Resveratrol downregulates PI3K/Akt/mTOR signaling pathways in human U251 glioma cells

Abstract

Resveratrol (trans-3,4', 5-trihydroxystilbene) is a naturally occurring polyphenolic compound that has antiinflammatory, antioxidant, neuroprotective properties and acts as a chemopreventive agent. Resveratrol causes cell cycle arrest and induces apoptotic cell death in various types of cancer cells. In the current studies, the effect of resveratrol on phosphoinositide kinase-3 (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway was examined in human U251 glioma cells. Resveratrol decreased both the expression and phosphorylation of Akt. Inhibitors of PI3K (LY294002) and Akt (SH-6) enhanced resveratrol-induced LDH release and caspase-3 activation. Resveratrol reduced phosphorylation of ribosomal protein S6 and the mTOR inhibitor rapamycin further enhanced resveratrol-induced cell death. These results suggest that the downregulation of PI3K/Akt/mTOR signaling pathways may be an important mediator in resveratrol-induced apoptosis in glioma cells.

Figure 1

Resveratrol induces dose-dependent increase of caspase-3 activation and decrease of cyclin D1 expression. U251 cells were treated with 0, 1, 10, 25, 50 and 100 µM of resveratrol for 24 h. Western blot analysis was performed using antibodies against cleaved caspase-3 and actin (A) or Cyclin D1 and actin (B). For densitometric analysis of cyclin D1 expression, **, p < 0.01, ***, p < 0.001, vs. Con, n=3.

Figure 2

PI3K/Akt inhibitors enhance the resveratrol-induced apoptosis. U251 cells were treated with 0 or 100 µM of Res for 24 h, in the absence or presence of 20 µM of PI3K inhibitor LY294002 or 20 µM of Akt inhibitor, SH-6. (A) Western blot analysis of phospho-Akt (ser473). (B) Western blot analysis of cleaved caspase-3 and PARP. (C) Caspase-3 activity assay; ***, p < 0.001, vs. Con; ***Δ, p < 0.001, vs. Res; n=3. (D) LDH release assay; **, p < 0.01, vs. Con; **Δ, p < 0.01, vs. Res; n=3.

Figure 3

Akt siRNA enhances resveratrol-induced apoptosis. U251 cells in 6-cm dishes were transfected with 50 nM of Akt siRNA for 48 h using a SignalSilence Akt siRNA kit (Cell Signaling), followed by treatment with 100 µM of Res for 24 h. (A) Western blot analysis of phospho-Akt (ser473), Akt, cleaved caspase-3 and actin. (B) LDH release assay; ***, p < 0.001, vs. Con; *Δ, p < 0.05, vs. Res; n = 3.

Figure 4

The mTOR inhibitor rapamycin enhances resveratrol-induced apoptosis. (A) U251 cells were treated with 100 µM of resveratrol for 0, 2, 4, 8 and 24 h. Western blot analysis was performed using antibodies against phospho-Akt (ser473), phospho-mTOR (ser2448) and actin. In a separate experiment, U251 cells were treated with 100 µM of resveratrol for 24 h, in the absence or presence of 10 nM rapamycin (Rapa). Western blot analysis was performed using PathScan multiplex Western cocktail I antibodies (B) or antibodies against phospho-Akt (ser473), Akt and actin (C). (D) LDH release assay was performed in U251 cells treated with vehicle or 100 µM of resveratrol for 24 h, in the absence or presence of 10 nM rapamycin (Rapa); **, p < 0.01, Vs. Con;* Δ, p < 0.05, vs. Res; n = 3.