Lipid nanocapsules (LNCs) are promising vehicles for drug delivery. However, since not much was known about cellular toxicity of these nanoparticles in themselves, we have here investigated the ...mechanisms involved in LNC-induced intoxication of the three breast cancer cell lines MCF-7, MDA-MD-231 and MDA-MB-468. The LNCs used were made of Labrafac™ Lipophile WL1349, Lipoid
S75 and Solutol
HS15.
High resolution SIM microscopy showed that the DiD-labeled LNCs ended up in lysosomes close to the membrane. Empty LNCs, i.e. without encapsulated drug, induced not only increased lysosomal pH, but also acidification of the cytosol and a rapid inhibition of protein synthesis. The cytotoxicity of the LNCs were measured for up to 72 h of incubation using the MTT assay and ATP measurements in all three cell lines, and revealed that MDA-MB-468 was the most sensitive cell line and MCF-7 the least sensitive cell line to these LNCs. The LNCs induced generation of reactive free oxygen species and lipid peroxidation. Experiments with knock-down of kinases in the near-haploid cell line HAP1 indicated that the kinase HRI is essential for the observed phosphorylation of eIF2α. Nrf2 and ATF4 seem to play a protective role against the LNCs in MDA-MB-231 cells, as knock-down of these factors sensitizes the cells to the LNCs. This is in contrast to MCF-7 cells where the knock-down of these factors had a minor effect on the toxicity of the LNCs. Inhibitors of ferroptosis provided a large protection against LNC toxicity in MDA-MB-231 cells, but not in MCF-7 cells.
High doses of LNCs showed a different degree of toxicity on the three cell lines studied, i.e. MCF-7, MDA-MD-231 and MDA-MB-468 and affected signaling factors and the cell fate differently in these cell lines.
The ether-lipid precursor sn-1-O-hexadecylglycerol (HG) can be used to compensate for early metabolic defects in ether-lipid biosynthesis. To investigate a possible metabolic link between ...ether-linked phospholipids and the rest of the cellular lipidome, we incubated HEp-2 cells with HG. Mass spectrometry analysis revealed major changes in the lipidome of HG-treated cells compared to that of untreated cells or cells treated with palmitin, a control substance for HG containing an acyl group instead of the ether group. We present quantitative data for a total of 154 species from 17 lipid classes. These species are those constituting more than 2% of their lipid class for most lipid classes, but more than 1% for the ether lipids and glycosphingolipids. In addition to the expected ability of HG to increase the levels of ether-linked glycerophospholipids with 16 carbon atoms in the sn-1 position, this precursor also decreased the amounts of glycosphingolipids and increased the amounts of ceramide, phosphatidylinositol and lysophosphatidylinositol. However, incubation with palmitin, the fatty acyl analogue of HG, also increased the amounts of ceramide and phosphatidylinositols. Thus, changes in these lipid classes were not ether lipid-dependent. No major effects were observed for the other lipid classes, and cellular functions such as growth and endocytosis were unaffected. The data presented clearly demonstrate the importance of performing detailed quantitative lipidomic studies to reveal how the metabolism of ether-linked glycerophospholipids is coupled to that of glycosphingolipids and ester-linked glycerophospholipids, especially phosphatidylinositols.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Animal studies have during the last years revealed a large potential for in vivo imaging with new metal-based nanoparticles and will certainly during the next years also continue to improve ...our understanding of basic biological processes. In the present article we discuss what is needed to bring such non–iron oxide particles into clinical imaging. For imaging agents it is essential to have a rapid clearance from blood so as to obtain low background signals and good images. The surface charge and hydrodynamic diameter of the nanoparticles in the presence of plasma proteins are important for their biodistribution, excretion. and a rapid clearance from blood. As discussed, some major challenges remain to be met regarding safety and metabolism issues. Measurements and optimization of the critical parameters will shorten the time needed for such particles to be accepted for widespread medical use. From the Clinical Editor This review article discusses what is needed to bring non-iron-oxide containing nanoparticles into clinical imaging, including the major challenges regarding safety and metabolism.
Clostridium botulinum C2 toxin is an ADP‐ribosyltransferase, causing depolymerization of the actin cytoskeleton in eukaryotic cells. The C2 toxin is a binary toxin consisting of the enzymatic subunit ...C2I and the binding subunit C2II. Proteolytical activation of the binding subunit triggers the formation of heptameric structures (C2IIa), which bind to cellular receptors. C2I is able to bind to C2IIa oligomers, and it has been suggested that the whole complex is internalized by a raft‐dependent mechanism. Here we analysed by which mechanism C2 toxin is endocytosed. In HeLa cells expressing a dominant‐negative dynamin mutant, cytotoxicity and C2 toxin uptake were blocked. Furthermore, siRNA‐mediated knockdown of flotillins or inhibition of Arf6 function, proteins suggested to be involved in dynamin‐independent endocytosis, did not affect C2 toxicity. Knockdown of caveolin did not inhibit endocytosis of C2 toxin, whereas inhibition of clathrin function reduced the uptake of C2 toxin and delayed the cytotoxic effect. Finally, we found evidence for a Rho‐mediated uptake of C2 toxin. In conclusion, C2 toxin is endocytosed by dynamin‐dependent mechanisms and we provide evidence for involvement of clathrin and Rho.
Cancer immunotherapy represents a promising approach to specifically target and treat cancer. The most common mechanisms by which monoclonal antibodies kill cells include antibody-dependent ...cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis, but also other mechanisms have been described. 14F7 is an antibody raised against the tumor-associated antigen NeuGc GM3, which was previously reported to kill cancer cells without inducing apoptotic pathways. The antibody was reported to induce giant membrane lesions in tumor cells, with apparent changes in the cytoskeleton. Here, we investigated the effect of humanized 14F7 on HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with LC-MS and live cell imaging. 14F7 did not kill the HeLa cells, however, it caused altered protein expression (MS data are available
via
ProteomeXchange with identifier PXD024320). Several cytoskeletal and nucleic-acid binding proteins were found to be strongly down-regulated in response to antibody treatment, suggesting how 14F7 may induce membrane lesions in cells that contain higher amounts of NeuGc GM3. The altered expression profile identified in this study thus contributes to an improved understanding of the unusual killing mechanism of 14F7.
•We here discuss how intravenously injected nanoparticles enter tumours and are taken up by tumour cells.•We discuss how nanoparticles can be transported across the endothelial cell layer.•More ...studies are required to conclude about the contribution of passive and energy dependant mechanisms for this transport.
Studies of how intravenously injected nanoparticles (NPs) enter tumours and are taken up by tumour cells have been discussed for more than three decades. Despite this, we still have much to learn about interactions between NPs and how they can be transported across the endothelial cell layer. More studies are required before firm conclusions can be drawn regarding the contribution of passive or energy dependent mechanisms for transport of NPs over the endothelial cells allowing the NPs to reach solid tumours.
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Here we discuss some pitfalls and challenges briefly when investigating which ...endocytic mechanisms are involved in the cellular uptake of nanoparticles. We specifically discuss some common misunderstandings regarding studies claimed to demonstrate uptake via caveolae. Scientists in the nanomedicine field should be aware that reducing the membrane content of cholesterol by adding methyl-β-cyclodextrin removes caveolae and inhibits other uptake mechanisms, such as macropinocytosis as well. Furthermore, the general tyrosine kinase inhibitor genistein is not a specific inhibitor of uptake from caveolae. Moreover, one can still see that scientists in the field write that they want to direct transport of their particles to caveolae and caveosomes to avoid lysosomal degradation. However, caveosomes are artifacts caused by overexpression of caveolin-1 constructs, and ligands or particles taken up by caveolae are transported to endosomes and lysosomes as reported for other types of endocytosis.
Extracellular vesicles contain a lipid bilayer membrane that protects the encapsulated material, such as proteins, nucleic acids, lipids and metabolites, from the extracellular environment. These ...vesicles are released from cells via different mechanisms. During recent years extracellular vesicles have been studied as possible biomarkers for different diseases, as biological nanoparticles for drug delivery, and in basic studies as a tool to understand the structure of biological membranes and the mechanisms involved in vesicular trafficking. Lipids are essential molecular components of extracellular vesicles, but at the moment our knowledge about the lipid composition and the function of lipids in these vesicles is limited. However, the interest of the research community in these molecules is increasing as their role in extracellular vesicles is starting to be acknowledged. In this review, we will present the status of the field and describe what is needed to bring it forward.
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Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of ...cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-beta-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However, confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus, our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.
The lipid composition of cellular membranes can impact a number of physiological processes such as signaling, cell migration, endocytosis and intracellular transport. In this article we focus on some ...aspects concerning analysis of lipids and research on lipid structure and function in mammalian cells that in our opinion have not obtained sufficient attention. This includes interleaflet coupling between the two layers of the membrane, and the role of lipid species, i.e. the role of the complete structure of the lipids, including lipid chain length and the position of double bonds. We highlight the role of PS species for membrane function. We also discuss the large diversity of PS species in different biological samples and the possible functional consequences, and we provide an overview of PS species from 40 different samples. Furthermore, recent studies show that there seems to be a coregulation concerning the levels of sphingolipids and ether lipids. We review and discuss the published data indicating such a coregulation. Moreover, we point to some of the pitfalls in the field of lipidomics and present suggestions for improvement. Finally, we discuss the importance of using asymmetric membrane models with a composition of lipid species that are common in biological membranes.
•The importance of quantifying both lipid classes and species are highlighted.•The large diversity of PS species in biology is described and discussed.•Data discussed support a coregulation between sphingolipids and ether lipids.•The importance of using asymmetric membrane models is discussed.•We propose some improvements in the software used for identifying lipid species.
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