Role of "sphingolipid rheostat" in mediating the apoptotic effect of luteolin in colon cancer cells

Luteolin, a common flavonoid present in different plants, exhibits a variety of biological effects, including antioxidant, anti-inflammatory, and anticancer actions. However, little is known on the anticancer potential of this bioflavonoid in colon cancer, one of the most commonly diagnosed and lethal cancers worldwide. The aim of this study was to investigate the effects and the molecular mechanisms of luteolin in colon cancer cells, focusing on the role of the sphingoid molecules ceramide and sphingosine-1-phosphate (S1P). These two lipids exert opposite roles on cell fate, with ceramide acting as a pro-apoptotic messenger, and S1P as a survival factor. Together, they form the “sphingolipid rheostat”, regulating the balance between survival and death in a variety of cell types. We first found that Luteolin exhibited a potent cytotoxic effect, by inducing apoptosis, in colon cancer cells, without affecting the viability of differentiated intestinal cells. This effect was found related to the increase of the cellular level of ceramide. Experimental conditions leading to enhance cell ceramide content mimicked the apoptotic effects of luteolin, thus suggesting the involvement of ceramide in the mechanism of luteolin-induced apoptosis. Further experiments demonstrated that luteolin was able to induce an alteration of ceramide metabolism, by inhibiting its utilization as precursor in the biosynthesis of complex sphingolipids. Additional studies demonstrated the presence of an aberrant traffic of ceramide during luteolin treatment resulted in its accumulation. In addition, experiments with a PI3K inhibitor showed that the down-regulation of PI3K/AKT signaling pathway led to the inhibition of ceramide transport and metabolism similar to that observed during luteolin treatment. Western blot analyses demonstrated that luteolin was able to act as a potent inhibitor of AKT phosphorylation. Taken together, the down-regulation of the PI3K/AKT pathway by luteolin emerged as crucial in the effect of luteolin on ceramide increase. We also found that luteolin was able to alter not only ceramide content but also that of S1P by acting as inhibitor of sphingosine kinase and thus reducing the level of this pro-survival signal. Finally, the addition of exogenous S1P resulted in the up-regulation of the PI3K/AKT pathway, which was able to rescue colon cancer cells from the apoptotic effect of luteolin. Taken together, our results demonstrate for the first time that the natural flavonoid luteolin can induce apoptosis in colon cancer cells by altering the “sphingolipid rheostat”, and directing its balance in favor of ceramide.