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. 2018 Jan;17(1):50-59.
doi: 10.1158/1535-7163.MCT-17-0173. Epub 2017 Oct 27.

Ceramide Nanoliposomes as a MLKL-Dependent, Necroptosis-Inducing, Chemotherapeutic Reagent in Ovarian Cancer

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Free PMC article

Ceramide Nanoliposomes as a MLKL-Dependent, Necroptosis-Inducing, Chemotherapeutic Reagent in Ovarian Cancer

Xuewei Zhang et al. Mol Cancer Ther. .
Free PMC article

Abstract

Ceramides are bioactive lipids that mediate cell death in cancer cells, and ceramide-based therapy is now being tested in dose-escalating phase I clinical trials as a cancer treatment. Multiple nanoscale delivery systems for ceramide have been proposed to overcome the inherent toxicities, poor pharmacokinetics, and difficult biophysics associated with ceramide. Using the ceramide nanoliposomes (CNL), we now investigate the therapeutic efficacy and signaling mechanisms of this nanoscale delivery platform in refractory ovarian cancer. Treatment of ovarian cancer cells with CNL decreased the number of living cells through necroptosis but not apoptosis. Mechanistically, dying SKOV3 ovarian cancer cells exhibit activation of pseudokinase mixed lineage kinase domain-like (MLKL) as evidenced by oligomerization and relocalization to the blebbing membranes, showing necroptotic characteristics. Knockdown of MLKL, but not its upstream protein kinases such as receptor-interacting protein kinases, with siRNA significantly abolished CNL-induced cell death. Monomeric MLKL protein expression inversely correlated with the IC50 values of CNL in distinct ovarian cancer cell lines, suggesting MLKL as a possible determinant for CNL-induced cell death. Finally, systemic CNL administration suppressed metastatic growth in an ovarian cancer cell xenograft model. Taken together, these results suggest that MLKL is a novel pronecroptotic target for ceramide in ovarian cancer models. Mol Cancer Ther; 17(1); 50-59. ©2017 AACR.

Figures

Figure 1
Figure 1. Cytotoxic effects of ceramide nanoliposomes in ovarian cancer cells
A, SKOV3 cells (1 × 105/well) were treated with 30 μM ceramide or ghost nanoliposomes for 24 h and then imaged by a phase-contrast microscopy. B, SKOV3 cells were treated with 30 or 100 μM nanoliposomes up to 48 h. The number of living cells was counted. The data represent the mean ± SD (n = 3). C, SKOV3 cells were treated with 1, 3, 10, 30, or 100 μM nanoliposomes. The cell viability was determined using a CellTiter-Glo luminescent cell viability assay according to the manufacturer’s protocol. The results are expressed as the percentages of 1 μM ghost nanoliposomes and the data represent the mean ± SD (n = 3). D, IC50 values were determined by GraphPad prism. The IC50 values of ceramide nanoliposomes in six kinds of ovarian cancer cell lines were shown.
Figure 2
Figure 2. Effects of ceramide nanoliposomes on cell death
A, SKOV3 cells were treated with 30 μM ceramide or ghost nanoliposomes, DMSO or 1 μM staurosporine for 18 h. Annexin V/7-AAD assay was performed for determining apoptotic cell death. B, SKOV3 cells were treated with ceramide nanoliposomes, ghost nanoliposomes or staurosporine for 24 h. Extracted cellular proteins were subjected to immunoblot analysis with antibodies for β-actin, GAPDH, LC3B, PARP, and procaspase 3 (apoptosis detection antibodies cocktail). Equal amounts of proteins were loaded in each lane. Three independent experiments were performed and representative images are shown. C, SKOV3 cells were incubated with the indicated concentrations of 3-methyladenine for 1 h and then treated with 30 μM ceramide or ghost nanoliposomes for 48 h. Cell viability was determined using a CellTiter-Glo luminescent assay according to the manufacturer’s protocol. The results are expressed as the percentages of 30 μM ghost nanoliposomes in non-3-methyladenine treatment group and the data represent the mean ± SD (n = 3).
Figure 3
Figure 3. Activation of MLKL by ceramide nanoliposomes
A, SKOV3 cells were transfected with MLKL-V5 vectors for 24 h followed by treatment with the indicated concentrations of ceramide or ghost nanoliposomes for 24 h. Cellular proteins extracted without reducing reagents were subjected to SDS-PAGE. Immunoblotting was performed using antibodies against V5 and β-actin. Three independent experiments were performed. Representative images are shown. B, SKOV3 cells were incubated with 30 μM ceramide or ghost nanoliposomes for 6 h and then were fixed followed by staining with TRITC-conjugated phalloidin (red), Hoechst 33342 (blue), MLKL (green). Imaging was performed by confocal microscopy, and representative images are shown. Arrows show the blebbing membranes.
Figure 4
Figure 4. MLKL-dependent cell death
A, SKOV3 cells were transfected with 5 nM control siRNA or two individual MLKL siRNA (sequence-1 and -2) for 24 h. The knockdown efficiency was confirmed by immunoblotting. B, Cells treated with siRNAs were further incubated with ceramide or ghost nanoliposomes for 48 h. Cell viability was determined using a CellTiter-Glo luminescent assay according to the manufacturer’s protocol. The results are expressed as the percentages of 10 μM ghost nanoliposomes in control siRNA group and the data represent the mean ± SD (n = 3). Statistical analyses were performed by unpaired, Student t-test. * p < 0.05 compared with control siRNA group. C, SKOV3 cells were incubated with the indicated concentrations of necrostatin-1 for 1 h and then treated with 30 μM ceramide or ghost nanoliposomes for 48 h. Cell viability was determined using a CellTiter-Glo luminescent assay according to the manufacturer’s protocol. The results are expressed as the percentages of 30 μM ghost nanoliposomes in non-necrostatin-1 treatment group and the data represent the mean ± SD (n = 3). D, SKOV3 cells were transfected with 5 nM control or RIPK3 siRNA for 24 h followed by treatment with ceramide or ghost nanoliposomes for 48 h. Cell viability was determined using a CellTiter-Glo luminescent assay according to the manufacturer’s protocol. The results are expressed as the percentages of 10 μM ghost nanoliposomes in control siRNA group and the data represent the mean ± SD (n = 3). E, proposed action mechanisms of ceramide nanoliposomes.
Figure 5
Figure 5. Significant correlation of MLKL expression with susceptibilities of ovarian cancer cells to ceramide nanoliposomes-induced cell death
A, Extracted cellular proteins from the indicated ovarian cancer cell lines were subjected to immunoblot analysis with antibodies for MLKL, RIPK-1, RIPK-3 and β-actin. Equal amounts of protein were loaded in each lane. Three independent experiments were performed and representative images are shown. The intensity of MLKL protein expression was quantified using Image Lab. IC50 values for ceramide nanoliposomes are plotted with MLKL protein expression (B), RIPK1 protein expression (C) and RIPK3 protein expression (D). R2 values were determined by GraphPad prism.
Figure 6
Figure 6. Effects of ceramide nanoliposomes on metastatic growth
A, SKOV3 cells were treated with 30 μM ceramide or ghost nanoliposomes for 6 h. Cells were fixed and counterstained with TRITC-conjugated phalloidin (red) and Hoechst 33342 (blue). Imaging was performed by confocal microscopy and the cell number forming lamellipodia were determined by counting more than 300 cells. The values represent the percentage of cells forming lamellipodia relative to total cells and the data represent the mean ± SD (n = 4). Four independent experiments were performed and yellow arrows show lamellipodia. Statistical analyses were performed by unpaired, student t-test. B and C, SKOV3 cells were treated with 30 μM ceramide nanoliposomes or ghost nanoliposomes for 18 h and then the assay for migration (B) and invasion (C) was performed as described in “Materials and Methods”. Data are represented as the percentage compared with ghost nanoliposomes’ group. The data represent are the means ± SD (n = 9). Statistical analyses were performed by unpaired, Student t-test. D, SKOV3 cells (5 × 106 / mouse) were injected intraperitoneally into peritoneal cavity of 4 weeks-old female nude mice. One day after implantation of cells, mice were intraperitoneally treated with ceramide or ghost nanoliposomes (40 mg/kg/day) continuously for 3 days. Four weeks later after inoculation, mice were euthanized to determine metastatic growth in the mesentery. The number of metastatic nodules was determined and mouse body weight was measured (n = 5). The data represent the mean ± SD and the representative images of specimens from mice were shown. White arrowheads indicate metastatic nodules. Statistical analyses were performed by unpaired, student t-test.

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