Article Figures & Data
Figures
- Figure 1.
NVP-BEZ235 treatment inhibits glioma proliferation and attenuates PI3K/mTOR signaling pathway. A, glioma cells in 96-well plates were treated with increasing concentrations of NVP-BEZ235 for 72 h and subjected to a sulforhodamine B assay, as described in Materials and Methods. Plot depicts the percentage growth of NVP-BEZ235–treated cells compared with the growth of the vehicle-treated control cells. Each culture was done in triplicate. Points, arithmetic mean from a single experiment; bars, SD. Similar results were obtained from three independent experiments. U251 and U87 cells are used as PTEN-negative cells and LN18 and LN229 as PTEN-positive cells. B, to evaluate the target inactivation by NVP-BEZ235 in a time-dependent manner, glioma cells were treated with a fixed concentration of NVP-BEZ235 for 72 h. At each indicated time point, cell extracts were subjected to immunoblotting analysis.
- Figure 2.
NVP-BEZ235 interrupts growth factor–induced PI3K/mTOR signaling. Under basal conditions in SFM, the PI3K signal as assessed by the phospho-Akt level was constitutively active in PTEN-negative U87 cells but not in wt-PTEN LN229 cells. On growth factor stimulation, levels of phospho-Akt in U87 cells remained unchanged, whereas a different pattern of phospho-Akt activation was noted in LN229 cells. Based on the extent of Akt activation, receptors for insulin-like growth factor-I (IGF-1) seemed to be more abundant than receptors for EGF and platelet-derived growth factor (PDGF) in LN229 cells. The VEGF receptor seemed to be scarce in LN229 cells. The activity of Akt and S6K1 in both cell lines was significantly inhibited by NVP-BEZ235, suggesting its effectiveness on blocking the PI3K/mTOR signal pathway. Expression of the corresponding total target protein and actin was used as control.
- Figure 3.
NVP-BEZ235 caused cell cycle arrest and induces autophagy. A, cell cycle analysis was done on cells treated or untreated with 50 nmol/L NVP-BEZ235 for 48 h. Cell cycle distribution was labeled on the histogram. NVP-BEZ235 treatment resulted in G1 cell cycle arrest. B, following NVP-BEZ235 treatment, glioma cells were stained with acridine orange for FACS analysis to assess autophagy or C, harvested for immunoblotting analysis, to assess LC3-II expression. It seems that glioma cells are prone to NVP-BEZ235–induced autophagy.
- Figure 4.
NVP-BEZ235 treatment prolongs survival of animals with intracranial xenografts. A, Kaplan-Meier survival curve for the U87 xenograft experiment, with a P value of 0.026. The P values in the plot determined by the log-rank test are for the comparison of the overall survival of the vehicle-treated mice with that of the NVP-BEZ235–treated mice. B, weight measurements of experimental animals at regular intervals. C, tumor volumes of intracranial tumors were measured at the time of sacrifice. The mean tumor volume was reduced in treated animal with NVP-BEZ235. D, immunoblotting analyses to assess LC3-II expression after 2- and 4-wk tumor cell extracts following NVP-BEZ235 treatment. The increase in ratio of LC3-II/LC3-I is an indication of autophagy in NVP-BEZ235–treated tumors in comparison with vehicle-treated animals. E, immunoblotting analyses of both the expression and activation of Akt and S6K1 in 2- and 4-wk tumors following NVP-BEZ235 treatment. Immunoblotting analyses showed that NVP-BEZ235 inhibited the activity of S6K1 in both sets of tumors, as assessed by the level of corresponding phosphorylated protein.
- Figure 5.
NVP-BEZ235 attenuates VEGF secretion and inhibits factor VIII expression in vivo. A, NVP-BEZ235 attenuated VEGF secretion by at least 40% in U87 cells. The VEGF level in the SFM was measured in triplicate, and the experiment was repeated at least twice to confirm results. The amount of secreted VEGF was rendered in pg/mL/105 cells/24 h, as described previously (26). B, detection of factor VIII. Staining of factor VIII was used to measure the effect of NVP-BEZ235 on tumor angiogenesis. A drastic reduction in factor VIII–positive staining was noted in the NVP-BEZ235–treated (45 mg/kg) tumor section compared with staining results in control cells.
Volume 8, Issue 8
