AFPep Enters Breast Cancer Cells via the Endocytic/Lysosomal Pathway

Abstract

AFPep, a therapeutic agent that has been shown to inhibit the growth of human breast cancer xenografts in mice and prevent the development of carcinogen‐induced mammary cancers in rats, is a cyclic 9‐amino acid peptide derived from the active site of alpha‐fetoprotein. However, the mechanism of action for AFPep is currently unknown. Understanding how this peptide enters breast cancer cells is critical to characterize its mechanism of action. We hypothesize that AFPep is internalized into human breast cancer cells via the endocytic pathway. We characterized the ability of AFPep‐Alexa conjugates to be internalized into T47D cells using confocal microscopy. Internalization of AFPep‐Alexa was tested by competition with unconjugated AFPep and co‐localization with differently labeled holo‐transferrin or dextran. AFPep‐Alexa internalization was significantly reduced in the presence of competing amounts of unconjugated AFPep, demonstrating a specific pathway of entry into breast cancer cells. AFPep‐Alexa colocalized with dextran, a fluid‐phase lysosomal marker, but not with holo‐transferrin, an early endosome marker. When T47D cells were treated with BafilomycinA1, a drug that disrupts lysosomal trafficking, AFPep‐Alexa appeared to be trapped in the early endosomes. We conclude that AFPep enters T47D cells via a specific endocytic pathway and is delivered to lysosomes, confirming our hypothesis.