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Background Quinones are reactive to proteins containing cysteine residues and the main protease in Covid-19 contains an active site that includes Cys145. Embelin, a quinone natural product, is known to have antiviral activity against influenza and hepatitis B. Preliminary studies by our group also indicate its ability to inhibit HSV-1 in cultured cells. Methods Docking and DFT methods applied to the protease target. Results a mechanism for this inhibition of the SARS-CoV-2 Mpro protease is described, specifically due to formation of a covalent bond between S(Cys145) and an embelin C(carbonyl). This is assisted by two protein amino acids (1) N(imidazole-His41) which is able to capture H[S(Cys145)] and (2) HN(His163), which donates a proton to embelin O(carbonyl) forming an OH moiety that results in inhibition of the viral protease. A similar process is also seen with the anti-inflammatory drugs methyl prednisolone and dexamethasone, used for Covid-19 patients. Methyl prednisolone and dexamethasone are methide quinones, and possess only one carbonyl moiety, instead of two for embelin. Additional consideration was given to another natural product, emodin, recently patented against Covid-19, as well as some therapeutic quinones, vitamin K, suspected to be involved in Covid-19 action, and coenzyme Q10. All show structural similarities with embelin, dexamethasone and methyl prednisolone results. Conclusions Our data on embelin and related quinones indicate that these natural compounds may represent a feasible, strategic tool against Covid-19.

The inflammatory protease caspase-1 is associated with the release of cytokines. An excessive number of cytokines (a “cytokine storm”) is a dangerous consequence of COVID-19 infection and has been indicated as being among the causes of death by COVID-19. The anti-inflammatory drug colchicine (which is reported in the literature to be a caspase-1 inhibitor) and the corticosteroid drugs, dexamethasone and methylprednisolone, are among the most effective active compounds for COVID-19 treatment. The SERM raloxifene has also been used as a repurposed drug in COVID-19 therapy. In this study, inhibition of caspase-1 by these four compounds was analyzed using computational methods. Our aim was to see if the inhibition of caspase-1, an important biomolecule in the inflammatory response that triggers cytokine release, could shed light on how these drugs help to alleviate excessive cytokine production. We also measured the antioxidant activities of dexamethasone and colchicine when scavenging the superoxide radical using cyclic voltammetry methods. The experimental findings are associated with caspase-1 active site affinity towards these compounds. In evaluating our computational and experimental results, we here formulate a mechanism for caspase-1 inhibition by these drugs, which involves the active site amino acid Cys285 residue and is mediated by a transfer of protons, involving His237 and Ser339. It is proposed that the molecular moiety targeted by all of these drugs is a carbonyl group which establishes a S(Cys285)–C(carbonyl) covalent bond.

The purpose of this study was to examine the free radical scavenging and antioxidant activities of ellagic acid (EA) and ellagic acid peracetate (EAPA) by measuring their reactions with the radicals, 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl using EPR spectroscopy. We have also evaluated the influence of EA and EAPA on the ROS production in L-6 myoblasts and rat liver microsomal lipid peroxidation catalyzed by NADPH. The results obtained clearly indicated that EA has tremendous ability to scavenge free radicals, even at concentration of 1 µM. Interestingly even in the absence of esterase, EAPA, the acetylated product of EA, was also found to be a good scavenger but at a relatively slower rate. Kinetic studies revealed that both EA and EAPA have ability to scavenge free radicals at the concentrations of 1 µM over extended periods of time. In cellular systems, EA and EAPA were found to have similar potentials for the inhibition of ROS production in L-6 myoblasts and NADPH-dependent catalyzed microsomal lipid peroxidation.

Integrin αvβ3, a cell surface receptor, participates in signaling transduction pathways in cancer cell proliferation and metastasis. Several ligands bind to integrin αvβ3 to regulate proliferation and metastasis in cancer cells. Crosstalk between the integrin and other signal transduction pathways also plays an important role in modulating cancer proliferation. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) activates the downstream integrin FAK to stimulate biological activities including cancer proliferation and metastasis. Blockage of signals related to integrin αvβ3 was shown to be a promising target for cancer therapies. 3,3′,5,5′-tetraiodothyroacetic acid (tetrac) completely binds to the integrin with the thyroid hormone to suppress cancer proliferation. The (E)-stilbene analog, resveratrol, also binds to integrin αvβ3 to inhibit cancer growth. Recently, nanotechnologies have been used in the biomedical field for detection and therapeutic purposes. In the current review, we show and evaluate the potentiation of the nanomaterial carrier RGD peptide, derivatives of PLGA-tetrac (NDAT), and nanoresveratrol targeting integrin αvβ3 in cancer therapies.

The most common cancer, lung cancer, causes deaths worldwide. Most lung cancer patients have non-small cell lung carcinomas (NSCLCs) with a poor prognosis. The chemotherapies frequently cause resistance therefore search for new effective drugs for NSCLC patients is an urgent and essential issue. Deaminated thyroxine, tetraiodothyroacetic acid (tetrac), and its nano-analogue (NDAT) exhibit antiproliferative properties in several types of cancers. On the other hand, the most abundant secondary metabolite in the sponge Hippospongia sp., heteronemin, shows effective cytotoxic activity against different types of cancer cells. In the current study, we investigated the anticancer effects of heteronemin against two NSCLC cell lines, A549 and H1299 cells in vitro. Combined treatment with heteronemin and tetrac derivatives synergistically inhibited cancer cell growth and significantly modulated the ERK1/2 and STAT3 pathways in A549 cells but only ERK1/2 in H1299 cells. The combination treatments induce apoptosis via the caspases pathway in A549 cells but promote cell cycle arrest via CCND1 and PCNA inhibition in H1299 cells. In summary, these results suggest that combined treatment with heteronemin and tetrac derivatives could suppress signal transduction pathways essential for NSCLC cell growth. The synergetic effects can be used potentially as a therapeutic procedure for NSCLC patients.

The properties and the antioxidant activity of a series of hydroxytyrosyl esters having different carbon chain lengths (C4, C8, C12 and C18) have been measured in phosphatidylcholine model membrane (liposomes) using specific probes for the bilayer and liposome lumen microenvironment, i.e., 1,6-diphenyl-1,3,5-hexatriene (DPH) and 2′,7′-dichlorodihydrofluorescein (H2DCF), respectively. Antioxidants self-assembly and their interaction with liposomes has been evaluated by light scattering, fluorescence, turbidimetry, gel filtration chromatography and microfiltration measurements, allowing the determination of critical aggregation concentration, bound fraction, capacity of crossing the lipid bilayer. The distribution of hydroxytyrosyl long chain esters has been proved to depend quite specifically on their lipophilic chain length, and this turns to have deep effects on their antioxidant behaviour. Shedding new light on the cut off effect and antioxidant behaviour of phenolipids, this study also put forward the relevance of cell-free liposome-based cellular models, like giant liposomes, for further characterization of analogous systems.

Embelin, a plant natural product found in Lysimachia punctata (Primulaceae), and Embelia ribes Burm (Myrsinaceae) fruit, possesses interesting biological and pharmacological properties. It is a unique chemical species as it includes both quinone and hydroquinone functional groups plus a long hydrophobic tail. By using hydrodynamic voltammetry, which generates the superoxide radical in situ, we show an unusual scavenging capability by embelin. Embelin as a scavenger of superoxide is stronger than the common food additive antioxidant 2,6-bis(1,1-dimethylethyl)-4-20 methylphenol, (butylated hydroxytoluene, BHT). In fact, embelin is even able to completely abolish the superoxide radical in the voltaic cell. Computational results indicate that two different types of embelin scavenging actions may be involved, initially through &pi, &ndash, &pi, interaction and followed by proton capture in the cell. A related mechanism describes embelin&rsquo, s ability to circumvent superoxide leaking by transforming the anion radical into molecular oxygen. In order to confirm its antioxidant properties, its biological activity was tested in a study carried out in THP-1 human leukemic monocytes and BV-2 mice microglia. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, proliferation curves and antioxidant activity by the use of a fluorescent probe showed good antioxidant properties at 24 h. This suggests that embelin&rsquo, s long alkyl C10 tail may be useful for cell membrane insertion which stimulates the antioxidant defense system, and cytoprotection in microglia. In conclusion, embelin could be an interesting pharmacological tool able to decrease the damage associated with metabolic and neurodegenerative diseases.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

New dihydroxytyrosyl esters 2a, 2c&ndash, 2j of dicarboxylic acids were synthesized from methyl orthoformate protected hydroxytyrosol 3 and diacyl chlorides. New compounds were characterized (HRMS, FT-IR, 1H- and 13C-NMR), and tested for antioxidant activity both in vitro (ABTS) and on L6 myoblasts and THP1 leukemic monocytes cell culture by DCF assay. According to the ABTS assay, compounds 2a, 2c&ndash, 2j showed a TEAC value of antioxidant capacity up to twice that of Trolox. Very high or complete ROS protections were obtained in the cell environment where lipophilicity and rigidity of dicarboxylic structure seem to facilitate the antioxidant effect. MTT assay and proliferation test were used for assessment of cell viability. These compounds can be envisaged as a new class of preservatives for food or cosmetic products.

Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination.

Cancer resistance to chemotherapeutic agents is a major issue in the management of cancer patients. Overexpression of the ribonucleotide reductase regulatory subunit M2 (RRM2) has been associated with aggressive cancer behavior and chemoresistance. Nano-diamino-tetrac (NDAT) is a nanoparticulate derivative of tetraiodothyroacetic acid (tetrac), which exerts anticancer properties via several mechanisms and downregulates RRM2 gene expression in cancer cells. Resveratrol is a stilbenoid phytoalexin which binds to a specific site on the cell surface integrin αvβ3 to trigger cancer cell death via nuclear translocation of COX-2. Here we report that resveratrol paradoxically activates RRM2 gene expression and protein translation in colon cancer cells. This unanticipated effect inhibits resveratrol-induced COX-2 nuclear accumulation. RRM2 downregulation, whether achieved by RNA interference or treatment with NDAT, enhanced resveratrol-induced COX-2 gene expression and nuclear uptake which is essential to integrin αvβ3-mediated-resveratrol-induced antiproliferation in cancer cells. Elsewhere, NDAT downregulated resveratrol-induced RRM2 expression in vivo but potentiated the anticancer effect of the stilbene. These findings suggest that RRM2 appears as a cancer cell defense mechanism which can hinder the anticancer effect of the stilbene via the integrin αvβ3 axis. Furthermore, the antagonistic effect of RRM2 against resveratrol is counteracted by the administration of NDAT.

The molecular pathogenesis of colorectal cancer encompasses the activation of several oncogenic signaling pathways that include the Wnt/β-catenin pathway and the overexpression of high mobility group protein A2 (HMGA2). Resveratrol – the polyphenolic phytoalexin – binds to integrin αvβ3 to induce apoptosis in cancer cellsviacyclooxygenase 2 (COX-2) nuclear accumulation and p53-dependent apoptosis. Tetraiodothyroacetic acid (tetrac) is a de-aminated derivative ofl-thyroxine (T4), which – in contrast to the parental hormone – impairs cancer cell proliferation. In the current study, we found that tetrac promoted resveratrol-induced anti-proliferation in colon cancer cell lines, in primary cultures of colon cancer cells, andin vivo. The mechanisms implicated in this action involved the downregulation of nuclear β-catenin and HMGA2, which are capable of compromising resveratrol-induced COX-2 nuclear translocation. Silencing of either β-catenin or HMGA2 promoted resveratrol-induced anti-proliferation and COX-2 nuclear accumulation which is essential for integrin αvβ3-mediated-resveratrol-induced apoptosis in cancer cells. Concurrently, tetrac enhanced nuclear abundance of chibby family member 1, the nuclear β-catenin antagonist, which may further compromise the nuclear β-catenin-dependent gene expression and proliferation. Taken together, these results suggest that tetrac targets β-catenin and HMGA2 to promote resveratrol-induced-anti-proliferation in colon cancers, highlighting its potential in anti-cancer combination therapy.

In line with our previous studies, we have developed through a rational design approach novel morpholine and benzoxa(or thia)zine lead compounds that modulate a multiplicity of targets against atherosclerosis. We have evaluated the most promising compounds for their efficiency to a) interact and scavenge free radicals, b) inhibit the metal ion (Cu2+)-induced LDL oxidation c) act intracellularly as antioxidants in THP-1 monocytes from a leukemic patient and d) inhibit the pro-inflammatory enzymes cycloxygenase- 1 (COX-1) and -2 (COX-2) in vitro. Furthermore, two representative compounds were tested for their potential to decrease lipidemic parameters (TC, LDL and TG) in hyperlipidemic mice. Most derivatives indicated a remarkable antioxidant activity, while at the same time exhibited a significant in vitro anti-inflammatory activity, inhibiting COX-1 or/and COX-2 activity at 20 μΜ. Furthermore, after their long-term administration, compounds 6 and 8 afforded considerable activity in a chronic experimental animal model of hyperlipidemia (after high fat diet administration). The multifunctional pharmacological profile exhibited by the compounds of this study renders them interesting lead compounds for the development of novel agents against atherosclerosis.

In order to better understand the antioxidant behavior of a series of polyphenolic 2'-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV-vis spectroscopic method indicate that a hydroxyl group in position 5' induces the highest antioxidant activity. Consequently, 2,2',5'-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2',5'-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa(-)/fa(-)) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2',5'-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.

BACKGROUND: Tetraiodothyroacetic acid (tetrac) and its nanoparticulate formulation (Nanotetrac) act at a cell surface receptor to block angiogenesis and tumor cell proliferation. OBJECTIVE: The complex anti-angiogenic properties of tetrac and Nanotetrac caused us to search in the literature and in certain of our unpublished mRNA experiments for evidence that these agents affect the early inflammatory response, perhaps through actions on specific cytokines and chemokines. RESULTS AND DISCUSSION: Tetrac and Nanotetrac inhibit expression in tumor cells of cytokine genes, e.g., specific interleukins, and chemokine genes, such as fractalkine (CX3CL1), and chemokine receptor genes (CX3CR1) that have been identified as high priority targets in the development of inflammation-suppressant drugs. The possibility is also examined that tetrac formulations have an effect on the function of inflammatory cells.

We report the results of in vivo studies in Caenorhabditis elegans nematodes in which addition of extra virgin olive oil (EVOO) to their diet significantly increased their life span with respect to the control group. Furthermore, when nematodes were exposed to the pesticide paraquat, they started to die after two days, but after the addition of EVOO to their diet, both survival percentage and lifespans of paraquat-exposed nematodes increased. Since paraquat is associated with superoxide radical production, a test for scavenging this radical was performed using cyclovoltammetry and the EVOO efficiently scavenged the superoxide. Thus, a linear correlation (y = -0.0838x +19.73, regression factor = 0.99348) was observed for superoxide presence (y) in the voltaic cell as a function of aliquot (x) additions of EVOO, 10 μL each. The originally generated supoeroxide was approximately halved after 10 aliquots (100 μL total). The superoxide scavenging ability was analyzed, theoretically, using Density Functional Theory for tyrosol and hydroxytyrosol, two components of EVOO and was also confirmed experimentally for the galvinoxyl radical, using Electron Paramagnetic Resonance (EPR) spectroscopy. The galvinoxyl signal disappeared after adding 1 μL of EVOO to the EPR cell in 10 minutes. In addition, EVOO significantly decreased the proliferation of human leukemic THP-1 cells, while it kept the proliferation at about normal levels in rat L6 myoblasts, a non-tumoral skeletal muscle cell line. The protection due to EVOO was also assessed in L6 cells and THP-1 exposed to the radical generator cumene hydroperoxide, in which cell viability was reduced. Also in this case the oxidative stress was ameliorated by EVOO, in line with results obtained with tetrazolium dye reduction assays, cell cycle analysis and reactive oxygen species measurements. We ascribe these beneficial effects to EVOO antioxidant properties and our results are in agreement with a clear health benefit of EVOO use in the Mediterranean diet.

The development of antioxidants as useful drugs for the treatment of neurodegenerative diseases such as Alzheimer’s is extremely challenging in medicinal chemistry. Coumarins have attracted great attention as possible therapeutic tools against oxygen radicals in human degenerative diseases. In order to establish the possible structure-antioxidant activity relationship, a series of twenty four 4-methylcoumarin derivatives were examined by employing reducing power measurements, and comparison with bond dissociation enthalpy and ionization potential calculations. Based on the reducing potency of 4-methylcoumarin derivatives with respect to trolox, these compounds were classified into five groups as “most active”, “more active”, “moderately active”, “less active” and “inactive” derivatives. The presence of hydroxyl groups is an essential requirement for the activity, and substitution of hydroxyl groups by methoxy groups leads to non-active derivatives. The results revealed that dihydroxyl groups in the ortho position show a better antioxidant activity with respect to dihydroxyl groups in the meta position. This is ascribed to the ability to construct more stable 4-methylcoumarin radical intermediates by rearrangement of intra-molecular hydrogen bonding. Our findings indicate that other important factors to enhance the antioxidant activity of coumarins are the number of hydroxyl groups, the presence of ester substitutions and a thiono functional group on the pyrone ring. However, bond dissociation enthalpy and ionization potential calculations alone are not sufficient to identify the best antioxidant structures. As a result, chemical and functional properties of molecules such as 4-methylcoumarins should be examined as a whole entity, considering all substitutions versus a single substitution to design functional compounds with good antioxidant activity.

Developing a rational strategy to control intracellular reactive oxygen species (ROS) requires understanding the mechanism of antioxidant activity. In this investigation the properties of a novel synthetic analog of vitamin E (IRFI005) with potent antioxidant activity are described. A mechanism is proposed for its efficient radical-scavenging effects. Cellular antioxidant and antitoxicity assays showed IRFI005 to freely permeate across cellular membranes, enabling it to be an effective suppressor of intracellular ROS and to protect cells against toxicity induced by free radical generating compounds. The free radical-scavenging activity of IRFI005 examined by UV-Vis and electron spin resonance (ESR) techniques clearly confirmed a "two electrons and/or H-atom" donation mechanism for each molecule of IRFI005. Reducing power assay as well as semi-empirical calculations revealed that under physiological conditions (pH∼7) almost all IRFI005 molecules are in the anionic state (IRFI005(-)). Data indicated that the electron donating ability of IRFI005(-) was dominant at physiological pH because of higher stability of quinine-IRFI005(-) and less barrier energy of IRFI005(-) than neutral IRFI005. Consequently, the efficient cellular protection of IRFI005 against toxic free radicals can be explained by a two electron-transfer process, because of reduced inter-frontier molecular orbital energy gap barrier at physiological pH. Our findings suggest that hydrophilic vitamin E-like antioxidants are good candidates in designing novel therapeutic strategies for inhibition of oxidative stress associated with different human diseases.

Nongenomic effects of thyroid hormones on Na+-K+-ATPase activity were studied in chick embryo hepatocytes at two different developmental stages, 14 and 19 days of embryonal age, and the signal transduction pathways involved were characterized. Our data showed the following. 1) 3,5,3′-Triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (3,5-T2) rapidly induced a transient inhibitory effect on the Na+-K+-ATPase; the extent and duration depended on the developmental age of the cells. 2) 3,5-T2behaved as a true hormone and fully mimicked the effect of T3. 3) Thyroxine had no effect at any of the developmental stages. 4) The inhibition of Na+-K+-ATPase was mediated by activation of protein kinase A, protein kinase C, and phosphoinositide 3-kinase, suggesting several modes of modulation of ATPase activity through phosphorylation at different sites. 5) The MAPK pathway did not seem to be involved in the early phase of hormone treatment. 6) The nonpermeant analog T3-agarose inhibited Na+-K+-ATPase activity in the same way as T3, confirming that hormone signaling initiated at a receptor on the plasma membrane. From these results, it can be concluded that the cell response mechanisms change rapidly and drastically within the early phase of embryo growth. The differences found at the two stages probably reflect the different roles of thyroid hormones during development and differentiation.

Nongenomic actions of thyroid hormone include several that involve or require calcium. Actions of thyroid hormone at the plasma or intracellular membranes include stimulation of membrane glucose transport and of the Na+/H+ antiporter (exchanger) by mechanisms that require liberation of intracellular calcium and stimulation of the cell membrane and sarcoplasmic reticulum calcium pumps (Ca2+-ATPases). These pumps not only transport Ca2+, but also are regulated by the intracellular calmodulin-Ca2+ complex (plasma membrane/sarcolemma) or calmodulin-dependent protein kinase II phosphorylation of phospholamban (sarcoplasmic reticulum). Intracellular calcium ion concentration may also be subject to regulation by other nongenomic effects of iodothyronines, such as those on the Na+/H+ antiporter or sodium current, that secondarily affect the Na+/Ca2+ exchanger. Certain of these nongenomic actions of thyroid hormone, e.g., Na+/H+ exchanger, Ca2+-ATPase, are now recognized to begin at a recently described hormone receptor on a heterodimeric structural membrane protein, integrin αvβ3. The thyroid hormone signal at this receptor is further transduced by the mitogen-activated protein kinase (MAPK; extracellular regulated kinase1/2, ERK1/2) pathway.

Nongenomic actions of thyroid hormone do not require a direct interaction of 3,5,30-triiodo-L-thyronine (T3) with the transcriptionally active nuclear receptors TRα and TRβ. A rapid response time is a characteristic of many nongenomic actions; the onset of the majority of these effects is within minutes because the action is independent of gene expression and protein synthesis. While only T3 is able to generate a genomic response, the different nongenomic effects may be activated by either T3 or T4 or by other iodothyronine derivatives such as T2. In the last decade, the discovery of a large number of nongenomic actions of thyroid hormones has increasingly attracted the interest of researchers, and different specific binding sites or receptors for these hormones/messengers have now been described in several cellular compartments, including the external surface of the plasma membrane. The function of nongenomic effects of these hormones has mainly been considered to relate to homeostasis, such as actions on plasma membrane ion transporters or maintenance of the cytoskeleton, but recent evidence supports the existence of crosstalk between nongenomic and genomic effects of the hormone. Further examination of such crosstalk may reveal hitherto unappreciated mechanisms underlying global illnesses, such as cardiovascular diseases, inflammatory and immune diseases, and mechanisms of tumor cell function. Indeed, these new aspects have already improved our understanding of the contributions of nongenomic thyroid hormone actions to the regulation of cancer-related angiogenesis and to cancer cell survival pathways. In the present report, we provide a concise overview of the main observations that define the nongenomic actions of thyroid hormones and give a description of the state of the art.

L-valine is a branched-chain amino acid (BCAA) largely used as dietary integrator by athletes and involved in some inherited rare diseases such as maple syrup urine disease. This pathology is caused by an altered BCAA metabolism with the accumulation of toxic keto acids in tissues and body fluids with consequent severe neurological symptoms. In animal models of BCAA accumulation, increased oxidative stress levels and lipid peroxidation have been reported. The aim of this study was to analyze both whether high BCAA concentrations in neurons induce reactive oxygen species (ROS) production and whether, by performing electrophysiological recordings, the neuronal functional properties are modified. Our results demonstrate that in primary cortical cultures, a high dose of valine increases ROS production and provokes neuronal hyperexcitability because the action potential frequencies and the persistent sodium current amplitudes increase significantly compared to non-treated neurons. Since Baicalein, a flavone obtained from the Scutellaria root, has been shown to act as a strong antioxidant with neuroprotective effects, we evaluated its possible antioxidant activity in primary cortical neurons chronically exposed to L-valine. The preincubation of cortical neurons with Baicalein prevents the ROS production and is able to revert both the neuronal hyperexcitability and the increase of the persistent sodium current, indicating a direct correlation between the ROS production and the altered physiological parameters. In conclusion, our data show that the electrophysiological alterations of cortical neurons elicited by high valine concentration are due to the increase in ROS production, suggesting much caution in the intake of BCAA dietary integrators.

Polyphenolic coumarins are known to act as antioxidants in biological systems, but it is difficult to distinguish their antioxidant activity from the many other effects they produce in cells. We have determined the radical scavenging capacity of 22 structurally related natural and synthetic 4-methylcoumarins, by measuring their reaction with radicals, galvinoxyl and 2,2-diphenyl-1-picrylhydrazyl, using electron paramagnetic resonance spectroscopy. Efficient antioxidant activity of 4-methylcoumarins in cells was verified using the DCF fluorescent probe assay for determination of intracellular reactive oxygen species levels. As expected, the o-dihydroxysubstituted coumarins were found to be excellent radical scavengers and better than the m-dihydroxysubstituted or monohydroxysubstituted analogues, but surprisingly the corresponding o-diacetoxy derivatives also turned out to be good scavengers, even in the absence of an esterase. Another unexpected result was that the anti-oxidant efficiency of 4-methylcoumarins could be modulated by introducing an ethoxycarbonyl-ethyl substituent at the C-3 position; this effect cannot be explained by simple electron donating/withdrawing properties. Coumarin concentrations of 10 μm or less were used in all experiments, corresponding to the levels relevant for therapeutic purposes. Considering that 4-methylcoumarins, in contrast to many other coumarins, are not metabolized to toxic epoxide intermediates, these results indicate promising new strategies for the design of non-toxic antioxidant coumarin-based drugs.

Integrin αvβ3 is generously expressed by cancer cells and rapidly dividing endothelial cells. The principal ligands of the integrin are extracellular matrix proteins, but we have described a cell surface small molecule receptor on αvβ3 that specifically binds thyroid hormone and thyroid hormone analogues. From this receptor, thyroid hormone (L-thyroxine, T4; 3,5,3’-triiodo-L-thyronine, T3) and tetraiodothyroacetic acid (tetrac) regulate expression of specific genes by a mechanism that is initiated nongenomically. At the integrin, T4 and T3 at physiological concentrations are pro-angiogenic by multiple mechanisms that include gene expression, and T4 supports tumor cell proliferation. Tetrac blocks the transcriptional activities directed by T4 and T3 at αvβ3, but, independently of T4 and T3, tetrac modulates transcription of cancer cell genes that are important to cell survival pathways, control of the cell cycle, angiogenesis, apoptosis, cell export of chemotherapeutic agents and repair of double-strand DNA breaks. We have covalently bound tetrac to a 200 nm biodegradable nanoparticle that prohibits cell entry of tetrac and limits its action to the hormone receptor on the extracellular domain of plasma membrane αvβ3. This reformulation has greater potency than unmodified tetrac at the integrin and affects a broader range of cancer-relevant genes. In addition to these actions on intracellular kinase-mediated regulation of gene expression, hormone analogues at αvβ3 have additional effects on intracellular protein-trafficking (cytosol compartment to nucleus), nucleoprotein phosphorylation and generation of nuclear coactivator complexes that are relevant to traditional genomic actions of T3. Thus, previously unrecognized cell surface-initiated actions of thyroid hormone and tetrac formulations at αvβ3 offer opportunities to regulate angiogenesis and multiple aspects of cancer cell behavior.

The extracellular domain of integrin αvβ3 contains a receptor for thyroid hormone and hormone analogs. The integrin is amply expressed by tumor cells and dividing blood vessel cells. The proangiogenic properties of thyroid hormone and the capacity of the hormone to promote cancer cell proliferation are functions regulated nongenomically by the hormone receptor on αvβ3. An L-thyroxine (T4) analog, tetraiodothyroacetic acid (tetrac), blocks binding of T4 and 3,5,3'-triiodo-L-thyronine (T3) by αvβ3 and inhibits angiogenic activity of thyroid hormone. Covalently bound to a 200 nm nanoparticle that limits its activity to the cell exterior, tetrac reformulated as Nanotetrac has additional effects mediated by αvβ3 beyond the inhibition of binding of T4 and T3 to the integrin. These actions of Nanotetrac include disruption of transcription of cell survival pathway genes, promotion of apoptosis by multiple mechanisms, and interruption of repair of double-strand deoxyribonucleic acid breaks caused by irradiation of cells. Among the genes whose expression is suppressed by Nanotetrac are EGFR, VEGFA, multiple cyclins, catenins, and multiple cytokines. Nanotetrac has been effective as a chemotherapeutic agent in preclinical studies of human cancer xenografts. The low concentrations of αvβ3 on the surface of quiescent nonmalignant cells have minimized toxicity of the agent in animal studies.

Non-Alcoholic Fatty Liver Disease (NAFLD) is nowadays considered one of the most diffused pathologic conditions affecting the liver especially in the Western world. The pathological mechanisms that induce the development of NAFLD are so far not completely clarified, even if now most researchers believe that an important condition that has to be satisfied is a significant accumulation of lipids inside the hepatocyte, principally as triglycerides. In the liver, such a lipid accumulation leads to an impairment of insulin signaling, which typically results in a failed phosphorylation of Akt, inducing the onset of insulin resistance. In the last years many researchers and clinicians focused their attention on the role played by fructose in metabolic diseases, such as obesity, type 2 diabetes, metabolic syndrome, as well as in liver specific pathologies, such as NAFLD. This huge debate about the role of fructose probably rose as a consequence of the recent increased diffusion of sweetened foods and beverages. In this context, the aim of this work was to develop an in vitro model for NAFLD, with particular reference to the role played by oleic acid and fructose in the onset of steatosis and insulin resistance. Moreover, we tested the effects of 3,5-diiodothyronine on steatosis and insulin resistance, trying to assess the potential mechanisms of action involved, considering the increasing interest about its role as a biologically active molecule. Our results show that, differently to oleic acid, fructose per se, in the range of physiological concentrations, is not able to induce neither triglycerides accumulation nor insulin resistance. Moreover we demonstrated that 3,5-diiodothyronine, at physiological levels, reduces triglycerides content and stimulates phosphorylation of Akt.