Natural killer-enhancing factor (NKEF) was identified and cloned on the basis of its ability to increase NK cytotoxicity. Two genes, NKEF-A and -B, encode NKEF
proteins and sequence analysis presented suggests that each belongs to a highly conserved family of
antioxidants. To examine the
antioxidant potential of NKEF, we transfected the coding region of NKEF-B
cDNA into the human endothelial cell line ECV304. The stable transfectant, B/1, was found to overexpress NKEF-B gene transcript and
protein. We subjected B/1 to oxidative stress by either culturing them with
glucose oxidase (GO), which continuously generates
hydrogen peroxide, or by direct addition of
hydrogen peroxide. We found that B/1 cells were more resistant than control cell lines. Resistance to
hydrogen peroxide was originally thought to be mediated mainly by
catalase and the
glutathione cycle. Therefore, we used inhibitors to block the two pathways and found that B/1 cells were more resistant to oxidative stress than control cells when we used inhibitors to preblock either pathway. We also examined the cellular inflammatory responses to
oxidized low-density lipoprotein (
LDL) and bacterial
lipopolysaccharide (LPS) by measuring monocyte adhesion to endothelial cells in vitro and found that B/1 cells were resistant to such responses. Lastly, we found that B/1 cells were more resistant to a novel chemotherapeutic agent
CT-2584, which appears to kill
tumor cells by stimulating production of
reactive oxygen intermediates in mitochondria. These results demonstrate that the NKEF-B is an
antioxidant that protects cells from oxidative stress,
chemotherapy agents, and
inflammation-induced monocyte adhesion. Furthermore, its expression may mediate cellular responses to proinflammatory molecules.