Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite

Abstract

High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancer cells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in the hepatocellular carcinoma cell line Huh7. Treatment with 20 μM of selenite reduced the viability of Huh7 cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold. Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxic effect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutral sphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 μM of SK1-II prior to treatment with 10 μM of selenite caused induction of apoptosis and gave rise to a 2.5-fold increase in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 μM of SK1-II combined with 10 μM of selenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides. The formation of reactive oxygen species (ROS) after treatment with 10 μM of selenite was maximally enhanced by 1 μM of SK1-II. Moreover, combined treatment with SK1-II and 10 μM of selenite synergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell line MIHA remained unaffected by the same treatment. These results raise the possibility that a combination of selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant, at doses that are non-toxic to normal liver cells.