Journal: The Journal of Biological Chemistry

Article Title: Inhibition of cell surface GRP78 on brain tumors reverses drug resistance and stops cancer stem cell expansion

doi: 10.1016/j.jbc.2026.111146

Figure Lengend Snippet: CBT300 reduces expression of cell surface GRP78 leading to increased doxorubicin internalization in glioma cells. A, U87 cells (20,000) were seeded in quadruplicate wells of tissue coated 96-well plates with either media (negative control – no doxorubicin), media (positive control – doxorubicin (2 μm)), media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to all the wells except for the negative control wells. 24 h after adding doxorubicin to the cells, cells were washed and flow cytometry analysis for red doxorubicin fluorescence was performed. B, U87 cells (3,000) were plated in four well chamber slides and allowed to attach overnight. Each chamber was treated with either media, media+GRP78 (5 μg/ml), or media+GRP78 (5 μg/ml)+CBT300 (100 nM) for 48 h. Doxorubicin (2 μM) was then added to each chamber and incubated for 24 h. Cells/chambers were then washed and stained with a mAb-FITC ( green ) to the C-terminal domain of GRP78, and 4′,6-diamidino-2-phenylindole ( blue ) to the DNA. Doxorubicin, which has a natural red fluorescence, was also measured for each treatment. A chart of three independent repeats was compiled for each marker, cell surface GRP78, and increase in doxorubicin internalized. Studies were performed with three independent replicates. The average mean and standard deviation for each set and marker are shown. Significance was determined between groups by two-way ANOVA analysis with post hoc Tukey’s test using GraphPad Prism 10.1.2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. C, the proposed mechanism of action of CBT300’s inhibition of surface expressed GRP78 leading to tumor regression and apoptosis. Cell surface GRP78 promotes tumor growth through stabilization of checkpoint protein PD-L1, oncofetal proteins ROR1, and Cripto (tumor promotion). CBT300 and CBT200 remove the surface GRP78, eliminating its stabilization of PD-L1, ROR1, and Cripto which then induces tumor apoptosis and reducing drug and immune resistance (tumor apoptosis). GBM, glioblastoma multiforme; GRP78, glucose-regulated protein 78; PD-L1, programed death-ligand 1; ROR1, receptor tyrosine kinase–like orphan receptor-1; CBT, Creative BioTherapeutics.

Article Snippet: Briefly, 5000 HGG (U118, U87, DIPG13, DIPG38, DIPG50, SF9402, SF9427, and SF8628) cells per well in Dulbecco’s modified Eagle’s medium (DMEM containing 10% FBS) were incubated in a 96-well plate (CELLTREAT, #229195) with and without GRP78 (5 μg/ml) overnight at 37 °C and 5% CO 2 .

Techniques: Expressing, Negative Control, Positive Control, Flow Cytometry, Fluorescence, Incubation, Staining, Marker, Standard Deviation, Inhibition