NSAIDs and NF-kB Inhibitors in Ovarian Cancer Figure 3. Reduction of tumor formation by NSAIDs. For each inoculation, 26106 SKOV-3 cells were injected subcutaneously into SCID mice. The mice were randomly divided into three groups (n = 7/group) and fed one of three diets through the entire experiment: AIN-93G as the control, the AIN-93G diet supplemented with 200 ppm Sulindac Sulfide or the AIN-93G diet supplemented with 100 ppm Diclofenac. The size of the tumors and tumor weight were measured after 2 months. Values not sharing the same letters are statistically significant with p values at least ,0.05. doi:10.1371/journal.pone.0024285.g003 measured by Western-blot, indicating that Sulindac Sulfide and Diclofenac are strong inducers of PARP cleavage (Figure S4a). Combinatorial treatment of pharmacological inhibitors of the NF-kB pathway with NSAIDs induce apoptosis in ovarian cancer cells We investigated the biological relevance of the NF-kB pathway in ovarian cancer cells and determined the functional consequenc- es of its inhibition. Instead of using adenoviral delivery of the IkB inhibitor we moved towards a more clinically relevant model and used pharmacological inhibitors of the NF-kB pathway. Inhibitors of the NF-kB pathway were tested for their abilities to induce apoptosis in ovarian cancer cells. Apoptosis was measured 24 hours after treatment of SKOV-3, CAOV-3 and SW626 ovarian cancer cells with four different inhibitors of NF-kB, 5 nM 6-Amino-4-(4-phenoxyphenylethylamino) quinazoline [53], 50 mM Isohelenin [54], 50 mM IKK-2 inhibitor SC-514 [55], and 200 mM IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12- trihydroxy-coumestan) [56] or DMSO (control). 6-Amino-4-(4- phenoxyphenylethylamino) quinazoline was an efficient inducer of apoptosis in all three cell lines, and the IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12-trihydroxy-coumestan) in- duced apoptosis in two of the three cell lines. Treatment with Isohelenin or IKK-2 inhibitor SC-514 resulted only in marginal or no apoptosis induction (Figure 5a). Additionally, Real-time PCR analysis indicates that Wedelolactone (7-Methoxy-5,11,12-trihy- droxy-coumestan) induces strong activation of the GADD45 a and c gene expression (Figure S4b), and promotes JNK phosphoryla- tion (Figure S4c) and cleavage of PARP (Figure S4a). As for the NSAIDs we performed a dose response analysis for 6- Amino-4-(4-phenoxyphenylethylamino) quinazoline and Wedelo- lactone (7-Methoxy-5,11,12-trihydroxy-coumestan) to determine the lowest dose that still induces programmed cell death of ovarian cancer cells. Reducing the concentration of 6-Amino-4-(4- phenoxyphenylethylamino) quinazoline from 5 nM to 1 nM still induced apoptosis, while reduced doses of Wedelolactone resulted in loss of apoptosis induction (Figure 5b). To determine whether the NF-kB inhibitors 6-Amino-4-(4- phenoxyphenylethylamino) quinazoline and Wedelolactone (7- Methoxy-5,11,12-trihydroxy-coumestan) enhance the pro-apo- ptotic activities of NSAIDs we combined the lowest doses of each of the four NSAIDs, Sulindac Sulfide, Diclofenac, Ebselen, and Naproxen with the lowest doses of the two NF-kB inhibitors that still induce apoptosis (Figure 1b and 5b, respectively). The NSAIDs and NF-kB inhibitors were tested for their abilities to induce apoptosis alone and in combination. SKOV-3, CAOV-3, and SW626 ovarian cancer cells were treated with 10 mM Sulindac Sulfide, 40 mM Diclofenac, 25 mM Ebselen, 40 mM Naproxen, 1 nM 6-Amino-4-(4 phenoxyphenylethylamino) qui- nazoline and 200 mM Wedelolactone. Apoptosis was measured 24 hours after treatment revealing that combinations of NSAIDs with the NF-kB inhibitor 6-Amino-4-(4 phenoxyphenylethyla- mino) quinazoline significantly enhanced apoptosis in ovarian cancer cells compared to either of the drugs alone (Figure 5c). In contrast, Wedelolactone enhanced the pro-apoptotic effects only of some of the NSAIDs and less efficiently in SKOV-3 cells (Figure 5c). Isobologram analysis indicates that the combination of 6-Amino-4-(4 phenoxyphenylethylamino) quinazoline with Sulindac Sulfide results in a synergistic effect (Figure 5d). Discussion NSAIDs have emerged as potential drugs for chemoprevention in cancer but their benefits are still in question. Some traditional NSAIDS such as Sulindac are currently being tested in clinical trials for various cancers. Indeed, preclinical studies provide PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24285