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Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long-term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high-risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti-proliferative effect of 13 cis -retinoic acid (RA) in HRNB cell lines. LA1-55n and SH-SY5Y cells were treated with TA (30 μM) or RA (20 μM) or both (optimized doses, derived from dose curves) for 48 h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter-Glo and Caspase-Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability ( p < 0.0001) when compared to individual agents. The anti-proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin-V positive cells, caspase 3/7 activity and c-PARP levels. Notably, TA + RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children. [ABSTRACT FROM AUTHOR]

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5-25μM) or TA (25-100μM) or combination of Cur (7.5μM) and TA (50μM). Cell viability was measured at 24-72h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur+TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur+TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G0/G1, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur+TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms. [ABSTRACT FROM AUTHOR]