Figure 7.

Mesothelioma cell apoptosis within spheroids is enhanced by blocking PI3K/Akt and mTOR pathways. (A) Apoptosis in treated spheroids quantified as the percentage of cytokeratin-positive cells with co-expression of cleaved, active caspase 3. Spheroids from three separate tumors were either not treated or treated with the strong apoptotic stimulus, TRAIL plus cycloheximide, for 48 h. To determine a possible contribution of known survival pathways, spheroids were also exposed to inhibitors of the proteasome (MG132), the mTOR pathway (rapamycin), or the PI3K/Akt pathway (LY294002). Apoptosis induced by TRAIL plus cycloheximide was enhanced by co-treatment with rapamycin or with LY294002, while LY294002 also had an effect on baseline apoptosis. (mean ± SEM; ^*different from untreated or inhibitor alone, ^†different from TRAIL plus cycloheximide without inhibitor; P < 0.05, n = 3). (B–E) Immunohistochemical confirmation of activity of PI3K/Akt and mTOR pathways and of effectiveness of inhibitors. In spheroids stained for p-Akt (B and C), spheroids treated with TRAIL plus cycloheximide alone (B) demonstrated staining for p-Akt that was reduced in spheroids co-treated with LY294002 (C). Similarly, in spheroids stained for p-S6 kinase (D and E), spheroids treated with TRAIL plus cycloheximide alone (D) demonstrated staining for p-S6 kinase, a target of mTOR, which was reduced in spheroids co-treated with rapamycin (E).