Antitumor effect of aspirin in glioblastoma cells by modulation of β-catenin/T-cell factor–mediated transcriptional activity

Laboratory investigation

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Object

The goal in this study was to investigate the antitumor effect of aspirin in glioblastoma cells and the molecular mechanism involved in its antineoplastic activities.

Methods

The authors used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method, flow cytometry, the annexin V method, and Transwell cell invasion test to detect the proliferation and invasive activity of U87 and A172 glioma cells before and after being treated with aspirin. To determine the effects of aspirin on β-catenin/T-cell factor (TCF) transcription activity, reporter constructs containing 3 repeats of the wild-type (TOPflash) or mutant (FOPflash) TCF-binding sites were used. Reverse transcriptase polymerase chain reaction and Western blot analyses were used to detect the expression of multiple β-catenin/TCF target genes following aspirin treatment.

Results

The transcriptional activity of the β-catenin/TCF complex was strongly inhibited by aspirin. Increasing the concentration of aspirin resulted in decreased expression of c-myc, cyclin D1, and fra-1 mRNA and protein in U87 and A172 cells in a dose-dependent manner. Aspirin inhibited glioma cell proliferation and invasive ability, and induced apoptotic cell death.

Conclusions

The results suggest that aspirin is a potent antitumor agent, and that it exerts its antineoplastic action by inhibition of the β-catenin/TCF signaling pathway in glioma cells.

Abbreviations used in this paper: COX = cyclooxygenase; FITC = fluorescein isothiocyanate; GBM = glioblastoma multiforme; LEF = lymphoid enhancer–binding factor; MMP = matrix metalloproteinase; MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI = propidium iodide; RT-PCR = reverse transcriptase polymerase chain reaction; TCF = T-cell factor.

Article Information

* Fengming Lan and Xiao Yue contributed equally to this work.

Address correspondence to: Chunsheng Kang, M.D., Ph.D., Laboratory of Neuro-Oncology, Tianjin Neurological Institute, 152 Anshan Road, Heping, Tianjin 300052, People's Republic of China. email: kang97061@yahoo.com.

Please include this information when citing this paper: published online July 1, 2011; DOI: 10.3171/2011.5.JNS113.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Bar graphs showing results of the MTT assay; the inhibitory effect of aspirin on growth of U87 and A172 cells is depicted. The U87 and A172 cells were treated with aspirin at concentrations of 0.5, 5, 10, and 20 mM, respectively, for 24, 48, and 72 hours. Aspirin inhibited the growth of U87 and A172 cells in a dose- and time-dependent manner. *p < 0.05, **p < 0.01 versus control group.

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    Graphs showing the effects of aspirin on cell cycle distribution in U87 and A172 cells. A: The U87 and A172 cells were treated with aspirin at a concentration of 10 mM, and cell cycle distributions were detected by flow cytometry 48 hours later. B: Percentages of cells in different phases of the cell cycle are shown in the histogram. **p < 0.01 versus control group.

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    Effects of aspirin on cell invasive ability in U87 and A172 cell lines. A: Cell invasive ability was assessed by a Transwell assay after 48 hours of treatment with aspirin at a concentration of 10 mM. B: The numbers of cells that could invade via the membrane are shown as a histogram. **p < 0.01 versus control group.

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    Aspirin induces apoptosis of U87 and A172 cells. A: Flow cytometry analyses of PI-stained cells were performed in triplicate. B: Percentages of apoptotic cells are shown in the histograms. **p < 0.01 versus control group.

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    Aspirin antagonized β-catenin/TCF transcriptional activity. A: The U87 and A172 cells were transiently transfected with TOP (wild-type) or FOP (mutant) luciferase reporter constructs, and after 16 hours the cells were treated with 10 mM aspirin for the indicated times. B: The U87 and A172 cells were transiently transfected with TOP or FOP luciferase reporter constructs, and after 16 hours the cells were treated with aspirin at concentrations of 0.5, 5, 10, and 20 mM for 24 hours. The TCF-dependent transcription was analyzed by measuring luciferase activity. “Fold” activation indicates the ratio of luciferase activity in each sample compared with that resulting from the mutated reporter (FOP) in untreated cells at time point 0. The average results of 2 repeated experiments performed in duplicate are shown; bars indicate SDs. TOP+/FOP+ = TOP/FOPflash with later addition of aspirin; TOP−/FOP− = TOP/FOPflash without later addition of aspirin. *p < 0.05, **p < 0.01 versus control group.

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    The impact of aspirin on the β-catenin/TCF pathway. A: The U87 and A172 cells were treated with aspirin at a concentration of 10 mM for 1, 6, 12, and 24 hours. B: Cells were treated with aspirin at concentrations of 0.5, 5, 10, and 20 mM for 24 hours. Change in expression of multiple members of the β-catenin/TCF pathway, including c-myc, cyclin D1, and fra-1, was measured by RT-PCR and Western blot. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Similar results were obtained in duplicate experiments.

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