IN-VITRO AND IN-VIVO INDUCTION OF APOPTOSIS BY SPHINGOSINE AND N,N-DIMETHYLSPHINGOSINE IN HUMAN EPIDERMOID CARCINOMA KB-3-1 AND ITS MULTIDRUG-RESISTANT CELLS

Sphingolipid breakdown products, including ceramide and sphingosine, r egulate cell growth, cell differentiation, and apoptosis, We examined the effect of various agents, including sphingolipids, on apoptosis in duction in human epidermoid carcinoma KB-3-1 and its multidrug-resista nt (MDR) subclone KB-C2 cells which express P-glycoprotein, Adriamycin (ADM) induced apoptosis in KB-3-1 cells but not in KB-C2 MDR cells at the concentration of 50 mu g/ml. On the other hand, 15 mu M sphingosi ne or its methylated derivative N,N-dimethylsphingosine (DR IS) induce d apoptosis in both cell types in vitro. These results suggested that KB-C2 MDR cells were resistant to apoptosis induction by ADM but sensi tive to that by sphingosine and DMS. Ceramide and sphingosine-1-phosph ate, the initial metabolites of sphingosine, failed to induce apoptosi s under the same experimental condition as sphingosine/DMS. The protei n kinase C (PKC) inhibitors H7 and staurosporine did not induce apopto sis in either cell line, suggesting that PKC-independent signaling is involved in apoptosis induced by sphingosine and DMS, although both sp hingosine and DMS have been shown to down-regulate PKC. Furthermore, D MS significantly inhibited the growth of KB-3-1 as well as KB-C2 MDR t umors in vivo, with evidence of increased apoptosis, The intracellular level of exogenously added [H-3]sphingosine or [C-14]DMS did not diff er between the KB-3-1 parent cell line and its MDR subclone KB-C2, whe reas that of [C-14]ADM was reduced in KB-C2 MDR cells compared to KB-3 -1 cells. These results suggest that P-glycoprotein acts as a transpor ter for ADM but not for sphingosine or DMS. Furthermore, DMS at the co ncentrations which induce apoptosis in KB-C2 cells did not affect the level of [C-14]ADM. Because sphingosine and DMS induce apoptosis regar dless of P-glycoprotein expression, they may provide a new strategy an d a promising approach to the treatment of anticancer drug-resistant c ancer.