A Small Molecule Derivative as a Targeting Agent for Sp1 and Survivin Effectively Suppresses Pancreatic Cancer Cell Growth

Background: Pancreatic cancer has one of the most fatal malignancies due to its poor prognosis. It currently has a one-year survival rate of 20%. Current standard forms of treatment contain a high level of toxicity, thus preventing an increase in dosage or frequency. This issue poses the need for more effective, yet less toxic agents for treatment. Tolfenamic acid (TA) is most commonly used to treat migraines but has recently been demonstrated to contain anti-cancer properties. It is known to downregulate the Specificity Protein (Sp) transcription factor, Sp1. Sp1 regulates several genes involved in cell proliferation and apoptosis, including survivin, an inhibitor of apoptosis protein. Interestingly, a recent discovery proposed that a copper(II) complex with TA as a ligand can result in higher therapeutic response; however its efficacy was not tested in gastro-intestinal cancers. Purpose: In this study, we assessed the therapeutic efficacy of a Cu(II)- containing complex of TA (Cu-TA) using human pancreatic cancer cell lines. Methods: MIA PaCa-2 and Panc1 cells were treated with increasing concentrations of DMSO (vehicle), equimolar CuCl2 (negative control), TA or Cu-TA and the cell viability was measured at 24 and 48 h post-treatment using CellTiter-Glo kit. CuTA was further tested for its effect on Sp1 and survivin expression by Western blot and quantitative PCR. The activation of apoptosis was determined by measuring the activity of effector caspases using the Caspase 3/7-Glo kit and the apoptotic cell population through flow cytometric analysis using Annexin-V staining. Cell cycle arrest was assessed by flow cytometry with propidium iodide staining. Results: While both TA and Cu-TA inhibited pancreatic cancer cell growth in a dose/time-dependent manner. Cu-TA was highly effective in inhibiting Sp1 and survivin protein expression and showed similar trend for inducing apoptotic markers and causing cell cycle arrest in G2/M phase. The results of qPCR demonstrated that the expression of survivin mRNA was significantly lower following both Cu-TA and TA treatment; however, the mRNA expression of Sp1 remained unchanged. This indicates that TA and Cu-TA could be affecting Sp1 by a similar mechanism. Conclusions: These results demonstrate that Cu-TA is more effective than TA and potentially useful for pancreatic cancer treatment after clinical testing. Studies to understand precise underlying mechanisms are currently under investigation.