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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Neurol Med Chir (Tokyo). Author manuscript; available in PMC 2010 June 28.
Published in final edited form as:
PMCID: PMC2892780

Induction of Cyclooxygenase-2 Expression by Interleukin-1β in Human Glioma Cell Line, U87MG


Cyclooxygenase-2 (COX-2) is up-regulated in most high-grade gliomas, and high COX-2 expression is associated with aggressive character and poor prognosis. However, the effect of COX-2 in human glioma cell lines is not well known. This study examined the effect of several stimuli, including interleukin-1β (IL-1β) and carcinogens, on COX-2 induction in normal astrocyte cells and human glioma cell lines U87MG, A172, and T98G. IL-1β-induced COX-2 expression strongly at both protein and messenger ribonucleic acid levels in only the U87MG cells of the glioma cell lines. Furthermore, carcinogen induced COX-2 expression. Similar findings were also observed in normal human astrocyte cells. The U87MG glioma cell line is a good model for COX-2 induction in glioma cell lines.

Keywords: cyclooxygenase-2, interleukin-1 beta, glioma


Prostaglandins and cytokine are associated with the pathogenesis of various pathological conditions in the brain, such as ischemia, seizure, infection, and injury.7,20) Interleukin-1β (IL-1β) is often expressed in human glioma, especially localized in the tumor cells and macrophages.25) Moreover, ILs may be related to tumor progression in some gliomas.24) Cyclooxygenases (COX-1 and COX-2), also known as prostaglandin H synthetases, catalyze the first rate-limiting step in the conversion of arachidonic acid into prostaglandins and thromboxanes.8) COX-1 is constitutively expressed in a wide variety of tissues, while COX-2 gene is highly inducible and expressed in a response to stimuli from various cytokines,17,20) growth factors,16) tumor promoters,10) and bacterial endotoxins.23) COX-2 is supposed to be associated with tumorigenesis, as studied in several cancers.13,16,23) Recent studies indicate that COX-2 is up-regulated in high-grade gliomas and that high COX-2 expression is associated with poor prognosis.12,27)

Our previous study demonstrated that many cytokines and carcinogens increase the expression of COX-2 but have little effect on the expression of COX-1 in normal human astrocyte cells.29) However, little is known about the induction of COX-2 in glioma cells.

The present study examined the effects of cytokines and carcinogens on COX-2 induction in human glioma cell lines.

Materials and Methods

Glioma cell lines (U87MG, A172, and T98G; American Type Culture Collection, Rockville, Md., U.S.A.) were grown in Eagle’s minimal essential medium containing 10% fetal bovine serum (HyClone, Logan, Utah, U.S.A.), 1 mM sodium pyruvate (Life Technologies, Inc., Rockville, Md., U.S.A.), and gentamicin (10 μg/ml), and cells from passages 5 to 15 were used. The normal human astrocyte cell line (Clonetics, San Diego, Calif., U.S.A.) was grown in astrocyte growth medium (Clonetics) and cells from passages 3 to 7 were used. IL-1β (R&D Systems, Minneapolis, Minn., U.S.A.) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (SIGMA, St. Louis, Mo., U.S.A.) were dissolved in dimethyl sulfoxide.

The effects of cytokines (lL-1β, interferon-γ, and tumor necrosis factor-α [TNF-α]; R&D Systems) and the carcinogens (TPA, benzo(a)pyrene [B(a)P], and the more potent metabolite benzo(a)pyrene-diol-epoxide [BPDE]; SIGMA) on COX-1 and COX-2 protein expression in normal human astrocyte cells were examined. Cells were incubated with the cytokines for 24 hours, then harvested. The effect of treatment with IL-1β on COX-2 expression was also examined in three glioma cell lines, A172, T98G, and U87MG. In addition, the effect of TPA on COX-2 expression was investigated in U87MG cells. The levels of COX-1 and COX-2 protein were analyzed by Western blot analysis as follows.

Semi-confluent cells on 100-mm diameter dishes were washed twice with ice-cold phosphate buffered saline and lysed in buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 1% Nonidet P-40 (SIGMA), 0.5% sodium deoxycholate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride). Cells were then sonicated on ice four times for 20 seconds at 50% power. Protein content was measured by the bicinchoninic acid method using bicinchoninic acid protein assay reagent (Pierce, Rockford, Ill., U.S.A.). Aliquots of the 20 μg protein were boiled in protein sample buffer (9% SDS, 15% glycerol, 30 mM Tris-HCl, pH 7.8, 0.05% bromphenol blue, 6% β-mercaptoethanol) and separated by SDS-polyacrylamide gel electrophoresis using 8% acrylamide gels. Ovine COX-1 purified protein (Cayman, Ann Arbor, Mich., U.S.A.) and murine recombinant COX-2 protein (Cayman) were also loaded on the gel as positive controls. After electrophoretic transfer of the protein from the polyacrylamide gel to nitrocellulose membrane, the membrane was blocked by incubating with 10% dry milk (Bio-Rad, Hercules, Calif., U.S.A.) in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) overnight at 4°C. After washing three times in TBS-T, the membrane was treated with monoclonal anti-human COX-1 mouse antibody (Cayman) diluted 1:4000 in TBS-T/1% dry milk, polyclonal anti-human COX-2 rabbit antibody (Oxford Biomedical Research, Oxford, Mich., U.S.A.) diluted 1:4000 in TBS-T/1% dry milk, polyclonal anti-human p53 mouse antibody (Oncogene, San Diego, Calif., U.S.A.) diluted 1:1000 in TBS-T/1% dry milk, or with polyclonal anti-human actin goat antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., U.S.A.) diluted 1:2000 in TBS-T/1% dry milk for 1 hour at room temperature. The membrane was then washed three times with TBS-T and incubated for 1 hour at room temperature with 1:5000 dilution of peroxidase conjugated anti-mouse (Amersham, Arlington Heights, Ill., U.S.A.), anti-rabbit (Amersham), or anti-goat (Santa Cruz Biotechnology) immunoglobulin antibody in TBS-T/1% dry milk. The membrane was washed three more times with TBS-T and the immunocomplex was visualized by enhanced chemiluminescence using the enhanced chemiluminescence kit (Amersham). Expression of actin was used as a control to confirm similar loading of protein in each cell line.

To confirm that COX-2 expression was induced at the transcriptional level in U87MG cells by treatment with IL-1β, COX-2 messenger ribonucleic acid (mRNA) was assayed by reverse transcription-polymerase chain reaction (RT-PCR) with specific primers for human COX-2 promoter and identified by sequenced analysis as follows.

First-strand complementary deoxyribonucleic acid (cDNA) was generated from 1 μg of total RNA using the Advantage RT-PCR kit (Clontech, Palo Alto, Calif., U.S.A.). The reaction solution was diluted to a final volume of 100 μl by adding diethyl pyrocarbonate-treated water. Ten μl of the cDNA solution were used for PCR. The PCR mixture consisted of 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 2.0 units of Taq DNA polymerase (Promega Corporation, Madison, Wis., U.S.A.), and 0.4 μM each of the following primers, which are specific for full-length human COX-2: for COX-2 (5′-CCCGCCGCTGCGATGCTCGCCC-3′,5′-GACTTCTACAGTTCAGTCGAACG-3′) and for glucose-3-phosphate dehydrogenase (G3PDH) (5′-ACCACAGTCCATGCCATCAC-3′,5′-TCCACCACCCTGTTGCTGTA-3′). The samples for COX-2 were then subjected to first denaturation (2 minutes at 94°C), and various cycles (24, 27, and 30 cycles) of denaturation (15 seconds at 94°C), annealing (30 seconds at 55°C), and extension (90 seconds at 72°C), followed by final extension at 72°C for 5 minutes. The samples for G3PDH were subjected to 25 cycles of denaturation (45 seconds at 94°C), annealing (45 seconds at 60°C), and extension (2 minutes at 72°C), followed by final extension at 72°C for 7 minutes. Analysis of the PCR product used 1.0% agarose gel electrophoresis and visualization under ultraviolet light.

The five prime untranslated regions of COX-2, from -954 to +28, and from -581 to +28 were generated by PCR amplification using genomic DNA from U87MG cells. PCR was performed using the Expand High Fidelity PCR system (Roche Diagnostics Corporation, Indianapolis, Ind., U.S.A.) according to the manufacturer’s instructions. Nucleotides illustrated here are relative to the transcription initiation site at +1 (GenBank accession number AF276953). The following primers were used: upstream primers, from -954, 5′-GCATCAGGGAGAGAAATGCC-3′; from -581, 5′-GCCTATTAAGCGTCGTCACT-3′; downstream primer, from +28, 5′-GACGCTCACTGCIVLGTCGTAT-3′. PCR product was cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif., U.S.A.) and sequenced by the ABI PRISM dRhodamine Terminator Cycle Sequencing Ready kit (Perkin-Elmer Applied Biosystems, Foster City, Calif., U.S.A.).


Investigation of the effect of cytokines on COX-1 and COX-2 expression in normal human astrocyte cells showed that none of the cytokines tested caused changes in the expression of COX-1 but both IL-1β and TNF-α increased COX-2 expression (Fig. 1). In addition, all carcinogens (TPA, B(a)P, and BPDE) increased the expression of COX-2 without changes in COX-1 expression.

Fig. 1
Induction of cyclooxygenase-2 (COX-2) protein in normal human astrocyte cells by treatment with cytokines and carcinogen. Semi-confluent cells were treated with cytokines (10 ng/ml interleukin-1β [IL-1β], 1 ng/ml interferon-γ [IFN-γ], ...

Expression of COX-2 was weak in the U87MG cell line, and only slight in the T98G cell line. After treatment with IL-1β, induction of COX-2 protein was slightly increased in the T98G cell line, and remarkably increased in the U87MG cell line (Fig. 2), suggesting that COX-2 gene is inducible in glioma cell lines, especially in the U87MG cell line. In addition, the carcinogen TPA caused strong induction of COX-2 protein at 24 hours after treatment (Fig. 3).

Fig. 2
Induction of cyclooxygenase-2 (COX-2) in glioma cell lines by treatment with interleukin-1β (IL-1β). Semi-confluent cells were treated with 10 ng/ml IL-1β or vehicle for 24 hours. Equal amounts (20 μg) of total protein ...
Fig. 3
Effect of carcinogen in cyclooxygenase-2 (COX-2) induction in U87MG cells. Semi-confluent cells were treated with 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA). Equal amounts (20 μg) of total protein were examined by Western blot analysis ...

IL-1β induced significant up-regulation of COX-2 mRNA in the U87MG cell line in a cycle-dependent manner (Fig. 4). After PCR, the 1-kb length of promoter region in COX-2 fragment was cloned into the TA vector (3.9-kb) and the was sequenced. The nucleotide sequence was determined (data not shown) as identical to that previously reported in human COX-2 promoter.14)

Fig. 4
Cyclooxygenase-2 (COX-2) induction in U87MG cells treated with interleukin-1β (IL-1β). Ribonucleic acid was isolated from U87MG cells after treatment with IL-1β and reverse transcription (RT)-polymerase chain reaction was performed ...


The present study showed that COX-2 induction was remarkably induced by IL-1β and TPA at both mRNA and protein levels in the U87MG glioma cell line. Such findings are in agreement with previous studies in other cell lines,20) and in normal human astrocyte cells.29)

Several factors may be associated with the regulation of COX-2. For example, epidermal growth factor (EGF) is known to increase the expression of COX-2 in several types of cell,18) and EGF receptor amplification is common in malignant glioma.34) Loss of heterozygosity of chromosome 10 together with EGF receptor amplification is often observed in glioblastoma multiforme. bcl-2, an antiapoptotic protein overexpressed in gliomas, interacts with COX-2.30) Moreover, p16, another tumor suppressor gene that might be associated with malignant progression, affects COX-2 expression.27) Therefore, the regulation of COX-2 expression in gliomas is likely to involve many factors, so further studies are required to fully elucidate the mechanisms.

Inactivation of tumor suppressor genes including p53 is considered to be one of the molecular mechanisms of tumor progression. The function of p53 as tumor suppressor molecule is not well known in glioma, but p53 sometimes works as a transcriptional factor which regulates the downstream product related to the tumor suppressor function.5,21) A recent study indicated that wild-type p53 inhibits COX-2 expression in vitro,28) but any relationship between COX-2 and p53 in vivo might not be evident. Approximately 30% of glioma cell lines lack wild-type p53.3l) Loss of normal functioning p53 results in disrupted regulation of the cell cycle such as growth arrest and apoptosis,32,33) which provides one of the mechanisms for the effects of the absence of wild-type p53 in gliomas. Therefore, regulation of p53 may affect the expression of COX-2 in gliomas. The U87MG glioma cell line expresses wild-type p53 and retains wild-type p53 function, whereas the T98G and A172 glioma cell lines contain mutant p53 alleles with loss of functional p53 activity.31) These results support the genetic subclassification of glioblastoma. Further, COX-2 can be induced by modulation of p53 with various stimuli in the U87MG cell line.

NS-398, a COX-2-specific inhibitor, reduces tumor cell proliferation and migration, and increases apoptosis in human glioblastoma cell lines.12) On the other hand, overexpression of eicosanoid-producing enzyme (e.g. COX-2, 5-lipoxygenase) may induce tumorigenesis by increasing the intracellular level of arachidonic acid.19) These results suggest that COX-2 inhibitors could be useful for the treatment of malignant glioma. Non-steroidal anti-inflammatory drugs suppress tumor formation and cell growth.3) Aspirin and indomethacin inhibit cell growth and induce apoptosis in glioma cell lines.1) Inhibition of cyclooxygenase suppresses growth in glioma cells.4,11,12) These non-steroidal anti-inflammatory drugs target COX and its metabolites of unsaturated fatty acids, among other target molecules, so act at the post-translational level of COX-2 in glioma cells.

Generally, expression of COX-2 differs between cell lines,6,9,26) and is correlated with the cell characteristics, but not directly with malignancy. In our study, normal cell lines (normal human astrocyte cells) also expressed COX-2 with the various stimuli, suggesting that prostaglandins and lipid metabolites formed by astrocytes may be involved in central nervous system pathology and physiology.2,15,22) The present study also indicates that glioma cell lines, such as the U87MG glioma cell line, with high induction of COX-2 are good candidates for modeling of COX-2 targeting in glioma cell lines.


This research project was supported, in part, by the Intramural Program NIH, NIEHS


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