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. 2017 Aug 12;8(44):76921-76934.
doi: 10.18632/oncotarget.20217. eCollection 2017 Sep 29.

Ceramide-containing Liposomes With Doxorubicin: Time and Cell-Dependent Effect of C6 and C12 Ceramide

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Ceramide-containing Liposomes With Doxorubicin: Time and Cell-Dependent Effect of C6 and C12 Ceramide

Anders Øverbye et al. Oncotarget. .
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Abstract

Doxorubicin, a widely used chemotherapeutic drug, has several potential high-risk side effects including cardiomyopathy. Furthermore, cellular resistance to this drug develops with time. By using liposomes as carrier vesicles both the side effects and drug resistance might be avoided. In this study we have investigated the cytotoxic effect of doxorubicin encapsulated in liposomes with and without ceramides containing 6 or 12 carbon atoms in the N-amidated fatty acyl chains. The short-chain ceramide species were included in the liposomal compositions due to their pro-apoptotic properties, which might cause a synergistic anticancer effect. We demonstrate that the ceramide species enhance the liposomal doxorubicin toxicity in a cell-specific manner. The C6-ceramide effect is most pronounced in cervical cancer cells (HeLa) and colon cancer cells (HCT116), whereas the C12-ceramide effect is strongest in breast cancer cells (MDA-MB-231). Moreover, the study reveals the importance of investigating cell toxicity at several time points and in different cell-lines, to assess drug-and formulation-induced cytotoxic effects in vitro. Furthermore, our data show that the cytotoxicity obtained with the nanocarriers in vitro, does not necessarily reflect their ability to inhibit tumor growth in vivo. We speculate that the larger effect of Caelyx® than our liposomes in vivo is due to a greater in vivo stability of Caelyx®.

Keywords: cell studies; ceramide; doxorubicin; liposomes; mice tumors.

Conflict of interest statement

CONFLICTS OF INTEREST None.

Figures

Figure 1
Figure 1. Description of liposomal formulations used in the study
(A) Illustrations of liposomes used. PL= phospholipids, PEG = polyethylene glycol, DOPE = dioleoylphosphatidyl-ethanolamine, DSPC = distearoyl-sn-glycero-3-phosphocholine, DSPE = 1,2-distearoylphosphatidyl-ethanolamine. (B) Liposomal stability in solution. DOX-containing liposomes (1 mM) were incubated in stock solution (5% (w/v) sucrose solution pH 5.0 or in growth medium (DMEM with 10% (v/v) FCS, pH 7.4 for 24, 48 or 72 h. The liposomes were filtered and DOX was quantified to determine the percent of DOX retained in liposomes. The values show average of three experiments with standard deviations. (C) Effect of ceramide on doxorubicin release from liposomes. HeLa cells were incubated with various concentrations (30 – 1000 nM) of four different DOX-loaded liposomes and Free-DOX. After 24 h the cell medium was removed, cells washed and lysed using 0.1% (v/v) Triton-X100. DOX still encapsulated in liposomes was separated from free/released DOX by solid phase extraction and quantified. The data show the percent of DOX being encapsulated in liposomes or free. Mean values obtained by analyzing 3 replicates are shown.
Figure 2
Figure 2. Dose-dependent effect of liposomal doxorubicin on cell proliferation
(A-C) Three different cell lines were incubated for 24 h with various concentrations of liposomal DOX (10-3000 nM) followed by measuring incorporation of [3H]thymidine. Free-DOX, empty liposomes with same lipid concentration or no treatment was used for comparison. A) HeLa, B) HCT116, C) MDA-MB-231. (D-F) Time-dependent effect of liposomal DOX on the cell proliferation. HeLa cells incubated with increasing liposomal DOX-concentrations (30, 300 and 3000 nM) or empty liposomes with same lipid concentration were incubated for up to 72 h and the cell proliferation was determined by measuring the incorporation of [3H]thymidine (Free-DOX used for comparison) at various time points (8 - 16 - 24 - 48 - 72 h). D) HeLa 30 nM, E) HeLa 300 nM, F) HeLa 3000 nM. The data show the mean values from at least three independent experiments and standard deviations. The total lipid concentration of the formulations is displayed on the secondary x-axis in A-C.
Figure 3
Figure 3. Dose-dependent effect of liposomal doxorubicin on cell viability
Cells were incubated for 72 h with various concentrations of DOX-containing (30 - 3000 nM DOX) liposomes and compared to Free-DOX and empty liposomes with the same lipid concentration. Cell viability was determined by using the MTT assay. (A) HeLa, (B) HCT116, (C) MDA-MB-231. The data show the mean values from at least three independent experiments and standard deviations. The total lipid concentration of the formulations is displayed on the secondary x-axis.
Figure 4
Figure 4. Dose-dependent effect of increased ceramide addition to liposomal doxorubicin on cell viability
(A) HCT116, (B) MDA-MB-231. Cells were incubated with various concentrations (30 nM - 1000 nM) of DOX-containing liposomes for 72 h. One part of the samples had an additional 1 μM of ceramide of same type of ceramide-liposomes without DOX added. Empty-Lip-C6 were added to DOX-Lip-C6 and Free-DOX, and Empty-Lip-C12 were added to DOX-Lip-C12 and Caelyx® and incubated for 72 h. The comparison was made between no addition (blue bars) and additional ceramide-containing liposomes (red bars). The cells’ viability was determined by using the MTT assay. The data show the mean values from three independent experiments and standard deviations. *p<0.05.
Figure 5
Figure 5. Influence of ceramide in liposomes on kinetics of DOX effect on cell proliferation
(A) Free-DOX, (B) DOX-Lip-C6, (C) DOX-Lip-C12, (D) Caelyx®, (E) DOX-Lip. HeLa cells were incubated with various concentrations (30 – 1000 nM) of four different DOX-loaded liposomes and Free-DOX. After 1 and 3 h, one third of the cells were washed twice, fresh medium was added and the incubation extended. After a total of 24 h incubation the cell proliferation were compared with that of continuously treated cells. The data show the mean values from three independent experiments and standard deviations.
Figure 6
Figure 6. Effects of ceramide and doxorubicin on cell death signaling
(A) HeLa cells were incubated with various concentrations (1-30 μM) of DOX-loaded liposomes and Free-DOX. Pan-caspase inhibitor zVADfmk (10 or 30 μM) was added to address the effect of caspase-activity on cell viability measured by the MTT assay after 24 h. Bar graphs show mean values from three independent experiments and standard deviations. (B) Immunoblotting of HeLa cells were performed to investigate influence of ceramide and DOX on cellular signaling pathways. HeLa cells treated with either Free-DOX (0.1 - 10 μM), Empty-Lip-C6 (0.3 - 30 μM) or DOX-Lip-C6 (0.3 – 30 μM) were lysed, the lysates separated on SDS-PAGE and immunoblotted against PARP, phosphorylated (Ser473) AKT, GAPDH, phosphorylated (Thr172) AMPK and gamma-tubulin in duplicate. Untreated cells, cells treated with Empty-Lip or Staurosporin (1 μM) were used as controls.
Figure 7
Figure 7. Effect of ceramide liposomes on tumor growth in mice bearing MAS98
12 breast cancer xenografts. The tumor volumes were measured from day 22, i.e. one day prior to injection day (arrow mark) and up to day 47, i.e. 24 days after intravenous injection of DOX-containing liposomes or Free-DOX (8 mg/kg DOX) or a similar amount of empty liposomes. Tumor volumes are shown as relative to the tumor volumes at start of treatment. Data show mean values and standard deviations (n = 7-11 tumors).

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