Combinatorial effect of non-steroidal anti-inflammatory drugs and NF-κB inhibitors in ovarian cancer therapy

Abstract

Several epidemiological studies have correlated the use of non-steroidal anti-inflammatory drugs (NSAID) with reduced risk of ovarian cancer, the most lethal gynecological cancer, diagnosed usually in late stages of the disease. We have previously established that the pro-apoptotic cytokine melanoma differentiation associated gene-7/Interleukin-24 (mda-7/IL-24) is a crucial mediator of NSAID-induced apoptosis in prostate, breast, renal and stomach cancer cells. In this report we evaluated various structurally different NSAIDs for their efficacies to induce apoptosis and mda-7/IL-24 expression in ovarian cancer cells. While several NSAIDs induced apoptosis, Sulindac Sulfide and Diclofenac most potently induced apoptosis and reduced tumor growth. A combination of these agents results in a synergistic effect. Furthermore, mda-7/IL-24 induction by NSAIDs is essential for programmed cell death, since inhibition of mda-7/IL-24 by small interfering RNA abrogates apoptosis. mda-7/IL-24 activation leads to upregulation of growth arrest and DNA damage inducible (GADD) 45 α and γ and JNK activation. The NF-κB family of transcription factors has been implicated in ovarian cancer development. We previously established NF-κB/IκB signaling as an essential step for cell survival in cancer cells and hypothesized that targeting NF-κB could potentiate NSAID-mediated apoptosis induction in ovarian cancer cells. Indeed, combining NSAID treatment with NF-κB inhibitors led to enhanced apoptosis induction. Our results indicate that inhibition of NF-κB in combination with activation of mda-7/IL-24 expression may lead to a new combinatorial therapy for 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 µM Ibuprofen, 1 mM Acetaminophen, 200 µM Naproxen, 200 µM NS-398, 200 µM Diclofenac, 50 µM Finasteride, 200 µM Flufenamic acid, 40 µM Meloxicam, 50 µM Ebselen, 20 nM Flurbiprofen, 50 µM Sulindac Sulfide and 50 µM Sulindac Sulfone or DMSO as control. (A) Apoptosis assay of ovarian cancer cells after NSAID treatment. Data means ± 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 µM of Sulindac Sulfide; 200, 100, 40 and 20 µM of Diclofenac; 200, 100, 40 and 20 µM of Naproxen and 50, 25, 10 and 5 µM of Ebselen or DMSO. Apoptosis was measured 24 hours post-treatment. Data means ± 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 ± s.d. of triplicate independent infection for each vector at each treatment.

Figure 2. Synergistic effects of NSAID used in combinations.

(A) Apoptosis assay of SKOV-3 cells after treatment with 10 µM Sulindac Sulfide, 40 µM Diclofenac, 25 µM Ebselen or 40 µM Naproxen and a combination thereof or DMSO. Apoptosis was measured 24 hours post-treatment. Data means ± s.d. of triplicate independent experiments for each treatment. (B) Normalised isobologram obtained by software Compusyn. CAOV-3 cells treated with a combination of 5 µM Sulindac Sulfide and 20 µM Diclofenac shows synergistic effect.

Figure 3. Reduction of tumor formation by NSAIDs.

For each inoculation, 2×10⁶ 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.

Figure 4. NSAID-treatment induces JNK activation.

(A) Total lysate before Immunoprecipitation. (B) Kinase assay showing induction of JNK kinase activity by NSAIDs. Induction of JNK activation by Sulindac Sulfide and Diclofenac was analyzed in cell lysates from SKOV-3 and CAOV-3 cells treated with 50 µM Sulindac Sulfide, 100 µM Diclofenac or DMSO using the SAPK/JNK assay Kit (Cell Signaling). (C) Western Blot analysis using anti-phospho JNK antibody of cell lysates from CAOV-3 cells treated with 50 µM Sulindac Sulfide, 100 µM Diclofenac or DMSO and infection with lentivirus encoding mda-7/IL-24 siRNA, GADD45α and GFP duplexes.

Figure 5. Combinatorial treatment of ovarian cancer cells with NSAIDs and NF-κB inhibitors.

(A) Pharmacological NF-κB 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 µM Isohelenin, 50 µM IKK-2 inhibitor SC-514, and 200 µM IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12-trihydroxy-coumestan) or DMSO as control. Data means ± s.d. of triplicate independent experiments for each treatment. (B) Dose-dependent induction of apoptosis by NF-κB 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 µM of Isohelenin, 50, 25, 10 and 5 µM of IKK inhibitor II Wedelolactone (7-Methoxy-5,11,12-trihydroxy-coumestan) (Wedelolactone), 50, 25, 10 and 5 µM of IKK-2 inhibitor SC-514 or DMSO. Apoptosis was measured 24 hours post-treatment. Data means ± s.d. of triplicate independent experiments for each treatment. (C) Apoptosis in ovarian cancer cells after NSAID treatment in combination with NF-κB inhibitors. SW626, CAOV-3, and SKOV-3 cells after treatment with 10 µM Sulindac Sulfide, 40 µM Diclofenac, 25 µM Ebselen, 40 µM Naproxen, 1 nM 6-Amino-4-(4 phenoxyphenylethylamino) quinazoline (6-amino), and 200 µM IKK inhibitor II Wedelolactone and a combination thereof. Apoptosis was measured 24 hours after treatment. Data means ± 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 µM Sulindac Sulfide and 2.5 nM 6-amino shows synergistic effect.