Abstract 4216: Combination of anti-cancer small molecule tolfenamic acid and curcumin or curcumin analog EF31 effectively inhibits pancreatic cancer cell growth
Authors: Basha Riyaz , Connelly Sarah F. , Nagaraju Ganji Purnachandra , Sankpal Umesh T. , Shoji Mamoru , Kayaleh Omar , El-Rayes Bassel 
Published: May 2015

Abstract
Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug is known to inhibit human cancer cells and mouse tumor growth in some cancer models. TA is known to target the transcription factor, specificity protein1 (Sp1) that mediates the expression of several genes associated with cancer including survivin, a key member of inhibitor of apoptosis proteins (IAP) family. It is currently in phase I clinical trials for treating upper gastro-intestinal cancer patients along with radiation. Curcumin (Cur) is an aromatic constituent of the plant curcuma longa (turmeric) which is extensively studied in some malignancies including breast, pancreatic and colon cancers. Even though, Cur shows a broad spectrum of anti-cancer activity in pre-clinical studies, its clinical application is greatly affected by its low bioavailability. Hence the strategies to improve the response to Cur soared by synthesizing and testing analogs. Recently our group showed that EF31 impacts the DNA methylation and causes anti-proliferative effect in pancreatic cancer cell liens and inhibits tumor growth in mice xenografts. We have evaluated the therapeutic efficacy of Cur and a Cur analog EF31 in combination with an anti-cancer small molecule, TA using human pancreatic cancer cell lines, MiaPaCa2 and L3.6 pl. MiaPaCa2 and L3.6 pl cells were treated with increasing concentrations of TA (25-100 µM) or Cur (5-25 µM) or EF31 (0.2-5 µM) or combination of optimized concentrations of TA (50 µM) and Cur (7.5 µM) or EF31 (0.5 µM) and the cell viability was measured at 24, 48, and 72 h post-treatment. All agents showed a steady and consistent decrease in cell viability following a clear dose and time-dependent response while the combination of TA and Cur or EF31 showed higher growth inhibition. Apoptosis and cell cycle analysis was performed using flow cytometry. Results showed a significant increase in the apoptotic fraction (annexin V positive) following combination treatment when compared to individual effect. TA caused cell cycle arrest in G0/G1 and the combination treatment showed both G0/G1 and G2 arrest. The activation of apoptosis was further confirmed by examining the activation of caspases (caspase 3/7, 8 and 9) and the expression of cleaved PARP. The Western blot results revealed that TA significantly decreased Sp1 and survivin expression and the combination of TA and Cur or TA and EF31 significantly modulated the expression of critical candidates associated with cell cycle. When compared to Cur, its synthetic analog EF31 showed higher efficacy in both individual and combination studies. These pre-clinical results demonstrate that the combination of anti-cancer NSAID, tolfenamic acid and curcumin or curcumin analogs may enhance the therapeutic efficacy in pancreatic cancer. Studies to better understand the underlying mechanisms by performing molecular profiling are currently under investigation.
Citation Format: Riyaz Basha, Sarah F. Connelly, Ganji Purnachandra Nagaraju, Umesh T. Sankpal, Mamoru Shoji, Omar Kayaleh, Bassel El-Rayes. Combination of anti-cancer small molecule tolfenamic acid and curcumin or curcumin analog EF31 effectively inhibits pancreatic cancer cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2014-4216