Small Molecule, Tolfenamic Acid Induces the Apoptotic Response of Cis-Retinoic Acid in High-Risk Neuroblastoma Cells

Neuroblastoma (NB) is an aggressive and highly heterogeneous extra-cranial solid tumor found in children. NB accounts for 15% deaths among pediatric cancer patients. 13-cis-retinoic acid (RA) is commonly used as adjuvant therapy during the remission maintenance phase of NB treatment. There is, however, a greater than 50% risk of relapse in high-risk neuroblastoma (HRNB) that necessitates the development of an alternative therapeutic strategy. Specificity protein 1 (Sp1) is a transcription factor that is involved in the regulation of various cellular functions including cell growth, differentiation, and apoptosis. The present study is aimed at investigating the effect of a small molecule and Sp1 inhibitor, Tolfenamic Acid (TA) for enhancing the anti-proliferative effect of RA in HRNB cell lines, LA1-55n and SH-SY5Y. The optimized doses obtained from dose/time-dependent response of individual agents were used for the combination treatment experiments. LA1-55n and SH-SY5Y cells were treated with TA (30 µM) or RA (20 µM) or both for 48 h and tested to assess 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 were evaluated by western blot analysis. Treatment with TA and RA resulted in significant inhibition of cell viability in dose/time- dependent manner in tested cell lines. Even though the individual agents showed anti-proliferative response, the combination of both agents increased cell growth inhibition in LA1-55n (91.8%), SH-SY5Y (78.2%) as compared to TA alone (LA1-55n: 65%; SH-SY5Y: 51%) and RA alone (LA1-55n: 50%; SH-SY5Y: 33.5%). This anti-proliferative effect is accompanied by a decline in Sp1 and survivin expression. Furthermore, TA and RA combination treatment resulted in a significant increase in apoptotic (Annexin-V positive) cells, caspase 3/7 activation (LA1-55n: 12 fold and SH-SY5Y: 9 fold; p