A novel mode of action of the putative sphingosine kinase inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II): induction of lysosomal sphingosine kinase 1 degradation.

Background: Sphingosine kinase 1 (SK1) is a key enzyme in the generation of sphingosine 1-phosphate (S1P) which critically regulates a variety of important cell responses such as proliferation and migration. Therefore, inhibition of SK-1 has been suggested to be an attractive approach to treat tumor growth and metastasis formation.

Results: We show here that the previously developed putative SK-1 inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (SKI II) displays an additional facet of action complementary to the known inhibition of enzymatic SK-1 activity. In various human cell lines including glomerular podocytes and mesangial cells, the human endothelial cell line EA.hy 926, and the lung cancer cell line NCI H358, SKI II reduced TGFbeta- and TPA-stimulated cellular SK-1 activity by downregulating SK-1 protein expression without affecting SK-1 mRNA expression. By using cycloheximide to block the de novo protein synthesis, the protein expression of SK-1 under untreated conditions was stable over 24h. Under SKI II treatment, the half-live drastically decreased to approximately 0.8h. Mechanistically, this degradation occurred through a lysosomal pathway and involved cathepsin B since the general lysosomal inhibitor chloroquine and the specific cathepsin B inhibitor CA-074ME were able to reverse the effect of SKI II. Surprisingly, in vitro SK-1 activity assays revealed only a very weak direct inhibitory effect of SKI II on SK-1 overexpressed HEK293 cell lysates.

Conclusion: These data show for the first time that the previously developed SK inhibitor SKI II hardly inhibits SK-1 directly but rather acts by triggering the lysosomal degradation of SK-1 in various cell types. This finding discloses a new mode of action of SKI II and strongly suggests that additional direct targets of SKI II may exist other than SK-1.