increased PTEN expression through the
activation of PPARγ To test whether the increase in the
PTEN level induced by rosiglitazone was mediated via the
activation of PPARγ, the Hep3B cells were pretreated for 1 h
with GW9662, a specific and irreversible inhibitor of the
PPARγ pathway, and exposed to rosiglitazone for an additional 24 h.
Transcription and the expression of PTEN were determined
by RT-PCR and Western blotting analyses, respectively. The
induced transcription and expression of PTEN were
completely blocked in the presence of GW9662, indicating that
the increase of the PTEN level was through PPARγ-mediated
transcription activation (Figure 2).
PTEN overexpression induced by rosiglitazone
inhibited the PI3K/Akt pathway The oncogene Akt has been
confirmed as the key cell survival kinase of the PI3K
pathway and is negatively regulated by the tumor suppressor
gene PTEN[13,14]. Functional PTEN decreases the Akt
phosphorylation level to inhibit the PI3K/Akt
pathway[15]. Our results showed that the phosphorylation level of Akt
(Ser-473 phosphorylation) was markedly reduced when the
expression of PTEN was increased when treated with
rosiglita-zone, indicating that rosiglitazone-increased PTEN was a
functional protein (Figure 3). To examine whether rosiglitazone
had a direct effect on the phosphorylated Akt level,
the PTEN gene in the Hep3B cells was silenced with the specific siRNA
and treated for 48 h, then the cells were incubated with
rosiglitazone for an additional 24 h. Unlike control siRNA,
PTEN siRNA almost completely eliminated endogenous PTEN
protein production, and the phosphorylated Akt level was
not reduced in the presence of rosiglitazone. It is
noteworthy that the total Akt protein level did not change (Figure 3).
Thus, all data indicated that rosiglitazone affected the
phosphorylation level of Akt indirectly and that the reduction in
phosphorylated Akt was due to the increase of the PTEN
expression. Therefore, rosiglitazone was found to increase
active PTEN protein expression, and the PTEN
overexpres-sion resulted in the inhibition of the PI3K/Akt pathway
activation.
Rosiglitazone inhibited Hep3B cells growth through
PPARγ activation Previous reports have demonstrated that
Akt plays a critical role in tumorigenesis and cancer
progression by stimulating cell proliferation and the inhibition of
apoptosis[16,17]. To further evaluate the role of Akt in Hep3B
cell growth, LY294002, a specific inhibitor of the PI3K/Akt
pathway, was used to block the activation of Akt. When the
cells were incubated in the medium containing 30 µmol/L
LY294002 or 20 μmol/L rosiglitazone for 48 h, cell viability
decreased to 46.6%±4.2% and 37.3%±8.1% of the control,
respectively (Figure 4A), which indicated the involvement
of activated Akt in Hep3B cell growth. Moreover, GW9662
completely abrogated the inhibitory effect of rosiglitazone
on cell growth, suggesting that rosiglitazone induced Hep3B
cell death through the PPARγ-dependent pathway (Figure
4B). To evaluate whether PTEN upregulation played a
crucial role in the effect of rosiglitazone on cell growth, we
silenced the PTEN gene and then the transfected cells were
exposed to rosiglitazone for 48 h. The effect of
rosiglitazone-decreased cell viability was dramatically reversed, whereas
no changes were observed in the control siRNA group (Figure
4B). Those results indicated that the overexpression of PTEN
was implicated in the inhibitory effect of rosiglitazone.
Rosiglitazone-induced apoptosis in cultured Hep3B
cells Rosiglitazone can cause apoptosis to Hep3B cells; the
DNA ladder was evoked after exposure to 20 μmol/L rosiglitazone for 12, 24, and 48 h (Figure 5A). Hoechst 33258
staining assay drew a similar conclusion: that rosiglitazone
(20 µmol/L for 24 h) evoked chromatin condensation in Hep3B
cells (Figure 5B). Those findings further supported
rosiglitazone-induced apoptosis of Hep3B cells. Moreover,
flow cytometry revealed that 20 µmol/L rosiglitazone
increased the population of the sub-G1 phase by 26.1%±
3.8% for 24 h, and was significantly higher than the 0.1%
DMSO-treated control group (Figure 5C). GW9662
completely abrogated the effect of rosiglitazone, suggesting that
rosiglitazone induced cell apoptosis via PPARγ activation.
Interestingly, the knockdown of PTEN reduced the
inductive effect of rosiglitazone on cell apoptosis, indicating that
PTEN was required for rosiglitazone-induced apoptosis of
Hep3B cells. The activation of PPARγ contributed to 6.4% of
the apoptotic rate via other signaling pathways. To further
elucidate the underlying molecular mechanism of the
apoptosis of Hep3B cells, caspase colorimetric assay was
used to assess the effect of rosiglitazone on the activation
of caspases-3, -8, and -9 (Figure 5D). Rosiglitazone (20
µmol/L) treatment induced the activation of caspases-3 and -9,
and its level reached the peak at 12 h, which was a 6.1- and
4.5-fold increase, respectively. However, the activation of
caspase-8 was not found with the same treatment. To
evaluate the role of caspases activated in rosiglitazone-induced
apopto-sis, we noted that 20 μmol/L Z-VAD-FMK, a
pan-caspase inhibitor, completely inhibited caspase activation
by rosiglitazone (Figure 5E). Those results suggested that
rosiglitazone-induced apoptosis was mediated by the
activation of caspases.
Discussion
The main results of our present study first demonstrated
that rosiglitazone increased PTEN expression through the
activation of PPARγ. PTEN overexpression decreased cell
viability and triggered cell apoptosis. Because the loss or
downregulation of PTEN and over-proliferation of tumor cells
play critical roles in the occurrence and progress of HCC,
those effects of rosiglitazone on PTEN and cell growth may
be antitumor mechanisms. Rosiglitazone could be a potent
chemopreventive or chemotherapeutic agent for treating
HCC.
Recently, the PI3K/Akt pathway has become a focus of
study. Activated PI3K converts the plasma membrane lipid
phosphatidylinositol -4,5-biphosphate (PIP2) to
phospha-tidylinositol-3,4,5-triphosphate (PIP3). This
phosphorylation then stimulates the catalytic activity of Akt, which
affects cell growth, cell cycle entry, and cell
apoptosis[18,19]. Previous studies have shown that PTEN is a dual-specificity
phosphatase possessing both lipid and protein phosphatase
activities. Activated PTEN affects a dephosphorylation of
PIP3, generates PIP2, and decreases the
phosphorylation level of Akt, which results in cell growth arrest and
apopto-sis[20]. PPARγ has been shown to inhibit the transcription of
genes related to tumor progression, such as
COX-2[21] and NF-κB[22]. We proposed that
PPARγ might have a specific relationship with PTEN as a tumor suppressor gene.
There-fore, we first confirmed that rosiglitazone upregulated
transcription and the expression of PTEN in Hep3B cells. The
effect was possibly correlated to PPARγ regulation. Patel
et al have found that 2 putative binding sites (PPRE1 and
PPRE2) are identified about 10 kb upstream of the minimal
promoter region of PTEN[11]. Moreover, it has been reported
that rosiglitazone increased PTEN expression in MCF-7 breast
cancer[23], AsPC-1 pancreatic
cancer[24], non-small-cell lung carcinoma (H1792 and
H1838)[25], and HT-29 colon cancer
cells[26] through the PPARγ-dependent pathway. Our present
results also supported this idea of HCC Hep3B cells.
Functional PTEN inhibits the PI3K/Akt mediated cell
survival pathway[20]. We found that Akt activation was required
for Hep3B cell growth. This result was consistent with
previous reports[27,28]. Rosiglitazone and LY294002 had similar
effects on HCC cell death, indicating that their mechanisms
in blocking cell growth may be similar. Our data
demonstrated that rosiglitazone upregulated the most effective
functional PTEN expression to reverse higher levels of Akt
phosphorylation in Hep3B cells. The overexpression of PTEN
with the treatment of rosiglitazone markedly inhibited Hep3B
cell growth, which has been identified as a
PPARγ-dependent effect. However, PTEN knockdown did not completely
abolish the inhibitory effect of rosiglitazone, suggesting that
rosiglitazone could also activate other signaling pathways
to inhibit Hep3B cell growth. Thus, further investigation of
these effects is needed. Han et al have demonstrated that
rosiglitazone inhibits non small cell lung cancer (NSCLC)
cell growth not only by the upregulation of PTEN, but also
the downregulation of mTOR/p70S6K[25].
Recent reports have shown that the activation of
PPARγ strongly induced cell apoptosis of HCC both
in vitro and in
vivo[29,30]. However, much remains to be learnt about this
process. The present results showed that rosiglitazone
decreased Hep3B cell viability via cell apoptosis, and the
effects were mediated by PPARγ activation. Moreover, we
first found the involvement of PTEN upregulation in the
apoptotic process of HCC, which might be one of
underlying anticancer mechanisms of rosiglitazone. Because PTEN
knockdown dramatically reversed the effect of
rosiglitazone-induced apoptosis, suggesting that apoptosis induction was
not only through the upregulation of PTEN, but also through
other signaling pathways, which required further
investiga-tion. Of note, the rosiglitazone-induced cleavage of
procas-pase to caspases-3 and -9 (Figure 5D) occurred as early as
the increase in PTEN expression (Figure 1B) in apoptotic
activities. Thus, caspase cleavage might not be absolutely
required for PTEN upregulation, at least in the initiation of
apoptosis. This also indicates that other pathways via
PPARγ activation as early as PTEN upregulation are involved in
Hep3B cell apoptosis. This result is consistent with Figure
5C (eighth histogram plot). Because the apoptosis
induction via the other pathways had only minor effects (6.4% of
apoptotic rate), the PTEN upregulation was considered an
important role in the apoptotic process of Hep3B cells.
PPARγ activation-mediated apoptosis through c-JNK
activation[30] or ERK
activation[31] has been described in tumor cell lines,
and those effects might be involved in rosiglitazone-induced
apoptosis. Interestingly, we further observed that
rosiglita-zone induced apoptosis by caspase activation since caspase
inhibitor Z-VAD-FMK totally abrogated the effect. In general,
2 main apoptotic pathways are confirmed as the mitochondrial
pathway (involving caspase-9) and the death-receptor
pathway (involving caspase-8). We found that caspases-9 and
-3 were activated by rosiglitazone to produce apoptotic
activities. Likewise, previous studies have reported that
PPARγ ligands (troglitazone and
15-d-PGJ2) induced apoptosis in hepatoma cell lines through initiating
caspases-9 and -3[32,33]. It has been shown that the accumulation of
PTEN in the mitochondria mediates rat hippocampal cell
apoptosis by inhibiting the PI3K/Akt pathway, the release
of cytochrome c, and the activation of caspases-9 and
-3[34]. Taken together, those data confirm that rosiglitazone via
PPARγ activation induces the Hep3B cell mitochondria
apoptosis pathway mainly through the upregulation of PTEN,
the inhibition of the PI3K/Akt pathway, and the activation
of caspases-9 and -3. Whether rosiglitazone has the same
effects in vivo needs to be further observed.
In summary, our results demonstrate that rosiglitazone, a
synthetic ligand of PPARγ, increases PTEN expression
through the activation of PPARγ, which in turn inhibits the
PI3K/Akt pathway and cell growth and induces apoptosis
of HCC cells. Additionally, our results suggest that
rosiglitazone-induced apoptosis is mediated by caspases-9
and -3 activation. Those observations may explain the
underlying mechanisms of the effects of PPARγ agonists on
hepatocellular carcinoma, including the effects of
rosiglita-zone, and constitute potential novel therapies for the
treatment or prevention of liver cancer.
Acknowledgements
The authors appreciate the Surgical Laboratory
Department of the First Affiliated Hospital of Sun Yat-Sen
University for providing experimental instruments and equipment,
and Miao-rong SHE and Xin-hui FU for language editing
and technology support.
References
1 Parkin DM. Global cancer statistics in the year 2000. Lancet
Oncol 2001; 2: 533_43.
2 Koeffler HP. Peroxisome proliferators-activated receptor gamma
and cancers. Clin Cancer Res 2003; 9: 1_9.
3 Meirhaeghe A, Tanck MW, Fajas L, Janot C, Helbecque N, Cottel
D, et al. Study of a new PPARgamma2 promoter polymorphism
and haplotype analysis in a French population. Mol Genet Metab
2005; 85: 140_8.
4 Koeffler HP. Peroxisome proliferator-activated receptor gamma
and cancers. Clin Cancer Res 2003; 9: 1_9.
5 Wang T, Xu J, Yu X, Yang RC, Han ZC. Peroxisome
proliferator-activated receptor gamma in malignant disease. Crit Rev Oncol
Hematol 2006; 58: 1_14.
6 Young PW, Buckle DR, Cantello BC, Chapman H, Clapham JC,
Coyle PJ, et al. Identification of high-affinity binding sites for
the insulin sensitizer rosiglitazone (BRL-49653) in rodent and
human adipocytes using a radioiodinated ligand for peroxisomal
proliferator activated receptor gamma. J Pharmacol Exp Ther
1998; 284: 751_9.
7 Kim KY, Kim SS, Cheon HG. Differential anti-proliferative
actions of peroxisome proliferators-activated receptor-gamma
agonists in MCF-7 breast cancer cells. Biochem Pharmacol 2006;
72: 530_40.
8 Leslie NR, Bennett D, Lindsay YE, Stewart H, Gray A, Downes
CP. Redox regulation of PI3-kinase signalling via inactivation
of PTEN. EMBO J 2003; 22: 5501_10.
9 Nakamura N, Ramaswamy S, Vazquez F, Signoretti S, Loda M.
Forkhead transcription factors are critical effectors of cell death
and cell cycle arrest downstream of PTEN. Mol Cell Biol 2000;
20: 8969_82.
10 Zhang LN, Yu Q, He JY, Zha XL. Study of the PTEN gene
expression and FAK phosphorylation in human hepatocarcinoma
tissues and cell lines. Mol Cell Biol 2004; 262: 25_33.
11 Patel L, Pass I, Coxon P, Downes CP, Smith SA, Macphee CH.
Tumor suppressor and anti-inflammatory actions of
PPARg agonists are mediated via upregulation of PTEN. Curr Biol 2001;
11: 764_8.
12 Zhou BR, Gumenscheimer M, Freudenberg, M, Galanos C. A
striking correlation between lethal activity and apoptotic DNA
fragmentation of liver in response of
D-galactosamine-sensitized mice to a non-lethal amount of lipopolysaccharide. Acta
Pharmacol Sin 2003; 24: 193_8.
13 Stoll V, Calleja V, Vassaux G, Downward J, Lemoine NR.
Dominant negative inhibitors of signalling through the phosphoinositol
3-kinase pathway for gene therapy of pancreatic cancer. Gut
2005; 54: 109_16.
14 Pedrero JM, Carracedo DG, Pinto CM, Zapatero AH, Rodrigo JP,
Nieto CS, et al. Frequent genetic and biochemical alterations of
the PI3-K/AKT/PTEN pathway in head and neck squamous cell
carcinoma. Int J Cancer 2005; 114: 242_8.
15 Kreisberg JI, Malik SN, Prihoda TJ, Bedolla RG, Troyer DA,
Kreisberg S, et al. Phosphorylation of Akt (Ser473) is an
excellent predictor of poor clinical outcome in prostate cancer.
Cancer Res 2004; 64: 5232_6.
16 Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKT
pathway in human cancer. Nat Rev Cancer 2002; 2: 489_501.
17 Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis.
Proc Natl Acad Sci USA 2001; 98: 10 983_5.
18 Cantley LC. The phosphoinositide 3-kinase pathway. Science
2002; 296: 1655_7.
19 Chan TO, Rittenhouse SE, Tsichlis PN. Akt/PKB and other D3
phosphoinositide-regulated kinases: kinase activation by
phosphoinositide-dependent phosphorylation. Annu Rev
Biochem 1999; 68: 965_1014.
20 Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1,
dephosphorylates the lipid second messenger, phosphatidyl-
inositol 3,4,5-trisphosphate. J Biol Chem 1998; 273: 13 375_8.
21 Yim HW, Jong HS, Kim TY, Choi HH, Kim SG, Song SH,
et al. Cyclooxygenase-2 inhibits novel ginseng metabolite-mediated
apoptosis. Cancer Res 2005; 65: 1952-60.
22 Okano H, Shiraki K, Inoue H, Yamanaka Y, Kawakita T, Saitou
Y, et al. 15-deoxy-delta-12-14-PGJ2 regulates apoptosis
induction and nuclear factor-kappaB activation via a peroxisome
proliferators-activated receptor-gamma-independent mechanism
in hepatocellular carcinoma. Lab Invest 2003; 83: 1529_39.
23 Teresi RE, Shaiu CW, Chen CS, Chatterjee VK, Waite KA, Eng C.
Increased PTEN expression due to transcriptional activation of
PPARgamma by lovastatin and rosiglitazone. Int J Cancer 2006;
118: 2390_8.
24 Farrow B, Evers BM. Activation of PPARgamma increases PTEN
expression in pancreatic cancer cells. Biochem Biophys Res
Commun 2003; 301: 50_3.
25 Han S, Roman J. Rosiglitazone suppresses human lung
carcinoma cell growth through PPARgamma-dependent and
PPARgamma-independent signal pathways. Mol Cancer Ther 2006; 5: 430_7.
26 Chen WC, Lin MS, Bai X. Induction of apoptosis in colorectal
cancer cells by peroxisome proliferators-activated receptor
gamma activation up-regulating PTEN and inhibiting PI3K
activity. Chin Med J (Engl) 2005; 118: 1477_81.
27 Chang HC, Tsai LH, Chuang LY, Hung WC. Role of Akt kinase
in sphingosine-induced apoptosis in human hepatoma cells. J
Cell Physiol 2001; 188: 188_93.
28 Leng J, Han C, Demetris AJ, Michalopoulos GK, Wu T.
Cyclooxygenase-2 promotes hepatocellular carcinoma cell growth
through Akt activation: evidence for Akt inhibition in
celecoxib-induced apoptosis. Hepatology 2003; 38: 756_68.
29 Yu J, Qiao L, Zimmermann L, Ebert MP, Zhang H, Lin W,
et al. Troglitazone inhibits tumor growth in hepatocellular carcinoma
in vitro and in vivo. Hepatology 2006; 43: 134_43.
30 Bae MA, Song BJ. Critical role of c-Jun N-termial protein kinase
activation in troglitazone-induced apoptosis of human HepG2
hepatoma cells. Mol Pharmacol 2003; 63: 401_8.
31 Li M, Lee TW, Yim AP, Mok TS, Chen GG. Apoptosis induced
by troglitazone is both peroxisome proliferator-activated
receptor-gamma- and ERK-dependent in human non-small lung
cancer cells. J Cell Physiol 2006; 209: 428_38.
32 Toyoda M, Takagi H, Horiguchi N, Kakizaki S, Sato K, Takayama
H, et al. A ligand for peroxisome proliferators activated receptor
gamma inhibits cell growth and induces apoptosis in human liver
cancer cells. Gut 2002; 50: 563_7.
33 Date M, Fukuchi K, Morita S, Takahashi H, Ohura K.
15-Deoxy-delta 12, 14-prostaglandin J2, a ligand for peroxisome
proliferators-activated receptor-gamma, induces apoptosis in human hepatoma
cells. Liver Int 2003; 23: 460_6.
34 Zhu Y, Hoell P, Ahlemeyer B, Krieglstein J. PTEN: a crucial
mediator of mitochondria-dependent apoptosis. Apoptosis 2006;
11: 197_207. |
