Pancreatic adenocarcinoma (PAC) is chemoresistant and challenging to treat due to lack of knowledge about how the human homolog of the Kirsten Rat Sarcoma viral oncogene (KRAS) mutated in 90% patients causes the disease. Available drugs such as gemcitabine and FOLFIRINOX remain ineffective. We present evidence that repurposing the FDA-approved raloxifene to potentiate the cytotoxicity of gemcitabine is a significant strategy for combating PAC’s chemoresistance. We are exploring the efficacy of gemcitabine (GEM), raloxifene (RAL) and the combination of raloxifene and gemcitabine 1:1 (RG) in three different pancreatic cancer cell lines including the <i>KRAS</i> mutant MIA PaCa-2 and PANC-1 and wild-type BxPC-3. The IC50 values for GEM, RAL, and RG in PANC-1 cells are 0.05 + 0.03 µM, 24.2 + 6.3 µM, and 14.4 + 0.2 µM respectively with similar trends in MIA PaCa-2 and BxPC-3 cells. Combination Index analysis are underway to assess if the combination of RG is synergistic and various concentrations of RG are being evaluated to determine the ideal ratio to achieve synergism. To investigate the mechanism of cytotoxicity, we treated the MIA PaCa-2 cells with raloxifene (0.05, 0.5, 5.0, and 20 μM) for 20 minutes, and 24 and 48 hours and measured the production of reactive oxidative species (ROS) using a dihydrofluorescein-based fluorescence assay. Our data show that RAL induces ROS production in 20 minutes and consistently for up to 48 hours. As ROS production is associated with a loss of mitochondrial membrane potential (mtMP), using a tetramethylrhodmaine-based fluorescence assay, we measured mtMP for the same dose and time points and found that RAL treatment compromises mtMP. Using Western blot, we ascertained that suppression of the catalse enzyme contributed in part to the oxidative stress response. We are extending these experiments using GEM and RG combinations to test the mechanism of the cytotoxicity of these drugs.

Repurposing the FDA-approved raloxifene to potentiate gemcitabine cytotoxicity in pancreatic cancer

255th National Meeting (2018)

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