Growth inhibition of human malignant glioma cells induced by the PI3-K—specific inhibitor

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Object. The phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a tumor suppressor by negatively regulating the growth/survival signals of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The PI3-K/Akt pathway in PTEN-deficient tumors may be one of the key targets for anticancer therapy. The authors examined the effects of the PI3-K inhibitor 2-(4-morpholinyl)-8-phenylchromone (LY294002) on human malignant glioma cells, and compared these effects on PTEN-deficient cells with those on PTEN—wild-type (PTEN-wt) cells.

Methods. Using human malignant glioma cell lines, including the PTEN-deficient cells A172 and U87MG and the PTEN-wt cells LN18 and LN229, the effects of LY294002 on cell growth, apoptosis, and chemotherapeutic agent—induced cytotoxicity were evaluated. The LY294002 inhibited the growth of U87MG cells associated with reduced phosphatidylinositol 3,4,5,-trisphosphate and phosphorylated Akt, and also induced growth inhibition in three other cell lines. Although LY294002 caused apoptosis in all four cell lines, apoptosis seemed to contribute to only a small portion of growth inhibition induced by LY294002. There was no link between the status of PTEN and the median inhibitory concentration values for LY294002 or between the gene status and the extent of LY294002-induced apoptosis. The LY294002 significantly augmented the cytotoxicity induced by etoposide in PTEN-deficient cells, but not in PTEN-wt cells. Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea— and cisplatin-induced cytotoxicity by LY294002 was not linked to the status of PTEN. No marked difference in the amounts of phosphorylated Akt was found between PTEN-deficient and PTEN-wt cells.

Conclusions. The findings show that PI3-K is a possible target for therapy in patients with gliomas, and PI3-K inhibitors in combination with chemotherapeutic agents could be potent therapeutic modalities for patients with malignant gliomas.

Article Information

Address reprint requests to: Takashi Shingu, M.D., Department of Neurosurgery, Shimane Medical University, Izumo 693–8501, Japan. email: nogeka@shimane-med.ac.jp.

© AANS, except where prohibited by US copyright law.

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Figures

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    Suppression of PIP3 production and Akt phosphorylation induced by LY294002 in U87MG cells. A: Thin-layer chromatography analysis of phospholipids extracted from 32P-labeled U87MG cells treated with the indicated concentrations of LY294002. B: Immunoblot analysis of lysates from cells treated with the indicated concentrations of LY294002 for 10 minutes (anti—phospho-Akt and anti-Akt antibodies were used). Phospho-Akt = phosphorylated Akt.

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    Graph demonstrating LY294002-induced growth inhibition of malignant glioma cells. The relative number of viable U87MG cells treated with the indicated concentrations of LY294002 was determined using MTT. Representative data from four independent experiments are presented and expressed as mean values ± standard deviations (SDs) of five studies. *p < 0.05; **p < 0.01.

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    Gels showing LY294002-induced oligonucleosomal DNA fragmentation in malignant glioma cells. A DNA fragmentation assay was performed after treatment of A172 (lanes 1–3), LN18 (lanes 4–6), and LN229 (lanes 7 and 8) cells with 0 µM (lanes 1, 4, and 7), 20 µM (lane 8), or 50 µM (lanes 2, 3, 5, and 6) LY294002 in the absence (lanes 1, 2, 4, and 5) or presence (lanes 3 and 6–8) of 10% FBS for 24 to 72 hours.

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    Bar graphs showing increases in anticancer drug—induced cytotoxicity in human malignant glioma cells in response to LY294002. Four cell lines including U87MG (A), A172 (B), LN18 (C), or LN229 (D) cells were treated with vehicle (—), 20 µM etoposide (ET), 100 µM BCNU, or 5 µM cisplatin (CDDP) in the presence (hatched bars) or absence (stippled bars) of 20 µM LY294002 for 3 days, and cytotoxicity was determined using MTT. Data shown represent the mean values of three independent experiments performed in triplicate assays. Error bars indicate SDs. *p < 0.05; **p < 0.01.

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    Immunoblots showing Akt phosphorylation and PTEN in human malignant glioma cells. The cells were serum-starved for 2 days (3 days for LN18; A) and then stimulated with 10% FBS for 1 hour (B). Cells cultured with serum were treated with 20 µM LY294002 for 1 hour (C). Lysates of these cells were analyzed by immunoblotting with anti—phospho-Akt and anti-Akt antibodies. Lysates from tumor cells cultured for 3 days in DMEM containing serum were analyzed by immunoblotting with the anti-PTEN antibody (D). U87 = U87MG cells.

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