Figure 8
Phospho-aspirin-2 suppresses the thioredoxin (Trx) system and activation of NF-кB. A: Left panel: PA-2 1.5 × IC50 reduced TrxR activity in MDA-MB-231 cells after 1 h treatment. *, p < 0.02, compared to control. Immunoblots of TrxR and Trx-1 showed that PA-2 reduced the expression of Trx-1 in MDA-MB-231 cells. Right panel: PA-2 reduced TrxR activity in the protein lysates from MDA-MB-231 xenografts (treatment protocol) from animals treated with vehicle or PA-2 for 25 days. *, p < 0.04, compared to vehicle. B: Left panel: PA-2 inhibited constitutive NF-κB activation. EMSA for NF-κB of nuclear fractions isolated from MDA-MB-231 (upper) and BT-20 (lower) cells after 4 h treatment with or without PA-2 1.5 × IC50. To determine the specificity of the NF-κB transcription factor-DNA complex, the control nuclear fraction was incubated in the presence of 100-fold molar excess of unlabeled oligonucleotide containing the consensus sequence for either the specific (+NF-κB) or an unspecific (+AP-1) transcription factor. Right panel: NF-κB (p-p65) levels from MDA-MB-231 tumors, determined by immunohistochemistry using an anti-p-p65 antibody, were reduced in PA-2 treated group compared to the vehicle control. The percentage of p-p65-positive cells in various fields was determined and averaged for each xenograft. *, p < 0.0009, compared to vehicle. Representative images are shown; magnification 200X. All values are mean ± SEM.