Bp1 splicing were significantly higher than what was demonstrated to induce

Bp1 splicing were significantly higher than what was demonstrated to induce GRP78 cleavage (Fig. 2A) and cytotoxicity (Fig. 3A); therefore, these findings suggest that this pathway does not play a significant role in the observed anti-tumor activity of EGF-SubA. Next, the cytotoxicity of EGF-SubA and SubA were evaluated in these models using a K162 biological activity clonogenic assay. In these studies, the respective glioblastoma cell lines were plated as singe cells, and exposed to either EGF-SubA or SubA for 24 h; culture plates were then replaced with fresh media and placed back into the incubator to allow for colony formation. As demonstrated in Fig. 3, EGFSubA demonstrated potent cytotoxicity, with IC50 values corresponding to the concentrations required for GRP78 cleavage, ranging from 0.5 pM (in U251) to 2.5 pM (in T98G; Fig. 3 A/B). Importantly, these concentrations were several orders of magnitude more potent than SubA toxin alone, which again corresponds to the increased ability of the fusion protein to target and cleave GRP78. Furthermore, U87 cells demonstrated relative resistance to EGF-SubA cytotoxicity when compared to the other lines (Fig. 3C), as predicted by its limited capacity of cleaving GRP78 in this specific line. Western blot was performed to define the mode of cell death following EGF-SubA. As demonstrated in Fig. 3D, exposing U251 cells to EGF-SubA for 24 h lead to an increase in apoptosis, as determined by cleaved caspase. As GRP78 has been previously reported to contribute towards Dimethylenastron custom synthesis therapeutic resistance [5,8,10,11,12,13,19], we next examined the potential of EGF-SubA to enhance the anti-tumor activity of standard cytotoxics in glioblastoma, including temozolomide and ionizing radiation [1]. In these experiments, U251 cells were exposed to EGF-SubA (1.0 pM) 16 h prior to either temozolomide or ionizing radiation. As demonstrated in Fig. 4, in addition to potent independent activity, EGF-SubA demonstrated the capacity to enhance both temozolomide-induced cytotoxicity (Fig. 4A) and response to therapeutic doses of ionizing radiation (Fig. 4B), further supporting this strategy in the treatment of glioblastoma. As described above, the UPR represents an important adaptive process that allows cells to survive microenvironmental stresses, including hypoxia, acidosis, and nutrient deprivation [4]. Although cells growing in such conditions have been previously associated with therapeutic resistance [20], we hypothesized that they would be more reliant on the UPR for survival, and therefore, particularly sensitive to UPR modulation. As an initial investigation, we studied the role acidosis may play in UPR activation [21]. U251 cells serially maintained in acidic conditions (pH 6.7) demonstrated UPR activation when compared to cells grown in standard media (pH 7.4), including PERK phosphorylation (Fig. 5A), Xbp1 splicing, and increased GRP78 transcription (Fig. 5B). Further, as we hypothesized, U251 cells grown in acidicconditions demonstrated increased sensitivity to EGF-SubA cytotoxicity, as determined by the clonogenic assay (Fig. 5C). In an effort to evaluate the cytotoxicity of EGF-SubA in both normal human astrocytes and GNS cells, which have limited capacity of growing as viable colonies, we applied the xCELLigence system, which allows for a real-time, label-free analysis of cellular growth by monitoring electrical impedance using specialized culture plates [22]. As an initial investigation, we sought to first confirm the anti-tumor acti.Bp1 splicing were significantly higher than what was demonstrated to induce GRP78 cleavage (Fig. 2A) and cytotoxicity (Fig. 3A); therefore, these findings suggest that this pathway does not play a significant role in the observed anti-tumor activity of EGF-SubA. Next, the cytotoxicity of EGF-SubA and SubA were evaluated in these models using a clonogenic assay. In these studies, the respective glioblastoma cell lines were plated as singe cells, and exposed to either EGF-SubA or SubA for 24 h; culture plates were then replaced with fresh media and placed back into the incubator to allow for colony formation. As demonstrated in Fig. 3, EGFSubA demonstrated potent cytotoxicity, with IC50 values corresponding to the concentrations required for GRP78 cleavage, ranging from 0.5 pM (in U251) to 2.5 pM (in T98G; Fig. 3 A/B). Importantly, these concentrations were several orders of magnitude more potent than SubA toxin alone, which again corresponds to the increased ability of the fusion protein to target and cleave GRP78. Furthermore, U87 cells demonstrated relative resistance to EGF-SubA cytotoxicity when compared to the other lines (Fig. 3C), as predicted by its limited capacity of cleaving GRP78 in this specific line. Western blot was performed to define the mode of cell death following EGF-SubA. As demonstrated in Fig. 3D, exposing U251 cells to EGF-SubA for 24 h lead to an increase in apoptosis, as determined by cleaved caspase. As GRP78 has been previously reported to contribute towards therapeutic resistance [5,8,10,11,12,13,19], we next examined the potential of EGF-SubA to enhance the anti-tumor activity of standard cytotoxics in glioblastoma, including temozolomide and ionizing radiation [1]. In these experiments, U251 cells were exposed to EGF-SubA (1.0 pM) 16 h prior to either temozolomide or ionizing radiation. As demonstrated in Fig. 4, in addition to potent independent activity, EGF-SubA demonstrated the capacity to enhance both temozolomide-induced cytotoxicity (Fig. 4A) and response to therapeutic doses of ionizing radiation (Fig. 4B), further supporting this strategy in the treatment of glioblastoma. As described above, the UPR represents an important adaptive process that allows cells to survive microenvironmental stresses, including hypoxia, acidosis, and nutrient deprivation [4]. Although cells growing in such conditions have been previously associated with therapeutic resistance [20], we hypothesized that they would be more reliant on the UPR for survival, and therefore, particularly sensitive to UPR modulation. As an initial investigation, we studied the role acidosis may play in UPR activation [21]. U251 cells serially maintained in acidic conditions (pH 6.7) demonstrated UPR activation when compared to cells grown in standard media (pH 7.4), including PERK phosphorylation (Fig. 5A), Xbp1 splicing, and increased GRP78 transcription (Fig. 5B). Further, as we hypothesized, U251 cells grown in acidicconditions demonstrated increased sensitivity to EGF-SubA cytotoxicity, as determined by the clonogenic assay (Fig. 5C). In an effort to evaluate the cytotoxicity of EGF-SubA in both normal human astrocytes and GNS cells, which have limited capacity of growing as viable colonies, we applied the xCELLigence system, which allows for a real-time, label-free analysis of cellular growth by monitoring electrical impedance using specialized culture plates [22]. As an initial investigation, we sought to first confirm the anti-tumor acti.