Effect of phospho-ibuprofen on the thioredoxin system in breast cancer. (A) Upper panel: Thioredoxin-1 (Trx-1) levels in MCF-7 cell lysates were determined by immunoblot. Lower panel: The redox status of Trx-1 in MCF-7 cells was assayed as in Materials and methods after 1 hour of treatment with phospho-ibuprofen (P-I). IC50, concentration that inhibits cell growth by 50%; NAC, N-acetyl-cysteine; ox, Trx-1 oxidized form; red, Trx-1 reduced form. (B) P-I reduces thioredoxin reductase (TrxR) activity. The effect of P-I on the enzymatic activity of TrxR was assayed in vitro using TrxR purified from rat liver (left). The TrxR activity was determined in protein extracts from MCF-7 cells treated with P-I for 1 hour (right). Representative images (200× magnification) of Trx-1 in (C) MCF-7 or (D) MDA-MB231 xenografts by immunohistochemistry staining and quantifications are shown. Lipo-P-I, liposome-encapsulated phospho-ibuprofen. (E) TrxR activity was determined in MCF-7 tumor lysates.