Effect of phospho-ibuprofen on reactive oxygen and nitrogen species and cyclooxygenase-2 levels in breast cancer. (A) Pretreating MCF-7 cells with 15 mM N-acetyl-cysteine (NAC) suppressed phospho-ibuprofen (P-I)-induced apoptosis. (B) After 1 hour of treatment, MCF-7 cells as indicated were stained with 2',7'-dichlorofluorescein diacetate (DCFDA) and the fluorescent intensity was determined by flow cytometry. IC50, concentration that inhibits cell growth by 50%. (C) Left: After 1 hour of treatment with P-I 1.5×IC50, MCF-7 cells were stained with MitoSOX Red or dihydroethidium (DHE) and the fluorescent intensity was determined by flow cytometry. Right: Confocal microscopy of MitoSOX Red-stained MCF-7 cells after same treatment as above. Upper panel: MitoSOX Red alone. Lower panel: MitoSOX Red merged with DIC images. (D) Cyclooxygenase-2 (COX-2) levels in MDA-MB231 cells were detected by immunoblot; loading control, β-actin. After a 6-hour treatment with P-I, prostaglandin E2 (PGE2) levels were assayed in the culture medium of MDA-MB231 cells using a kit from Cayman Chemicals. Results are folds over control. (E) Representative images (200× magnification) from immunohistochemistry-stained COX-2 in MDA-MB231 xenografts (from Figure 1C) and their quantification are shown. Insets: 600× images of COX-2-positive cells.