PMC

TuBEC constitutively overexpress GRP78 without concomitant induction of other major UPR targets. A. Western blot analysis of two different specimens of TuBEC and BEC for proteins induced by the UPR: ATF4, CHOP and spliced-variant (S) of XBP-1; GRP78 expression was also analyzed. BEC stimulated with 30 nM of thapsigargin for 24 h served as the positive control. B. The relative levels of the proteins, normalization to respective GAPDH levels which served as loading control, were plotted.

Overexpression of GRP78 in BEC promotes chemoresistance. A. BEC were uninfected (UI), infected with lentivirus expressing either the control green fluorescence protein (GFP) or GRP78. Five days post-infection cells were harvested for Western blot analysis, and probed for GRP78 and GAPDH. B. BEC, uninfected or infected with lentivirus GFP or GRP78, were treated with Eto [50 μM] for 0, 5 or 7 days. At the termination of the experiments, cell death was analyzed using the Cell Death Elisa Assay. Percent death was calculated based on total cell death control. Statistical comparisons were made between BEC/GRP78 and BEC/GFP on day 7; the star (*) represents p<0.05. C. BEC, uninfected or infected with lentivirus GFP or GRP78, were treated with CPT-11 [100 μM] for 0, 5 or 7 days. At the termination of the experiments, cell death was analyzed using the Cell Death Elisa Assay. Statistical comparisons were made between BEC/GRP78 and BEC/GFP on day 7; the star (*) represents p<0.05. D. BEC, uninfected or infected with lentivirus GFP or GRP78, were either untreated, treated with EGCG [40 μM] alone, CPT-11 [100 μM] alone, or EGCG [40 μM] plus CPT-11 [100 μM] for 7 days. At the termination of the experiments, cell death was analyzed using the Cell Death Elisa Assay. Statistical comparisons were made between BEC/GRP78 treated with CPT-11 and BEC/GRP78 treated with CPT-11 and EGCG; the star (*) represents p<0.05.

Overexpression of GRP78 protein in tumor-associated brain endothelial cells (TuBEC) and tumor vasculature. A. Cytocentrifuge cell preparations of primary cultures from two different specimens of TuBEC (upper panel) and two different specimens of control brain endothelial cells (BEC) (lower panel) were immunostained with anti-GRP78 antibody. The red precipitate designates positive staining; hematoxylin was used as the nuclear stain. B. Intensity of immunostaining from 20 specimens was quantitated: 0 = no staining; 4 = intense staining; the star (*) represents significance between groups p<0.001. C. Two representative samples of TuBEC and BEC were subjected to Western blot analysis. The relative GRP78 protein expression levels, after normalization to β-actin level, were plotted below. D. Cryostat sections of glioma tissues (upper panel) or normal brain tissues (lower panels) were stained with anti-GRP78 antibody (red), anti-CD31 antibody (green) and DAPI (blue) nuclear staining; the images were merged in the last panel. The arrows designate blood vessels. Bar = 100 microns.

Chemoresistance is reversed in TuBEC with reduced GRP78 protein. A. TuBEC and BEC were exposed to 1, 10, 50 μM etoposide, and vehicle control (DMSO) for 72 h, then examined for cell viability using the MTT assay. Vehicle treatment served as 100% viable control. Significance (p<0.05) is calculated by comparing drug treated (50 μM) cells and vehicle control treated cells. B. TuBEC were infected with control siRNA (siCtrlA) or siRNA specifically targeted against human GRP78 (siGRP78A); 5 days post-infection, cells were stained with anti-GRP78 antibody. Red precipitate identified positive staining; hematoxylin stained nuclei blue; 200X magnification. C. TuBEC were infected with lentivirus siCtrlA or lentivirus siGRP78A constructs, or left uninfected. Five days post-infection, cells were treated with control media, CPT-11 [100 μM], Eto [50 μM] or TMZ [300 μM] for another 7 days. At the conclusion of the experiments, cells were analyzed using the Cell Death Elisa Assay. The star (*) represents p<0.05; comparisons were made between TuBEC/siGRP78 and TuBEC/siControl groups for each drug treatment.

TuBEC with reduced GRP78 are susceptible to caspase-dependent apoptotic cell death when treated with chemotherapeutic agents. A. A second lentivirus containing siRNA targeted against human GRP78 (siGRP78B) also reduced GRP78 expression in TuBEC. Cells were infected with siCtrlB or siGRP78B, and analyzed for GRP78 staining after infection; TuBEC infected with control siRNA (siCtrlB) did not affect GRP78 expression. Red color denotes positive staining. 400X magnification. B. Uninfected TuBEC or cultures infected with siCtrlB or siGRP78B were treated with media or drugs (CPT-11 [100 μM], Eto [50 μM] or TMZ [300 μM]) alone, or incubated with the caspase inhibitor (Q-VDOPH) (C.I.) [10 μM] for 7 days. Cultures were then analyzed using the Cell Death Elisa Assay. The star (*) represents p<0.05; comparisons were made between TuBEC/siGRP78 and TuBEC/siControl groups for each drug treatment. C. TuBEC were uninfected or infected with lentivirus expressing siCtrlA or lentivirus expressing siGRP78B. After 5 days, cells were treated with etoposide (Eto) [50 μM] for 7 days and stained using the TUNEL assay. Apoptotic death was calculated as percent positive in drug treated compared to cells incubated in media alone. Statistical comparisons were made between TuBEC/siGRP78 and TuBEC/siControl groups; the star (*) represents p<0.05. D. TuBEC were treated with EGCG (20 μM) alone or in combination with TMZ (300 μM), CPT-11 (100 μM), or etoposide (50 μM), then incubated with drugs for 7 days. Media containing DMSO, served as the vehicle control. Statistical comparisons were made between cells treated with EGCG + drugs and EGCG alone; the star (*) represents p<0.05.