NSAIDs and NF-kB Inhibitors in Ovarian Cancer Figure 5. Combinatorial treatment of ovarian cancer cells with NSAIDs and NF-kB inhibitors. (A) Pharmacological NF-kB inhibitors induce apoptosis in ovarian cancer cells. SW626, CAOV-3, and SKOV-3 cells after treatment with 5 nM 6-Amino-4-(4-phenoxyphenylethylamino) quinazoline (6-amino), 50 mM Isohelenin, 50 mM IKK-2 inhibitor SC-514, and 200 mM IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12-trihydroxy-coumestan) or DMSO as control. Data means 6 s.d. of triplicate independent experiments for each treatment. (B) Dose-dependent induction of apoptosis by NF-kB inhibitors in ovarian cancer cells. Apoptosis assay of SKOV-3 ovarian cancer cells. Cells were treated with 5, 2.5, 1 and 0.5 nM of 6-Amino-4-(4- phenoxyphenylethylamino) quinazoline (6-amino) 50, 25, 10 and 5 mM of Isohelenin, 50, 25, 10 and 5 mM of IKK inhibitor II Wedelolactone (7- Methoxy-5,11,12-trihydroxy-coumestan) (Wedelolactone), 50, 25, 10 and 5 mM of IKK-2 inhibitor SC-514 or DMSO. Apoptosis was measured 24 hours post-treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (C) Apoptosis in ovarian cancer cells after NSAID treatment in combination with NF-kB inhibitors. SW626, CAOV-3, and SKOV-3 cells after treatment with 10 mM Sulindac Sulfide, 40 mM Diclofenac, 25 mM Ebselen, 40 mM Naproxen, 1 nM 6-Amino-4-(4 phenoxyphenylethylamino) quinazoline (6-amino), and 200 mM IKK inhibitor II Wedelolactone and a combination thereof. Apoptosis was measured 24 hours after treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (D) Normalised isobologram obtained by software Compusyn. CAOV-3 cells treated with a combination of 10 mM Sulindac Sulfide and 2.5 nM 6-amino shows synergistic effect. doi:10.1371/journal.pone.0024285.g005 adverse and off-target effects. Indeed a modified version of Sulindac has recently been reported to be more active against cancer cells without inhibiting COX 1 and 2 [61]. Diclofenac has previously been shown to induce apoptosis in colon and squamous cell carcinoma and to inhibit pancreatic tumor growth [62,63]. However, there are no reports about its use in ovarian cancer. Here, we demonstrate that Diclofenac as well as Sulindac Sulfide induce apoptosis and inhibit tumor growth of ovarian cancer. These compelling data reinforce the notion of the potential benefits of NSAID treatment for ovarian cancer. We also identified Naproxen and Ebselen as moderate inducers of apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While Naproxen helps to prevent urinary bladder and colon carcinogenesis [64], Ebselen has been shown to reduce cisplatin treatment toxicity in rat ovarian cancer models, enhancing anti- tumor activity and improving mortality, morbidity and outcome [65]. As mentioned before, we have reported that induction of mda-7/IL-24 by structurally different NSAIDs is crucial for apoptosis induction of breast, prostate, renal and stomach cancer cells [23]. However, in this previous study, Naproxen and Ebselen had only marginal effects on apoptosis induction. In this report, we observed different drug activities for Naproxen and Ebselen. Ebselen and Naproxen induced apoptosis and mda-7/IL-24 expression in ovarian cancer cells and also synergized with the more potent NSAIDs, Diclofenac and Sulindac Sulfide, suggesting potential clinical utility in ovarian cancer therapy. We have previously shown that inhibition of NF-kB in cancer cells increases apoptosis without promoting mda-7/IL-24 produc- tion [23]. One of the major transcriptional circuits implicated in inflammation is the NF-kB/IkB pathway [66]. Furthermore, NF- PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24285