Abstract
Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics.
Highlights
MAY 6, 2016 VOLUME 291 NUMBER 19 tractant protein-1 (MCP-1)3 is required for endothelial cell tumor formation and that Nox-4 is the primary source of oxidants inducing MCP-1 expression in EOMA cells [5,6,7]
As would be evident from the higher GSSG/GSH ratio of EOMA presented in Fig. 2, it is clear that high GSSG reductase in EOMA was ineffective in correcting the high GSSG/GSH noted in this cells
Pharmacologic inhibition of GSSG reductase (Fig. 3B) of EOMA resulted in loss of cell viability (Fig. 3C), which is not observed in murine aortic endothelial (MAE) cells (Fig. 3, B and C)
Summary
MAY 6, 2016 VOLUME 291 NUMBER 19 tractant protein-1 (MCP-1) is required for endothelial cell tumor formation and that Nox-4 is the primary source of oxidants inducing MCP-1 expression in EOMA cells [5,6,7]. Given the high abundance of cellular GSH, rapid accumulation of cellular GSSG is one of the early responses to oxidant insult [9] Such excessive GSSG may be recycled to GSH in the presence of appropriate levels of NADPH (10 –12). Our previous work has shown that high intracellular GSSG is lethal in neurons [13] These cells must rely on rapid clearance of GSSG to ensure their survival in an oxidant-rich scenario [13]. We tested the hypothesis that EOMA cells rely on an extraordinary mechanism to efflux cellular GSSG, which acts as a critical survival factor Inhibition of such a survival factor would make these cancer cells fall prey to excessive oxidants of their own making, which they otherwise use to support extraordinary growth
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