NSAIDs and NF-kB Inhibitors in Ovarian Cancer Figure 4. NSAID-treatment induces JNK activation. (A) Total lysate before Immunoprecipitation. (B) Kinase assay showing induction of JNK kinase activity by NSAIDs. Induction of JNK activation by Sulindac Sulfide and Diclofenac was analyzed in cell lysates from SKOV-3 and CAOV-3 cells treated with 50 mM Sulindac Sulfide, 100 mM Diclofenac or DMSO using the SAPK/JNK assay Kit (Cell Signaling). (C) Western Blot analysis using anti-phospho JNK antibody of cell lysates from CAOV-3 cells treated with 50 mM Sulindac Sulfide, 100 mM Diclofenac or DMSO and infection with lentivirus encoding mda-7/IL-24 siRNA, GADD45a and GFP duplexes. doi:10.1371/journal.pone.0024285.g004 consistent evidence that NSAIDs can effectively inhibit tumori- genesis in particular through inhibition of cyclooxygenase-2 (COX-2). Importantly, aspirin use has been associated with a decreased risk of distant breast cancer recurrence and breast cancer death [57]. Epidemiologic studies indicate inverse associations between use of nonsteroidal anti-inflammatory drugs (NSAID) and the incidence of ovarian cancer [5,6,7]. Several reports suggest that certain NSAIDs induce apoptosis and cell cycle arrest in human ovarian cancer cells but the exact molecular mechanism by which NSAIDs induce antitumorigenic activity is not clear. Previously, our groups described a novel pathway by which NSAIDs induce apoptosis and growth arrest in cancer cells. We demonstrated that induction of the pro-apoptotic cytokine MDA-7/IL-24 by NSAIDs is crucial for programmed cell death induced by NSAIDs [23]. However, this study did not include ovarian cancer cells. Several reports using an adenovirus encoding the mda-7/IL-24 gene (Ad-mda-7) show its profound and selective anticancer activity in animal models [26,27,50,58,59,60] including a report of selectively induced cell death of ovarian cancer cells that results in suppression of tumor growth in vivo [58]. However, transient expression, potentially adverse immune reactions (mediated by adenovirus) and problems with systemic delivery restrict the generalized use of adenoviral delivery of mda-7/IL-24, particularly when administered systemically as a non-replicating adenovirus. In this context, our findings that NSAIDs with anti-cancer activity induce high levels of mda-7/IL-24 in ovarian cancer cells provide a new therapeutic strategy to enhance mda-7/IL-24 levels on a systemic level. Indeed, we have obtained a comprehensive overview of the consequences of a whole panel of NSAIDs on ovarian cancer cell survival by comparing their efficacies to induce apoptosis and mda-7/IL-24 expression. The most potent inducers of mda-7/IL-24 gene expression include Sulindac Sulfide and Diclofenac. Our finding corresponds with previous reports that demonstrated that treatment of human lung tumor xenografts in nude mice with Ad-mda-7 in addition to Sulindac reduced tumor growth more efficiently than Ad-mda-7 [50]. Moreover, these results corroborate our previous findings that apoptosis induction of the pro-apoptotic cytokine mda-7/IL-24 mediates induction of GADD45 a and c expression and JNK activity in other types of cancer [23]. While Sulindac Sulfide and Diclofenac themselves may not be the ideal drugs to induce mda-7/IL-24 and apoptosis in ovarian cancer cells, and particularly Diclofenac elicits many adverse effects in patients that limit its use in cancer patients, it should be feasible to generate modified versions of these drugs that are more potent in their anti-cancer activities and with reduced PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e24285