GRP-78 is up-regulated and secreted from bortezomib-resistant tumor cells. (A) Western blot analysis of intracellular protein levels of GRP-78 in solid tumor cell lines (U373, MCF-7, PC-3, and HRT-18), myeloma cells (U266, OPM-2), and human endothelial cells (HUVECs). GADPH served as internal loading control. (B) GRP-78 analysis in conditioned supernatants of human solid tumor cell lines and HUVECs. (C) Myeloma (U266) and prostate carcinoma (PC-3) cells were stimulated with 5 ng/mL bortezomib for 24 hours. GRP-78 protein levels were analyzed by Western blot in cytosolic extracts (CT) and supernatant (SN). (D) Different concentrations of recombinant GRP-78 ranging from 0 to 100nM were used to inhibit the antiangiogenic action of bortezomib. Bortezomib-sensitive MCF7 and myeloma cells (U266 and OPM-2) were stimulated with 10 ng/mL bortezomib. Apoptotic cells (percentage) were determined by flow cytometry after annexin V/7-AAD staining. Bars represent mean ± SEM. *P < .05.