Figure 1.
Immunohistochemical staining was performed on a glioma tissue microarray using an anti-GRP78 antibody and expression levels (0, 1+, 2+, and 3+) were quantified based on the intensity of staining. Representative staining patterns (A) and tumor grade-specific distributions of identified staining intensities (B) are provided.
Figure 2.
SubA and EGF-SubA cleaves GRP78 and activates the UPR.
Exponentially growing glioblastoma cell lines and normal human astrocytes (NHA) were (A) treated with SubA or EGF-SubA at the specified picomolar concentrations for 24 h or (B) exposed EGF-SubA (1 pM) for the specified time periods. Total cellular protein was isolated and immunoblotting was performed with anti-GRP78 antibody. SubA and EGF-SubA cleaved the endogenous GRP78 (78 kDa) resulting in an additional smaller fragment of 28 kDa (cGRP78). (C-E) Total cellular protein and RNA were isolated from U251 cells exposed to EGF-SubA at the stated concentrations for 24 h. EGF-SubA induced GRP78 cleavage resulted in nuclear localization of ATF6 (C; nATF6), a dose-dependent phosphorylation of PERK (D; pPERK), and Ire1 activation, determined by Xbp1 mRNA splicing (E). Each figure is a representative of three independent experiments.
Figure 3.
The influence of SubA and EGF-SubA on glioma cell survival.
A clonogenic assay was performed to study the cytoxicity of SubA and EGF-SubA in U251 (A), T98G (B) and U87 cells (C). Cells were seeded as single cell suspensions in six well culture plates, allowed to adhere, and treated with the stated concentrations of SubA or EGF-SubA for 24 h. Plates were then replaced with fresh culture media and surviving fractions were calculated 10 to 14 d following treatment. Cell survival was significantly different between SubA and EGF SubA treatment in U251 (p<0.0001) and T98G (p<0.0001 at concentrations ≥0.5 pM) and not significant in U87 cells (p = 0.2112). (D) Immunoblotting of total cellular protein from U251 cells treated with EGF-SubA at the stated concentrations for 24 h demonstrates EGF-SubA induced apoptosis, as determined by cleaved caspase 3. Each figure is a representative of three independent experiments.
Figure 4.
EGF-SubA enhances anti-tumor activity of temozolomide and ionizing radiation.
A clonogenic assay was performed to evaluate the potential of EGF-SubA to enhance temozolomide (A) (statistically significant p<0.0001) and radiation-induced (B) cytotoxicity (statistically significant p<0.0024). U251 cells were seeded in six well culture plates and exposed to 1 pM of EGF-SubA 16 h prior to the addition of temozolomide or radiation exposure. Fresh media was then replaced in the culture plates after 8 h, and surviving fractions were calculated 10 to 14 d following treatment, normalizing for the individual cytotoxicity of EGF-SubA. Each figure is a representative of three independent experiments.
Figure 5.
Acidic pH activates the UPR pathway and enhances EGF-SubA cytotoxicity.
U251 cells grown in RPMI media whose pH was adjusted to 6.7 and 7.0 with 1N HCl for 3 passages prior to performing experiments demonstrated UPR activation, as determined by PERK phosphorylation (A; pPERK), Xbp1 splicing and increased GRP78 transcription (B). (C) To determine if cells grown in acidic conditions influenced EGF-SubA cytotoxicity, a clonogenic assay was performed with U251 cells grown in normal (pH 7.4) or acidic (pH 6.7) conditions at the stated concentrations. Cell survival was significantly different between cells grown in normal and acidic pH at higher doses of EGF SubA (p<0.0001 at 2.5 pM). Each figure is a representative of three independent experiments.
Figure 6.
EGF-SubA delays tumor growth in mice.
U251 cells were injected s.c in a mouse flank model (A). When tumors reached ∼150 mm3 in size, mice were randomized into two groups: vehicle control (PBS) or EGF-SubA (125 µg/kg) delivered s.c. on the stated days (arrow). To obtain a tumor growth curve, perpendicular diameter measurements of each tumor were measured with digital calipers, and volumes were calculated using the formula (L × W × W)/2. Tumor volumes (A) and weight of mice (B) were measured every other day. Tumor volumes were normalized to their volume at randomization. Each group contained six mice. *p = 0.0009. (C) U251 cells were injected s.c. in a mouse flank model. When tumors reached ∼500 mm3 in size, mice were exposed to either PBS alone or EGF-SubA (125 µg/kg). Mice were then sacrificed 24 h after treatment and stated tissue was dissected, flash frozen, and tissue lysates were generated to evaluate for GRP78 cleavage by immunoblot.