Bovine Lactoferrin-Induced CCL1 Expression Involves Distinct Receptors in Monocyte-Derived Dendritic Cells and Their Monocyte Precursors.

Lactoferrin (LF) exhibits a wide range of immunomodulatory activities including modulation of cytokine and chemokine secretion. In this study, we demonstrate that bovine LF (bLF) up-modulates, in a concentration- and time-dependent manner, CCL1 secretion in monocytes (Mo) at the early stage of differentiation toward dendritic cells (DCs), and in fully differentiated immature Mo-derived DCs (MoDCs). In both cell types, up-modulation of CCL1 secretion is an early event following bLF-mediated enhanced accumulation of CCL1 transcripts. Notably, bLF-mediated up-regulation of CCL1 involves the engagement of distinct surface receptors in MoDCs and their Mo precursors. We show that bLF-mediated engagement of CD36 contributes to CCL1 induction in differentiating Mo. Conversely, toll-like receptor (TLR)2 blocking markedly reduces bLF-induced CCL1 production in MoDCs. These findings add further evidence for cell-specific differential responses elicited by bLF through the engagement of distinct TLRs and surface receptors. Furthermore, the different responses observed at early and late stages of Mo differentiation towards DCs may be relevant in mediating bLF effects in specific body districts, where these cell types may be differently represented in physiopathological conditions.

Linking estrogen receptor ß expression with inflammatory bowel disease activity.

Crohn disease (CD) and ulcerative colitis (UC) are chronic forms of inflammatory bowel disease (IBD) whose pathogenesis is only poorly understood. Estrogens have a complex role in inflammation and growing evidence suggests that these hormones may impact IBD pathogenesis. Here, we demonstrated a significant reduction (p < 0.05) of estrogen receptor (ER)ß expression in peripheral blood T lymphocytes from CD/UC patients with active disease (n = 27) as compared to those in remission (n = 21) and healthy controls (n = 29). Accordingly, in a subgroup of CD/UC patients undergoing to anti-TNF-α therapy and responsive to treatment, ERß expression was higher (p < 0.01) than that observed in not responsive patients and comparable to that of control subjects. Notably, ERß expression was markedly decreased in colonic mucosa of CD/UC patients with active disease, reflecting the alterations observed in peripheral blood T cells. ERß expression inversely correlated with interleukin (IL)-6 serum levels and exogenous exposure of both T lymphocytes and intestinal epithelial cells to this cytokine resulted in ERß downregulation. These results demonstrate that the ER profile is altered in active IBD patients at both mucosal and systemic levels, at least in part due to IL-6 dysregulation, and highlight the potential exploitation of T cell-associated ERß as a biomarker of endoscopic disease activity.

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

Cellular effects of extremely low frequency (ELF) electromagnetic fields.

PURPOSE: The major areas of research that have characterised investigation of the impact of extremely low frequency (ELF) electromagnetic fields on living systems in the past 50 years are discussed. In particular, selected studies examining the role of these fields in cancer, their effects on immune and nerve cells, and the positive influence of these ELF fields on bone and nerve cells, wound healing and ischemia/reperfusion injury are explored. CONCLUSIONS: The literature indicates that there is still no general agreement on the exact biological detrimental effects of ELF fields, on the physical mechanisms that may be behind these effects or on the extent to which these effects may be harmful to humans. Nonetheless, the majority of the in vitro experimental results indicate that ELF fields induce numerous types of changes in cells. Whether or not the perturbations observed at the cellular level can be directly extrapolated to negative effects in humans is still unknown. However, the myriad of effects that ELF fields have on biological systems should not be ignored when evaluating risk to humans from these fields and, consequently, in passing appropriate legislation to safeguard both the general public and professionally-exposed workers. With regard to the positive effects of these fields, the possibility of testing further their efficacy in therapeutic protocols should also not be overlooked.

Hypoxia increases adhesion and spreading of MG-63 three-dimensional tumor spheroids.

The effects of hypoxia on adhesion and spreading of MG-63 human osteosarcoma spheroids were investigated. Hypoxia was induced in 2-day-old, small spheroids and verified by HIF-1alpha expression. Changes in adhesion were examined on both tissue culture plates and plates coated with fibronectin or collagen while spreading was analyzed in cocultures of MG-63 spheroids seeded on primary fibroblasts grown as a monolayer. In order to better distinguish the two different cell types, MG-63 cells were previously stably transfected with the green fluorescent protein EGFP-vector. Changes in the expression of molecules involved in tumor adhesion and spreading, such as two key integrins (fibronectin receptor, alpha5, and collagen receptor, alpha2) and fibronectin were also examined. The results indicate that hypoxia increases adhesion of spheroids and enhances their ability to spread into the surrounding fibroblast cell culture. These changes in adhesion and spreading are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins and fibronectin.

Metabolomics using 1H-NMR of apoptosis and Necrosis in HL60 leukemia cells: differences between the two types of cell death and independence from the stimulus of apoptosis used.

High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.

Three-dimensional cell organization leads to a different type of ionizing radiation-induced cell death: MG-63 monolayer cells undergo mitotic catastrophe while spheroids die of apoptosis.

The type of cell death occurring in the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as three-dimensional spheroids after exposure to 5 Gy of ionizing radiation was determined. Morphological analyses using the chromatin dye Hoechst 33258 demonstrated that spheroids showed the typical characteristics of apoptosis, while monolayer cells revealed those typical of mitotic catastrophe. In order to better characterize these two types of cell death, the role of caspases was examined in irradiated monolayer cells and spheroids using the broad spectrum caspase inhibitor zVAD-fmk. Death in monolayer cells was caspase-independent, whereas spheroid death was characterized by caspase dependence. Members of the Bcl-2 family of proteins and survivin involved in cell death processes were also studied by Western blot analysis. The pro-apoptotic protein Bax increased in spheroids, whereas this protein remained unchanged in monolayer cells after the same 5-Gy irradiation. The anti-apoptotic protein Bcl-2, on the other hand, remained unchanged in both monolayer cells and spheroids. Finally, survivin increased significantly after irradiation in both cells in monolayer and spheroids. The results presented suggest that three-dimensional cell organization leads to a different type of cell death after exposure to ionizing radiation. Thus, the use of spheroids, a cell model which mimics in vivo solid tumors more closely than cells grown in monolayer, is more appropriate when investigating the effects of antineoplastic treatments such as ionizing radiation.

Temporal dynamics of 1H-NMR-visible metabolites during radiation-induced apoptosis in MG-63 human osteosarcoma spheroids.

The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.

Hypoxia and ionizing radiation: changes in adhesive properties and cell adhesion molecule expression in MG-63 three-dimensional tumor spheroids.

The effects of chemically induced hypoxia and ionizing radiation on the adhesive properties of MG-63 human osteosarcoma three-dimensional spheroids were investigated. Hypoxia was induced by addition of CoCl2 to small, nonhypoxic spheroids and verified by HIF-1alpha expression. In addition, the possible role of important cell adhesion molecules involved in tumor dissemination in inducing adhesive changes were also studied. In particular, two key integrins (i.e., the alpha chain of the fibronectin receptor, alpha5, and the alpha chain of the collagen receptor, alpha2), an important member of the immunoglobulin superfamily (CD54 or ICAM-1) and the strategic molecule CD44 (H-CAM, the principal receptor for hyaluronan) were examined. Because of the important role of fibronectin in adhesive processes, variations in this extracellular matrix component were also examined. The results seem to indicate that CoCl2-induced hypoxia greatly increases adhesion of MG-63 spheroids to both tissue culture plates and plates coated with fibronectin or collagen when compared to controls, while ionizing radiation induces a great decrease in this attachment. Furthermore, chemically induced hypoxia also partially inhibits the effects of ionizing radiation. The data also show that these adhesive changes are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins, CD44, and CD54 and fibronectin.

1H-NMR evidence for a different response to the same dose (2 Gy) of ionizing radiation of MG-63 human osteosarcoma cells and three-dimensional spheroids.

High resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to examine the response of the MG-63 osteosarcoma cell line grown in monolayer and as 3-dimensional tumor spheroids to the same low dose (2 Gy) of ionizing radiation. The MG-63 cells and spheroids were irradiated at 24 h of growth and the 1H-NMR spectra of whole control and irradiated monolayer cells and of whole control and irradiated multicellular spheroids collected after another 24 h were compared. The 1H-NMR spectra of the perchloric acid extracts as well as the 2-dimensional 1H-NMR spectra of both pairs of cell systems were also obtained. Possible radiation-induced cell damage was determined by lactate dehydrogenase (LDH) release and variations in cell growth, while cell death was evaluated by chromatin dye Hoechst staining and DNA fragmentation assays. The results demonstrated that no cell damage took place, but that significant variations in numerous metabolites occured in both the monolayer cells and the spheroids after irradiation. Most of the changes observed were very similar in nature. In fact, significant increases in lactate, alanine, creatine and phosphocreatine and choline-containing metabolites and a significant decrease in glutathione (GSH) were observed in both cells and spheroids. However, while significant increases in CH2 and CH3 mobile lipids, glutamine/glutamate, taurine and inositol were seen in the spheroids, no variations in CH2 or CH3 lipids, glutamine/glutamate or taurine were recorded in the MG-63 cells grown in monolayer after irradiation. In addition, a significant decrease rather than a significant increase in inositol was also noted in the monolayer cells. The data presented seem to suggest that, although neither monolayer cells nor spheroids show apparent signs of damage after exposure to the same dose of ionizing radiation, very different cell death responses as well as very diverse antioxidant/osmoregulatory reactions were triggered by this stressing agent.

Increases in 1H-NMR mobile lipids are not always associated with overt apoptosis: evidence from MG-63 human osteosarcoma three-dimensional spheroids exposed to a low dose (2 Gy) of ionizing radiation.

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.

Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation.

One of the most important questions in tumor biology is the understanding of the mechanisms responsible for the resistance of cancer cells to radiotherapy. In the present study, the possible role played by cell-cell interactions in determining the response of tumor cells to ionizing radiation was investigated. HT-29 colon adenocarcinoma spheroids were irradiated with a dose of 15 Gy in two different stages of growth characterized by diverse degrees of compaction: loosely organized spheroids (early spheroids) and compacted spheroids (late spheroids). Morphological analyses demonstrated that late spheroids were less damaged than early spheroids. Moreover, analyses of the cell cycle and cell death showed that ionizing radiation induced necrosis in early spheroids and apoptosis in late ones. From these results it can be concluded that late, compacted spheroids are more resistant to ionizing radiation than early, loose spheroids. In order to understand the mechanisms regulating this compaction-induced resistance of late spheroids, E-cadherin/beta-catenin complex expression and distribution were analyzed. In late spheroids, E-cadherin/beta-catenin complexes were shown to be tethered to the cytoskeleton, and since this organization has been demonstrated to strengthen cell-cell adhesion in other systems, it can be postulated that the same is true in HT-29 spheroids. In conclusion, it can be hypothesized that compaction of HT-29 spheroids is mediated by the reorganization of E-cadherin/beta-catenin complexes on the plasma membrane and that this compaction may be responsible for the increase in resistance of HT-29 spheroids to ionizing radiation.

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.

Environmental fine particulate matter (PM 2.5) activates the RAW 264.7 macrophage cell line even at very low concentrations as revealed by 1H NMR.

Because of the association between inhalation of airborne particulate matter (PM) and human respiratory and cardiovascular disease, it is necessary to understand the tissue damage induced by these particles. One of the cell types principally involved in the body's reaction to PM are macrophages, which remove particles in the airway passages and the lungs through phagocytosis. In fact, when macrophages are exposed to a toxic agent such as PM, they undergo a series of changes (including variations in morphology, an increase in glycolysis, and consequent lactate production and the release of cytokines such as interleukin-6 and tumor necrosis factor-alpha) necessary to transform them from "resting" to "activated" macrophages. Because (1)H NMR is extremely useful in monitoring, noninvasively, macrophage metabolism and because this technique has never been utilized to examine macrophage activation after exposure to PM, it was the purpose of the present study to investigate the effects of PM exposure on the RAW 264.7 stabilized macrophage cell line using (1)H NMR spectroscopy. PM with a diameter <2.5 microm (PM 2.5) was utilized because a closer association to mortality and adverse respiratory health effects has been found with this fraction than with particles of a larger size. Measurements were conducted on whole cells at both 500 and 700 MHz as well as on perchloric acid extracts at 700 MHz. Significant variations in numerous metabolites were seen at very low concentrations of PM 2.5. Many of these changes point to activation of RAW 264.7 macrophages even at doses of PM 2.5 much lower than those commonly employed in cell studies. These results are particularly significant since the same concentrations of PM did not induce changes in morphology and release of cytokines in these cells. Therefore, (1)H NMR spectroscopy is an extremely sensitive probe in observing subtle variations in macrophages after exposure to PM 2.5.

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study.

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.

Influence of thiol balance on micellar cholesterol handling by polarized Caco-2 intestinal cells.

The in vitro thiol redox modulation of cholesterol homeostasis was investigated in polarized Caco-2 intestinal cells. Cells were pre-incubated with the pro-oxidant compound CuSO4 or with the antioxidant N-acetylcysteine (NAC), to induce a mild shift of the intracellular redox potential toward, respectively, a more oxidizing or a more reducing equilibrium, via a manipulation of intracellular soluble thiols (glutathione). Then, monolayers were exposed to micellar cholesterol and both the cholesterol uptake and export, as well as the cholesteryl ester cycle, were analyzed. We found that pro-oxidizing conditions induced a significant cholesterol retention within the cells, particularly in the unesterified form (FC), as a result of an augmented sterol incorporation coupled with a reduced rate of FC esterification. A reduction in FC export was also evident. Furthermore, the combination of FC retention and the oxidative imbalance leads to significant alterations of the monolayer integrity, evidenced by both the enhanced tight junction permeability and the lactate dehydrogenase release into the basolateral medium. In contrast, a more reducing environment generated by NAC pre-treatment favors the limitation of the resident time of FC into the cells, via a reduced cholesterol uptake and a concomitant increased cholesterol esterification. In addition, a significant higher FC extrusion into the basolateral medium was also appreciable. Our results indicate that the thiol balance of intestinal cells may be critical for the regulation of cholesterol homeostasis at the intestinal level, influencing the lipid transport throughout the intestinal barrier.

Induction of apoptosis or necrosis by ionizing radiation is dose-dependent in MG-63 osteosarcoma multicellular spheroids.

This study investigates cell growth and death (apoptosis and necrosis) in actively proliferating MG-63 osteosarcoma spheroids in response to two doses of ionizing radiation (5 and 30 Gy). Cell growth was examined by growth curves and by cell cycle analyses utilizing flow cytometry. Death was examined in both disaggregated and whole spheroids by the chromatin dye Hoechst and by Western blot analysis of the bcl-2 family of proteins known to be involved in the apoptotic process. The results indicate that after exposure to 5 Gy MG-63 spheroids die by apoptosis, while after exposure to 30 Gy they die by necrosis. The analysis of the bcl-2 family of proteins demonstrates that bcl-2, bax and bcl-XL are involved in triggering apoptosis in spheroids exposed to 5 Gy. The possibility of studying the mechanisms responsible for radiation-induced cell death in three-dimensional spheroids (which represent much more closely in vivo tumors) is extremely important if the foundations for more selective and controllable clinical radiotherapeutic protocols are to be laid.

Effects of a 50 Hz sinusoidal magnetic field on cell adhesion molecule expression in two human osteosarcoma cell lines (MG-63 and Saos-2).

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 0.5 mT can induce variations in the expression of cell adhesion molecules (CAMs) in two human osteosarcoma cell lines (MG-63 and Saos-2) was investigated. In particular, the expression of two important integrins, VLA-2, the receptor for collagen, and VLA-5, the receptor for fibronectin, as well as CD44, were examined in both cell lines after these had been exposed for 7 and 14 days to a 50 Hz, 0.5 mT field. Cell surface morphology (scanning electron microscopy), cell growth characteristics (growth curves and cell cycle phase distribution), and cell death (necrosis and apoptosis) were also examined. The results demonstrate that no variations in surface morphology and cell death occurred between control and exposed cells in both MG-63 and Saos-2 cells, while significant changes were noted in cell growth and fibronectin and CD44 expression in MG-63 cells. The results are discussed in view of the important role that CAMs play in controlling various cancer cell functions, particularly proliferation and metastasis.

Differential control of cholesterol and fatty acid biosynthesis in sensitive and multidrug-resistant LoVo tumor cells.

Multidrug resistance (MDR) describes the decrease in sensitivity of tumor cells to a wide variety of cytotoxic compounds. Although a central role has been ascribed to the P-glycoprotein (Pgp) pump in MDR, lipids also appear to be extremely important. However, their precise role in MDR is not yet fully understood. It was the aim of the present paper to gain a deeper understanding of intracellular lipid equilibrium in both sensitive and MDR tumor cells. In particular, intracellular cholesterol biosynthesis and cholesterol esterification were examined in LoVo-sensitive and Pgp-overexpressing resistant cells. The data presented seem to suggest that the higher synthesis of cholesteryl ester and triglyceride observed in resistant with respect to wild-type cells is due to a greater production of fatty acids in these cells. The results are discussed in view of the possible roles of sterol regulatory element-binding proteins and Pgp in these phenomena.