In vitro tumor cell culture models have illuminated the potential therapeutic utility of elevating the intracellular concentration of the antimitogenic and proapoptotic sphingolipid, ceramide. However, although cell-permeable, short-chain ceramide is an effective apoptotic agent in vitro, its use as an in vivo, systemically delivered therapeutic is limited by its inherent lipid hydrophobicity and physicochemical properties. Here, we report that the systemic i.v. delivery of C 6 -ceramide (C 6 ) in a pegylated liposomal formulation significantly limited the growth of solid tumors in a syngeneic BALB/c mouse tumor model of breast adenocarcinoma. Over a 3-week treatment period, a well-tolerated dose of 36 mg/kg liposomal-C 6 elicited a >6-fold reduction in tumor size compared with empty ghost liposomes. Histologic analyses of solid tumors from liposomal-C 6 -treated mice showed a marked increase in the presence of apoptotic cells, with a coincident decrease in cellular proliferation and in the development of a microvessel network. Liposomal-C 6 accumulated within caveolae and mitochondria, suggesting putative mechanisms by which ceramide induces selective cancer cell cytotoxicity. A pharmacokinetic analysis of systemic liposomal-C 6 delivery showed that the pegylated liposomal formulation follows first-order kinetics in the blood and achieves a steady-state concentration in tumor tissue. Confirming the therapeutic utility of i.v. liposomal-C 6 administration, we also shown diminution of solid tumor growth in a human xenograft model of breast cancer. Together, these results indicate that bioactive ceramide analogues can be incorporated into pegylated liposomal vehicles for improved solubility, drug delivery, and antineoplastic efficacy.Sphingolipids not only serve a structural role in membranes but also are substrates for the generation of bioactive second messengers that influence mitogenesis and apoptosis. Metabolism of sphingomyelin, the major sphingolipid in membranes, forms ceramide, a potent lipid-derived second messenger that modulates the induction of cell differentiation, cell cycle arrest, and/or apoptosis (1 -4). In addition, chemotherapeutic agents (5 -7) and ionizing radiation (8, 9) are two of the multiple cellular stressors (10 -16) that lead to the accumulation of ceramide within membranes. We and others have shown that ceramide-mediated signaling cascades induce apoptosis in part via the inhibition of Akt prosurvival pathways, mitochondrial dysfunction, and the stimulation of caspase activity, which ultimately leads to DNA fragmentation and cell death (17 -21).Although short-chain, cell-permeable ceramides, such as C 6 -ceramide (C 6 ), have been shown to be antiproliferative and proapoptotic in numerous cancer cell types in vitro (22, 23), there are obstacles to the delivery of ceramide for systemic applications, such as cancer chemotherapy. Despite being more efficacious than physiologic long-chain ceramides (C 18 -C 24 -ceramide), the effectiveness of cell-permeable ceramide analogues re...

It is therapeutically desirable to effectively deliver ceramide, an antimitogenic and proapoptotic lipid second messenger, to transformed cell types. However, the targeted delivery of cellpermeable ceramide analogs, including C 6 -ceramide, to cells may be impeded by the hydrophobicity of these bioactive lipids, resulting in reduced efficacy. The objective of this study is to develop and optimize liposomal vehicles to augment ceramide delivery to a breast adenocarcinoma cell line. We designed conventional, cationic, and pegylated drug release vesicles to efficaciously deliver ceramide to MDA-MB-231 breast adenocarcinoma cells. In vitro pharmacokinetic analysis demonstrated that liposomal ceramide delivery resulted in significantly greater accumulation of ceramide in MDA-MB-231 cells. Ceramide-formulated liposomes significantly inhibited MDA-MB-231 cell proliferation as compared with nonliposomal administration of ceramide. Ceramide-induced apoptosis correlated with the pharmacokinetic profile and the diminished proliferation in this highly aggressive, metastatic cell line. Liposomal ceramide formulations inhibited phosphorylated Akt levels and stimulated caspase-3/7 activity more effectively than nonliposomal ceramide, events consistent with apoptosis. Together, these results indicate that bioactive ceramide analogs can be incorporated into conventional, cationic, or pegylated liposomal vehicles for improved drug delivery and release.Sphingolipid metabolites are a unique class of lipids that have been shown to control the balance between mitogenesis and apoptosis. These lipids serve both a structural role in membranes and an intracellular signaling role within a cell. Ceramide is one of many sphingolipid metabolites known to have biological activity, serving as a lipid-derived second messenger that modulates the induction of cell differentiation, cell cycle arrest, and/or apoptosis (

We have previously demonstrated that hexanoyl-D-erythrosphingosine (C 6 -ceramide), an anti-mitogenic cell-permeable lipid metabolite, limited vascular smooth muscle growth by abrogating trauma-induced Akt activity in a stretch injury model of neointimal hyperplasia. Furthermore, ceramide selectively and directly activated protein kinase C (PKC) to suppress Akt-dependent mitogenesis. To further analyze the interaction between ceramide and PKC, the ability of ceramide to localize within highly structured lipid microdomains (rafts) and activate PKC was investigated. Using rat aorta vascular smooth muscle cells (A7r5), we now demonstrate that C 6 -ceramide treatment results in an increased localization and phosphorylation of PKC within caveolin-enriched lipid microdomians to inactivate Akt. In addition, ceramide specifically reduced the association of PKC with 14-3-3, a scaffold protein localized to less structured regions within membranes. Pharmacological disruption of highly structured lipid microdomains resulted in abrogation of ceramide-activated, PKC-dependent Akt inactivation, whereas molecular strategies suggest that ceramide-dependent PKC phosphorylation of Akt3 at Ser 34 was necessary for ceramide-induced vascular smooth muscle cell growth arrest. Taken together, these data demonstrate that structured membrane microdomains are necessary for ceramide-induced activation of PKC and resultant diminished Akt activity, leading to vascular smooth muscle cell growth arrest.

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