NSAIDs and NF-kB Inhibitors in Ovarian Cancer Figure 2. Synergistic effects of NSAID used in combinations. (A) Apoptosis assay of SKOV-3 cells after treatment with 10 mM Sulindac Sulfide, 40 mM Diclofenac, 25 mM Ebselen or 40 mM Naproxen and a combination thereof or DMSO. Apoptosis was measured 24 hours post-treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (B) Normalised isobologram obtained by software Compusyn. CAOV-3 cells treated with a combination of 5 mM Sulindac Sulfide and 20 mM Diclofenac shows synergistic effect. doi:10.1371/journal.pone.0024285.g002 interact with the upstream kinase of JNK, MTK1 [52], we evaluated the activation of JNK during NSAID-mediated apoptosis. JNK kinase activity was tested in protein extracts obtained from CAOV-3 and SKOV-3 cells treated with Sulindac Sulfide (50 mM), Diclofenac (200 mM) or DMSO for 24 hours by an in vitro kinase assay. Western blot analysis revealed very little JNK activity in untreated control cells and a strong increase in JNK activity in both cell lines upon treatment with Sulindac Sulfide and Diclofenac (Figure 4a and 4b). Corroborating this evidence a weak inducer of mda-7/IL-24, GADD45 a and c and apoptosis, Flurbiprofen, demonstrated only a marginal induction of JNK activity (data not shown). To elucidate the functional relevance of GADD45 a and c and mda-7/IL-24 for NSAID- mediated JNK induction and apoptosis in ovarian cancer, JNK kinase activity was tested in protein extracts obtained from CAOV-3 cells treated with Sulindac Sulfide and Diclofenac and infected with lentivirus encoding siRNA against GADD45 a and mda-7/IL-24 genes. Western blot analysis revealed JNK kinase activation by Sulindac Sulfide and Diclofenac was markedly dependent on GADD45 a and mda-7/IL-24 induction, since JNK kinase activity in Sulindac Sulfide and Diclofenac treated mda-7/ IL-24-/- cells was abolished when compared to mda-7/IL-24+/+ cells (Figure 4b). In order to further characterize the effect of NSAIDS in inducing apoptosis, the levels of PARP activation were PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24285