kB has been implicated in cancer cell survival and escape from programmed cell death and is activated by chemotherapeutic agents in cancer cells [30,31,33]. Mutations in different genes of the NF-kB pathway and constitutively active NF-kB are frequently observed in various types of cancer [33]. Indeed, ovarian cancer cells frequently contain activated NF-kB prior to therapy and are, therefore, expected to be resistant to chemotherapy a priori. On the other hand, we have demonstrated that inhibition of activated NF- kB in cancer cells induces apoptosis without the addition of a chemotherapeutic agent indicating the central role of NF-kB in cell survival of many cancer cells. These findings also suggest the possibility of enhancing therapeutic outcomes by combining NF- kB inhibitors with chemotherapy or other drugs such as NSAIDs. At high concentrations, NSAIDs have been shown to inhibit the TNF-mediated activation of NF-kB [67]. However, we have previously shown that at achievable plasma concentrations, NSAIDs Sulindac Sulfide has no effect on the NF-kB signaling pathway [23]. Indeed here we demonstrate that NF-kB inhibitors strongly induce apoptosis in ovarian cancer cells. Importantly, NF- kB inhibitors markedly enhanced the efficacy of NSAIDs to induce apoptosis, corroborating our hypothesis that these combinatorial regimens can be utilized even though animal model studies are still necessary to prove its efficacy in vivo. In summary, our results strongly support the hypothesis that drug treatment regimens that lead to enhanced mda-7/IL-24 expression in cancer cells and block NF-kB may have significant efficacy against ovarian cancer. These results also provide a rationale to screen for additional small molecules, natural compounds or even chemically modified NSAIDs, which selec- tively and efficiently induce mda-7/IL-24 expression in order to obtain more potent anti-cancer drugs. Materials and Methods Cell cultures The ovarian cancer cell lines SKOV-3, CAOV-3, and SW626 were obtained from American Type Culture Collection (Rockville, MD). 36M2 cells were kindly provided by Dr. Dimitrios Spentzos, Beth Israel Deaconess Medical Center. SKOV-3 were grown in McCoy’ 5a modified medium (Life Technologies, Carlsbad, CA), SW626 were grown in L-15 medium; and CAOV-3 and 36M2 were grown in DMEM (Life Technologies, Carlsbad, CA). The medium was supplemented with 10% fetal bovine serum (FBS), 50 units of penicillin/mL, and 50 mg streptomycin/mL (all from Life Technologies). The cells were maintained in a 5% CO2- humidified incubator at 37uC. Reagents Sulindac Sulfide, Sulindac Sulfone, Ibuprofen, Aspirin, Acet- aminophen, and Naproxen were obtained from Sigma-Aldrich (St. Louis, MO). Meloxicam, Diclofenac, Finasteride, and Flufenamic acid were obtained from LKT Laboratories (St. Paul, MN). NS- 398, Ebselen, and Flurbiprofen were purchased from Calbiochem (San Diego, CA). The drugs were dissolved in DMSO or ethanol. Cancer cells were treated in their particular medium for 24 hours. The concentrations of the different NSAIDs were chosen to be physiologically achievable doses. The final concentrations for each compound were as follows: 5 mM Aspirin, 200 mM Ibuprofen, 1 mM Acetaminophen, 200 mM Naproxen, 200 mM NS-398, 200 mM Diclofenac, 50 mM Finasteride, 200 mM Flufenamic acid, 40 mM Meloxicam, 50 mM Ebselen, 20 nM Flurbiprofen, 50 mM Sulindac Sulfide and 50 mM Sulindac Sulfone. For the controls, cells were treated with an equal amount of DMSO or ethanol, which was ,0.1% of the final concentration. NSAIDs and NF-kB Inhibitors in Ovarian Cancer NF-kB inhibitors: 5 nM 6-Amino-4-(4-phenoxyphenylethyla- mino) quinazoline, 50 mM Isohelenin, 50 mM IKK-2 inhibitor SC- 514, and 200 mM IKK inhibitor II Wedelolactone (7-Methoxy- 5,11,12-trihydroxy-coumestan) were purchased from Calbiochem (San Diego, CA). Real-time PCR Total RNA was harvested using QIAshredder (Qiagen, Valencia, CA) and RNeasy Mini kit (Qiagen). Real-time PCR was performed as described [23]. The full description of the method is described in Methods S1. The sequences of the primers are as follows: mda-7/IL- 24, sense 59-CAAAGCCTGTGGACTTTAGCC-39; antisense 59- GAATAGCAGAAACCGCCTGTG- 39; hGAPDH, sense 59- CAAAGTTGTCATGGATGACC-39; antisense 59-CCATGGA- GAAGGCTGGGG-39. GADD45a, sense 59-GCCTGTGAGT- GAGTGCAGAA- 39; antisense 59-ATCTCTGTCGTCGTC- CTCGT-39; GADD45c, sense 59-CTGCATGAGTTGCTGC- TGTC- 39; antisense 59-TTCGAAATGAGGATGCAGTG –39. Kinase assay JNK kinase activity was measured by using the SAPK/JNK assay kit (Cell Signaling Technology) according to the manufac- turer’ protocol. Briefly, CAOV-3 and 36M2 cells were treated with Sulindac Sulfide, Diclofenac or DMSO for 16 hours. Whole- cell lysates of treated and control cells were prepared in lysis buffer [20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium PPi, 1 mmol/L bGlycerophosphate, 1 mmol/L Na3VO4, 1 m g/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluo- ride]. 500 mg of the clarified lysate were immunoprecipitated using 2 mg agarose-conjugated anti-c-jun monoclonal antibody (Cell Signaling, Beverly MA) overnight at 4uC. The beads were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris (pH 7.5), 5 mM b-Glycerophosphate, 2 mM DTT, 0.11 mM Na3VO4, 10 mM MgCl2) and subjected to the kinase assays. The beads were suspended in 50 ml kinase buffer supplemented with 200 mM ATP and incubated for 30 minutes at 30uC. The reactions were terminated by adding 25 ml 3X SDS sample buffer and proteins were resolved by SDS-10% PAGE and probed with phospho-c-jun antibody (Cell Signalling). Western-blot Western-blot were performed as described in supplementary information by using antibodies against total SAPK/JNK (New England Biolabs), phospho JNK (Cell Signaling), mda-7/IL-24 (kindly provided by Paul B. Fisher, Virginia Commonwealth University, School of Medicine, Richmond, VA), PARP and GAPDH (Santa Cruz Biotechnology). Apoptotic assays Apoptosis was assayed by using the Apoptotic Cell Death Detection ELISA (Roche) according to the manufacturer’ protocol. Significant statistical difference of control samples against samples treated with NSAIDs was determined by Student’ t-test. Isobologram The isobologram was calculated using Calcusyn software (Biosoft). The drugs were applied across a range of concentrations and cell proliferation was evaluated using the MTT assay at each dosage. Data from cell proliferation was expressed as the fraction of cells inhibited by drug treatments compared with untreated cells. Interaction between pairs of drugs was determined using the Calcusyn software, which generated the isolobogram. The PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24285