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The synthetic triterpenoid CDDO and/or its derivatives, CDDO-Me and CDD0-Im, have been shown to induce apoptosis in acute myelogenous leukemia cell lines in association with suppression of MAPK pathways. Here, we report pre-clinical investigations on the cytotoxicity and associated biochemical changes induced by CCDO, and derivatives, in pediatric Acute Lymphoblastic Leukemia (ALL) cell lines. Methods. Cytotoxicity assay in 5% oxygen (bone marrow equivalent oxygen tension) at +96 hrs was by a fluorescence-based assay (DIMSCAN) in T-cell ALL lines, MOLT-4 and CEM, and in four newly-established pediatric ALL cell lines, COG-LL-317 (T-cell), COG-LL-319 (pre-B), COG-LL-327 (T-cell), and COG-LL-x-330. Sphingolipid analysis employed [14C]-serine labeling, TLC separation, and phosphoimaging quantitation. Reactive Oxygen Species (ROS) analysis employed DCFDA dye and flow cytometry analysis. Results. For CDDO, LD99 values were: CEM, 3.2 μM; MOLT-4, 2.2 μM; COG-LL-317, 2.7 μM; COG-LL-319, 6 μM; COG-LL-327, 4.2 μM; and COG-LL-x-330, 5.5 μM; for CDDO-Me, LD 99 values were: CEM, >1 μM; MOLT-4, 0.86 μM; COG-LL-317, 0.75 μM; and COG-LL-319, >1 μM; COG-LL-327, 1 μM; COG-LL-x-330, >1 μM; for CDDO-Im, LD99 values were: CEM, 0.3 μM; MOLT-4, 0.37 μM; COG-LL-317, 0.14 μM; and COG-LL-319, 0.23 μM; COG-LL-327, 0.3 μM; COG-LL-x-330, 0.4 μM. Treatment of MOLT-4 cells with approximately equally cytotoxic (LD90-LD99) doses of CDDO (5 μM), CDDO-Me (1 μM), and CDDO-Im (0.4 μM), increased total labeled ceramide species at +16 hrs by 2.4, 2.1, and 8 -fold, respectively. Pretreatment with myriocin or Fumonisin B1, inhib itors of de novo ceramide synthesis, completely prevented ceramide increase. In MOLT-4 cells with sphingomyelin (SM) prelabeled with [14C]serine for 24 hours followed by washout, CDDO-Im (0.4 μM) only increased labeled ceramides by 1.6 -fold, with a decrease in total labeled SM by ∼25%. Together, these data suggest that ceramide increased by CDDO-Im was predominantly by de novo synthesis. Interestingly, co-treatment of MOLT-4 cells with CDDO-Im (0.4 μM) and fenretinide (10 μM) increased total ceramides greater than either drug alone but shifted ceramide species distribution from unsaturated ceramides to saturated dihydroceramides as observed with fenretinide-alone, suggesting that both drugs affect a common de novo ceramide synthetic pathway. No increase in ROS was noted in CDDO-Im (0.4 μM) treated MOLT-4 cells out to +16 hrs. Conclusions. Our data suggest that CDDO, and its derivatives, increase ceramides (desaturated > saturated) in pediatric ALL cells through de novo synthesis and may have potential applications in the treatment of pediatric ALL.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]