Diacetyl Effects on Cancer Cell Lines

Human embryonic kidney 293 (HEK293) cells were grown in 100 mm cell culture dishes with Dulbecco’s modified Eagle’s medium (DMEM) (10–013, Corning, Manassas, VA), supplemented with 10% fetal bovine serum (FBS) (26140–079, Gibco, Carlsbad, CA) at 37°C with 5% CO₂. Cells that were ~80% confluent were treated with freshly prepared medium supplemented with diacetyl at concentrations indicated. The cells for mock controls were handled in the same manner without adding diacetyl to the medium. In order to prevent diffusion of diacetyl odor from the treatment dishes to the ones of mock control, the cell culture dishes in different conditions were cultured in separate CO₂ incubators.
Cell proliferation assays were performed to assess effects of compounds against cancer cell lines. Cells were maintained in a 37°C incubator with 5% CO₂ throughout the whole experiment. Compound stocks are dissolved in 10% DMSO, and final concentrations made fresh in complete media as needed. 10% DMSO solvent is used as control in complete media. Cells were seeded onto 12-well plates and allowed 12–24 hours until treatment to allow cells to adhere to wells. This is followed by minimum 5-day treatment of compounds, changing the media with treatment every 48 hours. Cell lines A549, PC3, and SK-MEL-5 will be seeded at 8000 cells/well, which was determined to provided 80–100% confluency after 6-day growth. SH-SY5Y was seeded at 8000 cells/well and 16,000 cells/well due to their known slower division rate and given at least 10 days for sufficient growth. After allotted growth times, cell counts were performed using the CountessTM automated cell counter. All assays had a minimum of three replicates for all conditions and analyzed through GraphPad Prism One-way ANOVA for significance.