growth arrest- and DNA damage-inducible proteins, GADD45 a, b and c play a role in the G2/M checkpoint in response to DNA damage [24]. Under normal physiological conditions, mda-7/IL- 24 is expressed in cells of the immune system and normal melanocytes [25]. High levels of mda-7/IL-24 have been demonstrated to specifically induce apoptosis of cancer cells and therefore mda-7/IL-24 has been referred as a ‘‘magic bullet’’ [26,27]. Moreover, several studies indicated that overexpression of mda-7/IL-24 by a recombinant adenovirus results in cancer cell apoptosis and therapeutic benefits in ovarian cancer [28,29]. NF-kB/IkB signaling is another pathway that has been implicated in drug resistance and survival in ovarian cancer [30,31]. The NF- kB/IkB pathway is emerging as key player in tumorigenesis, invasion and metastasis for various cancers [30,31] and is a critical step for cancer cells to escape programmed cell death [32]. Furthermore, resistance of cancer cells to chemotherapeutic agents has been associated with deregulated NF-kB activation [33]. Moreover, we have shown that inhibition of constitutively active NF-kB by adenoviral expression of IkB induces apoptosis in prostate cancer cells and inhibits tumor formation in SCID mice [34]. In this report, we compared a broad set of NSAIDs for their efficacies to induce apoptosis of ovarian cancer cells. Since our previous study demonstrated a role for mda-7/IL-24 in NSAID- mediated cell death, we also evaluated whether apoptosis induction by NSAIDs is due to induction of mda-7/IL-24 expression. Viral delivery of mda-7/IL-24 is currently used in clinical trials for various cancers [27]. Identification of drugs that are most efficient in mda-7/ IL-24 induction may provide an alternative to viral delivery for exploiting the anti-neoplastic effects of mda-7/IL-24. We also hypothesized that combining NSAIDs with inhibitors of the NF-kB pathway may enhance the effects of NSAIDs against ovarian cancer. We, therefore, tested pharmacological inhibitors of the NF-kB pathway for their abilities to induce apoptosis in ovarian cancer cells. Here, we demonstrate that Sulindac Sulfide and Diclofenac are the most potent NSAIDs that induce ovarian cancer apoptosis via mda-7/ IL-24 expression and also reduce tumor growth in vivo. mda -7/IL-24 expression leads to GADD45a and c upregulation and JNK kinase activation. Several pharmacological NF-kB inhibitors also induce apoptosis of ovarian cancer cells and in combination with NSAIDs potentiate the apoptotic effect of NSAIDs. Results NSAIDs are potent inducers of mda-7/IL-24 and apoptosis in ovarian cancer cells A broad panel of NSAIDs was tested for their abilities to induce apoptosis and mda-7/IL-24 gene expression in four ovarian cancer cell lines, SKOV-3, CAOV-3, SW626 and 36M2. The concen- trations for all NSAIDs drugs used in this study were selected to be comparable to achievable physiological plasma concentrations [35–49]. Apoptosis was measured 24 hours after treatment of SKOV-3, CAOV-3, SW626 and 36M2 ovarian cancer cells with this set of NSAIDs, revealing that a variety of, but not all NSAIDs induced apoptosis (Figure 1a and S1a). Consistently strong inducers of apoptosis in all four-cell lines included Sulindac Sulfide, Diclofenac, Ebselen and Naproxen when compared to the solvent controls. Some NSAIDs resulted in significant induction of apoptosis in a subset of the ovarian cancer cell lines including Sulindac Sulfone, Acetaminophen, Aspirin and Flurbiprofen whereas treatment with NS-398, Ibuprofen, Finasteride, Flufe- namic Acid and Meloxicam resulted only in marginal or no apoptosis induction (Figure 1a and S1a). For each of the four most consistent inducers of apoptosis we determined the lowest dose that still induces programmed cell death NSAIDs and NF-kB Inhibitors in Ovarian Cancer of ovarian cancer cells. The concentrations of the selected NSAIDs were tested at 2, 5 and 10 times lower concentrations than the physiologically achievable doses used in the experiments for Figure 1a and S1a. Apoptosis was measured in ovarian cancer cells 24 hours after treatment with different doses of the four NSAIDs. Our results show that Sulindac Sulfide, Diclofenac and Naproxen concentrations even at 5 times lower dose still effectively induce apoptosis, while Ebselen can be reduced only 2-fold (Figure 1b). Based on our previous observations in other types of cancer we determined whether apoptosis induction by NSAIDs correlates with mda-7/IL-24 induction. We measured mRNA expression levels of mda-7/IL-24 mRNA in response to different NSAIDs in SKOV-3 cells by real time PCR analysis demonstrating that mda- 7/IL-24 (maximum of 12-fold induction) expression is commonly induced by NSAIDs that promote apoptosis in ovarian cancer cells (Figure 1c). These results were also corroborated in two additional ovarian cancer cell lines. In CAOV-3 and SW626 ovarian cancer cell lines mda-7/IL-24 is also strongly induced (maximum of 77- fold induction in CAOV-3 cells) (Figure S1b). NSAIDs that strongly enhanced apoptosis such as Sulindac Sulfide, naproxen, ebselen, and diclofenac (Figure 1a) significantly induced mda7/IL- 24 expression (Figure 1c), whereas NSAIDs that only marginally induced apoptosis (Figure 1a) did not significantly enhance mda-7/ IL-24 expression except ibuprofen which induced mda-7/IL-24 but did not induce apoptosis (Figure 1c). We and others have shown that overexpression of mda-7/IL-24 following infection with an adenovirus carrying the mda-7/IL-24 gene induces apoptosis and inhibits cell proliferation in cancer cells [50,51]. In order to evaluate whether induction of apoptosis in cancer cells by NSAIDs is dependent on mda-7/IL-24 upregula- tion, we conducted experiments with a lentivirus encoding a siRNA against mda-7/IL-24 that was previously generated by our group [23]. Infection of SKOV-3 cells with the mda-7/IL-24 siRNA lentivirus reduced apoptosis induced by NSAIDs by 40– 70% relative to the control lentivirus (Figure 1d), further supporting the notion that NSAID-mediated apoptosis is at least partially dependent on mda-7/IL-24 induction. The same results were observed in CAOV-3 cells (data not shown). Synergistic effects of NSAID combinations Using the lowest dose of each NSAID that affected apoptosis of ovarian cancer cells (Figure 1b), we systematically analyzed apoptosis induction upon combining low doses of NSAIDs. A panel of NSAIDs including Diclofenac, Sulindac Sulfide, Naproxen and Ebselen were tested for their abilities to induce apoptosis alone and in combination. SKOV-3 ovarian cancer cells were treated with 10 mM Sulindac Sulfide, 40 mM Diclofenac, 25 mM Ebselen or 40 mM Naproxen and combinations thereof. Apoptosis was measured 24 hours after treatment revealing that the majority of the combinations of NSAIDs tested induced apoptosis significantly more than either of the NSAIDs alone in ovarian cancer cells (Figure 2a). Certain combinations such as Sulindac Sulfide and Diclofenac, Sulindac Sulfide and Naproxen and Diclofenac and Naproxen were more effective in apoptosis induction than others (Figure 2a). We validated these results in CAOV-3 and SW626 cell lines (Figure S2). Isobologram analysis using combinations of Diclofenac and Sulindac Sulfide indicates that combinations of the drugs results in a synergistic effect (Figure 2b). NSAID treatment reduces ovarian cancer xenograft growth in SCID mice To determine whether NSAIDs reduce tumor growth in vivo, SKOV-3 ovarian cancer cells were injected subcutaneously in SCID mice. The mice were randomly divided into 3 groups and PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24285