Abstract 3267: Novel combination treatment with 13-cis-retinoic acid and tolfenamic acid induces anti-proliferative response in high-risk neuroblastoma cells

Abstract

Abstract High-risk neuroblastoma (HRNB) is an aggressive extra-cranial solid tumor of childhood. 13-cis-retinoic acid (RA) is commonly used in adjuvant therapy and helpful in reducing the reoccurrence of this malignancy. Due to the very poor prognosis, there is an urgent need to establish more effective strategies for treating NB. In this study we evaluated the effect of the small molecule Tolfenamic Acid (TA) for enhancing the anti-proliferative effect of RA in NB cells, LA1-55n and SH-SY5Y. TA is known to induce degradation of Specificity protein1 (Sp1) transcription factor which regulates several genes associated with cell proliferation and cell cycle. After testing the dose/time-dependent response of individual agents, the optimized doses were used for the combination experiments. LA1-55n and SH-SY5Y cells were treated with TA (30 uM) or RA (20 uM) or both for 48 hour and tested to assess the effect on cell growth, apoptosis and selected molecular markers including Sp1, survivin, AKT and ERK1/2. Cell viability and caspase activity were measured with Cell TiterGlo and Caspase Glo kits. The apoptotic cell population was determined by flow cytometry using Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP were evaluated by western blot analysis. Combination treatment with TA and RA caused significant inhibition of cell viability (p <0.001) causing an anti-proliferative response in a time/dose-dependent manner. 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 (p <0.001) and c-PARP levels. Notably, our results also revealed that TA and RA combination treatment caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways. Even though the individual agents also showed anti-proliferative response, the combination of both agents’ effect was significantly higher. TA and RA combination treatment-induced cell growth inhibition was 40-50% more when compared to TA alone and almost doubled as compared to RA alone. These results indicate that combination treatment was highly effective and exhibited anti-cancer activity at doses lower than known physiological concentrations of individual drugs. Together, this study marks the importance for the development of a novel strategy involving TA and RA combination for the effective treatment of high-risk neuroblastoma in children. Citation Format: Sagar Shelake, Don Eslin, Robert Sutphin, Anmol Wadhwani, Laura E. Kenyon, W. Paul Bowman, Riyaz Basha. Novel combination treatment with 13-cis-retinoic acid and tolfenamic acid induces anti-proliferative response in high-risk neuroblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3267. doi:10.1158/1538-7445.AM2015-3267