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.


CONCLUSIONS
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.
Ren
Shuyu
S
Institute of Pharmacology, University of Bern, Bern, Switzerland.
Xin
Cuiyan
C
Pfeilschifter
Josef
J
Huwiler
Andrea
A
D002401:Cathepsin B / Q000378:metabolism; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D000099157:Sphingosine Kinase
D002401:Cathepsin B; D003513:Cycloheximide; D004791:Enzyme Inhibitors; D017853:Phosphotransferases (Alcohol Group Acceptor); D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D013844:Thiazoles; D016212:Transforming Growth Factor beta; D000099157:Sphingosine Kinase; C525025:4-(4-(4-chloro-phenyl)thiazol-2-ylamino)phenol