Thioredoxin-1 mediates the anti-cancer effect of phospho-ibuprofen in vitro and in vivo. (A) MCF-7 cells transfected with thioredoxin-1 (Trx-1) siRNA (TrxsiRNA) or control siRNA (CtlsiRNA) for 72 hours were treated with phospho-ibuprofen (P-I) for 16 hours. Cell death was assayed after annexin V staining. Immunoblots on the right shows the Trx-1 level in these cells. (B) Transient transfected MCF-7 cells were assayed for mitochondrial superoxide anion (O2-) after treatment with P-I for 1 hour. (C) Permanent Trx-1 knockdown (MCF-7shTrx-1) and its control (MCF-7shControl) cells were xenografted into nude mice, which were treated with P-I or vehicle. Tumor growth curves are shown. (D) At the end point, Trx-1 expression in vehicle-treated tumors was determined by immunoblot for protein level or by quantitative PCR for mRNA level. (E) Cell death (TUNEL) and active NF-κB xenografts as above were determined by immunohistochemistry. Representative images (200×) from each group and quantification results are shown as indicated.