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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Pharmacology, Pennsylvania State College of Medicine, Hershey, Pennsylvania
Received for publication
May 7, 2003
Accepted
August 6, 2003.
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Abstract |
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; Spiegel and Merrill, 1996; Merrill et al., 1997; Kolesnick and Kronke, 1998). Intracellular ceramide accumulation results from multiple stimuli, such as growth factor deprivation (Ito and Horigome, 1995), cytokines (Ballou et al., 1992; Dbaibo et al., 1993; Tepper et al., 1995), chemotherapy and other cytotoxic agents (Strum et al., 1994; Bose et al., 1995; Lucci et al., 1999), ionizing radiation (Haimovitz-Friedman et al., 1994; Bruno et al., 1998), heat shock (Chang et al., 1995), and various environmental factors (Hannun, 1996; Basu and Kolesnick, 1998). These stimuli have been observed to initiate ceramide-mediated signaling cascades, including the inhibition of Akt prosurvival pathways and the stimulation of caspase activity, which ultimately leads to DNA fragmentation and cell death (Jarvis et al., 1994; Tepper et al., 1999).
Due to its potent regulation of cell growth, differentiation, and death and the fact that it is a natural molecule that targets discrete kinases and signaling pathways linked to proliferation and/or survival, ceramide has been identified as a putative therapeutic agent in cancer (Radin, 2001) and cardiovascular disease (Johns et al., 1998). Our laboratory has demonstrated the potential clinical utility of local delivery of a cell-permeable ceramide analog (C6) from drug-eluting platforms. Specifically, ceramide-coated balloon catheters induced cell cycle arrest in stretch-injured vascular smooth muscle cells (Charles et al., 2000). Although the delivery of C6 from coated and distended balloons allows for direct delivery to the vasculature, there are several obstacles to the delivery of ceramide for systemic applications, such as cancer chemotherapy. Three significant barriers to systemic ceramide delivery exist. First, even though short-chain, cell-permeable ceramide analogs (C2-, C6-, or C8-ceramide) are more efficacious than physiological long-chain ceramides (C18-C24-ceramide), their effectiveness remains limited due to their hydrophobicity and possible precipitation as fine lipid micelle suspensions when administered in aqueous solutions (Radin, 2001). Second, the existence of a second aliphatic chain, albeit a short-chain derivative, hinders cellular permeability. Finally, because ceramide is a natural cellular lipid, circulating levels of free ceramide is susceptible to enzymatic degradation by ceramide-specific enzymes, such as ceramidase. Thus, there is a critical need for improved delivery systems to maximize intracellular ceramide accumulation upon systemic administration. We hypothesize that the development and optimization of ceramide-formulated liposomal vehicles will augment ceramide delivery, translating to enhanced growth inhibition and/or apoptosis in cancer. To test this hypothesis, we compared the in vitro pharmacokinetics, efficacy, and intracellular bioactivity of liposomal and nonliposomal C6 formulations in an in vitro model of human metastatic breast adenocarcinoma.
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Materials and Methods |
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Liposome Formulation and Extrusion. Lipids, dissolved in chloroform (CHCl3), were combined in specific molar ratios, dried under a stream of nitrogen above lipid transition temperatures, and hydrated with sterile phosphate-buffered saline (PBS). The resulting solution underwent sonication for 2 min followed by extrusion through 100-nm polycarbonate membranes. Incorporation efficiency was determined by incorporating trace amounts of [3H]C6 in the formulation, extracting constituent lipids in CHCl3/MeOH (2:1), and comparing radioactivity before and after extrusion using a scintillation counter.
The composition of formulated liposomes was validated by extracting constituent lipids in CHCl3/MeOH (2:1), followed by resolution on preheated silica gel 60 thin layer chromatography plates using a CHCL3/MeOH/ddH2O (60:25:4) solvent system. Lipids were visualized in an iodine chamber.
Transmission Electron Microscopy (TEM). To characterize the size and morphology of the formulated liposomes, we utilized TEM. Initially, formvar carbon-coated 400 mesh copper grids were coated with poly-L-lysine for 10 min to promote vesicular binding to the hydrophobic grids. Liposomal samples were next applied to the dried grids and allowed to adhere for 5 min. Negative staining was performed by applying 1% phosphotungstic acid (pH 7.0) to the dried grid for an additional 5 min. The sample was observed at 21,500x magnification with an accelerating voltage of 60 kV.
In Vitro Pharmacokinetics. MDA cells were seeded at 3.5 x 104 cells/well in 24-well plates and grown overnight in media containing 10% FBS. Cells were then treated with liposomal or nonliposomal C6 containing trace amounts of either [3H]C6 or [3H]CHE in media supplemented with 1% FBS for various time intervals. In this and each of the following experiments, liposomal C6 was added directly to cell media, and nonliposomal C6 was added in dimethylsulfoxide (DMSO) vehicle to a final concentration of 
0.1% (v/v). At the indicated time points, the media was removed, and cells were washed once with cold PBS to dissociate liposome/membrane-nonspecific interactions. The cells were then solubilized with 1% SDS, and either [3H]C6 or [3H]CHE accumulation into MDA cells was assessed with a scintillation counter.
[3H]Thymidine Proliferation Assay. MDA cells were seeded at 3.5 x 104 cells/well in 24-well plates and grown overnight prior to 24 h of serum starvation. At hour 12 of serum starvation, cells were treated with liposomal or nonliposomal C6 for the remainder of serum starvation. Following serum starvation, media was then supplemented with FBS (10% final concentration) for an additional 12 h, and cellular proliferation was assayed with the addition of 0.5 mCi/ml [3H]thymidine for the final 4 h of treatment. Cells were washed once with cold PBS and then twice with 10% trichloric acetic acid for 10 min. Cells were solubilized with 0.3 N NaOH, and [3H]thymidine incorporation into acid-insoluble DNA was assessed with a scintillation counter.
Caspase Assay. MDA cells were seeded to a density of 6.0 x 103 cells/well in 96-well plates and grown for 48 h in culture media containing 10% FBS. Cells were then treated with liposomal or nonliposomal C6 for 24 h in media containing 1% FBS. Caspase-3/7 enzymatic activity levels were measured using the Apo-ONE homogenous caspase-3/7 assay and performed following the instructions of the manufacturer. The kit provides a caspase-3/7 substrate and rhodamine 110, bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide), which is cleaved by enzymatically active caspase-3/7 resulting in a fluorogenic cleavage product.
Apoptosis Detection. To determine DNA fragmentation, the TUNEL apoptosis detection kit was performed according to the instructions of the manufacturer. Briefly, MDA cells were seeded at a density of 1.0 x 104 cells/well in eight-well chamber slides and grown for 48 h in culture media containing 10% FBS. Cells were then treated with liposomal or nonliposomal C6 for 8 and 16 h in media supplemented with 1% FBS. Fragmented DNA of apoptotic cells was stained in situ using terminal deoxynucleotidyl transferase to transfer biotin-dUTP to the free 3'-OH end of cleaved DNA, which were visualized by reaction with FITC-avidin.
In addition to TUNEL, apoptosis was assessed and quantified by flow cytometry analysis of annexin V-stained cells using the Vybrant apoptosis assay kit according to the instructions of the manufacturer. Briefly, MDA cells were seeded at 1.0 x 106 cells/plate in 100-mm tissue culture dishes and grown for 48 h in culture media containing 10% FBS. Cells were then treated with liposomal or nonliposomal C6 for 24 h in media supplemented with 1% FBS. Following treatment, adherent cells were harvested by trypsinization, and all cells (floating and adherent) were washed once with cold PBS. Pelleted cells were then stained with FITC-labeled annexin V and PI according to the instructions of the manufacturer. Labeled cells were immediately analyzed using flow cytometry. Viable cells were double negative, early apoptotic cells were positive for annexin V staining and negative for PI staining, and late apoptotic cells were double positive.
Apoptosis and DNA degradation was confirmed by performing cell cycle analysis of PI-stained cells. MDA cells were plated and treated similar to the annexin V experiment above. Following treatment, adherent cells were harvested by trypsinization, and all cells (floating and adherent) were washed once with cold PBS and fixed with 70% ethanol for 30 min. Fixed cells were pelleted and stained with a PI solution (50 µg/ml PI, 0.1 mg/ml Rnase A, and 0.05% Triton X-100) for 40 min at 37°C in the dark and analyzed immediately using flow cytometry. Apoptotic cells were identified in the subdiploid (subGo/G1) region. Cell debris was excluded from analysis using forward scatter/side scatter live gate.
Western Blot Analysis. MDA cells were seeded at 4.0 x 105 cells/well in 60-mm plates and grown overnight prior to 24-h serum starvation. At h 16 of serum starvation, cells were treated with liposomal or nonliposomal C6 for the remainder of serum starvation. At h 24 of serum starvation, IGF-1 (20 ng/ml) was added to cell media for a 15-min period. Cells were washed once with cold PBS followed by the addition of 150 µl of cold lysis buffer (1% Triton X-100, 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 mM Na4P2O7, 1 mM 
-glycerolphosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin in ddH2O, pH 7.5) on ice. Cells were lysed for 15 min on ice, and cell lysate was harvested and centrifuged at 15,000g for 15 min. Thirty-five micrograms of protein were loaded in 4 to 12% precasted SDS-polyacrylamide gel electrophoresis gradient gels and probed for pAkt. Blots were stripped and reprobed for Akt-1,2,3 to demonstrate equal loading. Protein bands were visualized using enhanced chemiluminescence and quantified by densitometry.
Statistical Analysis. Differences among treatment groups were statistically analyzed using a two-tailed Student's t test for statistical analyses. A statistically significant difference was reported if p < 0.05 or less. Data are reported at the mean ± S.E. from at least n = 3 separate experiments.
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Results |
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). TEM analysis confirmed that C6-incorporated liposomal vehicles were produced with a homogenous size distribution between 85 and 140 nm in diameter for all formulations (Fig. 1A). With the incorporation of trace amounts of [3H]C6 into conventional formulations, we observed that there was no significant loss of ceramide during the extrusion process (Fig. 1B). Additionally, lipid extracts from conventional liposomes were run on thin layer chromatography plates, confirming that there was no visual diminution of lipid constituents during the extrusion process (Fig. 1C).
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In Vitro Pharmacokinetics. We incorporated trace amounts of [3H]C6 into liposomal formulations to quantify the amount of C6 liposomal delivery compared with nonliposomal administration. In vitro pharmacokinetic studies showed that liposomal formulations delivered C6 more effectively and efficiently than nonliposomal administration of C6 in the presence of 1% FBS (Fig. 2A). Cationic liposomal delivery resulted in a 2-fold increase in ceramide accumulation by MDA cells, with a maximal accumulation observed at approximately 16 h. Conventional and pegylated liposomes were observed to have similar in vitro pharmacokinetic profiles (data not shown).
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We next investigated the mechanism by which C6 is released or transferred from liposomal vehicles into cellular membranes. We used a nontransferable cholesterol lipid marker, [3H]CHE, as a probe for liposome/cell membrane association. In these experiments, liposomes were tagged with either [3H]C6 or [3H]CHE and incubated with MDA cells for the indicated time periods. In Fig. 2B, it is evident that liposomes mediated the transfer of C6, but not cholesterol, from drug vehicle to cellular membrane. In fact, as C6 accumulation increased over time, CHE accumulation failed to significantly increase above background levels. The disparity between ceramide and cholesterol accumulation is also observed in a dose-dependent manner (Fig. 2C). These studies suggest that C6 is delivered via lipid transfer processes that permit C6 to partition out of the liposomal bilayer into the plasma membrane bilayer without associated liposome/cell membrane fusion.
Cell Proliferation. Several types of liposomal vehicles can be used for drug delivery. We initially investigated whether conventional lipid formulations would be useful for the delivery of short-chain ceramide to MDA cells. In Fig. 3A, a conventional liposome containing EPC and CH (solid lines) supplemented with C6 displayed a significant dose-dependent inhibition of MDA cell proliferation. The addition of a vesicle-destabilizing lipid, DOPE, into a conventional formulation did not further enhance the bioactivity of C6. MDA cells, in the presence of 10% FBS for 12 h of treatment, were completely growth inhibited when treated with liposomal C6 at 25 µM or greater. The delivery of C6 in liposomal formulations reduced the IC50 approximately 3-fold, decreasing from 15 to 5 µM, nonliposomal C6 to liposomal C6, respectively. These conventional formulations displayed an improved dose-response inhibition of growth in MDA cells compared with nonliposomal administration of C6 in DMSO vehicle (dashed line, open circle), indicating improved potency and efficacy. Liposomes without C6 (Ghost; dashed line, open square) as well as PBS controls did not display significant growth inhibition, implicating C6 as the only bioactive agent. This experiment demonstrates that C6-formulated conventional liposomes are more effective as an antiproliferative than freely administered C6.
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We next investigated whether C6 could also be efficacious when incorporated into cationic lipid formulations (Fig. 3B). Even though Ghost cationic liposomes (dashed line, open triangles) formulated with a positively charged lipid, DOTAP, enhanced MDA cell proliferation alone, C6-incorporated cationic liposomes (solid line, open triangle) dose dependently reduced MDA cell proliferation. This cationic formulation was more effective than nonliposomal C6 administration (dashed line, open circle) but not as effective as a conventional formulation (solid line, open square). This experiment indicates that cationic liposome formulations could also be used to deliver bioactive ceramide to dose dependently inhibit cell proliferation.
In the next set of experiments, we tested the role of pegylated lipid to further enhance the bioactivity of C6. We chose PEG-C8 for the additional potential benefit of membrane destabilization and subsequent liposome/membrane fusion (Webb et al., 1998). Additionally, the inclusion of PEG-C8 has been observed to facilitate time-release properties of liposomal bilayers, with the added benefit of bioavailability extension. Pegylated liposomes (stealth) did not significantly effect MDA cell proliferation, demonstrating that PEG-C8 is biochemically inert. Yet, C6-incorporated pegylated liposomes were as effective at inhibiting proliferation as conventional at 10 and 25 µM (Fig. 3C). This study indicates that a pegylated liposomal formulation designed for systemic drug delivery is also an effective vehicle for C6-mediated inhibition of MDA cell proliferation. Similar results were also observed in two highly aggressive and metastatic murine adenocarcinoma cell lines, 410.4 and T41 (McEarchern et al., 2001; Kundu and Fulton, 2002) (data not shown), alluding to the potential applicability of liposomal C6 delivery as an experimental therapeutic. Taken together, C6 delivered in multiple liposomal formulations displays an improved dose-response inhibition of growth compared with nonliposomal C6, indicating improved potency and efficacy.
Induction of Apoptosis. We next investigated whether C6-dependent growth inhibition correlates with enhanced apoptosis in MDA cells. To confirm that C6 delivery leads to MDA cell apoptosis, we initially performed TUNEL analysis, which stains cleaved DNA, a hallmark of cellular apoptosis (Fig. 4A). TUNEL staining of cycling, serum-fed MDA cells treated with liposomal and nonliposomal C6 demonstrated no DNA fragmentation at 8 h. Liposomal and nonliposomal C6 treatment induced DNA fragmentation in a similar manner to the DNase-positive control. Staining of cleaved 3'-OH DNA was observed at 16 h of treatment, a time point consistent with the in vitro pharmacokinetic profile of C6 delivery. No apoptosis was observed with the Ghost formulation.
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To quantitate the C6-induced apoptosis, we performed annexin V staining of treated cycling MDA cells. Annexin V stains phosphatidylserine on the outer leaflet of apoptotic cells, another hallmark of apoptosis. Following a 24-h treatment, liposomal C6 induced a significantly greater amount of annexin V staining compared with nonliposomal C6, whereas the Ghost formulation had no effect (Fig. 4B). Additionally, cell cycle analysis utilizing propidium iodide-stained MDA cells demonstrated a significant increase in the G2/M population (untreated, 5.56 ± 0.11; nonliposomal C6, 7.15 ± 0.28; and liposomal C6, 10.71 ± 0.84) and in subGo/G1 cellular debris (untreated, 9.95 ± 0.13; nonliposomal C6, 14.55 ± 3.94; and liposomal C6, 18.22 ± 1.07) following liposomal treatment compared with nonliposomal treatment. Liposomes formulated with the "inactive" analog, DHC6, had a marginal effect on the G2/M population (nonliposomal DHC6, 5.90 ± 0.13 and liposomal DHC6, 6.80 ± 0.16) and on subGo/G1 cellular debris (nonliposomal DHC6, 7.94 ± 0.40 and liposomal DHC6, 11.05 ± 0.11). Together, these proliferative and apoptosis studies indicate that liposomal delivery of C6 limits clonal expansion by promoting apoptosis in transformed cell lines.
Cell Signaling Analysis. As we have previously shown that cell-permeable ceramides inhibit the phosphorylation of Akt and extracellular signal-regulated kinase, in various cell types, to limit cell proliferation and/or survival (Bourbon et al., 2001, 2002), we examined ceramide-regulated Akt signaling pathways in MDA cells treated with liposomal and nonliposomal C6 formulations. Pegylated liposomal C6 was more effective at reducing IGF-1-stimulated pAkt levels than was nonliposomal ceramide, whereas the Ghost formulation had no effect on pAkt levels (Fig. 5, A and B). Liposomal DHC6 displayed a marginal inhibition of IGF-1-stimulated Akt phosphorylation compared with nonliposomal DHC6. We selected 8 h of C6 treatment, as this time point corresponds to near maximal accumulation of C6 into MDA cells. These studies support the fact that liposomal C6 induces cell growth inhibition and apoptosis through long-term inhibition of Akt signaling cascades.
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As apoptosis is associated with the up-regulation of caspase activity, we assessed caspase-3/7 activity following treatment of MDA cells with pegylated liposomes. Cells that were treated with liposomal ceramide displayed significantly greater caspase-3/7 activity than cells treated with nonliposomal ceramide (Fig. 5C). No significant change in caspase-3/7 activity was observed with Ghost treatments. Taken together, these results indicate that C6-formulated liposomes are more effective than nonliposomal administration of C6, resulting in significant inhibition of MDA cell proliferation and eventual apoptotic death.
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Discussion |
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). In addition, although it is a more cell-permeable analog of long-chain physiological ceramide, C6 retains two aliphatic chains, which limits its intercalation into plasma membranes. Moreover, the existence of circulating and intracellular ceramidase promotes the conversion of bioactive ceramide to inactive metabolites. Thus, to realize the therapeutic benefits of ceramide bioactivity, there is a need for improved delivery systems to assist with solubility, cell permeability, and protection from enzymatic degradation.
Nonliposomal organic solvent systems have been investigated to augment the delivery of ceramide to cells (Radin, 2001). It has been proposed that a dodecane/ethanol solvent system, which is insoluble in culture media, precipitates out with the ceramide and forms very small droplets, or micelles, that fuse with the plasma membrane (Ji et al., 1995). The use of such precipitating solvents may be limited by the variability in particle size and access to cellular membranes. Protein adjuvants, such as bovine serum albumin, may also assist in vitro ceramide delivery via nonspecific lipid/protein interactions but would not permit the efficient delivery of sufficient quantities of C6 to systemic targets. For this reason, we investigated a novel approach to optimize the delivery of C6 to cells utilizing a lipid-based carrier system that improves ceramide solubility, assists with cell permeability, and potentially protects against degradation.
Numerous studies have been conducted to demonstrate the physiological behavior of short- and long-chain ceramides in artificial phospholipid bilayers, or liposomes (Van Blitterswijk et al., 2003). Examining these systems in equilibrium, long-chain ceramides have been shown to enhance the degree of lipid acyl chain order within the bilayers (Holopainen et al., 1997), to separate into ceramide-rich lipid domains (Holopainen et al., 1998), and to contribute to the destabilization of lamellar phases (Veiga et al., 1999). These biophysical properties hinder long-chain ceramide exchange between lipid bilayers, thus limiting spontaneous transfer between bilayers (Simon et al., 1999). In fact, even though we have successfully formulated C16-ceramide incorporated liposomes, their biological efficacy is limited by inefficient transfer of C16-ceramide between lipid bilayers (unpublished data). However, short-chain ceramide analogs have different partitioning and behavior characteristics in biomembranes from physiological ceramides (Venkataraman and Futerman, 2000; Van Blitterswijk et al., 2003). Due to its amphilic nature, C6 labeled with N-(4-nitrobenzo-2-oxa-1,3-diazole)aminocaproic acid has been observed to spontaneously transfer out of lipid membranes and undergo transbilayer movement into other membranes via undefined lipid transfer mechanisms (Pagano and Martin, 1988; Van Blitterswijk et al., 2003). Additional studies have shown that short-chain analogs undergo similar transbilayer movement, with a correlation between lipid hydrophobicity (i.e., chain length) of the short-acyl chain of ceramide and the rate of transbilayer transfer (Bai and Pagano, 1997). However, these studies have not been expanded to clinical correlates, such as induction of apoptosis in transformed cell lines.
We observed that liposomal delivery ameliorates the primary problems associated with C6 delivery and efficacy, preventing the bioactive lipid from precipitating out of solution so that it can be delivered to cells more effectively. Utilizing [3H]C6 and [3H]CHE in the liposomal vehicles, we were able to determine the accumulation of ceramide into MDA cells in culture and to gain insights into the mechanism of drug delivery. In vitro pharmacokinetic results show that liposomal delivery leads to a significantly greater cellular accumulation of C6 than nonliposomal delivery. Peak accumulation occurred between 8 and 16 h, corresponding with the onset of cellular apoptosis. Using a nontransferable marker ([3H]CHE) for lipid association, we observed that an insignificant quantity of cholesterol lipid was transferred into MDA cells. [3H]CHE cannot transfer from one bilayer to another and thus cannot accumulate within a cell unless all liposome constituents are incorporated into the cell (i.e., via fusogenic processes). This observation supports the notion that C6 delivery from liposomes occurs via spontaneous interbilayer/lipid transfer from liposome to cell plasma membrane (Venkataraman and Futerman, 2000). We propose that liposomal delivery of C6, from multiple formulations (i.e., conventional, cationic, and pegylated) facilitates spontaneous interbilayer transfer upon transient liposome/cell interactions, resulting in efficient delivery of C6 to cellular membranes in vitro.
We validated the applicability and benefits of liposomal C6 delivery by examining the efficacy and bioactivity of intracellularly delivered ceramide. Ceramide accumulation has been shown to inhibit proliferation and/or induce apoptosis through several potential signaling pathways (Birbes et al., 2002; Gulbins and Kolesnick, 2002). The ability of ceramide to reduce pAkt levels suggests that C6 inhibits the Akt survival pathway, contributing to the observed apoptotic cell death (Zhou et al., 1998). Moreover, liposomal delivery leads to significantly more caspase activation and results in a greater induction in cellular apoptosis than nonliposomal C6. Critical observations that liposomal delivery results in greater ceramide accumulation, which is associated with improved Akt inhibition, caspase stimulation, growth diminution, and the induction of apoptosis, illustrate the advantages of liposomal delivery of this bioactive lipid. It is of interest that liposomal DHC6 delivery is somewhat effective in limiting Akt phosphorylation and inducing apoptosis in MDA cells. This may support the notion that the presumed "inactivity" of DHC6 may be a function of delivery and not bioactivity.
It is envisioned that liposomes may be applicable for both local and systemic delivery of therapeutic ceramide analogs. For example, our laboratory has demonstrated that the local and direct delivery of C6 from ceramide-coated balloons of embolectomy catheters limits neointimal hyperplasia (restenosis) in rabbits after stretch injury (Charles et al., 2000). The Hallenbeck laboratory has demonstrated that cell-permeable ceramide analogs, in DMSO vehicle, delivered both intracisternally and intravenously induced a neuroprotective effect in rats following focal cerebral ischemia (Furuya et al., 2001). The clinical potential for the packaged delivery of C6 with additional therapeutic agents in liposomal vesicles is significant. Studies have shown that ceramide may act synergistically with chemotherapeutic agents, such as paclitaxel (Mehta et al., 2000) and fenretinide (Maurer et al., 2000). Thus, delivery of chemotherapeutic agents in C6-formulated liposomes may further enhance apoptotic actions. Moreover, targeted immunoliposomes or stealth liposomes may also benefit from C6-ceramide incorporation. Nonetheless, the true benefits of liposomal ceramide delivery will be realized in ongoing and future in vivo studies aimed at assessing the efficacy of systemic delivery in rodent models of carcinogenesis or inflamed arteries.
Note Added in Proof. A recent article by Shabbits and Mayer has also demonstrated that ceramide-enriched liposomes enhanced cytotoxicity of a human breast cancer cell line [Shabbits JA and Mayer LD (2003) Intracellular delivery of ceramide via liposomes enhances apoptosis in vitro. Biochim Biophys Acta 1612:98-106].
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Acknowledgements |
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Footnotes |
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This work was supported by National Institutes of Health Grant HL66371. M.K. participates in a related but separate project sponsored in part by REVA Medical, Inc.
ABBREVIATIONS: EPC, egg phosphatidylcholine; DOPE, dioleoyl phosphatidylethanolamine; DOPC, dioleoyl phosphatidylcholine; CH, cholesterol; PEG-C8, polyethyleneglycol-450-C8-ceramide; DOTAP, dioleoyl-1,2-diacyl-3-trimethylammonium-propane; DHC6, di-hydro-erythro-hexanoyl-sphingosine; [3H]CHE, cholesteryl-1,2-3H(N) hexadecyl ether; IGF-1, insulin-like growth factor-1; PI, propidium iodide; MDA, human MDA-MB-231; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DMSO, dimethylsulfoxide; TEM, transmission electron microscopy; [3H]C6, N-hexanoyl-D-erythro-sphingosine [hexanoyl 6-3H]; pAkt, phosphorylated Akt; CHCl3, chloroform; ddH2O, double-distilled H2O; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; FITC, fluorescein isothiocyanate.
Address correspondence to: Dr. Mark Kester, Interim Chair and Professor, Department of Pharmacology, Penn State College of Medicine, P.O. Box 850, Hershey, PA 17033. E-mail: mxk38{at}psu.edu
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