Effects of aspirin on melanogenesis in B16F10 cells grown as 3D hanging drop tumor organoids.

Abstract

Aspirin (acetylsalicylic acid)has been shown to significantly reduce the melanin content in murine melanoma cells cultured in 2D. While this phenomenon has been attributed to lower tyrosinase expression and ERK‐induced degradation of MITF, the effect of aspirin treatment on the growth of melanoma cells in 3D, their morphology and melanosome secretion have yet to be characterized. Melanin secretion and gene expression data were generated from three and five, respectively, replicates of the following. Twelve hanging drop (HD) plates were seeded with B16F10 cells (5000 cells/well). Cells in six plates were cultured in DMEM+10% FBS+Pen/Strep (CONTROL; 50 ml/well) at 37C in a 5% CO2 in air environment. Cells in the remaining plates were cultured in the same medium supplemented with aspirin. Medium was changed daily (20 ml/well). Cells and supernatants were harvested (1 plate/treatment) at 24 hour intervals up to and including 144 hrs. of culture. Cells were washed in PBS and stored in either lysis buffer at −20C for Western analysis or RNA‐Later at −80C for qPCR. Supernatants were centrifuged in order to remove cells and 405l absorbance was measured. Aspirin visibly reduced the melanin content in B16F10 cells within 48 hrs. of initial exposure. Supernatants above both ASPIRIN and CONTROL cells contained significantly (P<0.05)more melanin at 120 hrs. of HD culture than any previous time point. The melanin content in ASPIRIN cell supernatants was also significantly (P<0.05) higher than that of CONTROL cell supernatants at 120 hrs. of HD culture. Tyrosinaseexpression and tyrosinaseprotein content were lower (P<0.03) in ASPIRIN as compared to CONTROL cells during the last 72 hrs. of HD culture. MITFexpression was also reduced (P<0.05)in ASPIRIN cells during the last 48 hrs. of HD culture. We have demonstrated that aspirin can reducethe melanin content in B16F10 cells cultured in 3D. This phenomenon may be due to aspirin's downregulation of two genes, tyrosinase and MITF, associated with the melanogenic pathway. Aspirin, however, did not affect melanin secretion by B16F10 cells grown in this HD plate format.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.