Copper Tolfenamic Acid as a Novel Survivin Inhibitor for Suppressing Pancreatic Cancer Cell Growth via Downregulating Sp1 and Sp3 Transcription Factors

Despite medical advancements, PaCa unfortunately still remains a lethal malignancy. Patients are typically diagnosed once the cancer is advanced and metastatic, making them ineligible for surgery. This cancer is usually aggressive at the time of diagnosis, so chemo and radiation offer little benefit. Therefore, PaCa urgently requires more effective and sensitizing agents for treatment. Two targets of interest for PaCa have been transcription factors Sp1 and Sp3. Both Sp1 and Sp3 are involved with regulating cell proliferation, differentiation and apoptosis. Their overexpression has been found to contribute to the progression, advancement, and poor prognosis of many types of cancers, including pancreatic. Survivin, an inhibitor of apoptosis protein, is a gene known to be regulated by both these Sp proteins. Survivin has also been found to be overexpressed in various tumor types and adds to the cancer's resistance to cytotoxic therapies. For these reasons, Sp1, Sp3, and survivin have been targets of interest for PaCa and researchers have becoming interested in finding drugs that inhibit their expression. Tolfenamic Acid (TA) is a generic migraine medicine sold in Europe. The small molecule TA has been gaining popularity for its anti-cancer properties such as inhibition of cell growth and induction of apoptosis in various tumor models. TA has been shown to work by downregulation of Sp proteins and survivin. TA has also been demonstrated to sensitize PaCa cells to radiation treatment. Although the results seen with TA thus far seem promising, its IC50 value is slightly high. Recently, it has been suggested that a copper(II) complex of TA (Cu-TA) can produce a higher therapeutic response; however, its efficacy has yet to be tested in gastro-intestinal cancers. Here, we used human PaCa cell lines (MIA PaCa-2 and Panc1) to evaluate the therapeutic efficacy of Cu-TA in an effort to increase TA's efficacy. This project contains three specific aims. Aim 1: Characterization of Cu-TA and determining its stability and anti-proliferative activity in human pancreatic cancer cell lines. Aim 2: Evaluate the effect of Cu- TA on potential markers associated with PaCa cell growth. Aim 3: Use gene expression analysis to precisely elucidate the underlying mechanisms of Cu-TA's anticancer activity in PaCa cells. Cu-TA was found to have an anti-proliferative effect in PaCa cells and its IC50 value was half that of TA's. Characterization of Cu-TA demonstrated its stability as a compound and its biological stability. Importantly, treatment of Cu-TA on cardiomyocytes did not affect cell viability. This is significant since NSAIDs can potentially cause cardiotoxicity. Cu-TA also downregulates expression of Sp proteins and survivin, molecular markers involved with PaCa growth and progression. Additionally, Cu-TA induces apoptosis and cell cycle arrest in PaCa. Finally, RNA sequencing and subsequent pathway analysis of treatment revealed Cu-TA affects pathways involved with cancer progression and metastasis. Thus, these results demonstrate Cu-TA as a potential anti-cancer agent for PaCa.