Modulation of the Cellular Phenotype in Human Colon Adenocarcinoma Cells by Folic Acid and Polyamine Pools Nathan W. Sweeney, Julie A. Buckmeier, Christina R. Preece, Patricia A. Thompson Cancer Biology Graduate Interdisciplinary Program, University of Arizona Cancer Center, Tucson, Arizona. Background Human colon adenocarcinoma is the most common type of gastrointestinal cancer and ranks third in occurrence and death in both males and females. Polyamines are small, positively charged molecules derived from endogenous biosynthesis or exogenous sources (diet, gut flora, cellular excretions). Polyamines have demonstrated to be involved in a large number of cellular processes such as receptor-ligand interactions, transcription, translation, DNA stabilization, cell growth, proliferation, and migration. Folate is a naturally occurring form of the water-soluble vitamin B9. Folic acid is a synthetic form of the vitamin used in fortified foods. Folic acid is crucial for proper brain function. As well as the synthesis, methylation and repair of DNA. Polyamines and Folic acid are required for both normal and cancer cell growth. Methods Cell Proliferation Assay: Treated human colon adenocarcinoma cell lines HCT116 and Caco2 with DFMO and/or MTX for 72 hours. DFMO uptake experiments: Cells were serum starved for 24 hours. Treated HCT116 cells with DFMO for 72 hours and approximately three doublings. Treated Caco2 cells with DFMO for 120 hours and approximately one doubling. Real Time PCR: HCT116 and Caco2 cells were treated with DFMO and/or MTX for 72 hours. Analyzed EGFR and KRT20 mRNA expression level in treated cells. Preliminary Results Questions 1.Difluoromethylornithine (DFMO), a cytostatic drug and effective inhibitor of polyamine production, alone is sufficient to arrest colon cancer cells but not induce senescence nor apoptosis. Methotrexate (MTX) is a cytotoxic drug capable of inhibiting folic acid. Therefore, will inhibition of both pools result in a complimentary effect? 2.Oncogene induced senescence (OIS) has been determined to be a form of tumor growth inhibition. What involvement does polyamine depletion have on preventing OIS? 3.DFMO, through polyamine depletion has shown to induce differentiation in cancer stem cells. Although the nature of the cell post treatment is unknown. Identification of the genes will reveal colon adenocarcinoma cell modulators. Figure 1. Overview of Polyamine and Folic Acid Pathways Summary of Preliminary Results: The cell proliferation assay revealed an IC50 for DFMO and MTX in HCT116 and Caco2 cell lines. DFMO uptake experiment indicated that DFMO requires cell division and that uptake occurs at approximately each estimated doubling time point. Real Time PCR revealed that treatment with DFMO and/or MTX modulates EGFR and KRT20 expression level in HCT116 and Caco2 cell lines. Results could demonstrate an induced differentiation initiated by DFMO. Discussion & Future Directions: Uncomplimentary effect of DFMO/MTX in combination during the cell proliferation assay leads us to believe an “either/or” result due to early cell cycle incorporation of DFMO and arrest preventing the incorporation of MTX and its cytotoxic effects. The DFMO treatment experiments indicated when drug is likely taken into the cell and that it is effective at each cell doubling. Flow cytometry will allow us to pinpoint at which cell cycle stage DFMO is active. Additionally, it seems prudent to utilize an agent that is not cell cycle dependent, in combination with DFMO, in order to perhaps achieve an enhanced or synergistic effect. Its been shown previously that cells treated with DFMO can be rescued. This rescue indicates that cells are temporally arrested and not in a permanent state of senescence. It’s thought that depletion of polyamines inhibits an oncogene induce senescence but the mechanisms are unknown and require investigation. Its been shown previously that DFMO can induce differentiation in cancer stem cells. KRT20 is an intermediate filament protein found in colonic epithelial cells and its 2-fold increased expression in Caco2 cells by DFMO may indicate a shift to a more differentiated phenotype. Further profiling of genes could potentially reveal colon adenocarcinoma cell modulators as well as targets. Figure 3. Effect of Serum Starvation and DFMO on Cellular Proliferation in HCT116 and Caco2 Cell Lines. Figure 2. Effect of DFMO and/or MTX on Cellular Proliferation in Human Colon Adenocarcinoma Cell Lines Figure 4. Differential Effect of DFMO and/or MTX on EGFR and KRT20 mRNA Expression in Human Colon Adenocarcinoma Cells Lines. Acknowledgements and Contact*: Thompson Lab CBIO Department Initiative for Maximizing Student Diversity (IMSD) Chahta Foundation & The Choctaw Nation of Oklahoma