Presentation Title (IN ALL CAPS)

COPPER-TOLFENAMIC ACID INHIBITS BREAST CANCER CELL GROWTH VIA CELL CYCLE ARREST AND APOPTOSIS

Departmental Affiliation and City, State, Zip for All Authors

Riyaz Basha, Umesh Sankpal: UNTHSC, Fort Worth, Texas, 76107; Alvin Holder: Old Dominion University, 5115 Norfolk, VA 23529

Scientific Abstract

Copper-Tolfenamic Acid Inhibits Breast Cancer Cell Growth via Cell Cycle Arrest and Apoptosis Rafid Patel1, Alvin A. Holder2, Umesh Sankpal1, Jaya Chhabra2, Deondra T. Brown2, Raj K. Gurung2, Riyaz Basha1 1Center for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 2Department of Chemistry and Biochemistry, Old Dominion University,, Norfolk, VA; Breast cancer (BC) is the most commonly diagnosed cancer in women and is the second leading cause of cancer related deaths among women. BC is among the cancers found to exhibit Health Disparities in the United States. Identifying effective strategies is important to treat these cancers thereby addressing cancer-related Health Disparities. The objective of this study was to identify an agent to target an Inhibitor of apoptosis (IAP) protein, Survivin to induce therapeutic efficacy in BC cells. Survivin is overexpressed in most human cancers including BC and its overexpression is linked to the aggressiveness and poor prognosis of the cancers. Results from our laboratory and others have demonstrated the anti-cancer activity of a small molecule, Tolfenamic acid (TA) by targeting Survivin. Recently, it has been suggested that the pharmacological effects of TA could be enhanced when linked with Copper (Cu) metal. In this investigation, we manufactured a TA and Cu derivative (Cu-TA) and evaluated its effect on Survivin expression and anti-cancer activity using BC cell lines, MDA231 and MCF7. The Cu-TA was synthesized and characterized by FTIR spectroscopy. Its purity and stability was assessed by HPLC. The purity was >99% and the compound was stable for 6 months in a solution (dissolved in DMSO). Both BC cell lines were treated with increasing concentrations of Cu-TA and the cell viability was measured at 24 and 48 hours post treatment using the CellTiter-Glo Kit. For each cell line, IC50 value was determined. Further studies were performed using the IC50 value of Cu-TA and an equimolar dose of TA. The protein expression of Survivin was determined by Western Blot analysis. The effect of Cu-TA on apoptosis was analyzed by flow cytometry using Annexin V staining. Cell cycle arrest was also measured by flow cytometry using Propidium Iodide. While both TA and Cu-TA showed a steady and consistent anti-proliferative effect, Cu-TA proved to be more effective. Western blot analysis confirmed a higher inhibition of Survivin expression following Cu-TA treatment when compared to TA. At the same time Cu-TA proved to be more effective in causing both cell cycle arrest [G2M arrest] as well as apoptosis [2-fold increase]. These results suggest that Cu-TA is highly effective and the anti-cancer activity can be achieved with a minimal dose when compared to TA. Since, Survivin is associated with drug resistance, future studies will focus on using this compound in combination treatment along with chemotherapeutic agents or radiation to induce sensitivity in cancer cells against standard care. Keywords: Breast Cancer, Survivin, Apoptosis, Copper Tolfenamic Acid

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COPPER-TOLFENAMIC ACID INHIBITS BREAST CANCER CELL GROWTH VIA CELL CYCLE ARREST AND APOPTOSIS

Copper-Tolfenamic Acid Inhibits Breast Cancer Cell Growth via Cell Cycle Arrest and Apoptosis Rafid Patel1, Alvin A. Holder2, Umesh Sankpal1, Jaya Chhabra2, Deondra T. Brown2, Raj K. Gurung2, Riyaz Basha1 1Center for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 2Department of Chemistry and Biochemistry, Old Dominion University,, Norfolk, VA; Breast cancer (BC) is the most commonly diagnosed cancer in women and is the second leading cause of cancer related deaths among women. BC is among the cancers found to exhibit Health Disparities in the United States. Identifying effective strategies is important to treat these cancers thereby addressing cancer-related Health Disparities. The objective of this study was to identify an agent to target an Inhibitor of apoptosis (IAP) protein, Survivin to induce therapeutic efficacy in BC cells. Survivin is overexpressed in most human cancers including BC and its overexpression is linked to the aggressiveness and poor prognosis of the cancers. Results from our laboratory and others have demonstrated the anti-cancer activity of a small molecule, Tolfenamic acid (TA) by targeting Survivin. Recently, it has been suggested that the pharmacological effects of TA could be enhanced when linked with Copper (Cu) metal. In this investigation, we manufactured a TA and Cu derivative (Cu-TA) and evaluated its effect on Survivin expression and anti-cancer activity using BC cell lines, MDA231 and MCF7. The Cu-TA was synthesized and characterized by FTIR spectroscopy. Its purity and stability was assessed by HPLC. The purity was >99% and the compound was stable for 6 months in a solution (dissolved in DMSO). Both BC cell lines were treated with increasing concentrations of Cu-TA and the cell viability was measured at 24 and 48 hours post treatment using the CellTiter-Glo Kit. For each cell line, IC50 value was determined. Further studies were performed using the IC50 value of Cu-TA and an equimolar dose of TA. The protein expression of Survivin was determined by Western Blot analysis. The effect of Cu-TA on apoptosis was analyzed by flow cytometry using Annexin V staining. Cell cycle arrest was also measured by flow cytometry using Propidium Iodide. While both TA and Cu-TA showed a steady and consistent anti-proliferative effect, Cu-TA proved to be more effective. Western blot analysis confirmed a higher inhibition of Survivin expression following Cu-TA treatment when compared to TA. At the same time Cu-TA proved to be more effective in causing both cell cycle arrest [G2M arrest] as well as apoptosis [2-fold increase]. These results suggest that Cu-TA is highly effective and the anti-cancer activity can be achieved with a minimal dose when compared to TA. Since, Survivin is associated with drug resistance, future studies will focus on using this compound in combination treatment along with chemotherapeutic agents or radiation to induce sensitivity in cancer cells against standard care. Keywords: Breast Cancer, Survivin, Apoptosis, Copper Tolfenamic Acid

Manuscript Number

1016