NOV-002, a redox-modulating glutathione disulfide mimetic, leads to calcium influx and nitric oxide generation through eNOS activation in myeloid c

Abstract 3: Session: NF-kB, Oxidative Stress, and Other Pathways in Cellular Drug Responses


NOV-002, a glutathione disulfide mimetic, is currently in a pivotal Phase 3 clinical trial in advanced non-small cell lung cancer. In clinical trials conducted to date, NOV-002 administered in combination with standard chemotherapeutic regimens has resulted in increased efficacy (survival, tumor response) and improved tolerance of standard chemotherapy (e.g. enhanced hematological recovery, immune stimulation). Recently, we showed that the myeloproliferative effect of NOV-002 in the pre-myeloid HL-60 cell line occurs in parallel with stress-induced S-glutathionylation and activation of kinase pathways (AKT, JAK2 and STAT5) that are known to regulate hematopoiesis. NOV-002 treatment produces oxidant signals intracellularly and at the cell surface. HL60 cells express the cell surface enzyme gamma-glutamyl transpeptidase (GGT). NOV-002 is a substrate for GGT and its cleavage alters the intracellular redox potential. Here we show that NOV-002 treatment in HL60 cells mediates changes of the plasma membrane electrical potential as detected by the fluorescent probe, bisOxonal. These changes were concurrent with time- and dose-dependent increases in the accumulation of intracellular Ca2+ as detected by Fura-2-AM and Fluo-3-AM fluorescent dyes. These effects were abolished by the addition of an extracellular Ca2+ chelator (EGTA) and decreased by the intracellular chelator, BAPTA-AM. These data suggest that NOV-002 induced calcium signaling involves entry of extracellular Ca2+ across the plasma membrane. Microarray analyses highlighted that NOV-002 treatment impacts expression of proteins involved in calcium signaling and nitric oxide (NO) metabolism pathways. NO generation following NOV-002 treatment was decreased by ~80% in HL60 cells transfected with siRNA to deplete eNOS levels. Overall, NOV-002 mediated alteration of cell surface redox status and transmembrane potential resulted in an induction of calcium influx, followed by NO generation through eNOS activation. These events may be pertinent to myeloproliferation that has been observed in cancer patients treated with NOV-002 plus standard chemotherapy.