Glioblastoma

The in vitro antiproliferative and apoptosis inducing properties of the nonsteroidal anti-inflammatory drugs (NSAIDs) like acetyl salicylic acid (aspirin) and indomethacin were investigated in T98G human glioblastoma cells to explore their potential role in the chemoprevention of human glioma. The biological effects induced by aspirin and indomethacin on T98G cells, in which the expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were confirmed by RT-PCR and immunostaining, were investigated by studying cell proliferation and apoptosis assays. The antiproliferative effects occurred in a dose- and time-dependent manner on T98G cells by the treatment with 0.1 -2 mM aspirin and 25-100 [mu]M indomethacin. Moreover, aspirin displayed the greatest growth inhibition within 24 h. Approximately 90% growth inhibition occurred following treatment either with 2 mM aspirin or 100 [mu]M indomethacin by 72 h and induction of apoptosis was confirmed by DNA laddering and TUNEL assay. Our in vitro findings indicate that aspirin and indomethacin have an antiproliferative effect on T98G human glioblastoma cells at toxic concentrations. [Neurol Res 2003; 25: 370-376]

Keywords: NSAIDs; T98G; COX; apoptosis; chemoprevention

INTRODUCTION

Gliomas are the most common form of primary brain tumors, with high grade gliomas (malignant gliomas) constituting the most serious as well as the most common group1. At present, malignant gliomas have been treated in combination with surgery, radiation, chemotherapeutic agents, and biological response modifiers such as interferons2. In spite of the progress of these strategies, gliomas still have a poor prognosis. A few patients survive more than two years despite large doses of radiation and systemic chemotherapy after surgery3. Even if an effective response is obtained by the primary therapy, it is very difficult to overcome the local recurrence and/or invasion of the tumors due to drug resistance and the heterogeneous characteristics of malignant gliomas4. Thus, chemopreventive agents are warranted to block, reverse or prevent the development of human malignant gliomas.

Recently, aspirin and indomethacin have attracted significant interest in oncology because of their potential role in the chemoprevention of different types of cancer cell lines in vitro and cancers in vivo especially in the case of colon cancers5-8. Recent studies have shown that NSAIDs including aspirin and indomethacin induce apoptosis in various human cancer cell lines8-11. The molecular target for NSAIDs is cyclooxygenases (COX). Cyclooxygenases are the rate-limiting enzymes for the synthesis of eicosanoids such as prostaglandin (PG) E^sub 2^ and -D^sub 2^ from arachidonic acid12. There are two isoforms of COX; a constitutive isoform, COX-1, expressed in most human tissues and an inducible isoform, COX-213. Cyclooygenase-2 is concentrated in the endoplasmic reticulum and perinuclear envelope, whereas COX-1 is localized primarily in the endoplasmic reticulum14. The COX isoforms are pharmacologically distinct such that they are differentially inhibited by individual NSAIDs15. Various cancers and cancer cell lines show expression of COX-1 and 213,16 with a few reports regarding gliomas17,20. The cyclooxygenase activity in the gliomas might be a potential target for chemopreventive NSAIDs in the future. Based upon the above background, we evaluated the in vitro antiproliferative effects of the conventional NSAIDs, aspirin and indomethacin on T98G human glioblastoma cells. In addition, the expression of COX-1 and COX-2 at the protein and mRNA levels were investigated in T98G cells.

MATERIALS AND METHODS

Cell line and culture

The T98G cell line was purchased from Health Science Research Resources Bank, Osaka, Japan. Cells were grown in Dulbeco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (CS) (Life Technologies, Gaithersburg, MD, USA), 2 mM glutamine, penicillin (100 units ml^sup -1^) and streptomycin (100 [mu]g ml^sup -1^) at 37[degrees]C in a humidified incubator with an atmosphere of 5% CO^sub 2^/95% O^sub 2^

lmmunostaining for COX-1 and COX-2 expression in T98G cells

Expression of COX-1 and COX-2 at the protein level were investigated by immunocytochemistry. COX-1 and COX-2 immunostaining were clone according to Silverman laboratory protocol (Children's Hospital, Boston MA, USA). Briefly, T98G cells were grown on chamber slide up to near confluence; washed with phosphate-buffered saline (PBS) and fixed in 10% neutral buffered formalin for 10 min. Endogenous peroxidase activity was quenched with 3% H^sub 2^O^sub 2^ and nonspecific binding was blocked by normal goat serum. Goat polyclonal anti-bodies, COX-1 and COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were then applied at a dilution of 1 : 400 overnight at 4[degrees]C. All other steps were done using a Histofine SAB-PO (G) kit (Nichirei Corp., Tokyo, Japan) according to the manufacturer's instructions. The reaction product was visualized by diaminobenzidine (DAB) solution (Histofine, Tokyo, Japan). Counterstaining was done using hematoxylin. As a negative control, the primary antibody was omitted. The experiment was repeated several times to confirm reproducibility.

RT-PCR for COX-1 and COX-2 expression in T98G cells

Expression of COX-1 and COX-2 at the mRNA level were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) as described previously21. Briefly, total RNA was extracted from T98G cells by the acid guanidinium thiocyanate-phenol-chloroform single step extraction method with lsogen (Nippon gene, Toyama, Japan). Forty [mu]l cDNA was prepared from 2 [mu]g of total RNA. All reagents for cDNA synthesis and DNA amplification were purchased from TaKaRa, Otsu, Japan. DNA amplification was carried out with 5.0 [mu]l cDNA using 2.5U Taq DNA polymerase, TaKaRa Ex Taq. The primer sequences and PCR product sizes were as follows: for COX-1, 5'-GCACCCCAGCAGCCGCGCCATGA-3'(F), and 5'-GCTGCTTTCCTGCCCCTCAGAGCTC-3'(R), 1835 bp; for COX-2, 5'-CCCGCCGCTGCGATGCTCGCCC-S'tF), 5'-GACTTCTACAGTTCAGTCGAACG-3'(R), 1832 bp. Amplification of COX-1, COX-2 and G3PDH was performed at the same time using a Mini Cycler, Model PTC-150-16 (Funakoshi, Tokyo, Japan). The PCR conditions were: for COX-1, 4 min 94[degrees]C; 35[degrees]1 min/94[degrees]C, 1 min/70[degrees]C, 2min/72[degrees]C; 7 min 72[degrees]C. For COX-2, 2 min 94[degrees]C; 35x1 5sec/94[degrees]C, 30 sec/55[degrees]C, 90sec/72[degrees]C; 5 min 72[degrees]C. Amplified cDNA were run on 1.0% agarose gel and stained with ethidium bromide (0.5 [mu]g ml^sup -1^). No cDNA in the PCR reaction was used as negative control for COX-1 and COX-2. The RT-PCR experiment was repeated several times to confirm reproducibility.

Cell viability assay

The effects of aspirin and indomethacin (Sigma Chemical Co., St. Louis, MO, USA) on T98G cells were determined by using a cell proliferation assay previously described16. Briefly, cells were seeded at a density of 1x10^sup 3^/100 [mu]l DMEM containing 10% CS per well in 96-well plates. After 24 h, fresh medium was added containing either aspirin (0 to 2000/ [mu]M) or indomethacin (0 to 100 [mu]M) in 96-well plates. The final concentration of DMSO for all treatments (including controls, where no drug was added) was less than 0.1%. The MTS [3-(4,5-dimethylthiozol-2-yl)-5-(3-carboxymethoxyphenyl) -2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] assay (Promega, Madison, Wl, USA) was performed to estimate the number of viable cells by using 96-well plate reader (Lab Systems Multiskan MS-UV, Lab systems, Finland) according to the manufacturer's instruction. The results shown in the figures are indicated as represent experiments performed in triplicate.

TUNEL assay

Apoptotic cells induced either by aspirin or indomethacin were identified by TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) method using the Apop Taq^sup (R)^ peroxidase in situ apoptosis detection kit (lntergen Company, 2 Manhattanville Road, NY, USA) according to manufacturer's instructions. Briefly, cells were seeded at a density of 1x10^sup 4^/ml DMEM containing 10% CS in chamber slides. Aspirin and indomethacin were diluted with DMEM containing 10% CS and cells were incubated either with aspirin (500 to 2000 [mu]M) or indomethacin (25 to 100 [mu]M) for 24 to 72 h at 37[degrees]C in a humidified chamber. After incubation and washing with PBS, the adherent cells were fixed in 10% neutral buffered formalin for 30 min. Slides were then incubated in proteinase-K (40 [mu]g ml^sup -1) for 1h at 37[degrees]C. The endogenous activity was quenched with 3.0% H^sub 2^O^2 in methanol for 30 min at room temperature. After incubating in equlibration buffer for 5 min at room temperature, terminal deoxynucleotidyl transferase (TdT) enzyme was applied to the cells and incubated at 37[degrees]C for 2h. The reaction was stopped by incubating slides in stop buffer for 30 min at 37[degrees]C. Anti-digoxigenin-peroxidase was applied and incubated at 37[degrees]C for 30 min. Slides were stained with diaminobenzidine (DAB) for 3 to 6 min and subsequently counter-stained with hematoxylin. TdT was omitted for use in the negative control.

'DNA ladder' detection on agarose gel

DNA fragmentation of T98G cells induced either by aspirin or indomethacin were evaluated by agarose gel electrophoresis of genomic DNA to detect DNA ladder pattern. Cells were cultured in DMEM containing 10% CS either with aspirin (500 to 2000 [mu]M) or indomethacin (100 to 400 [mu]M) for 24 h to 72 h. After collecting the floating cells, about 5 million cells were pelleted and the pellet was lysed in 0.2ml lysis buffer [10mM Tris (pH 8.0), 10mM EDTA and 0.5% Triton X-100] for 10 min on ice. Microcentrifugation was done at 15,000 rpm for 20 min at 4[degrees]C to separate the nuclear DNA precipitate from the fragmented DNA present in the supernatant. The supernatant was treated with RNase A (100 [mu]g mh^sup -1^) at 37[degrees]C for 1 h and then proteinase-K (100 [mu]g ml^sup -1^) was added and incubated for another 1 h at 370C. Fragmented DNA from the supernatant was precipitated with two volumes of iso-propanol and 1/4 volume of 5 [mu]M NaCl at -20[degrees]C overnight. DNA pellets were washed twice in 70% ethanol and re-suspended in 20 [mu]M of TE buffer [10 mM Tris (pH 7.4) and 1 mM EDTA]. Finally, 10 [mu]l DNA preparations were electrophoresed on 2% agarose gels. Gels were stained with ethidium bromide and visualized under UV light

Statistical analysis

The data shown in this manuscript are the representative ones which were performed by at least three independent experiments. The values of the data were expressed as mean + or - SD. Comparison between different groups was done using the ANOVA test. A p-value was assessed using post-hoc test.

RESULTS

Expression of COX-1 and COX-2 in T98G cells

The expression of COX-1 and COX-2 were examined in T98G cells by immunocytochemistry and RT-PCR. The results of these two techniques yielded concordant results. The expressions of COX-1 and COX-2 proteins were detected in T98G cells by immunocytochemistry. By this technique, cytoplasmic and perinuclear COX-1 (Figure 1A) and COX-2 (Figure 1B) immunostaining were detected in T98G cells. The expressions of COX-1 and COX-2 proteins were then confirmed by RT-PCR. RT-PCR demonstrated that T98G cells express both COX-1 and COX-2 mRNA. Higher levels of COX-2 mRNA was detected in comparison to COX-1 mRNA. One percent agarose-gel electrophoresis of the RT-PCR products amplified from T98G cells using COX-1 and COX-2 primers resulted in an 1835 bp and 1832 bp band respectively (Figure 2). Both our COX-1 and -2 immunostaining and RT-PCR data were reproducible. Thus, these immunocytochemical and RT-PCR results confirmed that T98G cells express both COX-1 and COX-2.

Growth inhibitory effects of aspirin and indomcthacin on T98G cells

Aspirin less than 500 [mu]M and indomethacin less than 25 [mu]M concentrations did not show any growth inhibitory effect on T98G cells (data not shown). Aspirin and indomethacin inhibited the growth of T98G cells. This growth inhibition occurred in a dose- and time-dependent manner. However, aspirin showed greater inhibitory effect by 24 h. T98G cells were incubated with several doses of either aspirin (0 to 2000 [mu]M) or indomethacin (0 to [mu]M) for 72 h and cell viability was determined by the MTS assay. Both aspirin and indomethacin produced a concentration-dependent growth inhibition on T98G cells at 72 h, when compared with control conditions (no treatment with NSAIDs) (Figure 3A,B). The percentage of growth inhibition induced by 2 mM aspirin was increased from 78% at 24 h to 89% at 72 h (Figure 4A) whereas the percentage of growth inhibition induced by 100 [mu]M indomethacin was increased from 26% at 24 h to 88% at 72 h (Figure 4B).

Analysis of apoptosis

To evaluate whether the growth inhibitory effects of aspirin and indomethacin on T98G cells were associated with apoptosis, TUNEL and DNA ladder assays were performed. By the TUNEL assay, approximately 20%-40% apoptotic cells were found by either 1 mM aspirin (Figure 5A) or 100 [mu]M indomethacin (Figure 5B) in T98G cells at 48 h. We found clear DNA ladder pattern at 500 to 2000 [mu]M aspirin and at 100 to 400 [mu]M indomethacin at 48 h (Figure 6). To confirm the apoptosis inducing properties of aspirin and indomethacin, we repeated the TUNEL and DNA laddering experiments several times and achieved similar result every time. These results indicated that growth inhibition induced by aspirin and indomethacin on T98G cells were associated with apoptosis.

DISCUSSION

Numerous data concerning the in vitro and in vivo effects of NSAIDs in colorectal cancer have been accumulated. Recently, some clinical trials with selective COX-2 inhibitor like Celecoxib, have already been started in patients with familial adenomatous polyposis (FAP), a condition associated with a 100% lifetime risk of colon cancer in determining if selective COX-2 inhibitors are useful in cancer prevention22,23. A few studies have been reported concerning the effects of NSAIDs on glioma cell line19,24. The present study was undertaken to analyze the effects of aspirin and indomethacin on T98G human glioblastoma cells. In this study, aspirin and indomethacin exhibited growth inhibition on T98G cells in a dose- and time-dependent manner. Aas et al.26 found 65% and 11% growth inhibition of rat glioma RG 2 cells at 60 h after treatment with 2 mM aspirin and 50 [mu]M indomethacin respectively. Searching PubMed, we did not find any paper concerning the growth inhibitory effect of aspirin and indomethacin on human glioma cell lines. That is why we compared the growth inhibitory effects of aspirin and indomethacin on T98G cells with those on human colorectal carcinoma cells. T98G cells displayed a higher sensitivity for aspirin and indomethacin than human colorectal carcinoma cells in a previous report7. Smith et al.7 reported 44%, 35%, 47%, and 26% growth inhibition of HT29.Fu, HCA-7, SVV480 and HCT116 cells respectively, after 72 h treatment with 100 [mu]M indomethacin whereas we found 88% growth inhibition of T98G cells at the same time and same concentration. In addition, approximately 30% growth inhibition was reported in HT29.Fu, HCA-7, SW480 and HCT 116 cells respectively at 72 h after treatment with 1 mM aspirin whereas we observed 53% growth inhibition of T98G cells at the same time and same concentration. Data obtained in in vitro studies with cultured cells show that NSAIDs inhibit cell proliferation and increase cellular apoptosis/necrosis at concentrations 10 to 100 fold greater than the concentrations required for inhibition of both COX enzymes. For instances, 1-10mM aspirin26 and 600 [mu]M indomethacin7 were used to show antiproliferative effects in cancer cells. Such plasma concentrations, if achieved, will be above the documented limit of toxicity in the physiological condition for human27, making their use in clinical situations unlikely. In contrast, in epidemiological studies, NSAIDs were used in low doses over a long period to prevent colorectal cancer28. In case of in vivo study, NSAIDs exhibit anti-neoplastic effect at low dose29. Eli et al.29 reported that indomethacin was found to be highly effective in delaying the growth of both the primary tumor inoculate and of lung metastatic nodules at plasma concentrations of approximately 6[mu]M ml^sup -1^. Hence, before we interpret the results obtained in our in vitro study and correlate their relevance to humans, the in vivo effects of existing NSAIDs in a glioma model is needed. In this study, we also confirmed that cytotoxicity induced by aspirin and indomethacin was associated with apoptosis. Recently apoptosis is being discussed as a final pathway of cell death in tumors and several other diseases. Induction of apoptosis is required to arrest neoplasia.

We found both COX-1 and COX-2 expression in T98G cells by immunostaining and RT-PCR techniques. The expression of COX-1 in T98G cells was already reported by Roller et al.18. They pointed out that the increase of doxorubicin and vincristine cytotoxicity by NSAIDs correlates with COX-1 expression. IC^sub 50^ of aspirin is 1.7 [mu]M for COX-1 and 278 [mu]M for COX-2 and that of indomethacin is 0.03 [mu]M for COX-1 and 1.7 [mu]M for COX-2, respectively30. In our studies, we used 10 to 100 fold higher concentrations of aspirin and indomethacin than those required for inhibition of both COX enzymes. So, with Vincent et al.30 we can conclude in this study that aspirin and indomethacin show antiproliferative effects in T98G cells independent of COX inhibition. Although some data demonstrating that the anti-tumorigenic activity of NSAIDs is related to COX inhibition29,31 , many other data suggest that NSAIDs have COX-independent effects. The COX-independent effects are explained by the following pathways; p53 pathway11, activation of caspases pathway26, cyclin expression pathway8, modulation of the ras signal transducation pathway32 and TGF-[beta] family pathway33 etc. To date, the precise mechanisms by which NSAIDs exert their anti-neoplastic effect remains unclear.

CONCLUSION

This study provides the fundamental in vitro growth inhibitory findings of aspirin and indomethacin in T98G human glioblastoma cells. Our findings suggest that further studies regarding the in vivo chemopreventive effects of NSAIDs in glioma models and elucidation of their precise mechanism(s) of action should be conducted.

ACKNOWLEDGEMENTS

We thank Mr F. Bottone of the Laboratory of Molecular Carcinogenesis (National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA) for editing the manuscript.

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Ruhul Amin, Hideki Kamitani, Habiba Sultana, Seijiro Taniura, Azharul Islam, Atsuko Sho, Minako lshibashi, Thomas E. Eling* and Takashi Watanabe

Department of Neurosurgery, Institute of Neurological Sciences, Faculty of Medicine, Tottori University School of Medicine, Tottori, Japan

*Laboratory of Molecular Carcinogenesis, National Institutes of Environmental Health Sciences, Research Triangle Park, NC, USA.

Correspondence and reprint requests to: Hideki Kamitani, Department of Neurosurgery, Institute of Neurological Sciences, Faculty of Medicine, Tottori University School of Medicine, 36-1 Nishi-cho, Yonago, Tottori 683-8504, Japan, [kamitani@grape.med.tottori-u.ac.jp] Accepted for publication February 2003.

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