Jens Z. Pedersen

Nanotechnology promises a revolution in pharmacology to improve or create ex novo therapies. Cerium oxide nanoparticles (nanoceria), well-known as catalysts, possess an astonishing pharmacological potential due to their antioxidant properties, deriving from a fraction of Ce(3+) ions present in CeO(2). These defects, compensated by oxygen vacancies, are enriched at the surface and therefore in nanosized particles. Reactions involving redox cycles between the Ce(3+) and Ce(4+) oxidation states allow nanoceria to react catalytically with superoxide and hydrogen peroxide, mimicking the behavior of two key antioxidant enzymes, superoxide dismutase and catalase, potentially abating all noxious intracellular reactive oxygen species (ROS) via a self-regenerating mechanism. Hence nanoceria, apparently well tolerated by the organism, might fight chronic inflammation and the pathologies associated with oxidative stress, which include cancer and neurodegeneration. Here we review the biological effects of nanoceria as they emerge from in vitro and in vivo studies, considering biocompatibility and the peculiar antioxidant mechanisms.

Antioxidant therapy is the novel frontier to prevent and treat an impressive series of severe human diseases, and the search for adequate antioxidant drugs is fervent. Cerium oxide nanoparticles (nanoceria) are redox-active owing to the coexistence of Ce(3+) and Ce(4+) oxidation states and to the fact that Ce(3+) defects, and the compensating oxygen vacancies, are more abundant at the surface. Nanoceria particles exert outstanding antioxidant effects in vivo acting as well-tolerated anti-age and anti-inflammatory agents, potentially being innovative therapeutic tools. However, the biological antioxidant mechanisms are still unclear. Here, the analysis on two leukocyte cell lines undergoing apoptosis via redox-dependent or independent mechanisms revealed that the intracellular antioxidant effect is the direct cause of the anti-apoptotic and prosurvival effects of nanoceria. Doping with increasing concentrations of Sm(3+), which progressively decreased Ce(3+) without affecting oxygen vacancies, blunted these effects, demonstrating that Ce(3+)/Ce(4+) redox reactions are responsible for the outstanding biological properties of nanoceria.

BACKGROUND: Increasing evidence suggests that thyroid hormones, L-thyroxine (T(4)) and 3,3',5-triiodo-L-thyronine (T(3)), are modulators of the immune response. In monocytes, macrophages, leukocytes, natural killer cells, and lymphocytes, a wide range of immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species (ROS), and cytokine synthesis and release are altered under hypo- and hyperthyroid conditions. SUMMARY: Hyperthyroidism decreases the proinflammatory activities of monocytes and macrophages, whereas enhancement of phagocytosis and increased levels of ROS may occur during hypothyroidism. The expression of proinflammatory molecules such as macrophage inflammatory protein-1α and interleukin-1β increases in hypothyroidism. However, in Kupffer cells, proinflammatory activities such as the respiratory burst, nitric oxide synthase activity, and tumor necrosis factor-α expression may result from increased T(3) levels. Thyroid hormones also affect natural killer cell activity and cell-mediated immune responses. Still, for many immune cells no clear correlation has been found so far between abnormally high or low T(3) or T(4) levels and the effects observed on the immune responses. CONCLUSIONS: In this review we outline the contributions of thyroid hormones to different aspects of innate and adaptive immune responses. The relationship between thyroid hormones and immune cells is complex and T(3) and T(4) may modulate immune responses through both genomic and nongenomic mechanisms. Future studies of the molecular signaling mechanisms involved in this cross-talk between thyroid hormones and the immune system may support development of new strategies to improve clinical immune responses.

We characterized the pro-apoptotic activity of two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine: (Cu(isapn)) and (Cu(isaepy)2), respectively. We demonstrated that these compounds trigger apoptosis via the mitochondrial pathway. The early induction of the p53/p21 system indicates a role for p53 in cell death, however, experiments carried out with small interfering RNA against p53, or with cells lacking p53, support that a p53-independent mechanism can also occur. The extent of apoptosis mirrors the kinetics of intracellular copper uptake. Particularly, Cu(isaepy)2 enters the cells more efficiently and specifically damages nuclei and mitochondria, as evidenced by atomic absorption analysis of copper content and by the extent of nuclear and mitochondrial integrity. Conversely, Cu(isapn), although less permeable, induces a wide-spread oxidative stress, as demonstrated by analyses of reactive oxygen species concentration, and oxidation of proteins and lipids. The increase of the antioxidant defense, through the overexpression of Cu,Zn-SOD, partially counteracts cell death; whereas retinoic acid-mediated differentiation completely rescues cells from apoptosis induced by both compounds. The activation of JNK- and Akt-mediated phosphorylative pathways has been found to be not functional for apoptosis induction. On the contrary, apoptosis significantly decreased when the analogous zinc complex was used or when Cu(isaepy)2 was incubated in the presence of a copper chelator. Altogether, our data provide evidence for a dual role of these copper(II) complexes: they are able to vehicle copper into the cell, thus producing reactive oxygen species, and could behave as delocalized lipophilic cation-like molecules, thus specifically targeting organelles. We characterized the pro-apoptotic activity of two new synthesized isatin-Schiff base copper(II) complexes, obtained from isatin and 1,3-diaminopropane or 2-(2-aminoethyl)pyridine: (Cu(isapn)) and (Cu(isaepy)2), respectively. We demonstrated that these compounds trigger apoptosis via the mitochondrial pathway. The early induction of the p53/p21 system indicates a role for p53 in cell death, however, experiments carried out with small interfering RNA against p53, or with cells lacking p53, support that a p53-independent mechanism can also occur. The extent of apoptosis mirrors the kinetics of intracellular copper uptake. Particularly, Cu(isaepy)2 enters the cells more efficiently and specifically damages nuclei and mitochondria, as evidenced by atomic absorption analysis of copper content and by the extent of nuclear and mitochondrial integrity. Conversely, Cu(isapn), although less permeable, induces a wide-spread oxidative stress, as demonstrated by analyses of reactive oxygen species concentration, and oxidation of proteins and lipids. The increase of the antioxidant defense, through the overexpression of Cu,Zn-SOD, partially counteracts cell death; whereas retinoic acid-mediated differentiation completely rescues cells from apoptosis induced by both compounds. The activation of JNK- and Akt-mediated phosphorylative pathways has been found to be not functional for apoptosis induction. On the contrary, apoptosis significantly decreased when the analogous zinc complex was used or when Cu(isaepy)2 was incubated in the presence of a copper chelator. Altogether, our data provide evidence for a dual role of these copper(II) complexes: they are able to vehicle copper into the cell, thus producing reactive oxygen species, and could behave as delocalized lipophilic cation-like molecules, thus specifically targeting organelles. In the last years synthesis and characterization of novel anti-tumor compounds have represented a field of research that has aroused expectations for more specific and less toxic therapies. Besides DNA and cellular replication, which so far represented the principal targets of cancer treatment, other intracellular compartments and other cell functions, as well as the microenvironment of cancer cells, have become the targets of new and more specific therapies (1Blagosklonny M.V. Pardee A.B. Cancer Res. 2001; 61: 4301-4305PubMed Google Scholar, 2Shapiro G.I. J. Clin. Oncol. 2006; 24: 1770-1783Crossref PubMed Scopus (857) Google Scholar, 3Townsend D.M. Findlay V.L. Tew K.D. Methods Enzymol. 2005; 401: 287-307Crossref PubMed Scopus (98) Google Scholar, 4Kalinowski D.S. Richardson D.R. Pharmacol. Rev. 2005; 57: 547-583Crossref PubMed Scopus (617) Google Scholar, 5Lyko F. Brown R. J. Natl. Cancer Inst. 2005; 97: 1498-1506Crossref PubMed Scopus (421) Google Scholar, 6Wilson L. Jordan M.A. J. 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Copper is a micronutrient essential for cell survival because it functions as cofactor of several metalloenzymes (e.g. Cu,Zn-SOD 4The abbreviations used are: SOD, superoxide dismutase; AIF, apoptosis inducing factor; Cu(isaepy)2, bis-[(2-oxindol-3-yl-imino)-2-(2-aminoethyl)pyridine-N,N′]copper(II); Cu(isapn), [bis-(2-oxindol-3-yl-imino)-1,3-diaminopropane-N,N′,O,O′]copper(II); EPR, electron paramagnetic resonance; JNK, c-Jun N-terminal kinase; PARP, poly(ADP-ribose) polymerase; RA, retinoic acid; ROS, reactive oxygen species; TRIEN, triethylenetetramine; Zn(isaepy), [(2-oxindol-3-yl-imino)-2-(2-aminoethyl)pyridine-N, N′]zinc(II); zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; TES,2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; PBS, phosphate-buffered saline; siRNA, small interfering RNA. and cytochrome c oxidase), but it is also toxic when present at high concentrations (20Gaetke L.M. Chow C.K. 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Biochem. 2005; 99: 1433-1440Crossref PubMed Scopus (94) Google Scholar, 30Cerchiaro G. Micke G.A. Tavares M.F.M. Ferriera A.M.D.C. J. Mol. Catalysis A Chem. 2004; 221: 29-39Crossref Scopus (41) Google Scholar). In this study, we have investigated the molecular mechanisms underlying the activation of apoptosis upon treatment with two specific isatin-diimine copper(II) complexes: Cu(isapn), and Cu(isaepy)2, in SH-SY5Y neuroblastoma cells, demonstrating that their pro-apoptotic activity involves the mitochondrial pathway. Because of the different copper coordinations characterizing the two complexes, the molecular events upstream of the execution of apoptosis are peculiar of each compound and seem to be tightly associated with the relative kinetics of copper uptake inside the cells. Materials—Isatin-diimine copper(II) complexes bis-[(2-oxindol-3-yl-imino)-1,3-diaminopropane-N,N′,O,O′]copper(II) perchlorate ([Cu(isapn)](ClO4)2) and [bis-(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N′]copper(II) perchlorate ([Cu(isaepy)2](ClO4)2), named here Cu(isapn) and Cu(isaepy)2, respectively (Fig. 1, A and synthesized as previously G. Micke G.A. Tavares M.F.M. Ferriera A.M.D.C. J. Mol. Catalysis A Chem. 2004; 221: 29-39Crossref Scopus (41) Google Scholar). The analogous complex as Zn(isaepy), was zinc to in the (Fig. and from and was from was from other obtained from Cell neuroblastoma cells SH-SY5Y from the of Cell and in from the by from the Cancer of and in cells with p53 or with a of from the Cancer of and in cell with and the cells at in an of in SH-SY5Y cell lines with obtained as previously M.R. A. E. L. Rotilio G. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). the experiments cells at a of and or of Cu(isapn), Cu(isaepy)2, or was the experiments by the compounds in with a concentration of at in with This concentration was for the experiments because it a of apoptosis at the (29Cerchiaro G. Aquilano K. Filomeni G. Rotilio G. Ciriolo M.R. Ferriera A.M.D.C. J. Inorg. Biochem. 2005; 99: 1433-1440Crossref PubMed Scopus (94) Google Scholar). of to cells. The was used at a concentration of for the of Cu(isapn) and Cu(isaepy)2, and the TRIEN, at a concentration of was the of Cu(isaepy)2 and the A of copper in water was the experiments and to at a concentration of with the specific and the at concentrations of and because our they not to be the of Cu(isapn) or Cu(isaepy)2 and the was to at a concentration of and for of to differentiation of cells, which was by the of the differentiation of Cell and and cells with PBS, and with to analysis by a The of apoptotic cells to I. G. F. C. J. PubMed Scopus Google by of nuclei cells and by a Cell and protein obtained by cells with a on in and and at for at Cell obtained as previously reported G. Aquilano K. Rotilio G. Ciriolo M.R. Antioxid. Redox. Signal. 2005; 7: PubMed Scopus Google Scholar). cells incubated in and of on of and was and cells by in a obtained of at whereas further at a and For cell and each by the of cytochrome c and the of for Cu,Zn-SOD present into the and for data For nuclear nuclear obtained by the cells in and and the nuclear obtained further with by and and c and of used as The specific protein upon specific treatment, was a system with of ROS, and reduced and of the upon of of with by the of to by the with as previously G. Rotilio G. Ciriolo M.R. J. 2003; PubMed Scopus (119) Google Scholar). of intracellular ROS by analyses of protein content as well as and levels as previously G. Aquilano K. Rotilio G. Ciriolo M.R. Cancer Res. 2003; Google Scholar). on at with and they with PBS, with and incubated with c and with and with an and an by and further with an cells also incubated with with PBS, and by mitochondrial cells with of the mitochondrial and with the of cells also incubated with and by of cells with a to and Copper with at copper concentration was by atomic absorption an A with a with and an cell from and by in to at with an with a high sensitivity a and a of to the signal to SH-SY5Y cells with a against the p53 target cells with a which not present with other by a system to the and into of into SH-SY5Y cells was by p53 with and found to be by the of J. Biol. Chem. PubMed Google Scholar). experiments at different as and was by by with Cu(isapn) and Cu(isaepy)2 Cell and the mechanisms by which base copper(II) complexes cell death in tumor cell on the of the previously obtained (29Cerchiaro G. Aquilano K. Filomeni G. Rotilio G. Ciriolo M.R. Ferriera A.M.D.C. J. Inorg. Biochem. 2005; 99: 1433-1440Crossref PubMed Scopus (94) Google Scholar), we the most effective molecules, Cu(isapn) and Cu(isaepy)2 (Fig. 1, A and and used them at a concentration of from analyses of SH-SY5Y cells with Cu(isapn) and Cu(isaepy)2 for and a more effective increase in the apoptotic cells with Cu(isaepy)2 treatment was evidenced (Fig. analyses of p53 and a activation of these two proteins although with different for Cu(isaepy)2 and more for Cu(isapn) with a of induction at and respectively (Fig. that the p53/p21 is as response to cell The mitochondrial of the apoptosis inducing and that mechanism was our not for the mechanism underlying apoptotic we used the to evidenced by analyses of SH-SY5Y cells for with copper complexes, not cytochrome c (Fig. that cytochrome c was efficiently from into the and that the mitochondrial was our analyses of and as well as and that each of the apoptotic program was upon treatment with Cu(isapn) or Cu(isaepy)2 (Fig. In fact, of and PARP, with a of of evidence that a apoptotic response treatment with both copper complexes, we the cells with of the for and Cu(isapn) or that a of cell from the of activation upon treatment with both that apoptosis is the principal mechanism for cell death induction in our the role of p53 in apoptosis we SH-SY5Y cells with against p53 or with a that not present with other analyses of p53 levels that the concentration of the protein decreased we to the cells with Cu(isapn) or Cu(isaepy)2 from that the cells to the and p53 still in analyses show that cells significantly to apoptosis with (Fig. This was also by of that a more of the protein in than in cells (Fig. We also experiments on cells with a for p53 or with an analyses of apoptosis upon treatment with Cu(isapn) or previously for also for the of cells was significantly reduced upon p53 This was further by analyses of (Fig. suggesting that p53 activation to copper a of p53-independent apoptosis was also Cu(isapn) and Cu(isaepy)2 the Cell and the of Cu(isapn) and Cu(isaepy)2 to the cells and the kinetics of their we copper uptake by atomic absorption that with both compounds in a increase of intracellular copper content that a This was when with that obtained with copper used as of cellular of the Cu(isaepy)2 to be more efficiently the cells with to demonstrated a copper uptake and the extent of with Cu(isaepy)2 more and more in inducing cell death than Because the we have synthesized have (23Radulovic S. Tesic Z. Manic S. Curr. Med. Chem. 2002; 9: 1611-1618Crossref PubMed Scopus (49) Google Scholar), we the concentration of Cu(isaepy)2 by the of Cu(isaepy)2 in cell and its relative concentration to of The data that a in the content of copper most due to its uptake by cells, was our these also that the complex was the as of the further both we the experiments at a at which are show these in the concentration of Cu(isaepy)2 was The with Cu(isapn), for which, however, a uptake was not that the of in cell treatment was due to cell uptake of both complexes than their to the different of Cu(isaepy)2 uptake at and Copper could behave as by intracellular redox cycles with oxygen thus generating as the we previously reported that both Cu(isapn) and Cu(isaepy)2 are able to ROS in the presence of with the more than the in generating (29Cerchiaro G. Aquilano K. Filomeni G. Rotilio G. Ciriolo M.R. Ferriera A.M.D.C. J. Inorg. Biochem. 2005; 99: 1433-1440Crossref PubMed Scopus (94) Google Scholar). they still able to ROS in a cell we ROS content by SH-SY5Y cells with analyses in that Cu(isapn) and Cu(isaepy)2 as ROS production with to cells. In Cu(isapn) was more effective as an upstream ROS because the increase of was as early as On the other Cu(isaepy)2 not ROS production to the to an of apoptotic cells, to that this phenomenon could be a of the death damages to both proteins (Fig. and (Fig. as by of and analyses of by respectively. the of this compound in generating ROS was in with the of an of protein as well as and In Cu(isaepy)2 treatment in a oxidative protein We the concentration and the redox of as this represents the most important low molecular in ROS and of treatment with Cu(isapn) and Cu(isaepy)2, both and with an extent the kinetics of copper suggesting that the levels of due to its to copper and be with the intracellular content of copper compounds. Cu(isapn) and on apoptosis was by phosphorylative pathways of oxidative by Cu(isapn) and Cu(isaepy)2, we analysis of levels of the different of protein and protein also known as in the of and and upon treatment with the copper complexes not but the of and Cu(isaepy)2 treatment (Fig. suggesting a role for these proteins in the induction of the cell death because this increase early treatment or activation and ROS production can be as far as Cu(isaepy)2 treatment is the role of in cell response to Cu(isapn) and Cu(isaepy)2, we SH-SY5Y cells with a specific of the upstream and the cells with the copper that of treatment with Cu(isapn) and in a increase of cell analyses of cell that of the Akt-mediated (Fig. and on the of Cu(isapn) and Cu(isaepy)2 suggesting that and activation are with the specific not in cell survival to with Cu(isapn) or Cu(isaepy)2 (Fig. that activation could represent an of the copper which not to the apoptotic of or by SH-SY5Y from Cu(isapn) and so far a different and of the oxidative upon with Cu(isapn) or Cu(isaepy)2, which could the different of the copper complexes Particularly, the could represent a more ROS and the a more toxic and the of oxidative in the execution of we used SH-SY5Y cells with an of the superoxide or by of of with the obtained by of cells and that cells more to both compounds than to different The antioxidant cells be highly to whereas a in cells upon Cu(isaepy)2 treatment was at support to a different of of the two copper complexes, in which ROS production represents a upstream in apoptosis induced by Cu(isapn), but only a role in Cu(isaepy)2 The p53/p21 was also upon both although with different In fact, p53 and highly and of with Cu(isaepy)2 and Cu(isapn), but activation was (Fig. that Cu(isapn) and Cu(isaepy)2 on cell of SH-SY5Y and cells, a by cell of cells not such compounds as toxic or less cells, we induced differentiation with The obtained have a potential for a use of copper complexes in cancer with against both copper A of cells to to cells as in and activation of the p53/p21 system was these (Fig. The increase of and activity not with the of cell found in cells, are some of the induced by and may to cell against and Cu(isapn) and a and and Cu(isaepy)2 are lipophilic compounds that may vehicle copper and the of redox and ROS production to also specific intracellular organelles. We the copper content in cellular of SH-SY5Y cells of which is in data for different with the copper atomic absorption which a increase of copper in nuclear and mitochondrial upon treatment with both with a copper uptake and suggesting a the of the we investigated and nuclei upon treatment with the two copper SH-SY5Y nuclei with an against the which is on DNA and of treatment, the of the of the DNA by Cu(isaepy)2 to a extent by Cu(isapn) treatment, a phenomenon that was further by analyses of nuclear (Fig. we incubated SH-SY5Y cells with a that in cells and that depending upon potential that for Cu(isaepy)2 treatment, a of completely was only in and not a as in cells. to the that although Cu(isaepy)2 less ROS, it is more than Cu(isapn) in inducing because of its to specifically and such as the and further the of copper in we synthesized an analogous complex in which a copper in the Cu(isaepy)2 we incubated the cells with of the copper TRIEN, which is able to copper from the complex and its uptake by the cells. analyses in that upon treatment with (Fig. or by the cells with TRIEN, the extent of cell decreased with to cells, although a of apoptosis was still with to that copper a role in due to its as catalyst of redox however, the isatin-Schiff base could also to the toxicity Cu(isapn) and Cu(isaepy)2 via the in Cell to a pro-apoptotic activity of the more copper complex Cu(isaepy)2, we two other tumor cells, the and the previously with SH-SY5Y cells, we and cells with that that treatment with this copper complex induced apoptosis in both the tumor cell lines with cells more analyses of and PARP, at and further that the mitochondrial represents the for the induction of apoptosis (Fig. the of Cu(isaepy)2 in inducing cell death in different tumor cells, and a of the In this we that two synthesized isatin-Schiff base copper(II) complexes Cu(isapn) and Cu(isaepy)2 are able to apoptosis via the mitochondrial in neuroblastoma SH-SY5Y cells and in other tumor by oxidative and both compounds are of producing such effects, the in the of of pro-apoptotic and oxidative and the extent of cell death on their to the Cu(isapn), although less more to and oxidative stress, whereas easily cell and damages nuclear and mitochondrial compartments at early In with these we that the of a oxidative upon treatment with Cu(isaepy)2 the of a redox activation of the in the events leading to cell

The interdependence between thyroid hormones (THs), namely, thyroxine and triiodothyronine, and immune system is nowadays well-recognized, although not yet fully explored. Synthesis, conversion to a bioactive form, and release of THs in the circulation are events tightly supervised by the hypothalamic-pituitary-thyroid (HPT) axis. Newly synthesized THs induce leukocyte proliferation, migration, release of cytokines, and antibody production, triggering an immune response against either sterile or microbial insults. However, chronic patho-physiological alterations of the immune system, such as infection and inflammation, affect HPT axis and, as a direct consequence, THs mechanism of action. Herein, we revise the bidirectional crosstalk between THs and immune cells, required for the proper immune system feedback response among diverse circumstances. Available circulating THs do traffic in two distinct ways depending on the metabolic condition. Mechanistically, internalized THs form a stable complex with their specific receptors, which, upon direct or indirect binding to DNA, triggers a genomic response by activating transcriptional factors, such as those belonging to the Wnt/β-catenin pathway. Alternatively, THs engage integrin αvβ3 receptor on cell membrane and trigger a non-genomic response, which can also signal to the nucleus. In addition, we highlight THs-dependent inflammasome complex modulation and describe new crucial pathways involved in microRNA regulation by THs, in physiological and patho-physiological conditions, which modify the HPT axis and THs performances. Finally, we focus on the non-thyroidal illness syndrome in which the HPT axis is altered and, in turn, affects circulating levels of active THs as reported in viral infections, particularly in immunocompromised patients infected with human immunodeficiency virus.