Understanding the Mechanism of Action of Copper-Tolfenamic Acid’s Anti-cancer Activity in Pancreatic Cancer Cells

Prokai, Laszlo
Levesque, Blair
Umesh, Sankpal
Basha, Riyaz
Hurtado, Myrna
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Purpose: Non-steroidal anti-inflammatory (NSAID) agents have been proven to have anti-cancer activity. Our group is investigating the use of NSAIDs as sensitizing agents to use alongside standard chemotherapy. This combination increases the efficacy of chemotherapy to accomplish higher anti-cancer activity at relatively low doses, thereby reducing the side effects. It is desirable to find anti-cancer NSAIDs with low Inhibitory concentration (IC50) values. The NSAID, tolfenamic acid (TA), has been tested in preclinical studies for anti-cancer activity against pancreatic cancer (PaCa). Recently, it is shown that metal complexes of NSAIDs enhances the efficacy. Methods: We investigated the anti-proliferative activity of copper-tolfenamic acid (Cu-TA) using 12 cancer cells and reported that the Cu-TA complex had a stronger therapeutic response and lower IC50 values by 30-80% compared to TA. The goal of this investigation is to determine the mechanism by which Cu-TA induces anti-cancer activity in PaCa cell lines. MIAPaCa2 cells were treated with vehicle or Cu-TA (IC50 value) and processed by Next Generation Sequencing (NGS). Ingenuity Pathway Analysis was used to determine the functional significance of the altered gene expression. The top upstream regulators were confirmed by Western blot analysis. Results: Several networks, regulators, and molecular and cellular functions were found to be affected by the Cu-TA treatment. qPCR and Western blot analysis were used to assess and confirm the alterations in the expression of the candidate markers in PaCa cells. Previously, confirmatory studies were performed using MIAPaCa2 cells. Due to the heterogeneity of PaCa, in this study we used a second cell line PANC1 for similar experiments. Tumor protein p53, human epidermal receptor growth factor 2, Specificity protein 1 and signal transducer and activator of transcription 3 were the top upstream regulators confirmed by Western blot analysis. It was demonstrated by qPCR of selected genes, Centromere protein F, DNA damage inducible transcript 3 and S-phase kinase associated protein 2 that Cu-TA is efficacious at a lower dose than TA. Conclusion: NGS and Ingenuity Pathway analysis identified important pathways and genes effected by Cu-TA. In this investigation, PANC1 showed similar results as MIAPaCa2 cells. The genes and pathways that were altered by treatment with Cu-TA involved cell survival or apoptosis demonstrating that Cu-TA is modulating genes associated with cancer. This identifies the potential of Cu-TA as an effective anti-cancer agent.