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

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 mu M inhibited VEGF-induced tube formation by HUVEC. The viability of MCF-7 and MDA-MB-435s breast cancer cells exposed to 125 mu M CMAEPn for 48 h was reduced to 76 and 85%, respectively. The viability of MCF-7 and MDA-MB-435s cells exposed to 250 mu M CMAEPn for 48 h under the same conditions was reduced to 38 and 45%, respectively. CMAEPn at 125 mu M inhibited VEGF-induced MDA-MB-435s cell migration and invasion. CMAEPn at 125 mu M also decreased VEGF, EGF levels in the conditioned media, PI3K, I kappa B phosphorylation and degradation in the cytoplasmic extracts, and NF kappa B 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.