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Tumor Biology |
Brain Tumor Research Center, Brigham and Women’s Hospital, The Children’s Hospital [T. J., O. H., D. C. N., L. B., J. Z., N. T. S., R. S. C., P. M. B.], and Dana Farber Cancer Institute, Departments of Neurosurgery [T. J., O. H., D. C. N., L. B., J. Z., N. T. S., R. S. C., P. M. B.] and Cancer Biology [S-K. K., L. B. C.], Harvard Medical School, Boston, Massachusetts 02115, and Department of Neurosurgery, Jikei University School of Medicine, Tokyo, Japan [T. J., T. A.]; Institute of Neurosurgery, University Milano, Ospedale Maggiore Policlinico, IRCCS, Milano, Italy [L. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of the specific COX-2 inhibitor NS-398 on monolayer cell cultures and three-dimensional glioma spheroids was investigated using U-87MG and U-251MG human glioblastoma cell lines. The proliferation rate was assessed in monolayer cultures. In addition, a growth assay, a migration assay, an apoptosis assay, and a tumor invasion assay were performed in a three-dimensional spheroid culture system. NS-398 was able to reduce the proliferation of monolayer cell cultures, as well as the growth of spheroids and tumor cell migration, in a dose-dependent manner. There was also a moderate increase in the number of apoptotic cells in the treated spheroids. NS-398 did not have an inhibitory effect on tumor invasion in the coculture spheroid system. Our study provides evidence that COX-2 is up-regulated in the majority of high-grade gliomas and that a potential role of COX-2 inhibitors as an adjuvant therapy for brain tumors may exist.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies have led to the recognition of the importance of COX-2 in colorectal tumorigenesis (5, 6, 7, 8, 9) and in the development of intestinal neoplasms in genetically manipulated animal models (9 , 10) . COX-2 expression has also been detected in gastric (11) , esophageal (12 , 13) , and lung carcinomas (14 , 15) and brain tumors (16) .
Recently, NS-398, a selective COX-2 inhibitor has been reported to mediate growth-inhibitory effects in colon (17) , esophageal (13) and pancreatic carcinoma cell lines (18) . In addition, NS-398 was shown to inhibit proliferation and to induce apoptosis in colorectal and lung carcinoma cell lines (19 , 20) .
The expression of COX-2 in human glioma and the effects of selective COX-2 inhibitors on the growth of human glioma cell lines have not been analyzed in detail. In this study, we examine the expression of COX-2 protein in human glioma specimens and cell lines. In addition, the effects of the selective COX-2 inhibitor NS-398 on glioma proliferation, migration, apoptosis, and invasion are also examined in vitro using a three-dimensional culture assay.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human glioma cell line U-87MG was obtained from American Type Culture Collection (Rockville, MD). The U-251MG cell line was kindly provided by Dr. D. Bigner (Duke University, Durham, NC). All cell lines were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Life Technologies, Inc., Grand Island, NY).
Western Blot Assay.
Frozen tissue samples were homogenized in Tris-HCl buffer (pH 7.4)
containing 0.5% NP40 and protease inhibitors (Complete; Boehringer
Mannheim, Indianapolis, IN). Samples containing 100 µg of protein and
SDS-PAGE loading buffer with 5% ß-mercaptoethanol were heated for 5
min at 100°C, and loaded on a 12% polyacrylamide gel (Bio-Rad,
Hercules, CA). Electrophoretic transfer to nylon membranes (Millipore,
Bedford, MA) was followed by immunoblotting with an anti-COX-2 antibody
(Transduction Laboratories, Lexington, KY; Ref. 13
) or
tubulin (Neomarkers, Fremont, CA). This was followed by hybridization
with a secondary antibody conjugated with peroxidase (Amersham
Pharmacia Biotech, Piscataway, NJ). The signal was detected by
chemiluminiscence using the ECL-Plus detection system (Amersham
Pharmacia Biotech).
Immunohistochemistry.
Paraffin tissue sections (4–6 µm) of tumor and normal brain
specimens were used for COX-2 immunohistochemical studies. Slides were
deparaffinized, and endogenous peroxidase activity was blocked by
incubation in 1% H2O2 in
PBS for 30 min. Antigen retrieval was performed by incubating slides in
10 mM sodium citrate at 100°C in a microwave for 30 min.
A mouse monoclonal antibody against human COX-2 (Transduction
Laboratories; Ref. 13
) was then applied at a dilution of
1:50 overnight at 4°C. After rinsing with PBS, the biotinylated
secondary IgG antibody was applied for 30 min at room temperature.
Immunoperoxidase staining was performed using the Vectastatin Elite ABC
kit (Vector Laboratories, Inc., Burlingame, CA), and sections were
counterstained with hematoxylin (Sigma Diagnostics, St. Louis, MO). As
an additional negative control, primary antibody was omitted. Tissue
from a human colon carcinoma known to overexpress COX-2 was used a
positive control. The percentage of positive tumor cells was determined
semiquantitatively by assessing the whole tumor section, and each
sample was assigned to one of the following categories: 0 (0–4%), 1
(5–24%), 2 (25–49%), 3 (50–74%), or 4 (75–100%). The intensity
of immunostaining was determined as 0 (negative), 1+ (weak), and 2+
(strong). Additionally, an immunoreactive score was calculated by
multiplication of the percentage of positive cells and the staining
intensity, as proposed by Krajewska et al.
(21)
. For heterogenous staining patterns, each component
was scored independently and the results were summed. For example, a
specimen containing 25% tumor cells with strong intensity (1 x 2+ = 2), 25% tumor cells with weak intensity
(1 x 1+ = 1), and 50% tumor cells without
immnoreactivity received a score of 2 + 1 + 0 = 3.
Cell Proliferation Assay in Monolayer Cell Cultures.
The effect of NS-398 (BIOMOL, Plymouth Meeting, PA) on cell growth was
determined using a MTT cell proliferation assay. NS-398 was dissolved
in 100% DMSO (Sigma Diagnostics) as 1000x stock solutions and then
diluted further in DMEM for cell culture experiments. The final
concentration of DMSO for all experiments was maintained at 0.1%. All
drug solutions were prepared fresh on the day of testing. U-87MG and
U-251MG cells were seeded at a density of 5 x 103
per well for all cell lines in 96-well plates
in DMEM containing 10% fetal bovine serum. After 24 h, fresh
medium was added containing NS-398 at concentrations of 0–200
µM. After a 4-day preincubation, the cell proliferation
assay, MTT CellTiter 96 (Promega, Madison, WI), was performed.
Absorbance was measured at 560 nm using a microtiter plate reader.
Tumor Spheroids.
Tumor spheroids from U-87MG and U-251MG cells were initiated using the
agar overlayer culture method, as described by Yuhas et al.
(22)
. Briefly, spheroids were formed by seeding
2 x 106 cells in 6 ml of media in
25-cm2 cell culture flasks base-coated with an
agar medium substrate. After 6–7 days in culture, spheroids were
formed and selected for additional experiments using a
stereo-microscope.
Immunohistochemistry for Ki-67 was carried out on acetone-fixed 6-µm frozen sections of glioma spheroids using the Vectastain Elite ABC kit. Briefly, the sections were blocked with horse serum for 30 min and incubated with primary antibody against Ki-67 nuclear antigen (1:100; DAKO Corp., Carpinteria, CA) for 1 h at room temperature. Slides were then incubated with biotinylated antirabbit IgG antibody for 30 min and washed. Detection was carried out with avidin-coupled horseradish peroxidase in the presence of chromogen, which resulted in a dark brown staining of positive cells. Sections were counterstained with 1% methyl green (Sigma Diagnostics) and mounted in Gelmount (Biomeda, Foster City, CA). Slides where the primary antibody was omitted served as negative controls. The proliferation index (Ki-67 index) was defined as the percentage of positively stained cells from five randomly chosen fields/section, scored under light microscopy at x100 magnification using the MetaMorph version 4.1 image analysis system (Universal Image Corp., West Chester, PA).
For in situ detection of apoptosis, slides from spheroids were fixed in 1% paraformaldehyde with PBS for 10 min at room temperature and quenched in 3.0% hydrogen peroxide in PBS for 5 min at room temperature. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated nick end labeling method (Intergen Co., Purchase, NY), followed by counterstaining with 1% methyl green. Sections of postpartum rat mammary gland (Intergen Co.) served as the positive control, and slides where terminal transferase was omitted served as the negative control. The apoptotic index was defined as the number of positively stained 10 randomly chosen spheroids/fields, as described above for Ki-67.
Growth Assay and Migration Assay in a Three-Dimensional Culture
System.
For the growth assay, spheroids of U-87MG and U-251MG were divided into
five treatment groups (control and 50, 100, 150, 200 µM
NS-398). For each group 12 spheroids were transferred individually into
24-well plates using a stereomicroscope. The 24-well plates were
base-coated with 0.5 ml of DMEM-agar and filled with 1 ml of DMEM. The
diameters of the spheroids were measured daily over a 12-day period
using a phase contrast microscope, and the spheroid volumes were
calculated. On day 12, spheroids treated with 150 µM
NS-398 were frozen in optimal cutting temperature and
immunohistochemistry for Ki-67 and apoptosis (ApoTag kit) was performed
as described above.
For the migration assay, spheroids were divided into five groups (control and 50, 100, 150, 200 µM NS-398). For each group, 12 spheroids were transferred individually into uncoated 24-well plates filled with 1 ml of DMEM. Colony diameter (cellular outgrowth) was measured daily over a 5-day period, and the area was calculated. Immunohistochemistry for Ki-67 was performed on spheroids in glass chamber slides treated with 150 µM NS-398 for 5 days. All experiments were performed in triplicate.
Preparation and Characteristics of Brain Aggregates for Tumor
Invasion Assay.
Fetal rat brain cell aggregates were prepared according to the
procedure of Bjerkvig et al. (23)
. Fetal rat
brain cell aggregates were obtained from 15-day-old fetuses of inbred
Sprague Dawley rats (Taconic Farms, Inc., Germantown, NY). Rat brains
were extracted under aseptic conditions, and the meningeal coverings
were removed. The tissue was cut into small pieces and dissociated by
serial trypsination into a single cell suspension using 0.25%
trypsin (Life Technologies, Inc.). Brain aggregates were produced by
seeding 6 x 106 cells distributed
equally into 1 ml of DMEM in a 24-well plate coated with 0.5 ml of a
nonadherent agar-medium substrate. Immature brain cell aggregates
started to form after 4 days in culture. After 20 days in culture, the
cellular differentiation within the aggregates was completed and the
aggregates were used for coculture invasion experiments with tumor
spheroids.
Tumor-Cell Invasion Assay.
Prior to confrontation, the U87-MG and U-251MG tumor spheroids were
treated with 150 µM NS-398 for 7 days. This was done to
obtain effective growth inhibition, based on the results obtained from
the proliferation assay. The spheroids were then transferred to 96-well
dishes and confronted with fetal rat brain aggregates. The cocultures
were continuously exposed to 150 µM NS-398 during a 4-day
period and analyzed using a confocal microscope (LSM410 microscope;
Tissue-Tek, Sakura Finetek, Torrance, CA). All experiments were
done in triplicate.
Statistical Analysis.
All statistical analyses were performed by ANOVA analysis using
Statview 5.0.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. By immunohistochemistry, cytoplasmic COX-2 staining was detected in
neurons of histologically normal human brain (data not shown). Granular
staining was also observed in the cytoplasm of tumor cells in all
histological grades. COX-2-positive staining accumulated in tumor
cells, in infiltrative lymphocytes, and in endothelial cells. By
immunohistochemistry, the percentage of positive staining cells was
significantly higher in high-grade glioma specimens than low-grade
glioma specimens (Fig. 2)
. High-grade glioma specimens had a heterogeneous staining pattern
compared with low-grade glioma specimens. Thus, for high-grade
specimens the immunoreactive score had a larger range of distribution.
The details of expression patterns regarding the percentage of positive
cells, staining intensity, and immunoreactive score are shown in Table 1
. The immunoreactive score of COX-2 staining in human glioma specimens
was related to the WHO classification grade of glioma, with a higher
immunoreactive score in more malignant tumors.
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Inhibition of Cell Proliferation by NS-398 in Monolayer Cells.
The effect of the selective COX-2 inhibitor NS-398 on proliferation of
the tumor cells was studied by using the MTT cell proliferation assay.
Tumor cells were treated with increasing concentrations of NS-398, and
the effect on proliferation was determined after 4 days of treatment.
In cultures treated with NS-398 there was no increase in the number of
nonadherent cells relative to control cultures. NS-398 suppressed the
proliferation of the two glioma cell lines examined in a dose-dependent
manner (Fig. 3)
. The IC50 for NS-398 was 
80 µM
for U-251MG cells and 130 µM for U-87MG cells. The
proliferation assay was done in triplicate.
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. Ki-67 staining was also done on
spheroids grown on chamber slides and treated with 150
µM NS-398 for 5 days. Ki-67-positive cells were
seen in areas of colony-outgrowth in controls. In the treated
spheroids, the percentage of Ki-67-positive cells was significantly
less than in controls (84.4% ± 2.7 versus
58.0% ± 2.0, P < 0.0001; Fig. 4, C and D
). In addition, there was a significant
increase in the number of apoptotic cells in the treated spheroids
(1.7 ± 0.45 versus 12.6 ± 1.4, P < 0.001; Fig. 4, E and F
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increased levels of PGs are, in part, due to COX-2 overexpression. This has been previously reported in colon and human brain tumor tissues as compared with the normal tissue (28, 29, 30) , suggesting that PGs play a role in tumor development. Thus, NS-398 may lead to a reduction in PGs levels that in turn may inhibit proliferation, migration, and induce apoptosis in the glioma cell lines we examined. A similar effect has been reported in colon (17) , esophageal (13) , and pancreatic carcinoma cell lines (18) , where a selective COX-2 inhibitor was shown to inhibit proliferation and to induce apoptosis (19 , 20) .
The pathophysiological consequences of COX-2 expression in the brain are not presently known. Overexpression of COX-2 in rat intestinal epithelial cells was found to reduce the rate of apoptosis and increase the expression of the antiapoptotic proto-oncogene Bcl-2 (31) . Moreover, transfection of colon cancer cells with a COX-2 expression vector resulted in an increased metastatic potential of these cells (32) .
Growth factors, tumor promoters, cytokines, and other inflammatory mediators have been found to induce COX-2 expression (1 , 2) . EGF is known to up-regulate the expression of the COX-2 enzyme in several cell types, such as human squamous carcinoma cells (33 , 34) . Among the genetic changes that are commonly observed in GBMs and that seem to be specific for this tumor stage are loss of heterozygosity of chromosome 10 along with EGF receptor amplification (35, 36, 37) . Therefore, EGF receptor up-regulation may be one of the factors that contributed to the up-regulation of COX-2 we observed. Furthermore, COX-2 gene expression has been reported to be inhibited by wild-type p53 (38) . A recent study suggests an interaction between p53 and COX-2 expression. It shows that wild-type p53 leads to a decrease in COX-2 expression. In contrast, mutant p53 did not reduce COX-2 expression levels. The interactions between p53 and COX-2 may be important for understanding why levels of COX-2 are increasing with progression of tumor grade (39, 40, 41) . Some groups have reported more p53 mutations in high-grade than low-grade tumors (42) . The increase in mutation rate of p53 may contribute to the elevated levels of COX-2 immunostaining we observed.
We have shown that COX-2 levels are frequently elevated in high-grade gliomas. Selective inhibition of COX-2 by NS-398 in human gliomas in two different in vitro models reduces growth activity. This inhibition may be due, in part, to a decrease in the proliferation rate of tumor cells and an increase in the number of apoptotic cells. Local invasion into normal brain tissue is an inherent feature of malignant gliomas. NS-398 did not inhibit invasion of glioma cells, supporting the hypothesis that the mechanism of invasion is different from that of cell proliferation and migration (43) .
The present study suggests that COX-2 may perform an important function in human glioma tumorigenesis. In addition, nonsteroidal anti-inflammatory drugs may be an effective therapeutic option for high-grade gliomas.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from the Boston
Neurosurgical Foundation. T. J. is a recipient of a fellowship from
Jikei University School of Medicine. 
2 To whom requests for reprints should be
addressed, at Brain Tumor Research Center, Brigham and Women’s
Hospital, Harvard Medical School, LMRC Room 121, 221 Longwood Avenue,
Boston, MA 02115. Phone: (617) 278-0177; Fax: (617) 232-9029.
3 The abbreviations used are: COX, cyclooxygenase;
GBM, glioblastoma; AA, anaplastic astrocytoma; LOW, low-grade
astrocytoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; EGF, epidermal growth factor; PG, prostaglandin.
Received 12/22/99. Accepted 7/ 6/00.
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