Elucidating the Anti-Cancer Mechanism of Low Density Lipoprotein-Mediated Delivery of Docosahexaenoic Acid to Hepatocellular Carcinoma Cells
Hepatocellular carcinoma is a lethal malignancy with few effective therapy options. New selective treatments are urgently needed to destroy hepatocellular carcinoma cells without harming the surrounding normal hepatocytes. Recently, docosahexaenoic acid has been shown to possess promising anticancer properties. The Corbin laboratory has incorporated docosahexaenoic acid into low density lipoprotein nanoparticles (LDL-DHA) as a means to transport these fatty acids to cancer cells. To test LDL-DHA's efficacy, immortalized mouse normal liver (TIB-73) and isogeneic malignant liver (TIB-75) cell lines were compared. Cell viability and co-culture experiments demonstrated that TIB-75 cells were more sensitive to LDL-DHA than TIB-73 cells. LDL-DHA enters into cells through LDL receptor-mediated endocytosis to the lysosomes. LDL-DHA treatment increased dichlorofluorescein fluorescence in TIB-75 cells over TIB-73 cells, and generation of reactive oxygen species by menadione sensitized TIB-73 cells to LDL-DHA. Importantly, TIB-75 cells were rescued from LDL-DHA cytotoxicity when antioxidants specific for removing lipid peroxide species were added indicating that lipid peroxidation was critical for LDL-DHA cytotoxicity. LDL-DHA also caused lysosomal membrane permeability of only the TIB-75 cells. Subsequent studies showed that only the LDL-DHA treated TIB-75 cells lose their mitochondrial membrane potential. Mitochondrial reactive oxygen species were elevated in TIB-75 cells following LDL-DHA treatment, and TIB-73 cells were sensitized to LDL-DHA after decoupling of mitochondrial respiration. LDL-DHA treatment also caused DNA damage selectively in the TIB-75 cells. When the Fenton reaction, an iron-catalyzed reaction that generates hydroxyl radicals and lipid peroxide species, was blocked by iron chelation, TIB-75 showed less LDL-DHA cytotoxicity, lipid peroxidation, and lysosome leaking. Studies conducted in human hepatocellular carcinoma cells (FOCUS, Hep3B, and Huh7) on LDL-DHA cytotoxicity, lysosome permeability, and mitochondrial reactive oxygen species production confirmed the findings seen in TIB-75 cells following LDL-DHA treatment. Furthermore, primary human hepatocytes were not sensitive to LDL-DHA treatment. In conclusion, these studies have shown that LDL-DHA is selectively cytotoxic to hepatocellular carcinoma cells and that iron-catalyzed lipid peroxidation sets off a subcellular chain of events resulting in increased reactive oxygen species, lysosome permeability, mitochondrial dysfunction, DNA damage, and, ultimately, cell death in hepatocellular carcinoma cells.