Tolfenamic Acid And Curcumin Treatment Induces Pancreatic Cancer Cell Growth Inhibition Via Suppressing Sp1 Expression, Nf-Kb Translocation To Nucleus

Pancreatic cancer (PC) is an aggressive malignancy. The current treatment options have limited response in addressing poor prognosis and low survival rate. Hence it is important to identify novel agents and strategies for effective treatment. Previously the combination of phytochemical, curcumin (Cur) and cyclooxygenase (COX) inhibitor celecoxib was tested for improving therapeutic efficacy in PC models. The objective of current study is to identify a combination treatment involving a low toxic small molecule and a phytochemical with anti-cancer properties to inhibit PC cell growth. Experiments were also conducted to understand potential mechanisms associated with this combination. We tested the combination of an anti-cancer non-steroidal anti-inflammatory drug (NSAID), Tolfenamic Acid (TA) and Cur using PC cell lines, L3.6 and MIA PaCa-2. Cells were treated with 5-25 μM of Cur or 25-100 μM of TA or combination of Cur (7.5 μM) and TA (50 μM). Effect on cell viability was measured at 24, 48 and 72 h post-treatment using CellTiter-Glo kit. While the two agents showed anti-proliferative effect, Cur and TA combination caused higher growth inhibition. The cell growth inhibition was compared with two COX inhibitors, ibuprofen and celecoxib and the cardiotoxicity was assessed using cordiomyocytes (H9C2). TA showed significantly less cytotoxicity in cardiomyocytes when compared to celecoxib. The expression of transcription factors, Specificity protein1 (Sp1) and NF-kB, and an inhibitor of apoptosis family protein, survivin, were determined by Western blot analysis. The expression of NF-kB, Sp1 and survivin was decreased by combination treatment. The levels of reactive oxygen species (ROS) were also measured in flowcytometer. To evaluate the effect of these agents on apoptosis, the activity of caspase 3/7 was measured with caspase-Glo kit; apoptotic cell population was evaluated by Annexin-V staining (flow cytometry); and c-PARP expression was determined by Western blot analysis. When compared to individual agents, the combination treatment caused a significant increase in ROS levels and apoptotic markers. L3.6 and MIA PaCa-2 cells were treated with TNF-a to induce NF-kB translocation from cytoplasm to nucleus and the effect of individual (TA or Cur) and combined treatment (TA+Cur) on NF-kB translocation from cytoplasm to nucleus was evaluated by immunofluorescence. When compared to individual agents, the combination treatment caused a significant decrease in NF-kB translocation to nucleus. Cell cycle phase distribution was measured using flow cytometry. The combination treatment showed mostly DNA synthesis phase arrest; however TA caused cell cycle arrest in early phase (G0/G1). These results demonstrate that combination of Cur and TA effectively inhibits PC cell growth via inducing apoptosis and modulating cell cycle phase distribution. Citation Format: Riyaz M. Basha, Sarah F. Connelly, Ganji Purnachandra, Umesh T. Sankpal, Hassaan Patel, Jamboor K. Vishwanatha, Sagar Shelake, Leslie Tabor-Simecka1, Mamoru Shoji, Jerry Simecka W. Simecka, Bassel El-Rayes. Tolfenamic acid and curcumin treatment induces pancreatic cancer cell growth inhibition via suppressing Sp1 expression, NF-kB translocation to nucleus. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4818.