Ceramide limits phosphatidylinositol-3-kinase C2ß-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Lysosomal ceramide generated by acid sphingomyelinase triggers cytosolic cathepsin B-mediated degradation of X-linked inhibitor of apoptosis protein in natural killer/T lymphoma cell apoptosis.

We previously reported that IL-2 deprivation induced acid sphingomyelinase-mediated (ASM-mediated) ceramide elevation and apoptosis in an NK/T lymphoma cell line KHYG-1. However, the molecular mechanism of ASM-ceramide-mediated apoptosis during IL-2 deprivation is poorly understood. Here, we showed that IL-2 deprivation induces caspase-dependent apoptosis characterized by phosphatidylserine externalization, caspase-8, -9, and -3 cleavage, and degradation of X-linked inhibitor of apoptosis protein (XIAP). IL-2 re-supplementation rescued apoptosis via inhibition of XIAP degradation without affecting caspase cleavage. However, IL-2 deprivation induced ceramide elevation via ASM in lysosomes and activated lysosomal cathepsin B (CTSB) but not cathepsin D. A CTSB inhibitor CA-074 Me and knockdown of CTSB inhibited ceramide-mediated XIAP degradation and apoptosis. Inhibition of ceramide accumulation in lysosomes using an ASM inhibitor, desipramine, decreased cytosolic activation of CTSB by inhibiting its transfer into cytosol from the lysosome. Knockdown of ASM also inhibited XIAP degradation and apoptosis. Furthermore, cell permeable N-acetyl sphingosine (C2-ceramide), which increases mainly endogenous d18:1/16:0 and d18:1/24:1 ceramide-like IL-2 deprivation, induced caspase-dependent apoptosis with XIAP degradation through CTSB. These findings suggest that lysosomal ceramide produced by ASM mediates XIAP degradation by activation of cytosolic CTSB and caspase-dependent apoptosis. The ASM-ceramide-CTSB signaling axis is a novel pathway of ceramide-mediated apoptosis in IL-2-deprived NK/T lymphoma cells.

Phase transitions of rat stratum corneum lipids by an electron paramagnetic resonance study and relationship of phase states to drug penetration.

In order to relate barrier function to stratum corneum structure and the thermal transitions of corneum lipids, samples from hairless rat skin were investigated by using ESR and drug penetration techniques. The phase transition of stratum corneum lipids was estimated using a deeper probe (16-doxyl-stearic acid) inserted in the lipid bilayers and measuring the rotational correlation time, tau(c). Results of ESR study showed that stratum corneum lipids underwent thermal transitions at 39.3 +/- 1.6 degrees C and 63.6 +/- 2.6 degrees C roughly similar to the data obtained by differential scanning calorimetry measurements. Cholesterol oxidase treatment decreased the fluidity of the lipids at lower temperatures. The treatment of stratum corneum with laurocapram (1%) and isopropyl myristate (IPM, 2%) little changed both phase transition temperatures, although the treatment highly increased the molecular motion of the lipids. The flux (J(s)) of lipophilic drugs (beta-estradiol, indomethacin and betahistine) through the skin was enhanced with increasing temperatures, with an increase in the diffusion constant within skin and a decrease in the lag time. There was a good relationship between log J(s) or log permeability coefficient (K(p)) and 1/tau(c) in the temperature range of 45 to 64 degrees C. The calculated activation energy (delta E) for diffusion of these drugs across skin was 17-40 kcal/mol. Judging from our data, stratum corneum lipids of rat probably exist as the gel, crystalline state below 39 degrees C, the mesomorphic state between 39 and 64 degrees C and the fluid, liquid-crystalline state at temperatures of 64 degrees C or above. These results are in line with the permeability of these lipophilic drugs through the intercellular lipids disordered is highly increased.