Glutathione is implied in the control of 7‐ketocholesterol‐induced apoptosis, which is associated with radical oxygen species production

Abstract

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

“…In vitro, under treatment with 7-ketocholesterol, 7ß-hydroxycholesterol, cholesterol-5ß,6ß-epoxide, cholesterol-5α,6α-epoxide, and 25-hydroxycholesterol, important oxidative processes, sometimes associated with a complex mode of cell death with some of the characteristics of apoptosis, have also been described on various cell types (15,27,32). Thus, on 7-ketocholesteroltreated U937 cells, an abrupt drop in reduced glutathione associated with an increased production of ROS preceding caspase activation has been reported (46). In human aortic smooth muscle cells, 7-ketocholesterol also induces oxidative stress associated with apoptotic events, and it has been demonstrated that this specific effect of 7-ketocholesterol is mediated by a robust upregulation (3-fold from the basal level) of Nox-4, an ROS-generating NAD(P)H oxidase homologue (32).…”

7‐Ketocholesterol favors lipid accumulation and colocalizes with Nile Red positive cytoplasmic structures formed during 7‐ketocholesterol–induced apoptosis: Analysis by flow cytometry, FRET biphoton spectral imaging microscopy, and subcellular fractionation

“…For all experiments, a stock solution of 7KC was prepared extemporaneously at a concentration of 800 g/ml as previously described (36). To obtain the initial solution, 800 g of 7KC was dissolved in 50 l of absolute ethanol, 950 l of culture medium were added, and the solution was sonicated.…”

“…Apoptotic cells were characterized by nuclear condensation of chromatin and/or nuclear fragmentation (36). Hoechst 33342 was prepared in distilled water at 1 mg/ml and added to culture medium at a final concentration of 10 g/ml.…”

“…For each fraction, lipid contents of cholesterol (esterified and unesterified), 7KC, phosphatidylcholine, and sphingomyelin were determined. To this end, lipids were extracted and analyzed by capillary gas chromatography and mass spectrometry as previously described (36).…”

Effects of caspase inhibitors (z‐VAD‐fmk, z‐VDVAD‐fmk) on Nile Red fluorescence pattern in 7‐ketocholesterol‐treated cells: Investigation by flow cytometry and spectral imaging microscopy

“…For all experiments, a stock solution of 7KC was prepared at a concentration of 800 lg/ml as previously described (27). To obtain the initial solution, 800 lg of 7KC were dissolved in 50 ll of absolute ethanol, 950 ll of culture medium were added, and the suspension was sonicated.…”