Figure 6
From: Metformin-induced ablation of microRNA 21-5p releases Sestrin-1 and CAB39L antitumoral activities
Metformin synergized with everolimus. (a) Graphs indicating the change in impedance of SUM159PTcells pretreated or not with metformin (0.5 mM) for 24 h and subsequently treated with everolimus (10–100 nM) for 0–550 min to evaluate early changes in cell fitness. Statistics (t-test): P<0.05 (versus control). (b) Combination index (CI) versus the fractional effect obtained from SUM159PT cells treated simultaneously with metformin (0.5 mM) and everolimus (0-100 nM) for 72 h. (c) Histograms showing the average percentage of colonies formed by SUM159PT cells pretreated with vehicle or metformin (0.5 mM) and subsequently treated with everolimus (0–100 nM). Bars indicate the average±s.e. of triplicate experiments. Statistics (t-test): P<0.05. (d) Everolimus and metformin treatment synergistically affected mammalian target of rapamycin (mTOR) signaling. Representative western blotting (upper panel) and quantitative densitometry (lower panel) phospho-ser2448-mTOR, (phospho-ser235/236)-S6, (phospho-thr37/46)-4-EBP1 and (phospho-thr389)-p70 S6 normalized to their respective non-phosphorylated protein levels. Bars indicate the average of three independent experiments. Statistics (t-test): P<0.05. (e) Altering the levels of calcium-binding protein 39-like (CAB39L) and Sestrin-1 (SESN1) affected the response to everolimus. Graphs indicating the change in impedance of SUM159PT treated as indicated. (f) Metformin affected the clonogenic ability and potentiated the antitumorigenic effects of everolimus in both phosphatase and tensin homolog (PTEN) wt and PTEN K/D isogenic HCT116 cells. Briefly, HCT116 cells harboring either PTEN wt (left panel) or deleted for PTEN (right panel) were treated, 24 h after seeding, with metformin, in the presence or absence of everolimus. Histograms showing the average of three independent clonogenic assays. Colonies were stained after 9 days. Statistics (t-test): P<0.05. Please note that very similar results were obtained after altering the levels of the miR-21-5p, whose downregulation strictly mimicked the effect of metformin on the everolimus-treated cells (Supplementary Figure S5). (g) Invasion assay. HCT116 cells harboring either PTEN wt (left panel) or deleted for PTEN (right panel) were treated as from panel (e) for 24 h and the number of migrated cells was scored. Statistics (t-test): P<0.05.
