N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

Abstract

Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC 50 toxicity values seven-to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABCtransporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol -water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

Doxorubicinolone Formation and Efflux: A Salvage Pathway against Epirubicin Accumulation in Human Heart

“…7A). The observation that doxorubicinolone [in 3 out] or doxorubicinolone [out 3 in] augmented epirubicin fluxes bidirectionally suggests that doxorubicinolone acted as a membrane-permeabilizing agent (Veldman et al, 2004). The experiments with doxorubicinolone [in 3 out] offered an opportunity to approximate that the efflux of 1 mol doxorubicinolone caused the elimination of as many as ϳ40 mol epirubicin, which was conceptually in agreement with a permeation-like effect.…”

“…It is interesting that we also noticed that purified doxorubicinolone would have no effect on the partitioning of doxorubicin across plasma and strips (data not shown). This observation argued against analogindependent gross effects of doxorubicinolone on the integrity of myocardial strips; it also denoted differences between doxorubicinolone and permeabilizing agents, such as natural double-chain lipids or digitonin, that were shown to potentiate the uptake or efflux of both doxorubicin and epirubicin in different cell types (Wielinga et al, 2000;Veldman et al, 2004). As was said before, epirubicin is appreciably more lipophilic than doxorubicin; hence, the effect of doxorubicinolone was discrete enough to only favor membrane permeation to the more lipophilic epirubicin.…”

Molecular requirements for doxorubicin-mediated death in murine oocytes

“…As cytoplasmic 33 and nuclear localization of ceramide have been previously documented, 34 we hypothesize that once inside the oocytes, DXR takes residence in the cytosol, binds to ceramide (if present), and ultimately it (DXR) or its derivative(s) is transported to the nucleus where again complexing with ceramide enhances its association with the DNA. As there is published evidence 35 of the possible modulatory role of other lipids (e.g., sphingomyelin) in DXR mediations, we cannot exclude their contribution to the DXR pathway in this model. Experiments in our laboratory are currently in progress to address the molecular mechanism(s) involved and the impact on the intracellular trafficking/retention of DXR; investigations that ultimately may enhance our understanding of drug resistance and sensitivity.…”

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

“…*, P < 0.05, statistically significant. LASS1/C 18 -Ceramide Enhances Drug-Induced Cell Death due to the increased cellular uptake of doxorubicin through the plasma membrane (39).…”