Ceramide--antiestrogen nanoliposomal combinations--novel impact of hormonal therapy in hormone-insensitive breast cancer

Abstract

Although the sphingolipid ceramide exhibits potent tumor suppressor effects, efforts to harness this have been hampered by poor solubility, uptake, bioavailability, and metabolic conversion. Therefore, identification of avenues to improve efficacy is necessary for development of ceramide-based therapies. In this study, we used mutant p53, triple-negative breast cancer (TNBC) cells, a type of breast cancer highly refractory to treatment, and cell-permeable nanoliposomal C6-ceramide in conjunction with the antiestrogen tamoxifen, which has been shown to be an effective modulator of ceramide metabolism. We show for the first time that nanoliposomal tamoxifen enhances nanoliposomal C6-ceramide cytotoxicity in cultured TNBC cells, a response that was accompanied by induction of cell-cycle arrest at G(1) and G(2), caspase-dependent induction of DNA fragmentation, and enhanced mitochondrial and lysosomal membrane permeability at 18 and 2 hours, respectively. Tamoxifen metabolites were also effective. Only tamoxifen promoted lysosomal membrane permeability. In addition, we show for the first time that tamoxifen inhibits acid ceramidase, as measured in intact cell assays; this effect was irreversible. Together, our findings show that tamoxifen magnifies the antiproliferative effects of C6-ceramide via combined targeting of cell-cycle traverse and lysosomal and mitochondrial integrity. We adduce that C6-ceramide-induced apoptosis is amplified by tamoxifen's impact on lysosomes and perhaps accompanying inhibition of acid ceramidase, which could result in decreased levels of sphingosine 1-phosphate. This drug regimen could serve as a promising therapy for chemoresistant and triple-negative types of breast cancer, and thus represents an indication for tamoxifen, irrespective of estrogen receptor status.

Figure 1

Effect of tamoxifen on C6-ceramide cytotoxicity in TNBC. A-D. Cells were seeded in 96-well plates (3,000 cells/well) in RPMI-1640 medium containing 10% FBS and cultured at 37°C. The next day, growth medium was replaced with 2.5 % FBS medium, agents were added and cells were incubated for 96 hr. D. MDA-MB-468 cells treated with N-desmethyltamoxifen (dme-tam) and 4-hydroxytamoxifen (4-OH tam). Viability was evaluated by MTS assay. [μM] concentrations. Data are the mean (n=6) ± SE, relative to vehicle controls and/or the ghost. Experiments were repeated and yielded similar results.

Figure 2

Effect of tamoxifen and C6-ceramide on cell cycle progression in TNBC. Cells were treated with C6-ceramide, tamoxifen, and the mix for 24 hr, collected and fixed in ethanol, stained with propidium iodide, and analyzed by flow cytometry as detailed in Methods. The distribution of cells in the different phases of the cell cycle was analyzed using FCS express software. Data are mean ± SE from three independent experiments. [μM] concentrations.

Figure 3

Influence of C6-ceramide, tamoxifen, and caspase inhibition on apoptosis in TNBC. A. MDA-MB-468 cells. B. MDA-MB-231 cells. C. MDA-MB-468 cells with N-desmethyltamoxifen. Cells were treated with C6-ceramide, tamoxifen, N-desmethyltamoxifen, or the combination for 24 hr. Cells were then trypsinized, fixed in ethanol, stained with propidium iodide, and analyzed for DNA fragmentation by flow cytometry. SubG₀ (apoptosis marker) was analyzed using FCS express software. Data are means ± SE from three independent experiments. Concentrations [μM] noted in brackets. CaspI [50 μM], caspase inhibitor.

Figure 4

Effect of C6-ceramide and tamoxifen on mitochondrial and lysosomal integrity. A. Effect of C6-ceramide on mitochondrial depolarization. B. Effect of C6-ceramide and N-desmethyltamoxifen on mitochondrial depolarization. MDA-MB-468 cells were treated with C6-ceramide, tamoxifen, N-desmethyltamoxifen, or the combination for 18 hr. Cells were then stained with JC-1 for 15 min in phenol red-free RPMI, trypsinized, washed with PBS, and placed on ice until quantitation by FACS. Quantitative analysis of Δψm was detected by JC-1 at FL-1 (green) and FL-2 (red). Data are mean from 3-4 independent experiments. C. Effect of C6-ceramide and tamoxifen on lysosomal permeabilization. MDA-MB-468 cultures were stained with Acridine orange for 15 minutes followed by PBS washing, and treated with tamoxifen for 2 hr. After treatment, cells were trypsinized, washed with PBS, and placed on ice until FACS analysis. A shift in red fluorescence (FL-3) indicates lysosomal peremeabilization. Data was analyzed using FCS express software. Data are means ± SE from at least three independent experiments.

Figure 5

Effect of tamoxifen and DM102 on AC activity in MDA-MB-468 cell lysates. A. Cell-free assay. Cells were preincubated with tamoxifen for 4 hr, washed, lysed, and assayed for AC activity as described in Methods. B. Cell-free assays with tamoxifen [20 μM] and DM102 [20 μM] added after lysis. Data are means ± SE, n = 3 from two independent experiments.

Figure 6

Scheme for C6-ceramide-tamoxifen elicitation of apoptosis. Tamoxifen can block C6-ceramide glycosylation and hydrolysis, the latter mediated via enhanced LMP. LMP can also impact mitochondria as can C6-ceramide, directly. Blocking AC dampens S1-P mitogenic responses and potentiates lysosomal and mitochondrial driven apoptosis. AC, acid ceramidase; S1-P, sphingosine 1-phosphate.