PARENT SESSION

(T629) CERAMIDE MODULATES DXR-INDUCED APOPTOSIS BY ALTERING DRUG TRAFFICKING AND NUCLEAR RETENTION.

Perez, Gloria ¹, Nakad, Toufic², Kujjo, Loro¹, ¹ Michigan State University, East Lansing, MI² St. George's Hospital Medical Center, Beirut, Lebanon

ABSTRACT- Ceramide, a central molecule in sphingolipid metabolism, is intimately involved in the regulation of cell growth, differentiation, senescence and apoptosis. A significant body of literature now implicates defects in ceramide accumulation as a key component in the development of tumor resistance to chemotherapy and failure of clinical treatments. Therefore, we have directed our interest in developing strategies that either mimic/antagonize sphingolipids or modulate their levels in order to provide novel avenues for cancer therapy. Here we investigated the role of ceramide in the cellular uptake and trafficking of doxorubicin (DXR) in OVCAR3 (a carcinoma cell line resistant to DXR), granulosa cells (GC); and oocytes. OVCAR3 or granulosa cells were pretreated with C16-ceramide (C16) and then incubated with DXR for 6 h. After another 18 h the cells were fixed, stained with DAPI, and checked for apoptotic nuclei and subcellular localization of DXR. When exposed to DXR alone for 6 hours, OVCAR3 cells internalized the drug and concentrated it in the nuclei, however 18 h later the amount of DXR remaining in the nucleus dropped to undetectable levels and the percentage of apoptotic cells by this time was 3%. The death rate increased to 45% when the cells were pre-exposed to ceramide followed by DXR treatment. Moreover, DXR was retained by the nucleus for longer time compared to its kinetics in the absence of ceramide. Pre-exposure of GC to ceramide also enhanced nuclear retention of DXR and the ensuing apoptosis (100% death with C16+DXR, compared to 65% with DXR alone). This modulatory role of ceramide was also observed in studies with MII oocytes of Acid Sphingomyelinase gene knockout (ASMase-KO) mice which are deficient in ceramide. Oocytes of ASMase-KO mice internalized DXR and by 15 min it was mostly localized to the DNA. Nevertheless, 1 h later the DXR was no longer detectable in the DNA. In contrast, oocytes from wildtype mice retained DXR in the DNA for up to 24 h. Overall, these observations point to the role of ceramide in modulating DXR actions by sustaining the contact of the chemotherapeutic agent with the target site, not only in carcinoma cell lines but in normal cells as well. (Supported by the Department of Physiology, MSU; and the Vincent Center of Reproductive Biology, MGH).

KEY WORDS: ceramide, doxorubicin, apoptosis, trafficking