Anne Regazzetti

P2X7-pannexin-1 and amyloid β-induced oxysterol input in human retinal cell: Role in age-related macular degeneration?

Age-related macular degeneration (AMD) is the most common cause of severe vision loss worldwide. Amyloid β involvement in degenerative diseases such as AMD is well known and its toxicity has been related to P2X7 receptor-pannexin-1. Recently, oxysterols (oxidized derivatives of cholesterol) have been implicated in AMD pathogenesis. The aim of our study was to highlight amyloid β/oxysterols relationship and to describe P2X7 receptor-pannexin-1 role in oxysterols toxicity. Using retinal epithelial cells, we first quantified sterols levels after amyloid β incubation and second we investigated the cytotoxic effects induced by oxysterols. For the first time, our results showed that amyloid β induced oxysterols formation in human retinal pigmented epithelial cells. We showed that oxysterol toxicity is mediated by P2X7 receptor activation. This activation was dependent on pannexin-1 with 25-hydroxycholesterol whereas P2X7 receptor signaling pathway was pannexin-1-independent for 7-ketocholesterol. Taken together our data suggest a pivotal role of P2X7 receptor-pannexin-1 in oxysterols toxicity in retinal cells which could be an important target to develop new treatments for AMD.

Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes

Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3′-, 4′-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3′- and 4′-hydroxy-FAA, thus corresponding to the 3′,4′-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3′,4′-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3′- and 4′-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

Lipid deregulation in UV irradiated skin cells: Role of 25-hydroxycholesterol in keratinocyte differentiation during photoaging

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation.

Changing in lipid profile induced by the mutation of Foxn1 gene: A lipidomic analysis of Nude mice skin.

Nude mice carry a spontaneous mutation affecting the gene Foxn1 mainly expressed in the epidermis. This gene is involved in several skin functions, especially in the proliferation and the differentiation of keratinocytes which are key cells of epithelial barrier. The skin, a protective barrier for the body, is essentially composed of lipids. Taking into account these factors, we conducted a lipidomic study to search for any changes in lipid composition of skin possibly related to Foxn1 mutation. Lipids were extracted from skin biopsies of Nude and BALB/c mice to be analyzed by liquid chromatography coupled to a high resolution mass spectrometer (HRMS). Multivariate and univariate data analyses were carried out to compare lipid extracts. Identification was performed using HRMS data, retention time and mass spectrometry fragmentation study. These results indicate that mutation of Foxn1 leads to significant modifications in the lipidome in Nude mice skin. An increase in cholesterol sulfate, phospholipids, sphingolipids and fatty acids associated with a decrease in glycerolipids suggest that the lipidome in mice skin is regulated by the Foxn1 gene.

New in vitro biomarkers to detect toxicity in human placental cells: The example of benzo[A]pyrene.

Our aim was to study the toxicity of benzo(a)pyrene (BaP), an environmental pollutant that can reach placenta, on two human placental models in order to propose biomarkers in risk assessment for pregnancy. Ex vivo human placental cells isolated from term placenta and JEG-3 cancer cell line were incubated with BaP at 0.1-10 μM for 48 h or 72 h. BaP induced neither loss of cell viability nor apoptosis in ex vivo placental cells. To go further, we performed experiments on JEG-3 cell line that provides near-unlimited cells. The results we obtained in JEG-3 cells confirmed that BaP, in our experimental conditions, is neither necrotic nor apoptotic for placental cells. BaP toxicity on placental cells resulted in cell cycle arrest (G2/M phase) associated with inhibition of cell proliferation. Besides, we observed that BaP remodeled the protein content of membrane microdomains via increased expression of ZO-1, caveolin-1 and P2X7 cell degenerescence receptor. In conclusion, we identified nuclear and membrane potential biomarkers of risks for placenta and then pregnancy. These potential biomarkers detected on placental cell lines could represent useful tools for toxicological studies.

Resveratrol reverses the Warburg effect by targeting the pyruvate dehydrogenase complex in colon cancer cells

Resveratrol (RES), a polyphenol found in natural foods, displays anti-oxidant, anti-inflammatory and anti-proliferative properties potentially beneficial in cancers, in particular in the prevention of tumor growth. However, the rapid metabolism of resveratrol strongly limits its bioavailability. The molecular mechanisms sustaining the potential biological activity of low doses of resveratrol has not been extensively studied and, thus, needs better characterization. Here, we show that resveratrol (10 µM, 48 hr) induces both a cell growth arrest and a metabolic reprogramming in colon cancer cells. Resveratrol modifies the lipidomic profile, increases oxidative capacities and decreases glycolysis, in association with a decreased pentose phosphate activity and an increased ATP production. Resveratrol targets the pyruvate dehydrogenase (PDH) complex, a key mitochondrial gatekeeper of energy metabolism, leading to an enhanced PDH activity. Calcium chelation, as well as the blockade of the mitochondrial calcium uniport, prevents the resveratrol-induced augmentation in oxidative capacities and the increased PDH activity suggesting that calcium might play a role in the metabolic shift. We further demonstrate that the inhibition of the CamKKB or the downstream AMPK pathway partly abolished the resveratrol-induced increase of glucose oxidation. This suggests that resveratrol might improve the oxidative capacities of cancer cells through the CamKKB/AMPK pathway.

25-Hydroxycholesterol induces both P2X7-dependent pyroptosis and caspase-dependent apoptosis in human skin model: New insights into degenerative pathways

Degenerative diseases are characterized by both cell death and inflammation, which involve different pathways such as apoptosis and pyroptosis. Oxysterols, oxidized derivatives of cholesterol, are known to act as key actors in degenerative disorders such as skin photoaging. We hypothesize that oxysterols could be implicated in either apoptosis or pyroptosis, or both. The aim of our study was first to quantify oxysterol levels in keratinocytes as a function of aging and UV irradiation. Second, we evaluated the effects of 25-OH oxysterol on apoptosis and pyroptosis hallmarks in keratinocytes. Our results showed that 25-OH exhibited an increasing after UV irradiation, highlighting the pivotal role of this oxysterol in skin degeneration. In our model, 25-OH induced not only caspases-dependent apoptosis associated to granzyme B release but also P2X7 receptor-dependent pyroptosis in skin cells. 25-OH seems to be at the origin of the main toxic pathways responsible for degenerative disorders; therefore, it could be the target of antidegenerative treatments, opening new potential therapeutic strategies.

Synthesis of S-mono- and S,O-bis-1,2,3-triazole linked 1,5-benzodiazepine conjugates and evaluation of their cytotoxic, anti-tyrosinase, and anti-cholinesterase activities

GRAPHICAL ABSTRACT ABSTRACT A series of S-mono and S,O-bis-1,2,3-triazole linked 1,5-benzodiazepines (BZD) conjugates 6a-j and 7a-j were synthesized employing “click chemistry” under microwave irradiation. The synthesized derivatives have been evaluated for their cytotoxic (MCF-7, HeLa, A549 cell lines), anti-tyrosinase and anti-cholinesterase activities. These molecules exhibited moderate to good biological activity.

Weak protein-cationic co-ion interactions addressed by X-ray crystallography and mass spectrometry.

The adsorption of Rb(+), Cs(+), Mn(2+), Co(2+) and Yb(3+) onto the positively charged hen egg-white lysozyme (HEWL) has been investigated by solving 13 X-ray structures of HEWL crystallized with their chlorides and by applying electrospray ionization mass spectrometry (ESI-MS) first to dissolved protein crystals and then to the protein in buffered salt solutions. The number of bound cations follows the order Cs(+) < Mn(2+) ≃ Co(2+) < Yb(3+) at 293 K. HEWL binds less Rb(+) (qtot = 0.7) than Cs(+) (qtot = 3.9) at 100 K. Crystal flash-cooling drastically increases the binding of Cs(+), but poorly affects that of Yb(3+), suggesting different interactions. The addition of glycerol increases the number of bound Yb(3+) cations, but only slightly increases that of Rb(+). HEWL titrations with the same chlorides, followed by ESI-MS analysis, show that only about 10% of HEWL binds Cs(+) and about 40% binds 1-2 Yb(3+) cations, while the highest binding reaches 60-70% for protein binding 1-3 Mn(2+) or Co(2+) cations. The binding sites identified by X-ray crystallography show that the monovalent Rb(+) and Cs(+) preferentially bind to carbonyl groups, whereas the multivalent Mn(2+), Co(2+) and Yb(3+) interact with carboxylic groups. This work elucidates the basis of the effect of the Hofmeister cation series on protein solubility.