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Experimental Therapeutics |
Laboratory for Membrane Transport, Harvard Medical School [S. S. P., R. F., H. A., A. K. C., S. H., E. K., J. A. H.], and Department of Medicine, Brigham and Women’s Hospital [A. S., J. A. H.], Boston, Massachusetts 02115
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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, resulting in inhibition of protein synthesis at
the level of translation initiation, preferentially reducing the
synthesis and expression of growth-regulatory proteins, including G1
cyclins, and causes cell cycle arrest in G1. In a KLN-205
squamous cell carcinoma mouse model, daily oral administration of EPA
resulted in a significant reduction of tumor size and expression of
cyclin D1 in the tumor tissues. Furthermore, EPA-treated tumors showed
a significant increase in the proportion of diploid cells, indicative
of cell cycle arrest in G0-G1, and a
significant reduction of malignant hypertetraploid cells. These results
characterize EPA as a member of an emerging new class of anticancer
compounds that inhibit translation initiation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Recent work from our laboratory has established a distinct connection
between partial depletion of intracellular Ca2+
stores and inhibition of both cell proliferation and tumor growth
(12)
. Depletion of Ca2+ stores
activates IFN-inducible PKR, which phosphorylates and thereby inhibits
the translation initiation factor eIF2, resulting in inhibition of
translation initiation, reduced synthesis and expression of G1
cyclins, and cell cycle arrest in G1
(13)
. Indeed, regulation of translation initiation plays a
critical role in the control of cell growth and division
(14)
because translation of most growth-regulatory
proteins and oncogenes is highly inefficient because of the presence of
stable secondary structures in the 5' UTR of their mRNAs (15
, 16)
. The length and secondary structure of the 5' UTR of a mRNA
molecule is the most critical feature influencing its translation
efficiency. A moderately long, unstructured 5' UTR with low content of
G and C bases seems to be optimal to ensure high translational
efficiency. In contrast, long 5' UTRs with a high GC content are a
major barrier to translation because G and C bases tend to form bonds
that stabilize the secondary structure of the 5' UTR. Sequence analyses
of a large number of vertebrate cDNAs have shown that mRNAs with
complex, highly structured 5' UTRs include a disproportional high
number of proto-oncogenes such as the G1 cyclins, transcription and
growth factors, cytokines, and other proteins critical in cell growth
regulation. In contrast, mRNAs that encode housekeeping proteins rarely
have highly structured GC-rich 5' UTRs (14
, 15
, 17)
. These
structural differences in the 5' UTR of their mRNA explain why
translation of growth-regulatory proteins including cyclins but not of
housekeeping proteins is highly dependent on the activity of
translation initiation factors such as eIF2 and eIF4E. The critical
role of translation initiation in cell growth regulation is illustrated
by the finding that activating mutations of some translation initiation
factors cause malignant transformation. For example, malignant
transformation of cells can be induced by the expression of a
dominant-negative form of PKR, which regulates eIF2 activity
(18)
by a constitutively active, noninhibitable mutant of
eIF2 (eIF2-51A; Refs. 19
and 20
) or by the
overexpression of eIF4E (21)
. In contrast, repression of
eIF4E activity by overexpression of eIF4E binding proteins inhibits
cell growth and can reverse a malignant phenotype
(22, 23, 24)
. Furthermore, compounds such as clotrimazole that
activate PKR and phosphorylate eIF2 by causing partial depletion of
intracellular Ca2+ stores are potent inhibitors
of cell growth (13)
and tumor growth in experimental
cancer models (12)
.
EPA is likely to induce partial depletion of intracellular
Ca2+ stores because it releases
Ca2+ from IP3-sensitive
Ca2+ pools (25)
and inhibits
store-dependent capacitative Ca2+ influx
(26)
. This suggested to us that EPA might inhibit
translation initiation and thereby inhibit synthesis and expression of
cell growth-regulatory proteins. We report here that by releasing
Ca2+ from intracellular stores while preventing
their refilling via capacitative Ca2+ influx, EPA
causes PKR-mediated phosphorylation of eIF2, inhibition of
translation initiation, and preferential inhibition of synthesis and
expression of G1 cyclins with consequent cell cycle arrest in
G1. In a mouse KLN-205 squamous cell carcinoma
model, oral EPA administration significantly reduced tumor growth and
the expression of cyclin D1 in tumor cells while causing cell cycle
arrest in G0-G1. This work
defines EPA as an inhibitor of translation initiation, a new emerging
class of drugs for cancer therapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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-3 PUFA concentrate containing
300 mg of EPA, 200 mg of docosahexaenoic acid, and 1 IU vitamin
E/gelatin capsule.
Cell Culture and Transfection.
NIH 3T3 cells were cultured in DMEM/10% calf
serum/penicillin/streptomycin, except during the mitogenic
assays and in experiments for synthesis and expression of G1 cyclins
with EPA treatment, when the cells were in DMEM/0.1% calf serum/bFGF
(5 ng/ml). NIH 3T3 cells were transfected with pBABE (vector alone),
cotransfected pBABE either with eIF2-51A or dominant-negative PKR
(PKR-K296), and puromycin-resistant stable colonies were
constructed and maintained in DMEM/10% calf serum with 2.5 µg/ml
puromycin, as described (13)
. Human solid tumor-based cell
lines obtained from American Type Culture Collection (Rockville, MD)
were grown in RPMI 1640 with 5% FBS.
Cell Growth Assay.
Adherent human solid tumor cells plated in 96-well plates were treated
for 5 days in the presence and absence of various doses of EPA ranging
from 20 to 100 µM in RPMI 1640 containing 5% FBS. At the
time of termination, cells were stained with sulforhodamine B and
counted as described (27)
.
Mitogenic Assay.
Synthesis of DNA by incorporation of
[3
H]thymidine was determined as described
(12)
. Briefly, 4 x 103
NIH 3T3 cells, cells transfected with either
vector, PKR-K296, or eIF2-51A, were made quiescent by serum
withdrawal for 36 h (0.1% calf serum/DMEM) and then exposed to a
mitogenic stimulus (5 ng/ml bFGF) in the absence or presence of
different concentrations of EPA. After 15 h, 1 µCi/ml
[3
H]thymidine (DuPont NEN, Boston, MA) was
added, and the cells were incubated at 37°C in 5%
CO2 for 5 h. Cells were harvested and
counted in a Packard Top Count (Packard, CT).
Ca2+ Measurements.
Exponentially growing cells were harvested, washed with PBS, and
resuspended in Krebs-Ringer medium buffered with 25 mM
HEPES (pH 7.4 at 37°C). Cells were loaded in the same buffer with 5
µM Fura-2 AM (Molecular Probes, Eugene, OR), and
4 x 106 were used for cytosolic
Ca2+ measurement with a Photon Technology
International dual excitation spectrofluorometer (South Brunswick, NJ),
exactly as described (28)
.
Polysome Profile Analysis.
Exponentially growing NIH 3T3 cells were exposed to EPA (30
µM) for 2 h in DMEM/5 ng/ml bFGF/0.1% calf serum,
followed by addition of cycloheximide (25 µg/ml), and polysome
profiles were determined exactly as described (13
, 29)
.
Protein Synthesis.
Total protein synthesis was measured as described (13)
.
Briefly, NIH 3T3 cells and stable transfected cells were treated with
EPA for 1 h and pulse labeled with
[35S]Met-Cys for 15 min, and incorporation of
label into TCA-precipitated total protein determined.
Western and Northern Blot Analyses.
Protein extraction and Western blot analysis were done as described
(30)
. Total protein extract (40 µg/lane) was analyzed on
blots incubated overnight in specific antibodies (Santa Cruz
Laboratories, Santa Cruz, CA). Horseradish peroxidase-conjugated
secondary antibodies were detected by ECL kit (Pierce, Rockford, IL).
RNA (25 µg) was fractionated on a 1% formaldehyde agarose gel,
blotted onto nylon membranes, and hybridized with either cyclin D1 or
18S RNA cDNA probe labeled by random-prime labeling with
[-32P]dCTP as described (13)
.
Phosphorylation of eIf2-.
Exponentially growing NIH 3T3, PKR-K296, eIF2-51A, or vector
control stable transfected cells were labeled with
32P-orthophosphoric acid (200 µCi/ml) for
3.5 h. One-half of the cells were challenged with EPA (30
µM) for 0.5 h, lysed with IP buffer, and RNase-DNase
treated. TCA precipitable counts were determined, and equal number of
counts were immunoprecipitated with anti-eIF2 antibody (kindly
provided by Dr. N. Sonenberg, McGill University, Montreal, Quebec,
Canada) and separated by SDS-PAGE; phosphorylation of eIF2 was
quantified by PhosphorImager as described (13)
.
Cell Cycle Analysis.
NIH 3T3 cells (1 x 105) were
treated with 7.5 µM EPA for up to 48 h in DMEM with
2% calf serum and 5 ng/ml bFGF, changed each day. The cells were fixed
with ethanol and stained with propidium iodide for cell cycle analysis
by flow cytometry.
Animal Studies.
To establish the KLN-205 squamous cell carcinoma model, KLN-205 tumor
cells were grown in culture, harvested and injected as a suspension
(2.5 x 105 cells in 0.1 ml of
PBS) s.c. into 6-week-old female DBA/2J mice (Jackson Laboratories).
Four days after tumor implantation, mice received either vehicle or EPA
(2.5 g EPA/kg body weight) in the form of a lipid concentrate by gavage
daily for 5 weeks. Three-dimensional tumor size was measured by
calipers every week, and volumes were calculated using the equation:
v (in mm3
)= length x width x height. Statistical differences between tumor
volumes in control and the EPA-treated group was determined by
Student’s t test. The general health of the animals was
monitored during the treatment period. At the end of the study, animals
were sacrificed by CO2 asphyxiation, and tumors
were excised and fixed in 10% buffered formalin.
Expression of Cyclin D1 in Tumor Tissue.
To analyze for cyclin D1 expression, 4-µm sections of formalin-fixed,
paraffin-embedded tumor tissues from all of the animals in each group
were cut and fixed on slides. Sections were deparaffinized in graded
alcohol, immunostained with anti-cyclin D1 monoclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA), and counterstained with
hematoxylin. A minimum of three different areas from each slide per
animal were quantitated for cyclin D1 using BioQuant software. The
percent labeling index for cyclin D1 was calculated by the formula:
(cyclin D1 positive nuclei/total nuclei) x 100.
DNA Analysis for Ploidy Levels and Tumor Cell Kinetics.
Formalin-fixed, paraffin-embedded tumor sections were stained by
the Feulgen method and microscopically analyzed for DNA content using
the BioQuant system. The DNA content was measured in 350 tumor cell
nuclei selected randomly from distinct areas of all slides from each
treatment group. The proportion of cell populations with differing DNA
content were estimated by peak fitting using Jandel software that
identifies normally distributed populations in the data. The area
percent under the fitted curves represents cells with differing ploidy
content. A Feulgen-stained mouse liver sample was used to verify the 2C
DNA content. A 2C content characterizes cells in
G0-G1, a 2C-4C content
characterizes cells in S phase, and a 4C content, cells in
G2-M phase. Malignant tumors characteristically
show a cell population containing hypertetraploid (>>4C) DNA content,
considered an index of tumor malignancy (31
, 32)
.
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RESULTS AND DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. In quiescent NIH 3T3 cells stimulated in DMEM/bFGF (5 ng/ml)/0.1%
calf serum, EPA inhibited DNA synthesis, as measured by
[3
H]thymidine incorporation, with half-maximal
inhibition at 7.5 µM (Fig. 1
).
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), EPA abolished the
typical cytosolic Ca2+ transient elicited by bFGF
(Fig. 2a
). This is because of EPA-mediated depletion of
IP3-sensitive intracellular
Ca2+ pools rather than blocking of bFGF signaling
upstream of the IP3 receptor, because EPA:
(a) potently released Ca2+ from
intracellular stores (Fig. 2b
); (b) inhibited
thapsigargin-induced capacitative Ca2+ influx
(Fig. 2c
); and (c) did not affect bFGF-induced
transcription of cyclin D1 (see below). To investigate further the
relationship between the inhibitory effect of EPA on cancer cell growth
and its effect on intracellular Ca2+ pools, human
cancer cells were loaded with Fura-2 AM and challenged with EPA to
assess cytosolic Ca2+ transients. Fig. 2d
shows that at a concentration of 30
µM, EPA induced rapid
Ca2+ release in a representative EPA-sensitive
cancer cell line (U118-MG; IC50 for growth
inhibition, 36 µM) but not in a EPA-resistant
cancer cell line (HTB-174; IC50 for growth
inhibition, 80 µM). The inset to
Fig. 2d
shows that in the HTB-174 cell line, 120
µM EPA induced Ca2+
release in a manner comparable with that obtained in U118-MG cells with
30 µM. These results provide evidence for a
direct link between the inhibitory effect of EPA on cancer cell growth
and Ca2+ depletion.
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b, lower curve). In contrast, pretreatment
with vitamin E did not affect the Ca2+ release
induced by clotrimazole or thapsigargin (data not shown) indicating
that vitamin E does not affect the ability of the cellular
Ca2+ stores to release
Ca2+, and that peroxidation products of EPA
induce Ca2+ release. Together, these results
provide a direct evidence for a link between the effect of EPA on
Ca2+ pools and its growth-inhibitory activity and
indicate that these effects are related to a peroxidation product(s) of
EPA on the filling state of the cellular Ca2+
pools.
EPA Inhibits Protein Synthesis at the Level of Translation
Initiation.
The filling state of the Ca2+ pools influences
the rate of cellular protein synthesis (38)
. To
investigate whether EPA inhibits protein synthesis, NIH 3T3 cells were
treated with EPA and pulse labeled with
[35S]Met-Cys, and we determined the
incorporation of label into total protein. EPA inhibited protein
synthesis (Fig. 3a
) in a dose-dependent manner (Fig. 3
a, inset).
The rate of total protein synthesis as a function of EPA concentration
reaches a minimum plateau at 
33% of control (Fig. 3a
);
this is most likely because EPA only inhibits synthesis of a subset of
proteins whereas other proteins are not affected, as we will show
below. Fig. 3
shows that the concentration of EPA required for
half-maximal inhibition of protein synthesis is 
15
µM, a value comparable with the concentration
required for half-maximal inhibition of cell growth (Fig. 1
). Similar
protein synthesis inhibition was obtained with our cancer cell panel
(data not shown). To investigate whether EPA inhibits protein synthesis
as a direct result of Ca2+ store depletion,
exponentially growing NIH 3T3 cells were transiently incubated with or
without EPA for 1 h. The cells were washed with
Ca2+-free medium to remove EPA and also to render
the media Ca2+ free. Cells were then
pulse-labeled with [35S]Met-Cys in the presence
or absence of external Ca2+, and incorporation of
label was determined as above. Cells transiently exposed to EPA
reinitiated protein synthesis only when external
Ca2+ was supplied; pulse labeling in the absence
of external Ca2+ resulted in marked inhibition of
protein synthesis. In contrast, removal of external
Ca2+ during the pulse labeling period in control
cells not exposed to EPA had negligible effect on protein synthesis.
This experiment indicates that inhibition of protein synthesis by EPA
is the direct result of Ca2+ store depletion
(Fig. 3b
).
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and inhibits translation
initiation, the rate-limiting step of protein synthesis
(39, 40, 41)
. To investigate whether EPA inhibits protein
synthesis at the level of translation initiation, we determined the
polyribosome profiles in sucrose density fractions derived from
exponentially growing NIH 3T3 cells incubated with EPA for 2 h.
EPA shifted the ribosomal profile from heavy polyribosomes to lighter
polysomes, monosomes, and free ribosomal subunits (Fig. 4
). This shift toward lighter fractions clearly demonstrates that EPA
inhibits translation initiation.
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subunit of eIF2 by PKR inhibits
GDP-GTP exchange, suppressing the initiation of translation. To
investigate whether the inhibitory effect of EPA on translation
initiation is attributable to phosphorylation of eIF2 by PKR, cells
expressing either a nonphosphorylatable mutant of eIF2 (eIF2-51A;
Ref. 19
) or a dominant-negative mutant of PKR [PKR-K296;
which has no enzymatic activity and acts as a dominant-negative mutant
because of its ability to heterodimerize with endogenous protein
(43)
] were tested for their sensitivity to EPA. Compared
with vector-transfected cells, eIF2-51A and PKR-K296 transfected
cells were resistant to the inhibitory action of EPA on both cell
growth and protein synthesis (Fig. 5 and b
). This observation does not reflect a
general resistance of the eIF2-51A and PKR-K296 transfected cell
lines to antiproliferative agents because their sensitivity to
etoposide was similar to that of vector-transfected control cells (data
not shown). These results indicate that PKR-mediated phosphorylation of
eIF2 is responsible for the inhibitory effect of EPA on protein
synthesis and cell growth.
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in response to EPA treatment. Exponentially growing NIH 3T3
cells transfected with vector only, eIF2-51A, or PKR K296 were
incubated with [32P]Pi to
label the intracellular ATP pool and challenged with EPA.
Phosphorylation of eIF2 was determined by the amount of
32P incorporated into immunoprecipitated eIF2.
EPA treatment markedly increased eIF2 phosphorylation in both
parental NIH 3T3 (not shown) and vector-transfected cells; in contrast,
phosphorylation of eIF2 was minimal in eIF2-51A and PKR-K296
transfected cell lines (Fig. 5c
). It has been reported
recently that mouse embryonic fibroblasts that are nullizygous for PKR
are not impaired in their ability to inhibit protein synthesis in
response to endoplasmic reticulum stress (44)
. This is
because the endoplasmic reticular-resident kinase PERK that is also
activated by Ca2+ depletion phosphorylates
eIF2 on serine 51 and thereby inhibits mRNA translation. Our results
showing abrogation of eIF2 phosphorylation in PKR-K296 transfected
cells treated with EPA indicate that PERK is not involved in the
inhibition of protein synthesis that we observe in response to EPA.
Alternatively, PKR-K296 may also block PERK activity and/or compete for
a rate-limiting cofactor required for phosphorylation of eIF2.
EPA Preferentially Abrogates G1 Cyclin Synthesis and Expression and
Causes Cell Cycle Arrest in G1.
Reducing the rate of translation initiation preferentially inhibits the
synthesis of proteins that are inefficiently translated (45
, 46)
, mostly because they are coded for by mRNAs with highly
structured 5' UTR. Because the mRNAs of G1 cyclins contain highly
structured 5' UTRs, their synthesis and expression requires optimal
activity of translation initiation factors (16)
. Thus,
pharmacological agents that decrease the rate of translation initiation
are expected to inhibit preferentially synthesis and expression of G1
cyclins and other cell growth-regulatory proteins (15)
.
Exponentially growing NIH 3T3 cells were treated with EPA and
[35S]Met-Cys for 1 h and assayed for
incorporation of label into immunoprecipitated cyclin D1, cyclin E, and
Ras, as well as into ß-actin and ubiquitin. EPA inhibited synthesis
of cyclin D1, cyclin E, and Ras, all coded for by mRNAs with highly
structured 5' UTRs, but not of ß-actin or ubiquitin, proteins coded
for by mRNAs with simple 5' UTRs (Fig. 6a
). EPA treatment also resulted in a parallel reduction in
the expression of cyclin D1 and cyclin E, whereas expression of
ß-actin and ubiquitin was not affected (Fig. 6a
). Because
cyclin D1 has a short half-life of 30 min (47)
, the
effect of EPA on incorporation of [35S]Met-Cys
into cyclin D1 and on cyclin D1 expression could be attributable to
reduced synthesis or increased degradation. In contrast, the effect of
EPA on Ras is unlikely attributable to an increased degradation of this
protein because it has a longer half-life that is comparable with that
of ubiquitin or ß-actin (>16 h). These results support the
conclusion that EPA exerts a differential inhibitory effect on the
synthesis and expression of a subset of proteins including G1 cyclins.
This is further confirmed by the finding that in PKR-K296 and
eIF2-51A transfected cells that are resistant to its
growth-inhibitory effect, EPA did not affect cyclin D1 or cyclin E
expression (Fig. 6c
). EPA-induced inhibition of translation
initiation may explain the reduced expression of farnesyl protein
transferase, an enzyme essential for the transforming activity of ras
oncoproteins in EPA-treated mice with colon cancer (48)
.
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). Furthermore, Northern blot
analysis with a cyclin D1-specific probe of mRNAs isolated from
polysome fractions (described in Fig. 4
) showed a significant shift of
cyclin D1 mRNA from heavier to lighter polysomal fractions (Fig. 6d
). These experiments confirm that EPA inhibits cyclin D1
synthesis at the level of translation initiation. Importantly, they
also demonstrate that EPA does not affect cyclin D1 transcription,
indicating that EPA inhibits cell growth without interfering with
growth factor signaling upstream from cyclin D1 transcription. In eukaryotic cells, successive expression of cyclin D1 and E during the G1 phase of the cell cycle is critical for progression beyond the restriction point and into S phase. To test whether EPA blocks cell cycle progression before the restriction point, quiescent NIH 3T3 cells were stimulated with bFGF, and the effect of adding EPA at hourly intervals on entry into S phase was monitored. Quiescent cells enter S phase 12–13 h after bFGF stimulation (13) . EPA addition to these cells until late G1 (10–11 h after bFGF stimulation) prevented G1-S progression, even when cells were exposed to EPA for only 1 h before beginning of S phase. In contrast, the addition of EPA at later times failed to inhibit DNA synthesis (data not shown), indicating that EPA blocks cell proliferation specifically in G1, most probably before the restriction point. These results were further confirmed by FACS cell cycle analysis of propidium iodide-stained cells. Treatment with EPA (7.5 µM) for 48 h resulted in a significant accumulation of cells in G1: Control cells: G0-G1, 65%; S, 31%; G2-M, 4%; EPA-treated cells: G0-G1, 87%; S, 11%; G2-M, 2%. These results indicate that by reducing synthesis and expression of G1 cyclins, EPA blocks cell cycle progression in G1.
EPA Reduces Tumor Growth and Induces G0-G1
Arrest in Vivo.
The in vivo consequences of EPA-mediated inhibition of
translation initiation were elucidated in the KLN mouse squamous cell
carcinoma model. Starting 4 days after inoculation, either vehicle or
marine lipid concentrate containing EPA was administered daily by
gavage at a dose of 2.5 g/kg for 5 weeks (n = 10 animals/group). Tumors were measured on a weekly basis for 5 weeks
and then analyzed by immunohistochemistry for expression of cyclin D1
and by Feulgen stain for nuclear DNA content. EPA treatment
significantly reduced tumor growth (Fig. 7a
) while showing no signs of toxicity, as indicated by the
absence of weight loss or any behavioral abnormalities with treatment.
Consistent with the effect of EPA on G1 cyclin expression in cultured
cells, EPA-treated tumors showed a significant decrease in the
number of cells expressing cyclin D1 (Fig. 7b
). EPA reduced
the cyclin D1 labeling index from 29 ± 5 in
vehicle-treated animals to 4 ± 1 (mean ± SE, P < 0.001). Nuclear DNA content
analysis of tumors documented that EPA significantly increased the
number of cells with a diploid (2C) DNA content, indicating cell cycle
arrest in G0-G1. In
addition, it significantly decreased the number of hypertetraploid
cells (>4C DNA content), representative of the rapidly dividing
malignant cell population (Fig. 8
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by NIH Grant CA
78411. 
2 To whom requests for reprints should be
addressed, at Laboratory for Membrane Transport, Harvard Medical
School, C1–607, 240 Longwood Avenue, Boston, MA 02115. Phone:
(617) 432-2394; Fax: (617) 432-0933; E-mail: jose_halperin{at}hms.harvard.edu
3 The abbreviations used are: PUFA,
polyunsaturated fatty acid; EPA, eicosapentaenoic acid; PKR, protein
kinase R; eIF2, eukaryotic initiation factor 2; UTR, untranslated
region; IP3, inositol triphosphate; bFGF, basic fibroblast
growth factor; TCA, trichloroacetic acid.
Received 11/29/99. Accepted 3/31/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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 S. S. Palakurthi, H. Aktas, L. M. Grubissich, R. M. Mortensen, and J. A. Halperin Anticancer Effects of Thiazolidinediones Are Independent of Peroxisome Proliferator-activated Receptor {gamma} and Mediated by Inhibition of Translation Initiation Cancer Res., August 1, 2001; 61(16): 6213 - 6218. [Abstract] [Full Text] [PDF]
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, 2.5 g/kg body weight) or vehicle (
) administered
daily by gavage was started 4 days after tumor implant. Each
point represents mean of tumor volume measured at the
end of each week (n = 10;
bars, SE; when not shown, SE is smaller than the size of
the point); Ps were calculated using Student’s
t test. b, tumor sections immunostained
with monoclonal anti-cyclin D1 antibody and horseradish peroxidase
secondary antibodies.