ABT-737 increased intracellular ceramides through sphingomyelin hydrolysis in ALL cell lines

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Ceramides are thought to act as lipid second messengers in cell death pathways. Some antineoplastaic drugs, such as daunorubicin, Ara-C, or vincristine increase ceramides through sphingomyelin (SM) hydrolysis, while others (doxorubicin, etoposide, fenretinide) stimulate de novo synthesis of ceramides. We reported that the combinatoin of ABT-737 (a Bcl-2 family inhibitor) and the cytotoxic retinoid fenretinide (4-HPR) caused a greater increase in ceramides in acute lymphoblastic leukemia (ALL) cells than either drug alone (Proc AACR 47:1336, 2006). We have examined the mechanism by which ABT-737 increased ceramides in the ALL cell lines RS4-11 (pre-B) and CEM (T cell). Ceramides were measured by thin-layer chromatography and [¹⁴C]-L-serine. Cytotoxicity was determined using a fluorescence-based DIMSCAN assay. RNA expression of sphingmyelinases was determined by Taqman RT-PCR. In CEM cells pre-labeled overnight with [¹⁴C]-L-serine, ABT-737 caused a time-dependent increase in ceramides relative to controls (8-fold at 6 hours). Myriocin, a serine palmitoyltransferase (SPT) inhibitor did not affect the increase in ceramides in response to ABT-737, but GW4869, a neutral sphingomyelinase (nSMase) inhibitor, significantly decreased the generation of ceramides by ABT-737 (p= 0.007). Similar results were obtained with RS4-11 cells. These data indicate that synthesis of ceramides by ABT-737 is mainly due to sphingomyelin hydrolysis. In addition, real-time RT-PCR showed that mRNA for both neutral-SMase (1.51 ± 0.06-fold; p=0.007 at 2h) and acid-SMase (2.25 ± 0.24; p=0.09 at 4h) were up-regulated by ABT-737 in RS4-11 cells. However, cytotoxicity of ABT-737 for ALL cells was minimally affected by the nSMase inhibitor GW4869 or by desipramine (an acid-SMase inhibitor). Thus, ABT-737 increased intracellular ceramides in ALL cells via the sphingomyelin hydrolysis pathway in a time-dependent manner, but the role of ceramides in ABT-737-induced cytotoxicity appeared to be minimal.