Browsing by Subject "Cell Death"
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Item Elucidating the Anti-Cancer Mechanism of Low Density Lipoprotein-Mediated Delivery of Docosahexaenoic Acid to Hepatocellular Carcinoma Cells(2015-07-17) Moss, Lacy Reynolds; Brown, Kathlynn C.; Corbin, Ian R.; Minna, John D.; Repa, Joyce J.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.Item [Southwestern News](2001-07-05) Wren, Worth, Jr.Item [Southwestern News](1999-12-07) Stieglitz, HeatherItem [Southwestern News](2003-07-16) Shields, AmyItem [Southwestern News](2005-06-30) McKenzie, AlineItem [Southwestern News](2000-09-11) Shields, AmyItem Using C. Elegans as Model Organism to Study the Mode of Action of a Natural Toxin, Psymberin(2011-12-15) Wu, Cheng-Yang; Roth, Michael G.Psymberin is an extremely potent cytotoxin isolated from the marine sponges Psammocinia and Ircinia ramose. Several cancer cell lines are sensitive to psymberin, including breast, melanoma and colon cancer cell lines. Psymberin is the only member of the pederin natural product family that contains a dihydroisocoumarin side chain. The cytotoxicities of psymberin in various human tumor cell lines are between sub-nanomolar to nanomolar IC50. Like pederin, the first member of this natural product family, psymberin and mycalamide A inhibit translation in vivo and in vitro. This inhibition by psymberin is 40 to 100 fold more potent than cycloheximide, which inhibits >90% translation at 100 micromolar in vivo. In a SAR study, both the cytotoxicity of psymberin and psymberin-induced translation inhibition were attenuated by substituting the psymberin side chain with the pederin side chain. However, the attenuation of cytotoxicity was relatively greater than of translation. The stereo configuration and both side chains of psymberin are required for both inhibition of translation and cytotoxicity. The result of the SAR study suggests that additional bioactivity is contained in psymberin. Psymberin is at best a poor substrate for small molecule pumps in the cell. Two separate forward genetics screens in C. elegans isolated seven independent psymberin-resistant mutants. In each the mutation was a C361T point mutation in the rpl-41 gene that changes Pro65 to Leu65 in the protein coding sequence. The psymberin-resistant mutant strain DA2312 is resistant to psymberin only. This mutation did not appear to cause weaker binding of psymberin to the ribosome, but must allow translation to continue with the toxin bound. There are additional modes of actions of psymberin compared to mycalamide A. The endogenous protein level of LC3, an autophagy marker, is decreased faster with psymberin treatment than mycalamide A. In HT-29 cells, psymberin is capable of synergizing TNFa-induced necrotic cell death more efficiently than mycalamide A. The results from SAR study and from study of the psymberin-specific mutation in C. elegans suggest that psymberin may induce fast cell death through multiple pathways, including translation inhibition, apoptosis and necrosis. The structural uniqueness of psymberin has functional consequences suggesting that the mode of action of psymberin on the ribosome is different from other members of the pederin family.