Low-Density Lipoproteins Increase Proliferation, Invasion, and Chemoresistance via an Exosome Autocrine Mechanism in MDA-MB-231 Chemoresistant Cells.

Dyslipidemias involving high concentrations of low-density lipoproteins (LDLs) increase the risk of developing triple-negative breast cancer (TNBC), wherein cholesterol metabolism and protein translation initiation mechanisms have been linked with chemoresistance. Doxorubicin (Dox) treatment, a member of the anthracycline family, represents a typical therapeutic strategy; however, chemoresistance remains a significant challenge. Exosomes (Exs) secreted by tumoral cells have been implicated in cell communication pathways and chemoresistance mechanisms; the content of exosomes is an outcome of cellular cholesterol metabolism. We previously induced Dox resistance in TNBC cell models, characterizing a variant denominated as variant B cells. Our results suggest that LDL internalization in parental and chemoresistant variant B cells is associated with increased cell proliferation, migration, invasion, and spheroid growth. We identified the role of eIF4F translation initiation factor and the down-regulation of tumor suppressor gene PDCD4, an inhibitor of eIF4A, in chemoresistant variant B cells. In addition, the exomes secreted by variant B cells were characterized by the protein content, electronic microscopy, and cell internalization assays. Critically, exosomes purified from LDL-treated variant B cell promoted cell proliferation, migration, and an increment in lactate concentration. Our results suggest that an autocrine phenomenon induced by exosomes in chemoresistant cells may induce modifications on signaling mechanisms of the p53/Mdm2 axis and activation of p70 ribosomal protein kinase S6. Moreover, the specific down-regulated profile of chaperones Hsp90 and Hsp70 secretion inside the exosomes of the chemoresistant variant could be associated with this phenomenon. Therefore, autocrine activation mediated by exosomes and the effect of LDL internalization may influence changes in exosome chaperone content and modulate proliferative signaling pathways, increasing the aggressiveness of MDA-MB-231 chemoresistant cells.

eIF4A/PDCD4 Pathway, a Factor for Doxorubicin Chemoresistance in a Triple-Negative Breast Cancer Cell Model.

Cells employ several adaptive mechanisms under conditions of accelerated cell division, such as the unfolded protein response (UPR). The UPR is composed of a tripartite signaling system that involves ATF6, PERK, and IRE1, which maintain protein homeostasis (proteostasis). However, deregulation of protein translation initiation could be associated with breast cancer (BC) chemoresistance. Specifically, eukaryotic initiation factor-4A (eIF4A) is involved in the unfolding of the secondary structures of several mRNAs at the 5' untranslated region (5'-UTR), as well as in the regulation of targets involved in chemoresistance. Importantly, the tumor suppressor gene PDCD4 could modulate this process. This regulation might be disrupted in chemoresistant triple negative-BC (TNBC) cells. Therefore, we characterized the effect of doxorubicin (Dox), a commonly used anthracycline medication, on human breast carcinoma MDA-MB-231 cells. Here, we generated and characterized models of Dox chemoresistance, and chemoresistant cells exhibited lower Dox internalization levels followed by alteration of the IRE1 and PERK arms of the UPR and triggering of the antioxidant Nrf2 axis. Critically, chemoresistant cells exhibited PDCD4 downregulation, which coincided with a reduction in eIF4A interaction, suggesting a sophisticated regulation of protein translation. Likewise, Dox-induced chemoresistance was associated with alterations in cellular migration and invasion, which are key cancer hallmarks, coupled with changes in focal adhesion kinase (FAK) activation and secretion of matrix metalloproteinase-9 (MMP-9). Moreover, eIF4A knockdown via siRNA and its overexpression in chemoresistant cells suggested that eIF4A regulates FAK. Pro-atherogenic low-density lipoproteins (LDL) promoted cellular invasion in parental and chemoresistant cells in an MMP-9-dependent manner. Moreover, Dox only inhibited parental cell invasion. Significantly, chemoresistance was modulated by cryptotanshinone (Cry), a natural terpene purified from the roots of Salvia brandegeei. Cry and Dox co-exposure induced chemosensitization, connected with the Cry effect on eIF4A interaction. We further demonstrated the Cry binding capability on eIF4A and in silico assays suggest Cry inhibition on the RNA-processing domain. Therefore, strategic disruption of protein translation initiation is a druggable pathway by natural compounds during chemoresistance in TNBC. However, plasmatic LDL levels should be closely monitored throughout treatment.

LDL Promotes Disorders in ß-Cell Cholesterol Metabolism, Implications on Insulin Cellular Communication Mediated by EVs.

Dyslipidemia is described as a hallmark of metabolic syndrome, promoting a stage of metabolic inflammation (metainflammation) that could lead to misbalances in energetic metabolism, contributing to insulin resistance, and modifying intracellular cholesterol pathways and the renin-angiotensin system (RAS) in pancreatic islets. Low-density lipoprotein (LDL) hypercholesterolemia could disrupt the tissue communication between Langerhans ß-cells and hepatocytes, wherein extracellular vesicles (EVs) are secreted by ß-cells, and exposition to LDL can impair these phenomena. ß-cells activate compensatory mechanisms to maintain insulin and metabolic homeostasis; therefore, the work aimed to characterize the impact of LDL on ß-cell cholesterol metabolism and the implication on insulin secretion, connected with the regulation of cellular communication mediated by EVs on hepatocytes. Our results suggest that ß-cells can endocytose LDL, promoting an increase in de novo cholesterol synthesis targets. Notably, LDL treatment increased mRNA levels and insulin secretion; this hyperinsulinism condition was associated with the transcription factor PDX-1. However, a compensatory response that maintains basal levels of intracellular calcium was described, mediated by the overexpression of calcium targets PMCA1/4, SERCA2, and NCX1, together with the upregulation of the unfolded protein response (UPR) through the activation of IRE1 and PERK arms to maintain protein homeostasis. The LDL treatment induced metainflammation by IL-6, NF-κB, and COX-2 overexpression. Furthermore, LDL endocytosis triggered an imbalance of the RAS components. LDL treatment increased the intracellular levels of cholesterol on lipid droplets; the adaptive ß-cell response was portrayed by the overexpression of cholesterol transporters ABCA1 and ABCG1. Therefore, lipotoxicity and hyperinsulinism induced by LDL were regulated by the natural compound auraptene, a geranyloxyn coumarin modulator of cholesterol-esterification by ACAT1 enzyme inhibition. EVs isolated from ß-cells impaired insulin signaling via mTOR/p70S6Kα in hepatocytes, a phenomenon regulated by auraptene. Our results show that LDL overload plays a novel role in hyperinsulinism, mechanisms associated with a dysregulation of intracellular cholesterol, lipotoxicity, and the adaptive UPR, which may be regulated by coumarin-auraptene; these conditions explain the affectations that occur during the initial stages of insulin resistance.

Lipid Modulation in the Formation of ß-Sheet Structures. Implications for De Novo Design of Human Islet Amyloid Polypeptide and the Impact on ß-Cell Homeostasis.

Human islet amyloid polypeptide (hIAPP) corresponds to a 37-residue hormone present in insulin granules that maintains a high propensity to form ß-sheet structures during co-secretion with insulin. Previously, employing a biomimetic approach, we proposed a panel of optimized IAPP sequences with only one residue substitution that shows the capability to reduce amyloidogenesis. Taking into account that specific membrane lipids have been considered as a key factor in the induction of cytotoxicity, in this study, following the same design strategy, we characterize the effect of a series of lipids upon several polypeptide domains that show the highest aggregation propensity. The characterization of the C-native segment of hIAPP (residues F23-Y37), together with novel variants F23R and I26A allowed us to demonstrate an effect upon the formation of ß-sheet structures. Our results suggest that zwitterionic phospholipids promote adsorption of the C-native segments at the lipid-interface and ß-sheet formation with the exception of the F23R variant. Moreover, the presence of cholesterol did not modify this behavior, and the ß-sheet structural transitions were not registered when the N-terminal domain of hIAPP (K1-S20) was characterized. Considering that insulin granules are enriched in phosphatidylserine (PS), the property of lipid vesicles containing negatively charged lipids was also evaluated. We found that these types of lipids promote ß-sheet conformational transitions in both the C-native segment and the new variants. Furthermore, these PS/peptides arrangements are internalized in Langerhans islet ß-cells, localized in the endoplasmic reticulum, and trigger critical pathways such as unfolded protein response (UPR), affecting insulin secretion. Since this phenomenon was associated with the presence of cytotoxicity on Langerhans islet ß-cells, it can be concluded that the anionic lipid environment and degree of solvation are critical conditions for the stability of segments with the propensity to form ß-sheet structures, a situation that will eventually affect the structural characteristics and stability of IAPP within insulin granules, thus modifying the insulin secretion.

Preparation of Polymeric Films of PVDMA-PEI Functionalized with Fatty Acids for Studying the Adherence and Proliferation of Langerhans ß-Cells.

This study reports the synthesis of thin polymeric films by the layer-by-layer deposition and covalent cross-linking of polyvinyl dimethylazlactone and polyethylene imine, which were functionalized with lauric (12-C), myristic (14-C), and palmitic (16-C) saturated fatty acids, whose high levels in the bloodstream are correlated with insulin resistance and the potential development of type 2 diabetes mellitus. Aiming to assess the effect of the fatty acids on the adhesion and proliferation of Langerhans ß-cells, all prepared films (35 and 35.5 bilayers with and without functionalization with the fatty acids) were characterized in terms of their physical, chemical, and biological properties by a battery of experimental techniques including 1H and 13C NMR, mass spectrometry, attenuated total reflectance-Fourier transform infrared spectroscopy, field emission scanning electron microscopy, atomic force microscopy, cell staining, and confocal laser scanning microscopy among others. In general, the developed films were found to be nanometric, transparent, resistant against manipulation, chemically reactive, and highly cytocompatible. On the other hand, in what the effect of the fatty acids is concerned, palmitic acid was found to impair the proliferation of the cultured ß-cells, contrary to its homologues which did not alter this biological process. In our opinion, the multidisciplinary study presented here might be of interest for the research community working on the development of cytocompatible 2D model substrates for the safe and reproducible characterization of cell responses.

Protein translation associated to PERK arm is a new target for regulation of metainflammation: A connection with hepatocyte cholesterol.

Endoplasmic reticulum stress is a cellular phenomenon that has been associated with metabolic disorders, contributing to the development of obesity, fatty liver disease, and dyslipidemias. Under metabolic overload conditions, in cells with a high protein-secretory activity, such as hepatocytes and Langerhans ß cells, the unfolded protein response (UPR) is critical in to maintain protein homeostasis (proteostasis). UPR integrated by a tripartite signaling system, through activating transcription factor 6, protein kinase R-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1, regulates gene transcription and translation to resolve stress and conserve proteostasis. In the current study, we demonstrated in hepatocytes under metabolic overload by saturated palmitic and stearic fatty acids, through activation of PERK signaling and CCAAT-enhancer-binding protein homologous protein (CHOP) transcription factor, an association with the expression of cyclooxygenase 2. More important, isolated exosomes from supernatants of macrophages exposed to lipopolysaccharides can also induce a metainflammation phenomenon, and when treated on hepatocytes, induced a rearrangement in cholesterol metabolism through sterol regulatory element-binding protein 2 (SREBP2), low-density lipoprotein receptor (LDLR), apolipoprotein A-I, and ABCA1. Moreover, we demonstrate the cellular effect of terpene-derived molecules, such as cryptotanshinone, isolated of plant Salvia brandegeei, regulating metainflammatory conditions through PERK pathway in both hepatocytes and ß cells. Our data suggest the presence of a modulatory mechanism on specific protein translation process. This effect could be mediated by eukaryotic initiation factor-4A, evaluating salubrinal as a control molecule. Likewise, the protective mechanisms of unsaturated fatty acids, such as oleic and palmitoleic acid were confirmed. Therefore, modulation of metainflammation suggests a new target through PERK signaling in cells with a high secretory activity, and possibly the regulation of cholesterol in hepatocytes is promoted via exosomes.

Modulation of Amyloidogenesis Controlled by the C-Terminal Domain of Islet Amyloid Polypeptide Shows New Functions on Hepatocyte Cholesterol Metabolism.

The islet amyloid polypeptide (IAPP) or amylin maintains a key role in metabolism. This 37-residues-peptide could form pancreatic amyloids, which are a characteristic feature of diabetes mellitus type 2. However, some species do not form amyloid fibril structures. By employing a biomimetic approach, we generated an extensive panel of optimized sequences of IAPP, which could drastically reduce aggregation propensity. A structural and cellular characterization analysis was performed on the C-terminal domain with the highest aggregation propensity. This allowed the observation of an aggregative phenomenon dependent of the lipid environment. Evaluation of the new F23R variant demonstrated inhibition of ß-sheet structure and, therefore, amyloid formation on the native C-terminal, phenomenon that was associated with functional optimization in calcium and cholesterol management coupled with the optimization of insulin secretion by beta cells. When F23R variant was evaluated in microglia cells, a model of amyloidosis, cytotoxic conditions were not registered. In addition, it was found that C-terminal sequences of IAPP could modulate cholesterol metabolism in hepatocytes through regulation of SREBP-2, apoA-1, ABCA1, and LDLR, mechanism that may represent a new function of IAPP on the metabolism of cholesterol, increasing the LDL endocytosis in hepatocytes. Optimized sequences with only one residue modification in the C-terminal core aggregation could diminish ß-sheet formation and represent a novel strategy adaptable to other pharmacological targets. Our data suggest a new IAPP function associated with rearrangements on metabolism of cholesterol in hepatocytes.