Fig. 2.

The mTOR kinase inhibitor KU-0063794 and the dual PI3K/mTOR inhibitor PI-103 both block phosphorylation of Akt and induce autophagy. In comparison, the mTOCR1 inhibitor rapamycin-induced phosphorylation of Akt and was a less potent inducer of autophagy. (a) PTEN mutant U373:MG cells were treated with DMSO, PI3Kα inhibitor PIK-90 (1 μM), mTORC1 inhibitor rapamycin (100 nM), mTOR inhibitor Ku-0063794 (5 μM), or a dual PI3K/mTOR inhibitor PI-103 (1 μM) for 48 h and stained with acridine orange (1 μg/ml) for 15 min. Cells were analyzed by flow cytometry. Autophagy was quantified by the accumulation of acidic vescular organelles. Percentage of AVOs is indicated. (b) Percentages of cells positive for AVOs; mean ± S.E. for triplicate samples (top panel). Cells were treated as in (a) for 24 h and lysates examined by immunoblot using antibodies shown (bottom panel). In contrast to rapamycin, Ku-0063794 and PI-103 block both p-Akt and p-rpS6. Ku-0063794 and PI-103 also decreased the phosphorylation of 4EBP1 and induced autophagy more potent than rapamycin as indicated by increased levels of the autophagy marker LC3-II. The PI3Kα inhibitor PIK-90 blocked phosphorylation of Akt, but did not affect phosphorylation of 4EBP1 or of rpS6, demonstrating that inhibition of PI3K and Akt activation alone is not sufficient to block the phosphorylation of mTORC1 targets 4EBP1 and of rpS6. (Reproduced from ref. 13 with permission from AAAS).