Synergistic antiproliferative effect of C6-ceramide and 2´nitroflavone combination in murine mammary tumor cells.

FLORENCIA CELESTE GENTILCORE; MARIEL MARDER; LEONOR P ROGUIN; JULIETA MARINO; VIVIANA C. BLANK

Mar del Plata

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Sociedad Argentina de Investigación Clínica (SAIC)

Flavonoids are polyphenolic compounds that exhibit multiple biological activities, such as antiproliferative, antiallergic, antiangiogenic and antioxidant actions. We have previously demonstrated that the synthetic flavonoid 2´-nitroflavone (2NF) inhibited tumor growth in vitro and in vivo in a breast cancer murine model. On the other hand, existing evidences have suggested that sphingolipid metabolites are key molecules in regulating a number of cancerous behaviours. Thus, while sphingosine-1-phosphate promotes cancer cell survival and proliferation, ceramide and sphingosine accumulation could promote cell death. Moreover, certain flavonoids have been reported to increase the levels of ceramides, by inhibiting sphingosine kinase-1 (SphK-1), an enzyme involved in ceramide catabolism.The aim of our study was to investigate the effect of the combination of exogenously added short-chain cell-permeable ceramides and 2NF in LM3 murine mammary tumor cells. It was initially demonstrated by western blot analysis that LM3 cells over-expressed SphK-1 compared to normal murine NMUMG cells. In addition, the incubation of LM3 cells with different concentrations of 2NF or ceramides alone for 48 h inhibited cell growth, with IC50 values of 21±2 μM (2NF), 46±0.5 μM (C2-ceramide) and 35±1 μM (C6-ceramide). When 2NF and ceramides (C2 or C6) were simultaneously added in concentrations that individually inhibited cell growth approximately 30% (5 μM for 2NF, 40 μM for C2-ceramide and 30 μM for C6-ceramide) we found no significant differences in antiproliferative activity for the combination 2NF+C2-ceramide, but cell viability was drastically reduced for the combination 2NF+C6-ceramide. In order to quantitatively characterize the interaction between 2NF and C6-ceramide, dose-effect curves were analyzed by Compusyn software, being combination index of 0.68±0.1, indicative of synergism. In conclusion, our results demonstrated a synergistic antiproliferative effect of C6-ceramide and 2NF combination in LM3 tumor cells, suggesting that 2NF could affect ceramide catabolism, probably by SphK-1 inhibition.