Mitochondrial Metabolism Drives Low-density Lipoprotein-induced Breast Cancer Cell Migration.

Most cancer-related deaths are due to metastases. Systemic factors, such as lipid-enriched environments [as low-density lipoprotein (LDL)-cholesterol], favor breast cancer, including triple-negative breast cancer (TNBC) metastasis formation. Mitochondria metabolism impacts TNBC invasive behavior but its involvement in a lipid-enriched setting is undisclosed. Here we show that LDL increases lipid droplets, induces CD36 and augments TNBC cells migration and invasion in vivo and in vitro. LDL induces higher mitochondrial mass and network spread in migrating cells, in an actin remodeling-dependent manner, and transcriptomic and energetic analyses revealed that LDL renders TNBC cells dependent on fatty acids (FA) usage for mitochondrial respiration. Indeed, engagement on FA transport into the mitochondria is required for LDL-induced migration and mitochondrial remodeling. Mechanistically, LDL treatment leads to mitochondrial long-chain fatty acid accumulation and increased reactive oxygen species (ROS) production. Importantly, CD36 or ROS blockade abolished LDL-induced cell migration and mitochondria metabolic adaptations. Our data suggest that LDL induces TNBC cells migration by reprogramming mitochondrial metabolism, revealing a new vulnerability in metastatic breast cancer. Significance: LDL induces breast cancer cell migration that relies on CD36 for mitochondrial metabolism and network remodeling, providing an antimetastatic metabolic strategy.

Redox lipid reprogramming commands susceptibility of macrophages and microglia to ferroptotic death.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

Formation of type 4 resistant starch and maltodextrins from amylose and amylopectin upon dry heating: A model study.

Starch is one of the main components of human diet. During food processing, starch is submitted to high temperatures in the presence or absence of water. Thus, the main goal of this work was to identify structural modifications caused by dry heating in starch polysaccharides (amylose and amylopectin) and structurally related oligosaccharides, maltotetraose (M4) and glucosyl-maltotriose (GM3), simulating processing conditions. The structural modifications were evaluated by methylation analysis, electrospray mass spectrometry (ESI-MS), tandem mass spectrometry (ESI-MS/MS) and anionic chromatography after in vitro enzymatic digestion. Dry heating promoted dehydration, depolymerization, as well as changes in Glc glycosidic linkage positions and anomeric configuration. In oligosaccharides, polymerization was also observed. All these changes resulted in a lower in vitro digestibility, suggesting that dry heating of starch polysaccharides and related oligosaccharides may be associated with the formation of type 4 resistant starch and maltodextrins, non-digestible carbohydrates that are responsible for beneficial effects in human intestinal tract.

Characterization of cardiolipins and their oxidation products by LC-MS analysis.

Cardiolipins, a class of mitochondria-specific lipid molecules, is one of the most unusual and ancient phospholipids found in essentially all living species. Typical of mammalian cells is the presence of vulnerable to oxidation polyunsaturated fatty acid resides in CL molecules. The overall role and involvement of cardiolipin oxidation (CLox) products in major intracellular signaling as well as extracellular inflammatory and immune responses have been established. However, identification of individual peroxidized molecular species in the context of their ability to induce specific biological responses has not been yet achieved. This is due, at least in part, to technological difficulties in detection, identification, structural characterization and quantitation of CLox associated with their very low abundance and exquisite diversification. This dictates the need for the development of new methodologies for reliable, sensitive and selective analysis of both CLox. LC-MS-based oxidative lipidomics with high mass accuracy instrumentation as well as new software packages are promising in achieving the goals of expedited and reliable analysis of cardiolipin oxygenated species in biosamples.

Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,ß-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Structural analysis of linear mixed-linkage glucooligosaccharides by tandem mass spectrometry.

Dextrans and glucooligosaccharides (GLOS) are produced by lactic acid bacteria (LAB) during sourdough fermentation. The dextrans can act as hydrocolloids in sourdough bread, while the GLOS may have antistaling and prebiotic properties, depending on their structure. Development of high-throughput methods for screening the structural properties of dextrans and GLOS produced by different LAB in varying fermentation conditions is therefore of interest. In this study we explored the possibility of using electrospray ionisation tandem mass spectroscopy (ESI-MS/MS) to unequivocally determine the structures of underivatised GLOS. The emphasis was on linear mixed linked model GLOS, especially those containing (1→3) linkages that are common in dextrans. After evaluation of the model GLOS, the ESI-MS/MS method was used to determine the linkage positions of two mixed-linked tetrasaccharides obtained by hydrolysis of Weissella confusa and Leuconostoc citreum dextrans. In positive mode, only the reducing end linkage could be determined because isomeric fragment ions, present in subsequent MS(n) cycles, hindered assignment of the remaining linkages. By contrast, it was possible to unambiguously assign all the linkages in each GLOS using the negative mode spectra. The present study thus shows that negative mode is the preferred method for ESI-MS/MS structural analysis of underivatised GLOS. In combination with liquid chromatography this method will enable rapid profiling of the structural variation of dextrans and prebiotic GLOS.