Down-regulation of lipid raft-associated onco-proteins via cholesterol-dependent lipid raft internalization in docosahexaenoic acid-induced apoptosis

• DHA is known to be incompatible with cholesterol which is a critical component for lipid rafts, plasma membrane microdomains.
• DHA decreases cell surface levels of lipid rafts via their internalization, which is reversed by cholesterol.
• DHA down-regulates and inactivates lipid raft-associated onco-proteins such as EGFR and Hsp90.
• DHA decreases the levels of Hsp90 client proteins, including EGFR, HER2, Akt, and Src.
• DHA could exert its anti-cancer effect via modulation of lipid raft-associated signaling events.

Lipid rafts, plasma membrane microdomains, are important for cell survival signaling and cholesterol is a critical lipid component for lipid raft integrity and function. DHA is known to have poor affinity for cholesterol and it influences lipid rafts. Here, we investigated a mechanism underlying the anti-cancer effects of DHA using a human breast cancer cell line, MDA-MB-231. We found that DHA decreased cell surface levels of lipid rafts via their internalization, which was partially reversed by cholesterol addition. With DHA treatment, caveolin-1, a marker for rafts, and EGFR were colocalized with LAMP-1, a lysosomal marker, in a cholesterol-dependent manner, indicating that DHA induces raft fusion with lysosomes. DHA not only displaced several raft-associated onco-proteins, including EGFR, Hsp90, Akt, and Src, from the rafts but also decreased total levels of those proteins via multiple pathways, including the proteasomal and lysosomal pathways, thereby decreasing their activities. Hsp90 overexpression maintained its client proteins, EGFR and Akt, and attenuated DHA-induced cell death. In addition, overexpression of Akt or constitutively active Akt attenuated DHA-induced apoptosis. All these data indicate that the anti-proliferative effect of DHA is mediated by targeting of lipid rafts via decreasing cell surface lipid rafts by their internalization, thereby decreasing raft-associated onco-proteins via proteasomal and lysosomal pathways and decreasing Hsp90 chaperone function.