Search

The results of this study suggest that the well-documented loss of GSH and ascorbate in organisms under oxidative stress may be mainly due to their reactions with protein radicals and/or peroxides. Protein hydroperoxides were generated in HL-60 cells exposed to radiation-generated hydroxyl radicals. We found for the first time evidence of chain peroxidation of the proteins in cells, with each hydroxyl radical leading to the formation of about 10 hydroperoxides. Protein peroxidation showed a lag, probably due to the endogenous antioxidant enzymes, with simultaneous loss of the intracellular GSH. Enhancement of the GSH levels by N-acetylcysteine decreased the formation of hydroperoxides, while treatment with l-buthionine sulfoximine had the opposite effect. The effect of variation of GSH levels on the formation of the peroxidized proteins is explained primarily by reduction of the protein hydroperoxides by GSH. Loading of the cells with ascorbate resulted in reduction of the amounts of protein hydroperoxides generated by the radiation, which was proportional to the intracellular ascorbate concentration. In contrast to the GSH, inhibition of protein hydroperoxide formation in the presence of the high (mM) intracellular ascorbate levels achieved was mainly due to the direct scavenging of hydroxyl radicals by the vitamin.

FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na+/K+-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments. Expression of FXYD3 in MCF-7 breast cancer cells was ~8-fold and ~2-fold higher than in non-cancer MCF-10A cells and MDA-MB-468 cancer cells, respectively. A ~50% reduction in FXYD3 expression increased glutathionylation of the β1 Na+/K+-ATPase subunit and reduced Na+/K+-ATPase activity by ~50%, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the β1 subunit of Na+/K+-ATPase and glutathionylation-induced inhibition of Na+/K+-ATPase. Treatment of MCF-7 and MDA-MB- 468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments up-regulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na+/K+-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.