Kinase experiment:

Caspase 3 activity in the presence or absence of 200 ng/mL CD40L plus 1 μg/mL CD40L enhancer and 100 μM Oxaprozin is performed. The enzymatic activity is spectrophotometrically determined for 60 minutes at 405 nm assuming an extinction coefficient of 8.8×103 M1/cm[2].

Cell experiment:

Purified monocytes are resuspended at 106/mL and cultured for 48 hours. In selective experiments, cells are cultured in the presence or absence of 50 μM PD98059, 1 μM SB203580, 50 μM LY294002, 20 μM SN-50, 50 μM Ac-DEVD-CHO, different doses (5, 10, 50, 100 μM) of Oxaprozin, 100 μM ibuprofen,100 μM indomethacin, or 100 μM naproxene. Percentages of apoptotic cells are measured by both fluorescence microscope and flow cytometer[2].

Oxaprozin is an inhibitor of both COX-1 and COX-2 with IC50s of 2.2 μM and 36 μM for human platelet COX-1 and IL-1-stimulated human synovial cell COX-2, respectively. Oxaprozin also inhibits the activation of NF-κB.

Oxaprozin induces apoptosis in a dose-dependent manner. Oxaprozin increases caspase-3 activity in the activated but not in the resting condition. NF-κB activation is inhibited by 50 μM Oxaprozin. Oxaprozin inhibits activation of the IKK system induced by the reagent IκBα[1]. Oxaprozin dose dependently increase CD40L-treated monocyte apoptosis. 100 μM Oxaprozin induces the strongest proapoptotic effect. 100 μM Oxaprozin significantly increases CD40L-treated monocyte apoptosis. Oxaprozin treatment inhibits CD40L-induced Akt and NF-κB (p65) phosphorylation[2].

References:
[1]. Ottonello L, et al. Delayed apoptosis of human monocytes exposed to immune complexes is reversed byoxaprozin: role of the Akt/IkappaB kinase/nuclear factor kappaB pathway. Br J Pharmacol. 2009 May;157(2):294-306.
[2]. Montecucco F, et al. Oxaprozin-induced apoptosis on CD40 ligand-treated human primary monocytes is associated with the modulation of defined intracellular pathways. J Biomed Biotechnol. 2009;2009:478785.