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.
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 elucidation of toxicity determinants of multi-walled carbon nanotubes (MWCNT) is still incomplete. Functionalization with carboxyl groups is, however, commonly used to mitigate MWCNT toxicity, ...although the rationale for the mitigating effect has not been fully clarified yet. In this work, two optimized chemical vapor deposition methods were employed to obtain MWCNT of comparable length but different diameter, which were subsequently functionalized. For MWCNT of diameter larger than 40 nm, no detrimental effects on cell viability of macrophages were observed, while mild cytotoxicity was recorded for diameters between 15 and 40 nm, with a mitigating effect of functionalization. To investigate the factors responsible for the mitigation, we used the thinnest MWCNT preparation on different cell models, evaluating several endpoints, such as viability, production of nitric oxide (NO), expression of pro-inflammatory markers, the Trans-Epithelial Electrical Resistance (TEER), and clonogenic activity. Substantial mitigation of the changes caused by pristine MWCNT was observed not only with carboxyl- but also with amino-functionalized MWCNT, suggesting that negative or positive surface charge was not the main factor responsible for the effect. Instead, either functionalized preparation exhibited a stronger tendency to agglomerate that was strictly dependent on the presence of proteins. Moreover, we found that either carboxyl- or amino-functionalized MWCNT adsorbed a larger amount of serum proteins than pristine counterparts, with a distinctive pattern for each type of MWCNT. We propose, therefore, that the formation of larger agglomerates, dependent upon different protein coronae, contributes to mitigate the biological effects of functionalized MWCNT in protein-rich biological media.
It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be ...modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.
Amorphous silica nanoparticles (ASNP) can be synthetized via several processes, 2 of which are the thermal route (to yield pyrogenic silica) and the wet route from a solution containing silicate ...salts (to obtain precipitated, colloidal, mesoporous silica, or silica gel). Both methods of synthesis lead to ASNP that are applied as food additive (E551). Current food regulation does not require that production methods of additives are indicated on the product label, and, thus, the ASNP are listed without mentioning the production method. Recent results indicate, however, that pyrogenic ASNP are more cytotoxic than ASNP synthesized through the wet route. The present study was aimed at clarifying if 2 representative preparations of ASNP, NM-203 (pyrogenic) and NM-200 (precipitated), of comparable size, specific surface area, surface charge, and hydrodynamic radius in complete growth medium, had different effects on 2 murine macrophage cell lines (MH-S and RAW264.7 cells). Our results show that, when incubated in protein-rich fluids, NM-203 adsorbed on their surface more proteins than NM-200 and, once incubated with macrophages, elicited a greater oxidative stress, assessed from Hmox1 induction and ROS production. Flow cytometry and helium ion microscopy indicated that pyrogenic NM-203 interacted with macrophages more strongly than the precipitated NM-200 and triggered a more evident inflammatory response, evaluated with Nos2 induction, NO production and the secretion of TNF-α, IL-6 and IL-1β. Moreover, both ASNP synergized macrophage activation by bacterial lipopolysaccharide (LPS), with a higher effect observed for NM-203. In conclusion, the results presented here demonstrate that, compared to precipitated, pyrogenic ASNP exhibit enhanced interaction with serum proteins and cell membrane, and cause a larger oxidative stress and stronger proinflammatory effects in macrophages. Therefore, these 2 nanomaterials should not be considered biologically equivalent.
Abstract only
In a growing group of human cancers, glutamine (Gln) transport is considered a possible therapeutic target to achieve tumor control. However, Gln transport is mediated by several ...carriers in human cells, thus complicating the delineation of a strategy to obtain an effective intracellular shortage of the amino acid. To evaluate the feasibility of this approach, we have investigated the effects of Gln depletion on two cell lines (Hs683 and HOG) of human oligodendroglioma (OD), a rare brain tumors characterized by lack of expression of Glutamine Synthetase (GS). We have hypothesized that if OD cells do not express GS, they should exhibit a strict dependence upon extracellular Gln and, hence, on the activity of Gln transporters. When cultured under control conditions (DMEM supplemented with 4 mM Gln), OD cells did not express detectable GS (Western Blot) and, at variance with other cell models, GS expression was not enhanced by Gln starvation. Gln‐free incubation or the glutaminolytic enzyme L‐asparaginase, which converts Gln into glutamate and ammonium, completely suppressed cell viability, demonstrating the absolute requirement of OD cells for extracellular Gln. Radiolabeled
3
H‐Gln uptake was characterized under control conditions with or without Na
+
in the presence of specific transporter inhibitors: threonine (Thr) for ASCT2; MeAIB for SNAT1/SNAT2, histidine (His) for SNAT3 and leucine (Leu) for LAT1. All these transporters were found expressed in OD cells at mRNA and protein level. While Gln uptake in HOG cells was mainly Na
+
‐dependent and mostly inhibited by Thr, and thus attributable to ASCT2 activity, Hs683 cells transported Gln mainly through a Na
+
‐independent, Leu‐sensitive transporter, identifiable with LAT1. In both cell lines, GPNA, an amino acid analogue used to inhibit the ASCT2 transporter in several other tumor cell models not only suppressed the Na
+
‐dependent influx, but also affected the Na
+
‐independent uptake, thus behaving as an inhibitor of both ASCT2 and LAT1. In both cell lines, the Na
+
‐dependent influx was completely inhibited combining Thr with His, thus suggesting that SNAT3 also contributes to Gln uptake. Under conditions of amino acid deprivation, a MeAIB‐sensitive Gln transport also emerged, suggesting that during nutritional stress OD cells are able to concentrate Gln through the SNAT1/SNAT2 transporters. The effects of transporter inhibitors on cell viability were also tested. Viability was markedly hindered by GPNA in both OD cell lines, while it was only partially affected by high doses of Thr, His and MeAIB. In conclusion, we found that (1) OD cells are GS‐negative and strictly depend upon extracellular Gln; (2) OD cells display a complex transportome for Gln uptake; (3) the impressive effects of GPNA on OD cell viability should not be attributed only to a specific inhibition of the ASCT2 transporter but also to a shortage of essential amino acids due to LAT1 inhibition. On the basis of these results, depletion of extracellular Gln caused by L‐asparaginase seems a more straightforward approach than transport inhibition for the control of olidendrogliomas and, possibly, other GS‐negative tumors.
Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO
2
nanofibers (TiO
2
NF) cause a dose-dependent decrease of cell ...viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO
2
NF of industrial origin. Long TiO
2
NF (L-TiO
2
NF) were more cytotoxic than their shortened counterparts (S-TiO
2
NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO
2
NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO
2
NF, S-TiO
2
NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO
2
NF but not to S-TiO
2
NF, suggesting that short-term macrophage activation in vitro may not be always a reliable indicator of persistent inflammation in vivo. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence in vitro as well as inflammation development in vivo. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO
2
NF toxic effects.
Length and aspect ratio represent important toxicity determinants of fibrous nanomaterials. We have previously shown that anatase TiO
nanofibers (TiO
NF) cause a dose-dependent decrease of cell ...viability as well as the loss of epithelial barrier integrity in polarized airway cell monolayers. Herein we have investigated the impact of fiber shortening, obtained by ball-milling, on the biological effects of TiO
NF of industrial origin. Long TiO
NF (L-TiO
NF) were more cytotoxic than their shortened counterparts (S-TiO
NF) toward alveolar A549 cells and bronchial 16HBE cells. Moreover, L-TiO
NF increased the permeability of 16HBE monolayers and perturbed the distribution of tight-junction proteins, an effect also mitigated by fiber shortening. Raw264.7 macrophages efficiently internalized shortened but not long NF, which caused cell stretching and deformation. Compared with L-TiO
NF, S-TiO
NF triggered a more evident macrophage activation, an effect suppressed by the phagocytosis inhibitor cytochalasin B. Conversely, a significant increase of inflammatory markers was detected in either the lungs or the peritoneal cavity of mice exposed to L-TiO
NF but not to S-TiO
NF, suggesting that short-term macrophage activation
may not be always a reliable indicator of persistent inflammation
. It is concluded that fiber shortening mitigates NF detrimental effects on cell viability and epithelial barrier competence
as well as inflammation development
. These data suggest that fiber shortening may represent an effective safe-by-design strategy for mitigating TiO
NF toxic effects.
It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be ...modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO
NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO
NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of
by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO
NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account.
Although causing moderate cytotoxicity and inflammatory effects, TiO
2
nanoparticles (NPs) are considered relatively safe materials. However, it is known that TiO
2
NPs bind bioactive environmental ...contaminants, such as bacterial lipopolysaccharide (LPS, endotoxin), and it is possible that this interaction leads to increased biological activity. In this report we have investigated the pro-inflammatory responses of Raw264.7 murine macrophages exposed to two preparations of TiO
2
NPs, co-administered with LPS. The simultaneous exposure to NPs and LPS produced marked increases in
Nos2
mRNA, Nos2 protein and medium nitrite concentration (an indicator of NO production) well beyond the levels observed with LPS or TiO
2
NP alone. TiO
2
NPs also synergized LPS effects on
Ptgs2
expression and cytokine secretion. The cytoskeletal drug cytochalasin B lowered the amount of NPs internalized by the cells and suppressed the synergy between TiO
2
NPs and LPS in NO production and cytokine secretion. Pre-treatment with the TLR4 inhibitors polymyxin B and CLI-095 eliminated the synergy that was also partially hampered by the inhibition of p38 MAPK, but not of ERK1/2. Moreover, p38 phosphorylation was synergistically enhanced by the combined treatment at 6 h of incubation. It is concluded that TiO
2
NPs enhance macrophage activation by LPS
via
a TLR4-dependent mechanism that involves p38 and is mainly triggered from an intracellular site. These findings suggest that the simultaneous exposure to LPS and TiO
2
NPs may exacerbate the inflammatory response
in vivo
.
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