Inflammatory and cell death mechanisms induced by 7-ketocholesterol in the retina. Implications for age-related macular degeneration.

This review will focus on the inflammatory and toxic mechanism of action of 7-ketocholesterol (7KCh) and the potential implications of its accumulation, especially in the retina. 7KCh is a pro-inflammatory oxysterol usually associated with oxidized lipoprotein deposits present in aged retinas. High amounts of 7KCh can be generated in situ in these lipoprotein deposits possibly through a free radical-mediated mechanism catalyzed by iron. 7KCh seems to activate several kinase signaling pathways that work via multiple transcription factors to induce cytokines and intracellular effectors causing cell death. There seems to be a controversy in the literature in relation to the mechanisms of death induced by 7KCh. Some of the discrepancies arise from the way the oxysterol is delivered because different signaling pathways are activated in different experimental setups. The elucidation of the inflammatory and toxic mechanisms is crucial for the discovery and design of new therapies. Importantly, there is little evidence of 7KCh detoxifying mechanisms in the retina, although some potential enzymes have been described. Thus, continuous formation throughout life and potential toxicity of 7KCh points it out as an "age-related" risk factor in pathologies such as age-related macular degeneration.

Argan Oil-Mediated Attenuation of Organelle Dysfunction, Oxidative Stress and Cell Death Induced by 7-Ketocholesterol in Murine Oligodendrocytes 158N.

Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25-50 µM; 24 h) without and with argan oil (0.1% v/v) or α-tocopherol (400 µM, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, ß-amyrin and citrostadienol. α- and γ-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood-brain barrier.

Toxicity of 7-ketocholesterol on lethality, growth, reproduction, and germline apoptosis in the nematode Caenorhabditis elegans.

7-Ketocholesterol is one of the most abundant cholesterol oxides, and is known to be cytotoxic to various types of cultured mammalian cells; however, little is known regarding its effects in vivo. With the use of the nematode Caenorhabditis elegans as model organism, in vivo toxicity of 7-ketocholesterol was investigated. The aim of the study was to examine the effects on life span, as well as short-term effects on reproduction, thermotolerance, germline apoptosis, and reactive oxygen species (ROS) generation resulting from C. elegans exposure to 7-ketocholesterol at concentrations ranging from 0 to 200 µg/ml. Results indicated that 7-ketocholesterol reduced reproductive capacity, shortened the life span in a concentration-dependent manner, and impaired thermotolerance of the adult nematode. 7-Ketocholesterol also induced germline apoptotic cell death and increased ROS generation in adult worms. Thus, the model organism C. elegans is recommended for assessment of the safety and bioactivity of cholesterol oxides.

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3ß-hydroxysterol-Δ(7)-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4ß-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4ß-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d(7)-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25µmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.

Increased resistance to oxysterol cytotoxicity in fibroblasts transfected with a lysosomally targeted Chromobacterium oxidase.

7-Ketocholesterol (7KC) is a cytotoxic oxysterol that plays a role in many age-related degenerative diseases. 7KC formation and accumulation often occurs in the lysosome, which hinders enzymatic transformations that reduce its toxicity and increase the sensitivity to lysosomal membrane permeabilization. We assayed the potential to mitigate 7KC cytotoxicity and enhance cell viability by overexpressing 7KC-active enzymes in human fibroblasts. One of the enzymes tested, a cholesterol oxidase engineered for lysosomal targeting, significantly increased cell viability in the short term upon treatment with up to 50 µM 7KC relative to controls. These results suggest targeting the lysosome for optimal treatment of oxysterol-mediated cytotoxicity, and support the use of introducing novel catalytic function into the lysosome for therapeutic and research applications.

Lipid accumulation and lysosomal pathways contribute to dysfunction and apoptosis of human endothelial cells caused by 7-oxysterols.

Endothelial dysfunction and cell death play an important role in pathogenesis of atherosclerosis. 7-Oxysterols, the major cytotoxic component found in oxidized low-density lipoprotein, are toxic to endothelial cells. However, the pathways and molecular mechanism involved in the process remain incompletely understood. In this study, we first investigate whether 7ß-hydroxycholesterol (7ßOH) or 7-ketocholesterol (7keto) induces apoptosis of human endothelial cell line (HUVEC-CS). We then examine possible involved pathways by focusing on cellular lipid, lysosomal pathways, cellular oxidative stress and mitochondrial pathways. Our results for the first time showed that 7-oxysterols induced apoptotic cell death of HUVEC-CS after 24h, which was preceded by early lipid accumulation (6h) and lysosomal membrane permeabilization (6-12h). Afterward, levels of reactive oxygen species, mitochondrial membrane permeabilization, and lysosomal cathepsin were increased assayed by immuno-cytochemistry and blotting. Notably, the exposure to 7ßOH or 7keto induced expressions and secretion of isoforms of von Willebrand factor (VWF). We conclude that apoptosis of HUVEC-CS induced by 7ßOH or 7keto mediates by early lysosomal lipid accumulation and oxidative lysosomal pathways, which results in induction and release of VWF. The results suggest the cell death induced by 7-oxysterols may contribute to endothelial dysfunction and atherothrombosis.

Anti-inflammatory and Cytoprotective Effect of Clinacanthus nutans Leaf But Not Stem Extracts on 7-Ketocholesterol Induced Brain Endothelial Cell Injury.

Clinacanthus nutans (Lindau) (C. nutans) has diverse uses in traditional herbal medicine for treating skin rashes, insect and snake bites, lesions caused by herpes simplex virus, diabetes mellitus and gout in Singapore, Malaysia, Indonesia, Thailand and China. We previously showed that C. nutans has the ability to modulate the induction of cytosolic phospholipase A2 (cPLA2) expression in SH-SY5Y cells through the inhibition of histone deacetylases (HDACs). In the current study, we elucidated the effect of C. nutans on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induced a dose-dependent loss of hCMEC/D3 cell viability, and such damage was significantly inhibited by C. nutans leaf extracts but not stem extracts. 7KC also induced a marked increase in mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX-2) in brain endothelial cells, and these increases were significantly inhibited by C. nutans leaf but not stem extracts. HPLC analyses showed that leaf extracts have a markedly different chemical profile compared to stem extracts, which might explain their different effects in counteracting 7KC-induced inflammation. Further study is necessary to identify the putative phytochemicals in C. nutans leaves that have anti-inflammatory properties.

Effect of Ergothioneine on 7-Ketocholesterol-Induced Endothelial Injury.

Ergothioneine (ET) is a naturally occurring antioxidant that is synthesized by non-yeast fungi and certain bacteria. ET is not synthesized by animals, including humans, but is avidly taken up from the diet, especially from mushrooms. In the current study, we elucidated the effect of ET on the hCMEC/D3 human brain endothelial cell line. Endothelial cells are exposed to high levels of the cholesterol oxidation product, 7-ketocholesterol (7KC), in patients with cardiovascular disease and diabetes, and this process is thought to mediate pathological inflammation. 7KC induces a dose-dependent loss of cell viability and an increase in apoptosis and necrosis in the endothelial cells. A relocalization of the tight junction proteins, zonula occludens-1 (ZO-1) and claudin-5, towards the nucleus of the cells was also observed. These effects were significantly attenuated by ET. In addition, 7KC induces marked increases in the mRNA expression of pro-inflammatory cytokines, IL-1ß IL-6, IL-8, TNF-α and cyclooxygenase-2 (COX2), as well as COX2 enzymatic activity, and these were significantly reduced by ET. Moreover, the cytoprotective and anti-inflammatory effects of ET were significantly reduced by co-incubation with an inhibitor of the ET transporter, OCTN1 (VHCL). This shows that ET needs to enter the endothelial cells to have a protective effect and is unlikely to act via extracellular neutralizing of 7KC. The protective effect on inflammation in brain endothelial cells suggests that ET might be useful as a nutraceutical for the prevention or management of neurovascular diseases, such as stroke and vascular dementia. Moreover, the ability of ET to cross the blood-brain barrier could point to its usefulness in combatting 7KC that is produced in the CNS during neuroinflammation, e.g. after excitotoxicity, in chronic neurodegenerative diseases, and possibly COVID-19-related neurologic complications.

Methionine sulfoxide reductase B2 is highly expressed in the retina and protects retinal pigmented epithelium cells from oxidative damage.

Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial enzyme that converts methionine sulfoxide (R) enantiomer back to methionine. This enzyme is suspected of functioning to protect mitochondrial proteins from oxidative damage. In this study we report that the retina is one of the human tissues with highest levels of MSRB2 mRNA expression. Other tissues with high expression were heart, kidney and skeletal muscle. Overexpression of an MSRB2-GFP fusion protein increased the MSR enzymatic activity three-fold in stably transfected cultured RPE cells. This overexpression augmented the resistance of these cells to the toxicity induced by 7-ketocholesterol, tert-butyl hydroperoxide and all-trans retinoic acid. By contrast, knockdown of MSRB2 by a miRNA in stably transfected cells did not convey increased sensitivity to the oxidative stress. In the monkey retina MSRB2 localized to the ganglion cell layer (GLC), the outer plexiform layer (OPL) and the retinal pigment epithelium (RPE). MSRB2 expression is most pronounced in the OPL of the macula and foveal regions suggesting an association with the cone synaptic mitochondria. Our data suggests that MSRB2 plays an important function in protecting cones from multiple type of oxidative stress and may be critical in preserving central vision.

Tyrosine kinase inhibitor AG126 reduces 7-ketocholesterol-induced cell death by suppressing mitochondria-mediated apoptotic process.

The preventive effect of tyrosine kinase inhibitor AG126 against the 7-ketocholesterol toxicity was investigated in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, the mitochondrial membrane permeability changes, the formation of reactive oxygen species and the depletion of GSH, which leads to cell death in differentiated PC12 cells. Tyrphostin AG126 significantly attenuated the 7-ketocholesterol-induced decrease in cytosolic Bid and Bcl-2 levels, increase in cytosolic pro-apoptotic Bax levels, mitochondrial membrane potential loss, cytochrome c release and subsequent caspase-3 activation. The inhibitory effect of tyrphostin AG126 may be supported by the inhibitory effect on another oxysterol 25-hydroxycholesterol-induced cell death. The results show that tyrphostin AG126 may prevent the 7-ketocholesterol toxicity by suppressing the mitochondrial membrane permeability change that leads to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and the depletion of GSH.

7-ketocholesterol induces cell apoptosis by activation of nuclear factor kappa B in mouse macrophages.

We investigated the molecular mechanisms responsible for the induction of apoptosis in mouse monocytic macrophage cell line J774A.1 stimulated by 7-ketocholesterol (7-KC). Cell apoptosis was detected by Annexin V-propidium iodide (PI) staining. The DNA-binding activity of nuclear factor kappa B (NF-kappaB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that 7-KC-stimulation in J774A.1 cells activated NF-kappaB, which is involved in cell apoptosis, in a time- and dose-dependent manners. 7-KC was also found to increase the binding activity of NF-kappaB to specific DNA binding sites, a possible mechanism for the induction of the cell apoptosis. Moreover, these effects were partially inhibited by pyrrolidine dithiocarbamate (PDTC), an NF-kappaB inhibitor. Taken together, 7-KC may be an important factor in atherosclerosis due to the ability of 7-KC to induce cell apoptosis, which is at least partially mediated through the activation of NF-kappaB.

Mutagenicity of 7-ketocholesterol in CHO cells: The role of lipid peroxidation.

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 µM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

7-Ketocholesterol and 7ß-hydroxycholesterol: In vitro and animal models used to characterize their activities and to identify molecules preventing their toxicity.

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7ß-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7ß-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7ß-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches.

Glycyrrhizin prevents 7-ketocholesterol toxicity against differentiated PC12 cells by suppressing mitochondrial membrane permeability change.

Defects in mitochondrial function participate in the induction of neuronal cell injury. In neurodegenerative conditions, oxidative products of cholesterol are elevated and oxysterols seem to be implicated in neuronal cell death. The present work was designed to study the inhibitory effect of licorice compounds glycyrrhizin and 18beta-glycyrrhetinic acid against the toxicity of 7-ketocholesterol in relation to the mitochondria-mediated cell death process. 7-Ketocholesterol induced the nuclear damage, loss of the mitochondrial transmembrane potential, increase in the cytosolic Bax and cytochrome c levels, caspase-3 activation and cell death in differentiated PC12 cells. Glycyrrhizin and 18beta-glycyrrhetinic acid prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death. The results obtained show that glycyrrhizin and 18beta-glycyrrhetinic acid may prevent the 7-ketocholesterol-induced neuronal cell damage by suppressing changes in the mitochondrial membrane permeability.

Suppression of atherogenesis by overexpression of glutathione peroxidase-4 in apolipoprotein E-deficient mice.

Accumulation of oxidized lipids in the arterial wall contributes to atherosclerosis. Glutathione peroxidase-4 (GPx4) is a hydroperoxide scavenger that removes oxidative modifications from lipids such as free fatty acids, cholesterols, and phospholipids. Here, we set out to assess the effects of GPx4 overexpression on atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. The results revealed that atherosclerotic lesions in the aortic tree and aortic sinus of ApoE(-/-) mice overexpressing GPx4 (hGPx4Tg/ApoE(-/-)) were significantly smaller than those of ApoE(-/-) control mice. GPx4 overexpression also diminished signs of advanced lesions in the aortic sinus, as seen by a decreased occurrence of fibrous caps and acellular areas among hGPx4Tg/ApoE(-/-) animals. This delay of atherosclerosis in hGPx4Tg/ApoE(-/-) mice correlated with reduced aortic F(2)-isoprostane levels (R(2)=0.75, p<0.01). In addition, overexpression of GPx4 lessened atherogenic events induced by the oxidized lipids lysophosphatidylcholine and 7-ketocholesterol, including upregulated expression of adhesion molecules in endothelial cells and adhesion of monocytes to endothelial cells, as well as endothelial necrosis and apoptosis. These results suggest that overexpression of GPx4 inhibits the development of atherosclerosis by decreasing lipid peroxidation and inhibiting the sensitivity of vascular cells to oxidized lipids.

Interactions between cell death induced by statins and 7-ketocholesterol in rabbit aorta smooth muscle cells.

BACKGROUND AND PURPOSE: 7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. EXPERIMENTAL APPROACH: Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. KEY RESULTS: NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive. CONCLUSIONS AND IMPLICATIONS: 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.

Microbial degradation of 7-ketocholesterol.

7-Ketocholesterol (7KC) is an oxidized derivative of cholesterol suspected to be involved in the pathogenesis of atherosclerosis and possibly Alzheimer's disease. While some oxysterols are important biological mediators, 7KC is generally cytotoxic and interferes with cellular homeostasis. Despite recent interest in preventing the accumulation of 7KC in a variety of matrices to avoid adverse biological effects, its microbial degradation has not been previously addressed in the peer-reviewed literature. Thus, the rate and extent of biodegradation of this oxysterol was investigated to bridge this gap. A wide variety of bacteria isolated from soil or activated sludge, including Proteobacterium Y-134, Sphingomonas sp. JEM-1, Nocardia nova, Rhodococcus sp. RHA1, and Pseduomonas aeruginosa, utilized 7KC as a sole carbon and energy source, resulting in its mineralization. Nocardia nova, which is known to produce biosurfactants, was the fastest degrader. This study supports the notion that microbial catabolic enzymes could be exploited to control 7KC levels in potential biotechnological applications for agricultural, environmental, or medical use.

Redox regulation of 7-ketocholesterol-induced apoptosis by beta-carotene in human macrophages.

The aim of this study was to verify the hypothesis that beta-carotene may prevent 7-ketocholesterol (7-KC)-induced apoptosis in human macrophages. Therefore, THP-1 macrophages were exposed to 7-KC (5-50 microM) alone and in combination with beta-carotene (0.25-1 microM). 7-KC inhibited the growth of macrophages in a dose- and a time-dependent manner by inducing an arrest of cell cycle progression in the G0/G1 phase and apoptosis. Concomitantly, p53, p21, and Bax expressions were increased by 7-KC, whereas the levels of AKT, Bcl-2, and Bcl-xL were decreased. beta-Carotene prevented the growth-inhibitory effects of 7-KC in a dose- and time-dependent manner as well as the effects of 7-KC on the expression of cell cycle- and apoptosis-related proteins. 7-KC also enhanced reactive oxygen species (ROS) production through an increased expression of NAD(P)H oxidase (NOX-4). The effects of 7-KC were counteracted by the addition of the NAD(P)H oxidase inhibitor DPI or by cotransfection of siNOX-4 mRNA. beta-Carotene prevented 7-KC-induced increase in ROS production and in NOX-4 expression, as well as the phosphorylation of p38, JNK, and ERK1/2 induced by 7-KC. These data suggest a possible antiatherogenic role of beta-carotene through the prevention of 7-KC toxicity in human macrophages.

Cytotoxic effects of various stressors on PC12 cells: involvement of oxidative stress and effect of antioxidants.

In order to specifically elucidate the involvement of oxidative stress, the effects of various types of stressors and antioxidants on PC12 cells were examined. In this study, the following four stressors were studied in detail: free radicals generated from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 4-hydroxynonenal (HNE), 7-ketocholesterol (KC), and arsenic trioxide (As2O3). Undifferentiated PC12 cells were treated with 50% lethal concentration (LC50) of these stressors, and subsequently the viability, apoptosis/necrosis ratio, reactive oxygen species (ROS) production, caspase-3 activity, and protection by antioxidants were measured to elucidate the underlying mechanisms that determine the action of these stressors on PC12 cells. The cytotoxicity did not correlate directly with the intracellular formation of ROS. For example, as compared to AAPH, As2O3 produced considerably smaller amounts of ROS at LC50. As observed in the cells incubated with As2O3, KC and HNE exerted cell toxicity, but with a moderate production of ROS. With the exception of HNE, the apoptosis/necrosis ratio of all the stressors evaluated by annexin V and propidium iodide assays increased with an increase in the incubation time at the LC50 values of these stressors. In accordance with apoptosis ratio, caspase activity was detected in the cells incubated with AAPH, As2O3, and KC, but not HNE at LC50 for 24 h. The protective effect of alpha-tocopherol, 17beta-estradiol, 2,3-dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butylbenzofuran (BO653), glutathione, and N-acetylcysteine (NAC) against cytotoxicity depended on the type of stressors. These antioxidants were found to be effective against the abovementioned stressors, except As2O3 against which only NAC was effective. These results suggest that the involvement of ROS and the protective effect of antioxidants depend on the type of stressors.

Novel side chain modified Delta8(14)-15-ketosterols.

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).