Synergistic Augmentation of Rapamycin-Induced Autophagy in Malignant Glioma Cells by Phosphatidylinositol 3-Kinase/Protein Kinase B Inhibitors

Abstract

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway and a central modulator of cell proliferation in malignant gliomas. Therefore, the targeting of mTOR signaling is considered a promising therapy for malignant gliomas. However, the mechanisms underlying the cytotoxic effects of a selective mTOR inhibitor, rapamycin, on malignant glioma cells are poorly understood. The purpose of this study was thus to elucidate how rapamycin exerts its cytotoxic effects on malignant glioma cells. We showed that rapamycin induced autophagy but not apoptosis in rapamycinsensitive malignant glioma U87-MG and T98G cells by inhibiting the function of mTOR. In contrast, in rapamycin-resistant U373-MG cells, the inhibitory effect of rapamycin was minor, although the phosphorylation of p70S6 kinase, a molecule downstream of mTOR, was remarkably inhibited. Interestingly, a PI3K inhibitor, LY294002, and an Akt inhibitor, UCN-01 (7-hydroxystaurosporine), both synergistically sensitized U87-MG and T98G cells as well as U373-MG cells to rapamycin by stimulating the induction of autophagy. Enforced expression of active Akt in tumor cells suppressed the combined effects of LY294002 or UCN-01, whereas dominant-negative Akt expression was sufficient to increase the sensitivity of tumor cells to rapamycin. These results indicate that rapamycin exerts its antitumor effect on malignant glioma cells by inducing autophagy and suggest that in malignant glioma cells a disruption of the PI3K/Akt signaling pathway could greatly enhance the effectiveness of mTOR inhibitors. (Cancer Res 2005; 65(8): 3336-46)

Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells

“…mTOR inhibitors induce apoptosis in some types of tumor cells (Hosoi et al, 1999;Nepomuceno et al, 2003;Majumder et al, 2004;Avellino et al, 2005), whereas they trigger autophagy in other settings (Noda and Ohsumi, 1998;Gutierrez et al, 2004;Kanazawa et al, 2004;Ravikumar et al, 2004) as well as in malignant glioma cells as shown in this study and our previous investigation (Takeuchi et al, 2005). Autophagy is a process by which cells degrade and recycle proteins and intracellular components in response to stress or starvation (Klionsky and Emr, 2000;Levine and Klionsky, 2004;Shintani and Klionsky, 2004).…”

“…As shown in Figure 4a, although the phosphorylation of p70S6K was suppressed to an undetectable level in U87-MG cells by treatment with 100 nM rapamycin for 48 h, mTOR catalytic activity was not inhibited. A PI3K inhibitor LY294002 (5 mM, 24 h), which we used as a negative control (Takeuchi et al, 2005), remarkably suppressed the phosphorylation of p70S6K and the mTOR catalytic activity. These results suggested that the cellular effects of rapamycin are not mediated through the suppression of mTOR kinase activity.…”

“…Soluble proteins were isolated from treated or untreated tumor cells for Western blotting as described previously (Takeuchi et al, 2005). Equal amounts of protein (20-40 mg) from each sample were separated by electrophoresis through sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Richmond, CA, USA) and transferred to a Hybond-P membrane (Amersham, Piscataway, NJ, USA).…”

“…Tumor cells were grown on glass coverslips, treated as described above for the cell viability assay, and fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h. After fixation, the samples were post-fixed in 1% OsO 4 in the same buffer for 1 h, and then subjected to electron microscopic analysis as described previously (Takeuchi et al, 2005). Representative areas were chosen for ultrathin sectioning and viewed with a JEM 1010 transmission electron microscope (JEOL, Peabody, MA, USA) at an accelerating voltage of 80 kV.…”

Antitumour effect of polyoxomolybdates: induction of apoptotic cell death and autophagy in in vitro and in vivo models

“…To determine the effect of PM-17 on tumour cells, we assessed cell viability after treatment using the trypan blue dye exclusion assay (Takeuchi et al, 2005). AsPC-1 and MKN45 in exponential growth phase were harvested, seeded at 1.5 Â 10 5 cells per 6 cm cell culture dish (5 ml), and incubated at 371C in 5% CO 2 in air overnight.…”