Figure 1.
PI3K/Akt pathway activation status and inhibition of Akt phosphorylation by PI3K/Akt pathway inhibitors in primary SLGCs.
(A) Western blot analysis of basal Akt, p-Akt, and PTEN expression levels. Sox2 is shown as a stemness marker and β-Actin as a loading control. (B) Inhibition of phosphorylation of Akt (serine 473) and of S6 ribosomal protein by Akt inhibitor III (Akti), LY294002 and PI-103. The cells were treated with the inhibitors for 2 h before collecting the samples for analysis.
Figure 2.
PI3K/Akt pathway inhibitors do not generally promote γIR-induced cell death in primary SLGCs.
(A–C) Percentage of annexin V/PI positive cells 2, 4, or 6 d after irradiation with 0, 2, or 10 Gy. Prior to irradiation, the cultures were pretreated for 1 h with Akt inhibitor III, LY294002 or PI-103. (D) Example of flow cytometric cell death analyses. The population with decreased forward light scatter (FSC) in the 10 Gy-irradiated sample reflects cell shrinkage and fragmentation typical of apoptosis. Early and mid apoptotic cells are annexin V-positive but still exclude PI; late apoptotic cells with compromised membrane integrity are annexin V/PI-double positive. (E) Clonogenic survival 14 d after treatment with γIR +/− Akt inhibitor III (25 µM) or PI-103 (0.6 µM). Experiments were performed in triplicates. Data in A–C represent means ± SD of three independent experiments. Statistical significance is indicated by an asterisk (p<0.05).
Figure 3.
PI3K/Akt pathway inhibitors reduce G2M arrest and induce autophagy in γ-irradiated SLGCs.
(A) Cells were treated with Akt inhibitor III (25 µM), LY294002 (50 µM), or PI-103 (0.6 µM) for 1 h and then irradiated with 10 Gy. The cell cycle analyses were performed 24 h later. (B) Conversion of cytosolic LC3-I to autophagosome-associated LC3-II, and (C) kinetics of H2AX-phosphorylation, a measure of DNA damage; the SLGCs were pretreated with PI3K/Akt pathway inhibitors for 1 h before irradiation with 10 Gy. Representative Western blots are shown.
Figure 4.
CQ and γIR synergistically induce strong apoptosis in SLGCs.
(A) Upper panel: percentage of annexin V/PI positive cells 5 d after treatment with CQ alone (white bars) or CQ plus γIR (gray bars). The cells were pretreated with CQ for 1 h and then irradiated with 10 Gy. Lower left: example of the flow cytometric analysis of apoptotic and dead cells; lower right: apoptotic morphology observed after Hoechst 33342/annexin V staining at 3 d after combination treatment. (B) Reduced survival of clonogenic cells 13 d after combination treatment. (C) Size reduction of preformed neurospheres 7 d after combination treatment. Viability of neurospheres was determined after calcein-AM staining. (D, E) Representative Western blots showing the effect of CQ or CQ plus γIR on expression levels of activated caspase-3 and LC3-I/II conversion (D), as well as on phosphorylation of H2AX (γH2AX) (E). (F) Lack of cell death induction in human fibroblasts. In A and F, data represent means of three independent experiments. Experiments in B and C were performed twice in triplicates. Statistical significance is indicated by an asterisk (p<0.05).
Figure 5.
Triple combinations of γIR, a PI3K/Akt pathway inhibitor and low doses of CQ show additive to highly synergistic proapoptotic effects in primary SLGCs.
(A) Percentage of annexin V/PI positive cells 5 d after treatment with 10 Gy, a PI3K/Akt pathway inhibitor (25 µM Akt inhibitor III, 50 µM LY294002, and 0.6 µM PI-103) and CQ (at the doses indicated), alone, or in double or triple combinations. Lower panel: example of flow cytometric analysis of GBM22 SLGCs showing that most cells treated with the triple combinations die by apoptosis. (B) Western blot analyses showing a strong accumulation of LC3-II in SLGC samples with high proportions of annexin V/PI positive cells. In addition, expression levels of pro- and antiapoptotic molecules and cleaved caspase-3 are shown. Data in (A) are means of three independent experiments. Statistical significance is indicated by an asterisk (p<0.05).
Figure 6.
Low doses of γIR, CQ and PI-103 synergistically induce apoptosis in GBM22 SLGCs.
(A, B) Left: Percentage of annexin V/PI positive cells 5 or 13 d after treatment with either γIR (3.5 Gy), CQ (5 µM) or PI-103 (0.5 µM) alone, with double or triple combinations. Right: cell numbers after trypan blue staining. Lower panel in A: flow cytometry data showing that cell death occurred primarily through apoptosis. (C) Western blot analysis of LC3-I/II conversion and of p21 expression levels.
Figure 7.
Synergistic effects of a low dose triple combination including PI-103 in stem cell surrogate assays.
(A) Adherent monolayer cultures of GBM22 SLGCs were treated for 5 d as described in Fig. 6, then plated in serum-free medium to form neurospheres. Seven days later total neurospheres were counted and photographed. (B) Treatment of preformed 3D neurospheres. Cells were plated to form one neurosphere per well and 2 d later treated as indicated. 7 d after treatment, neurospheres were stained with calcein-AM to determine the viability and diameter of the spheres. Data in (A and B) are means of three independent experiments. Statistical significance is indicated by an asterisk (p<0.05).