In Vitro and in Vivo Anti-angiogenic Activities and Inhibition of Hormone-Dependent and -Independent Breast Cancer Cells by Ceramide Methylaminoethylphosphonate

Abstract

Ceramide methylaminoethylphosphonate (CMAEPn) was isolated from eastern oyster ( Crassostrea virginica ) and screened against in vitro and in vivo angiogenesis and against MCF-7 and MDA-MB-435s breast cancer cell lines. In vitro angiogenesis was evaluated by the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) tube formation assay. MCF-7 and MDA-MB-435s cell viability was evaluated by the CellTiter 96 AQ(ueous) One Solution Cell Proliferation assay. Apoptosis was evaluated by the caspase-9 assay, autophagy by acridine orange staining and beclin-1 level. Our study indicates that CMAEPn at 50 microM inhibited VEGF-induced tube formation by HUVEC. The viability of MCF-7 and MDA-MB-435s breast cancer cells exposed to 125 microM CMAEPn for 48 h was reduced to 76 and 85%, respectively. The viability of MCF-7 and MDA-MB-435s cells exposed to 250 microM CMAEPn for 48 h under the same conditions was reduced to 38 and 45%, respectively. CMAEPn at 125 microM inhibited VEGF-induced MDA-MB-435s cell migration and invasion. CMAEPn at 125 microM also decreased VEGF, EGF levels in the conditioned media, PI3K, IkappaB phosphorylation and degradation in the cytoplasmic extracts, and NFkappaB nuclear translocation. Both acridine orange staining and beclin-1 indicated autophagic cell death in MCF-7 and MDA-MB-435s cells, respectively. In vivo, CMAEPn at 30 mg/kg body weight inhibited bFGF-induced angiogenesis and caused a 57% reduction in hemoglobin levels in the matrigel plug assay within 7 days. This is the first report on CMAEPn-inhibited angiogenesis both in vitro and in vivo.

Ceramide-Based Therapeutics for the Treatment of Cancer

“…Unfortunately these short-chain analogs are still sufficiently insoluble so that in vivo delivery is restricted and must rely on novel delivery systems to improve solubility, permeability, and pharmacokinetics. CMAEPh also showed in vivo efficacy in an angiogenesis model [93], which suggests that these ceramide analogues could also limit tumor growth via dysregulation of the tumor vascular system. These substantially modified ceramide analogs have shown in vivo efficacy in preclinical cancer models [49,79].…”

“…The advantage of these analogs is that they behave very similar to natural ceramides in terms of their effects on cellular function, are metabolized similar to natural ceramides, and can even be deacylated and subsequently re-acylated to natural ceramides. Naturally occurring sphingadienes [91,92], and sphingatrienes from sea cucumbers [92], and ceramide methylaminoethylphosphonates (CMAEPh) found in oysters [93], have shown efficacy in various in vitro cancer models. An intriguing new approach has been the generation of water soluble pyridinium-ceramides [78,79].…”

Coupling In vitro and In vivo Paradigm Reveals a Dose Dependent Inhibition of Angiogenesis Followed by Initiation of Autophagy by C6-Ceramide

“…Putative mechanisms responsible for ceramide-induced apoptosis of mammary cancer cells include the accumulation of ceramides within the mitochondria which leads to mitochondrial dysfunction, cytochrome c release and caspase activation, dephosphorylation of ceramide-activated protein phosphatase, control of calcium homeostasis, and activation of cathepsin D and protein kinase C ζ 1, 3, 5, 7-9. We have recently demonstrated that ceramide methylaminoethylphosphonate (CMAEPn), a naturally occurring shingophospholipid inhibited the viability of hormone-dependent and -independent breast cancer cells by inactivating VEGF, EGF, and PI3K, which are some of the main signaling pathways associated with the progression of breast cancer 10. These data suggest that addition of exogenous ceramides to tumor cells provides an important mechanism for decreasing tumor cell survival and makes ceramide an attractive bioactive compound for cancer prevention and/or treatment.…”

Comparative Lipid Profiling of the Cnidarian Aiptasia pallida and Its Dinoflagellate Symbiont

“…These unusual lipids are widely observed in marine invertebrates [55] and have been observed in anemones previously [39], [56]. The presence of the C-P bond in the aminoethyl phosphonate head group is resistant to hydrolytic enzymes [57], [58]. This may indicate an important role for this lipid in the establishment of symbiosis.…”