Targeting Nitric Oxide Mediated Upregulation of Membrane-bound Glucose Regulated-protein 78 by Subtractive Single Chain Variable Fragment (scFv) Phage Display

Nitric oxide (NO) is a pleiotropic signaling molecule, and may be a contributing factor to the hypoxia encountered in a variety of solid tumors. Previously we reported that increased NOS expression resulted in a concomitant increase in expression of the glucose-regulated protein 78 (GRP78) in human embryonic kidney 293 cells. In the present study, we found that GRP78 was expressed on the cell membrane after nitric oxide induction. In order to isolate membrane-bound GRP78, we have designed a novel scFv library screening method, based on alternative screening with recombinant protein and cell surface-expressed antigen. After first routine screen using GRP78 protein, we used NO-induced cell surface GRP78 as a bait to subtract the none-membrane binding phages. We have successful isolated a panel of scFv specific for GRP78. Treatment of cells with one of the scFv (scFv-GRP78-H19) attenuated the GRP78-mediated protection against thapsigargin, a selective ER Ca 2+ -ATPase inhibitor. Furthermore, it synergized with ionomycin (IM) to promote apoptosis in prostate cancer cells and inhibited the glucose-deprivationmediated etoposide resistance in human colon and prostate cancer. The work presented here has highlighted the possibility that anti-GRP78 antibodies have the potential to be used therapeutically for the treatment of cancer.