P18-110 Investigation of Rutin's Effect on Murine Melanoma Cell Behavior and Response to treatment via Modulation of Melanin Production

Recent studies describe the relationship between melanin and melanoma initiation as intricate, melanin being considered “a double edge-sword”. Even though some light is shed on the role played by melanin in melanoma initiation, its part in the metastatic process is poorly understood. A natural compound that attracted interest in the last years, as potential active compound in melanoma, is rutin (RUT), a flavonoid, that exhibited multiple biological effects, including: antioxidant, anti-inflammatory, anticancer, and even anti-melanogenic effect. The present study was performed to assess the impact of rutin (RUT) in three different pigmented murine melanoma cells (B16F0, B16F10 and B164A5) in terms of cells’ behavior and response to treatment. To that end, the following methodology was employed: 1) standardized cytotoxicity assessment methods as MTT and Cell-Titer Glo to assess RUT (0-100 µM) effect at different time points (24 and 48 h); 2) bright field microscopy and immunofluorescence to evaluate cell morphology changes; 3) determination of intracellular and extracellular melanin content following RUT treatment via spectroscopy and NaOH method; 4) treatment with specific modulators (forskolin – as melanin stimulator and kojic acid as inhibitor) to evaluate the response to RUT treatment; 5) Scratch assay – for cell migration and invasion; 6) qPCR – for pro-apoptotic and anti-apoptotic markers, and 7) Western blot analysis for protein expression and mechanistic insight regarding the RUT and melanin interplay. RUT treatment induced a cell-type, dose- and time-dependent cytotoxic effect, characterized by a significantly reduced cell viability, changes in cellular morphology, nuclear modification and apoptoticlike features. Moreover, by increasing the passage of cells, the content of melanin has decreased and also the response to the treatment was different. RUT treatment stimulated the expression of pro-apoptotic markers and reduced the expression of anti-apoptotic ones. These results provide relevance to rutin's potential as a pigmentation modifier and its applicability to several investigations of melanoma biology.