NSAIDs and NF-kB Inhibitors in Ovarian Cancer Figure 1. Multiple NSAIDs induce apoptosis and mda-7/IL-24 gene expression in ovarian cancer cells. SKOV-3 cell line after treatment with 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 or DMSO as control. (A) Apoptosis assay of ovarian cancer cells after NSAID treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (B) Dose- dependent induction of apoptosis by NSAIDs in ovarian cancer cells. Apoptosis assay of SW626 ovarian cancer cells. Cells were treated with 50, 25, 10 and 5 mM of Sulindac Sulfide; 200, 100, 40 and 20 mM of Diclofenac; 200, 100, 40 and 20 mM of Naproxen and 50, 25, 10 and 5 mM of Ebselen or DMSO. Apoptosis was measured 24 hours post-treatment. Data means 6 s.d. of triplicate independent experiments for each treatment. (C) Real time PCR analysis of mda-7/IL-24 expression in SKOV-3 cells after 24 hours treatment with different NSAIDs. Each sample was normalized to hGAPDH. (D) Apoptosis assay of CAOV-3 ovarian cancer cells after NSAID treatment or DMSO and infection with lentivirus encoding mda-7/IL-24 siRNA duplexes. Data means 6 s.d. of triplicate independent infection for each vector at each treatment. doi:10.1371/journal.pone.0024285.g001 fed one of three diets through the entire experiment: AIN-93G as the control and the AIN-93G diet supplemented with either 200ppm Sulindac Sulfide or 100ppm Diclofenac. Two months later the animals were examined for tumor formation and tumor weight. All mice developed tumors indicating that this particular dose of NSAIDs did not prevent tumor formation. However, as seen in Figure 3, Sulindac Sulfide and Diclofenac treatment reduced the average tumor volume by 30% and 20%, respectively, when compared to the control diet with a p-value ,0.05, confirming its anti-tumor efficacy. Induction of GADD45 a and c gene expression and activation of JNK in ovarian cancer by NSAIDs We have previously reported that induction of apoptosis by NSAIDs is tightly linked to induction of mda-7/IL-24 expression and consequently to GADD45 a and c upregulation in several cancer cell lines [23]. As NSAIDs induce mda-7/IL-24 expression in ovarian cancer cells (Figure 1c and S1c), we evaluated changes in GADD45 a and c gene expression. To evaluate whether regulation of GADD45 genes is involved in NSAID-mediated apoptosis, expression of GADD45 a and c mRNAs was measured by real time PCR in SKOV-3, CAOV-3, and SW626 cells treated with NSAIDs. The NSAIDs with the strongest pro-apoptotic activity including Sulindac Sulfide, Diclofenac, Naproxen and Ebselen strongly enhanced GADD45 a and c expression, indicating that increased GADD45 a and c expression closely correlates with pro-apoptotic activity of NSAIDs (Figure S3a and S3b). Lentiviral expression of siRNA against mda- 7/IL-24 in ovarian cancer cells demonstrated that knockdown of mda-7/IL-24 reduces diclofenac-induced GADD45 a and c gene expression indicating that GADD45a and c induction is at least partially dependent on mda-7/IL-24 expression (Figure S3c). Since others and we had shown that JNK activation plays a role in apoptosis induction in cancer cells and GADD45 a and c PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24285