PMC

Western blot of total U251 cell lysates shows that DR4 and DR5 are up-regulated with (A) GSK3 inhibitors as indicated (all at a 0.5 μM concentration for 24 hours of treatment) (B) and by GSK3 siRNAs. (C) TRAIL is up-regulated by GSK3siRNA (48 hours of treatment).

(A) TRAIL acts synergistically with enzastaurin in glioma cell killing. (B) Overexpression of c-MYC kills glioma cells. TRAIL enhances cell death caused by c-MYC overexpression. (C) Combination of enzastaurin with carboplatin results in significantly reduced tumor volume in a mouse subcutaneous U87 xenograft model. (D) Combination of enzastaurin with carboplatin prolongs survival in U87 intracranial xenograft model.

NF-κB luciferase reporter assay shows that GSK3 siRNA (A) down-regulates NF-κB activity in U251. (B) Kenpaullone, LiCl and enzastaurin down-regulate NF-κB activity in a dose-dependent manner (24 hours of treatment). (C) NF-κB silencing by siRNA resulted in cytotoxicity of U251. Lower graph shows functional efficiency of NF-κB activity silencing by siRNA in U251 as measured by luciferase reporter assay (NS, non-silencing control siRNA). In all luciferase assays cells were cotransfected with luciferase reporters for NF-κB and b-gal. Luciferase activity levels from NF-κB reporter were divided by those from the b-gal reporter. To control for transfection efficiency and cell viability, a CMV-βgal plasmid was co-transfected. NF-κB and β-galactosidase reporter activities were detected 24 hours after transfection and NF-κB values were normalized compared to β-galactosidase levels. Western Blot shows the efficiency of NF-κB silencing by siRNA.

(A) GSK3 inhibition by siRNA and enzastaurin up-regulates c-MYC activity as revealed by ELISA-based DNA-binding assay (B) WB analysis of nuclear extracts prepared from cells treated with 5 μM enzastaurin or DMSO. c-MYC phosphorylation at T58 is down-regulated in U251. This correlates with reduced Y phosphorylation of GSK3, both at 4 and 24 hours (shown) of treatment with enzastaurin. Conversely, S62 phosphorylation of c-MYC was up-regulated at 24 hours (C) Western Blot of total cell lysates: c-MYC siRNA negatively regulates Bax, DR4 and DR5 proteins in U251. NS, non-silencing control siRNA. (D) Silencing of c-MYC in U251 by shRNA in two individual clones, c-MYC-shRNA_1 & 2, effectively protects cells from 0.5 μM 705701 or from 0.5 μM LY2064827 (24 hours treatment) compared to control cells. Cells were plated at a density of 50,000 per well in a 6-well plate.

(A) Western Blot analysis of GSK3 siRNAs treated cell lysates show that GSK3 siRNAs decrease S640 phosphorylation of GYS in U251 (48 hours after transfection). (B) Bar graph shows glucose levels decrease in U251 cells treated with enzastaurin, 5 μM for 4 hours. Immunocytochemistry staining with anti-glycogen antibody revealed that GSK3 inhibition resulted in increased glycogen stores in the glioma cells. U251 cells were treated with (a) DMSO (4hours), (b) enzastaurin, 5μM (4hours); (c) insulin (30 minutes); (d) glucoamylase (3 hours) Glucoamylase digests glycogen and therefore is used as a negative control. The similar data were obtained with 705701 and LY2064827. (C) Western blot analysis of Bax levels in U251 treated with control or GSK3 siRNA. (D) Ratio of Bax/HK activity in mitochondria fractions is increased with GSK3 siRNA (48 hours) and enzastaurin treatments (4 hours). MitoTracker® Green staining of the functional mitochondria in DMSO and 5μM enzastaurin treated living U251 cells. Red color in the right panel shows fluorescence of enzastaurin.

(A) Cytotoxicity of enzastaurin (5 μM) in U251 glioma cell line as determined by a cell-counting assay at different time points as indicated. Bar graph shows results of the cell cycle analysis of U251 cells exposed to enzastaurin for the indicated time, then labeled with10 μM BrdU for 2 hours The drug induces G2/M arrest at 18 hours and subsequent apoptosis. (B) Changes in phosphorylation of GSK3 at Y276/216 and S21 of GSK3α caused by different GSK3 inhibitors correlate with survival of U251. Cells were treated with 0.5 μM of the indicated drugs for 48 hours Cell survival was assessed by cell counting (upper panel, graph). Y216/279 activating phosphorylation and S21 inhibitory phosphorylation of GSK3α/β were measured by Western Blot (lower panel). Here and below the asterisks designate p-values (t-test) as follows: *, p<0.05 ; ** , p<0.005 and ***, p<0.00, the error bars represent standard deviation. (C) GSK3 siRNA inhibits proliferation of glioma cells in vitro and in vivo (upper left corner). GSK3-specific siRNAs were transfected into U251 cells. Cells were harvested and counted 48 hours after transfection. Western blot shows efficiency of protein silencing by siRNA. In vitro clonogenicity assay demonstrated reduced number of colonies derived from U251 cells treated with GSK3 siRNA compared to non silencing (NS) siRNA. (D) Subcutaneous xenograft model in nude mice. After treatment with control, GSK3, or NF-κB siRNAs, 0.5 million U251 cells were injected subcutaneously into the thighs of nude mice. Each group consisted of 6 animals. Tumors were measured twice a week. The results are presented as tumor volume.