Ceramides (CERs) are believed to act as lipid second messengers of cell death when induced by certain chemotherapeutic agents in cancer cells. Dihydroceramides (dhCERs) are converted to CERs by dihydroceramide desaturases, DES-1 and DES-2. The importance of DES activity to chemotherapy induced cytotoxicity is not known. Exogenous, long (native) chain dhCERs (C14+) are membrane inpenetrant, which has hindered investigation of their biological properties. Thus, cytotoxic potential of endogenous long chain dhCERs remain to be determined. We previously reported that fenretinide (4-HPR), has cytotoxicity in a variety of human cancer cell lines in association with an increase of dhCERs via de novo synthesis. In the present study, we attempt to clarify the biological role of DES activity in ceramide-mediated cytotoxicity, and whether endogenous long chain dhCERs may mediate a cytotoxic response.

METHODS. We previously reported that pediatric acute lymphoblastic leukemia (ALL) cell lines COG-LL-317 and COG-LL-319 cells have high baseline expression levels of DES-1 or DES-2, respectively. Cells were treated with sphinganine (1 to 10 μM) with or without DES inhibitor, GT11, for 96 hours prior to cytotoxicity assay. The effect of drug treatment on dhCER and CER synthesis at +6 hrs was investigated by TLC assay; myriocin was used to block serine palmitoyltransferase, and 14C-palmitic acid used to label sphinganine-derived ceramide species. COG-LL-319 cells were also treated with de novo CER synthesis stimulator, PSC833, with or without GT11. Cytotoxicity and TLC assay of dhCER and CER synthesis were also performed.

RESULTS. Sphinganine (2.5 and 5 μM) was non- to minimally- cytotoxic to COG-LL-317 and COG-LL-319 cells. Combining sphinganine with a non-toxic concentration of GT11 (0.5 μM) greatly increased cell kill (P< 0.001). In COG-LL-317, survival fraction (S.F.) decreased from 80% to ~0.5% at 5 μM sphinganine; in COG-LL-319, S.F. decreased from 70% to ~0.6% at 5 μM sphinganine. In COG-LL-319 cells, GT11 (0.5 μM) treatment decreased by ~60% the synthesis of shorter chain CER (C14 - C18); in contrast, the relative level of dhCERs (C18 - C24) induced by sphinganine increased by ~30%. Combining GT11 with PSC833 decreased shorter chain CER synthesis (by 50% at 5 μM, and by 74% at 10 μM of PSC833) while enhancing the synthesis of dhCERs (C18 - C24) (by ~1.7 fold for 5 μM, and ~2.5 fold for 10 μM of PSC833). The combination of GT11 and PSC833 also achieved a greater cytotoxic effect than did PSC833 or GT11 alone.

CONCLUSIONS. Inhibition of DES activity using GT11 enhanced the cytotoxicity of sphinganine and PSC833 in association with an increase of dhCER levels in ALL cell lines. Further investigations on the cytotoxic properties of dhCER using siRNA to DES1 and 2 to more specifically decrease DES activity are in progress.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA