Figure 4.

Natural compound inhibitors of the mTOR pathway and their effect on the levels of p53 and p21 alone or following nucleolar stress. (A) U2OS cells were pre-treated with rapamycin and natural compound inhibitors for 6 hours in duplicate samples and thereafter one of these sample sets was treated with Act D and incubated for an additional 18 hours. Cells were harvested, protein lysates prepared, and the relative levels of p53, p21, S6K1, and p-S6K1 (Thr389) were determined by immunoblotting. β-actin served as a loading control. Concentrations of compounds were as follows; rapamycin 2.74 μM; wortmannin 0.4 μM; caffeine 1 mM; resveratrol 25 μM; curcumin 20 μM; EGCG 25 μM; and Act D 5 nM. (B) Morphology of U2OS cells treated with the indicated compounds. (C) U2OS cells were treated for 18 hours with a low (5 nM) or high (1 μM) concentration of Act D and subsequently immunofluorescence stained for cleaved caspase-3. (D) Quantification of cleaved caspase-3 positive cells in different treatments. Approximately 1000 cells in 3 different low power fields were evaluated for each treatment and results are presented as % cleaved caspase-3 positive cells (mean ± SD). (E) U343MGa Cl2:6 glioma cells were pre-treated with rapamycin or various natural compounds for 6 hours and then actinomycin D (5 nM) was added as indicated. Cells were harvested, proteins extracted and the levels of p53 and p21 relative to the DMSO control were determined by immunoblotting. Concentrations of the compounds used were as follows; rapamycin 2.74 μM; wortmannin 0.4 μM; caffeine 1 mM; resveratrol 25 μM; curcumin 20 μM; EGCG 25 μM; dexamethasone 1 mM; and actinomycin D 5 nM. Dexamethasone (Dex) was included in this experiment as a possible regulator of p53.⁷³