Asparagine Synthetase (ASNS) catalyzes the synthesis of the non-essential amino acid asparagine (Asn) from aspartate (Asp) and glutamine (Gln). ASNS expression is highly regulated at the ...transcriptional level, being induced by both the Amino Acid Response (AAR) and the Unfolded Protein Response (UPR) pathways. Lack of ASNS protein expression is a hallmark of Acute Lymphoblastic Leukemia (ALL) blasts, which, therefore, are auxotrophic for Asn. This peculiarity is the rationale for the use of bacterial L-Asparaginase (ASNase) for ALL therapy, the first example of anti-cancer treatment targeting a tumor-specific metabolic feature. Other hematological and solid cancers express low levels of ASNS and, therefore, should also be Asn auxotrophs and ASNase sensitive. Conversely, in the last few years, several reports indicate that in some cancer types ASNS is overexpressed, promoting cell proliferation, chemoresistance, and a metastatic behavior. However, enhanced ASNS activity may constitute a metabolic vulnerability in selected cancer models, suggesting a variable and tumor-specific role of the enzyme in cancer. Recent evidence indicates that, beyond its canonical role in protein synthesis, Asn may have additional regulatory functions. These observations prompt a re-appreciation of ASNS activity in the biology of normal and cancer tissues, with particular attention to the fueling of Asn exchange between cancer cells and the tumor microenvironment.
Titanium dioxide (TiO2) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO2 nanoparticles have been extensively ...studied, the toxicological characterization of TiO2 nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO2 nanofibres using TiO2 nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO2 nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO2 NP did not decrease cell viability. TiO2 nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO2 nanoparticles or crocidolite. Moreover, TiO2 nanofibres and crocidolite, but not TiO2 nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO2 nanofibres, as well as by TiO2 nanoparticles and crocidolite. This study indicates that TiO2 nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO2 nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO2 nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation.
The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell ...lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138+ cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138+ cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138+ cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.
•Myeloma cells produce ammonium in the presence of glutamine, showing high glutaminase and low glutamine synthetase expression.•Myeloma cells show high expression of glutamine transporters and inhibition of ASCT2 transporter hinders myeloma growth.
l
-γ-Glutamyl-
p
-nitroanilide (GPNA) is widely used to inhibit the glutamine transporter ASCT2, although it is known that it also inhibits other sodium-dependent amino acid transporters. In a panel ...of human cancer cell lines, which express the system
l
transporters LAT1 and LAT2, GPNA inhibits the sodium-independent influx of leucine and glutamine. The kinetics of the effect suggests that GPNA is a low affinity, competitive inhibitor of system
l
transporters. In Hs683 human oligodendroglioma cells, the incubation in the presence of GPNA, but not ASCT2 silencing, lowers the cell content of leucine. Under the same conditions the activity of mTORC1 is inhibited. Decreased cell content of branched chain amino acids and mTORC1 inhibition are observed in most of the other cell lines upon incubation with GPNA. It is concluded that GPNA hinders the uptake of essential amino acids through system
l
transporters and lowers their cell content.
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•In two human cell lines, Cd toxicity varied depending on its form: nano or ionic.•Cells were more sensitive to ionic Cd than to Cd as quantum dots.•HepG2 cells were more sensitive ...than THP-1 but this did not correlate to Cd uptake.•Cell-type and Cd-type responses were correlated with the miRNome.•In silico and in vitro pathway analysis suggests apoptosis (HepG2) or autophagy (THP-1).
Cadmium is toxic to humans, although Cd-based quantum dots exerts less toxicity. Human hepatocellular carcinoma cells (HepG2) and macrophages (THP-1) were exposed to ionic Cd, Cd(II), and cadmium sulfide quantum dots (CdS QDs), and cell viability, cell integrity, Cd accumulation, mitochondrial function and miRNome profile were evaluated.
Cell-type and Cd form-specific responses were found: CdS QDs affected cell viability more in HepG2 than in THP-1; respective IC20 values were ∼3 and ∼50 μg ml−1. In both cell types, Cd(II) exerted greater effects on viability.
Mitochondrial membrane function in HepG2 cells was reduced 70 % with 40 μg ml−1 CdS QDs but was totally inhibited by Cd(II) at corresponding amounts. In THP-1 cells, CdS QDs has less effect on mitochondrial function; 50 μg ml-1 CdS QDs or equivalent Cd(II) caused 30 % reduction or total inhibition, respectively. The different in vitro effects of CdS QDs were unrelated to Cd uptake, which was greater in THP-1 cells.
For both cell types, changes in the expression of miRNAs (miR-222, miR-181a, miR-142-3p, miR-15) were found with CdS QDs, which may be used as biomarkers of hazard nanomaterial exposure. The cell-specific miRNome profiles were indicative of a more conservative autophagic response in THP-1 and as apoptosis as in HepG2.
Over the last decades, cerium oxide nanoparticles (CeO
NPs) have gained great interest due to their potential applications, mainly in the fields of agriculture and biomedicine. Promising effects of ...CeO
NPs are recently shown in some neurodegenerative diseases, but the mechanism of action of these NPs in Parkinson's disease (PD) remains to be investigated. This issue is addressed in the present study by using a yeast model based on the heterologous expression of the human α-synuclein (α-syn), the major component of Lewy bodies, which represent a neuropathological hallmark of PD. We observed that CeO
NPs strongly reduce α-syn-induced toxicity in a dose-dependent manner. This effect is associated with the inhibition of cytoplasmic α-syn foci accumulation, resulting in plasma membrane localization of α-syn after NP treatment. Moreover, CeO
NPs counteract the α-syn-induced mitochondrial dysfunction and decrease reactive oxygen species (ROS) production in yeast cells. In vitro binding assay using cell lysates showed that α-syn is adsorbed on the surface of CeO
NPs, suggesting that these NPs may act as a strong inhibitor of α-syn toxicity not only acting as a radical scavenger, but through a direct interaction with α-syn in vivo.
Oral administration of nanoparticles (NPs) is a promising strategy to overcome solubility and stability issues of many active compounds. However, this route faces major obstacles related to the ...hostile gastrointestinal (GI) environment, which impairs the efficacy of orally administered nanomedicines. Here, we propose nanocomposites as a promising approach to increase the retention time of NPs in the intestinal tract by using bio- and mucoadhesive matrixes able to protect the cargo until it reaches the targeted area. A microfluidic-based approach has been applied for the production of tailored nanoemulsions (NEs) of about 110 nm, used for the encapsulation of small hydrophobic drugs such as the anti-inflammatory JAK-inhibitor tofacitinib. These NEs proved to be efficiently internalized into a mucus-secreting human intestinal monolayer of Caco-2/HT29-MTX cells and to deliver tofacitinib to subepithelial human THP-1 macrophage-like cells, reducing their inflammatory response. NEs were then successfully encapsulated into alginate hydrogel microbeads of around 300 μm, which were characterized by rheological experiments and dried to create a long-term stable system for pharmaceutical applications. Finally, ex vivo experiments on excised segments of rats′ intestine proved the bioadhesive ability of NEs embedded in alginate hydrogels compared to free NEs, showing the advantage that this hybrid system can offer for the treatment of intestinal pathologies.
Multiple millennia of human evolution have shaped the chemical composition of breast milk toward an optimal human body fluid for nutrition and protection and for shaping the early gut microbiota of ...newborns. This biological fluid is composed of water, lipids, simple and complex carbohydrates, proteins, immunoglobulins, and hormones. Potential interactions between hormones present in mother's milk and the microbial community of the newborn are a very fascinating yet unexplored topic. In this context, insulin, in addition to being one of the most prevalent hormones in breast milk, is also involved in a metabolic disease that affects many pregnant women, i.e., gestational diabetes mellitus (GDM). Analysis of 3,620 publicly available metagenomic data sets revealed that the bifidobacterial community varies in relation to the different concentrations of this hormone in breast milk of healthy and diabetic mothers. Starting from this assumption, in this study, we explored possible molecular interactions between this hormone and bifidobacterial strains that represent bifidobacterial species commonly occurring in the infant gut using 'omics' approaches. Our findings revealed that insulin modulates the bifidobacterial community by apparently improving the persistence of the Bifidobacterium bifidum taxon in the infant gut environment compared to other typical infant-associated bifidobacterial species.
Breast milk is a key factor in modulating the infant's intestinal microbiota composition. Even though the interaction between human milk sugars and bifidobacteria has been extensively studied, there are other bioactive compounds in human milk that may influence the gut microbiota, such as hormones. In this article, the molecular interaction of the human milk hormone insulin and the bifidobacterial communities colonizing the human gut in the early stages of life has been explored. This molecular cross talk was assessed using an
gut microbiota model and then analyzed by various omics approaches, allowing the identification of genes associated with bacterial cell adaptation/colonization in the human intestine. Our findings provide insights into the manner by which assembly of the early gut microbiota may be regulated by host factors such as hormones carried by human milk.
Bifidobacteria are among the first microbial colonizers of the human gut, being frequently associated with human health-promoting activities. In the current study, an
methodology based on an ...ecological and phylogenomic-driven approach allowed the selection of a
prototype strain, i.e.,
PRL2023, which best represents the overall genetic content and functional features of the
taxon. Such features were confirmed by
experiments aimed at evaluating the ability of this strain to survive in the gastrointestinal tract of the host and its ability to interact with human intestinal cells and other microbial gut commensals. In this context, co-cultivation of
PRL2023 and several gut commensals revealed various microbe-microbe interactions and indicated co-metabolism of particular plant-derived glycans, such as xylan.IMPORTANCEThe use of appropriate bacterial strains in experimental research becomes imperative in order to investigate bacterial behavior while mimicking the natural environment. In the current study, through
and
methodologies, we were able to identify the most representative strain of the
species. The ability of this strain,
PRL2023, to cope with the environmental challenges imposed by the gastrointestinal tract, together with its ability to switch its carbohydrate metabolism to compete with other gut microorganisms, makes it an ideal choice as a
prototype and a member of the healthy microbiota of adults. This strain possesses a genetic blueprint appropriate for its exploitation as a candidate for next-generation probiotics.
The possibility of counteracting inflammation-related barrier defects with dietary compounds such as (poly)phenols has raised much interest, but information is still scarce. We have investigated here ...if (+)-catechin (CAT) and procyanidin B2 (PB2), two main dietary polyphenols, protect the barrier function of intestinal cells undergoing inflammatory stress. The cell model adopted consisted of co-cultured Caco-2 and HT29-MTX cells, while inflammatory conditions were mimicked through the incubation of epithelial cells with the conditioned medium of activated macrophages (MCM). The epithelial barrier function was monitored through trans-epithelial electrical resistance (TEER), and ROS production was assessed with dichlorofluorescein, while the expression of tight-junctional proteins and signal transduction pathways were evaluated with Western blot. The results indicated that MCM produced significant oxidative stress, the activation of NF-κB and MAPK pathways, a decrease in occludin and ZO-1 expression, and an increase in claudin-7 (CL-7) expression, while TEER was markedly lowered. Neither CAT nor PB2 prevented oxidative stress, transduction pathways activation, ZO-1 suppression, or TEER decrease. However, PB2 prevented the decrease in occludin expression and both polyphenols produced a huge increase in CL-7 abundance. It is concluded that, under the conditions adopted, CAT and PB2 do not prevent inflammation-dependent impairment of the epithelial barrier function of intestinal cell monolayers. However, the two compounds modify the expression of tight-junctional proteins and, in particular, markedly increase the expression of CL-7. These insights add to a better understanding of the potential biological activity of these major dietary flavan-3-ols at intestinal level.
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