Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.

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