Figure 6

mTORC2 is a better therapeutic target for cancer stem-like cells. (a) Stem-like cells were treated separately with mTORC2 inhibitors (mahanine and Ku-0063794) (Sigma-Aldrich), GANT61 (Gli2 inhibitor), cyclopamine (Shh inhibitor) (Sigma-Aldrich) and temozolomide (DNA intercalator) (Sigma-Aldrich) at different doses. Cell viability was checked using Cell Proliferation Reagent WST-1 (Sigma-Aldrich). Only mahanine and KU exhibited significantly decreased viability of cancer stem-like cells in a dose-dependent manner. (b) U87MG cells were treated with different doses of mahanine for 24 h and processed for western blot analysis. The result showed that mahanine inhibited Gli1 and Gli2 proteins. (c) Mahanine-treated GBM stem-like cells exhibited reduced neurosphere formation in a dose-dependent manner. (d) Mahanine-treated stem-like cells showed significantly reduced number of CD133-positive cells and its expression, as revealed by flow cytometric analysis. (e) Stem-like cells demonstrated enhanced sensitivity toward mahanine in a dose-dependent manner, as determined by PI positivity analysis using flow cytometry. (f) Mahanine showed minimal toxicity to normal glial cells compared with cancer stem-like cells. (g) Pictorial representation of summary of the events describing cross talk between mTORC2, Hh pathway and stemness characteristics in GBM. Statistical significance compared with the control is indicated by **P<0.01 and ***P<0.001