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3CL-Pro is the SARS-CoV-2 main protease (MPro). It acts as a homodimer to cleave the large polyprotein 1ab transcript into proteins that are necessary for viral growth and replication. 3CL-Pro has been one of the most studied SARS-CoV-2 proteins and a main target of therapeutics. A number of drug candidates have been reported, including natural products. Here, we employ elaborate computational methods to explore the dimerization of the 3CL-Pro protein, and we formulate a computational context to identify potential inhibitors of this process. We report that fortunellin (acacetin 7-O-neohesperidoside), a natural flavonoid O-glycoside, and its structural analogs are potent inhibitors of 3CL-Pro dimerization, inhibiting viral plaque formation in vitro. We thus propose a novel basis for the search of pharmaceuticals as well as dietary supplements in the fight against SARS-CoV-2 and COVID-19.

OXER1 is a recently identified receptor, binding the arachidonic acid metabolic product 5-oxo-ETE, considered an inflammatory receptor, implicated in chemoattraction of circulating mononuclear cells, Ca2+ surge in neutrophils, inflammation and cancer. Recently, we have shown that OXER1 is also a membrane androgen receptor in various cancer tissues. It was reported that the presence of OXER1 in leucocytes and the production and release of 5-oxo-ETE by wounded tissues is a wound sensing mechanism, leading to lymphocyte attraction. In view of the similarity of hallmarks of cancer and wound healing, we have explored the role of OXER1 and its endogenous ligand in the control of cell migration of human cancer epithelial cells (DU-145, T47D and Hep3B), mimicking the activation/migration phase of healing. We show that OXER1 is up-regulated only at the leading edge of the wound and its expression is up-regulated by its ligand 5-oxo-ETE, in a time-related manner. Knock-down of OXER1 or inhibition of 5-oxo-ETE synthesis led to decreased migration of cells and a prolongation of healing, in culture prostate cancer cell monolayers, with a substantial modification of actin cytoskeleton and a decreased filopodia formation. Inhibition of cell migration is a phenomenon mediated by Gβγ OXER1 mediated actions. These results provide a novel mechanism of OXER1 implication in cancer progression and might be of value for the design of novel OXER1 antagonists.

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

OXER1 (oxoeicosanoid receptor 1) was deorphanized in 1993 and found to be the specific receptor for the arachidonic acid metabolite 5-oxo-ETE. Recently, we have reported that androgen binds to this receptor also, being a membrane androgen receptor, triggering a number of its membrane-mediated actions (cell migration, apoptosis, cell proliferation, Ca2+ movements). In addition, our previous work suggested that a number of natural monomeric and oligomeric polyphenols interact with OXER1, acting similar to testosterone. Here, we interrogated the natural product chemical space and identified nine polyphenolic molecules with interesting in silico pharmacological activities as putative OXER1 antagonists. The molecule with the best pharmacokinetic-pharmacodynamic properties (ZINC15959779) was purchased and tested on OXER1, in prostate cancer cell cultures. It showed that it has actions similar to those of testosterone in inhibiting cAMP, while it had no action in intracellular Ca2+ mobilization or actin cytoskeleton rearrangement/migration. These results are discussed under the prism of structure-activity relationships and in silico models of the OXER1 binding groove. We suggest that these compounds, together with the previously reported (poly)phenolic compounds, can be lead structures for the exploration of the anti-inflammatory and antiproliferative effects of OXER1 antagonists.

Drug development is an arduous procedure, necessitating testing the interaction of a large number of potential candidates with potential interacting (macro)molecules. Therefore, any method which could provide an initial screening of potential candidate drugs might be of interest for the acceleration of the procedure, by highlighting interesting compounds, prior to in vitro and in vivo validation. In this line, we present a method which may identify potential hits, with agonistic and/or antagonistic properties on GPCR receptors, integrating the knowledge on signaling events triggered by receptor activation (GPCRs binding to Gα,β,γ proteins, and activating Gα, exchanging GDP for GTP, leading to a decreased affinity of the Gα for the GPCR). We show that, by integrating GPCR‐ligand and Gα‐GDP or ‐GTP binding in docking simulation, which correctly predicts crystallographic data, we can discriminate agonists, partial agonists, and antagonists, through a linear function, based on the ΔG (Gibbs‐free energy) of liganded‐GPCR/Gα‐GDP. We built our model using two Gαs (β2‐adrenergic and prostaglandin‐D2), four Gαi (μ‐opioid, dopamine‐D3, adenosine‐A1, rhodopsin), and one Gαo (serotonin) receptors and validated it with a series of ligands on a recently deorphanized Gαi receptor (OXER1). This approach could be a valuable tool for initial in silico validation and design of GPRC‐interacting ligands.

Background Nuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so far Methods An unsupervised in silico approach was used, followed by experimental validation. Results We identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation. Conclusions The NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members. General significance Elucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.

Many epidemiological studies revealed an association of dietary consumption of fatty acids and prostate cancer. Linoleic acid and alpha-linolenic acid and their derivatives such as arachidonic acid and eicosapentanoic acid are important polyunsaturated fatty acids in animal fats and in many vegetable oils. Their metabolism at the cellular level by enzymes such as lipoxygenases and cycloxygenases produces the group of eicosanoids molecules with many biological roles and activities in a variety of human diseases including cancer. In this review, we describe the biological activities of lipids with focus in eicosanoids and prostate cancer.

In prostate cancer, calcium homeostasis plays a significant role in the disease's development and progression. Intracellular calcium changes are an important secondary signal, triggered by a variety of extracellular stimuli, that controls many cellular functions. One of the main events affecting calcium is androgen signaling. Indeed, via calcium changes, androgens regulate cell processes like cell growth, differentiation and motility. In the present work we explored the nature of the receptor involved in calcium response induced by membrane-acting testosterone in prostate cancer cells. We report that testosterone, independently of the presence of the classical androgen receptor, can rapidly increase intracellular calcium from calcium stores, through the oxoeicosanoid receptor 1 (OXER1) and a specific signaling cascade that triggers calcium release from the endoplasmic reticulum. These findings reveal for the first time the receptor involved in the rapid calcium changes induced by androgens. Moreover, they further support the notion that androgens, even in the absence of AR, can still exert specific effects that regulate cancer cell fate.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.

The levels of expression and membrane localization of programmed cell death ligand 1 (PD-L1), an immune checkpoint type I transmembrane glycoprotein, are related to the clinical response of anti-PD-L1/PD-1 therapy. Although the biologically relevant localization of PD-L1 is on the plasma membrane of cancer cells, it has also been reported to be in the cytoplasm and sometimes in the nucleus. Furthermore, it has been claimed that chemotherapeutics can modify PD-L1 expression and/or its nuclear localization. Data from our group suggest that the nuclear localization of PD-L1, and other plasma membrane proteins as well, could be an artifact resulting from inadequate experimental conditions during immunocytochemical studies. Mild detergent and rigorous fixation conditions should be used in order to preserve the membrane localization and to prevent an erroneous translocation of PD-L1 and other non-interconnected membrane proteins, such as CD24, into other cellular compartments including the nucleus, of untreated and chemotherapeutically treated breast cancer cells. We propose that well-specified and rigorously followed protocols should be applied to immunocytochemical diagnostic techniques, especially to those related to individualized diagnosis and treatment.

The beneficial role of olive oil consumption is nowadays widely recognized. However, it is not clear whether its health effects are due to the presence of monounsaturated lipids and/or to the antioxidant fraction of microconstituents present in olive oil. The aim of the present study was to analyze the exact role of olive oil in the modification of metabolic factors (glucose and circulating lipids) and explore the role of its antioxidant polyphenols. In the present work, we have performed a network meta-analysis of 30 human intervention studies, considering direct and indirect interactions and impact of each constituent. Interestingly, we show that the impact of olive oil on glucose, triglycerides, and LDL-cholesterol is mediated through an adherence to the Mediterranean diet, with the only notable effect of olive oil polyphenols being the increase of HDL-cholesterol, and the amelioration of the antioxidant and inflammatory status of the subjects. Additionally, we report for the first time that lower antioxidant polyphenol levels may be sufficient for the beneficial effects of olive oil, while we show that the lipid fraction of olive oil may be responsible for some of its beneficial actions. In all parameters examined the beneficial effect of olive oil was more pronounced in subjects with an established metabolic syndrome or other chronic conditions/diseases. In conclusion, all these findings provide new knowledge that could lead to re-establishment of the role of olive oil in human nutrition.

Background & aims TGFβ superfamily member Activin-A is a multifunctional hormone/cytokine expressed in multiple tissues and cells, where it regulates cellular differentiation, proliferation, inflammation and tissue architecture. High activin-A levels have been reported in alcoholic cirrhosis and non-alcoholic steatohepatitis (NASH). Our aim was to identify the cell types involved in the fibrotic processes induced by activin-A in liver and verify the liver diseases that this molecule can be found increased. Methods We studied the effect of activin-A on mouse primary Kupffer cells (KCs) and Hepatic Stellate cells (HSCs) and the levels of activin-A and its inhibitor follistatin in the serum of patients from a large panel of liver diseases. Results Activin-A is expressed by mouse hepatocytes, HSCs and Liver Sinusoid Endothelial cells but not KCs. Each cell type expresses different activin receptor combinations. HSCs are unresponsive to activin-A due to downregulation/desensitization of type-II activin receptors, while KCs respond by increasing the expression/production of TNFα και TGFβ1. In the presence of KCs or conditioned medium from activin-A treated KCs, HSCs switch to a profibrogenic phenotype, including increased collagen and αSMA expression and migratory capacity. Incubation of activin-A treated KC conditioned medium with antibodies against TNFα and TGFβ1 partially blocks its capacity to activate HSCs. Only patients with alcoholic liver diseases and NASH cirrhosis have significantly higher activin-A levels and activin-A/follistatin ratio. Conclusions Activin-A may induce fibrosis in NASH and alcoholic cirrhosis via activation of KCs to express pro-inflammatory molecules that promote HSC-dependent fibrogenesis and could be a target for future anti-fibrotic therapies.

Dyslipidemia and impaired glucose metabolism are the main health issues of growing prevalence and significant high healthcare cost, requiring novel prevention and/or therapeutic approaches. Epidemiological and animal studies revealed that olive oil is an important dietary constituent, inducing normolipidemia. However, no studies have specifically investigated the polyphenol-rich water extract of olives (OLWPE), generated during olive oil production. In the present work, we initially examined the effect of OLPWE on animals’ metabolic parameters. Rats fed with a high-fat diet were treated with three different doses of OLPWE for 4 months. Additionally, bioavailability was explored. Afterwards, OLWPE’s metabolic effect was explored in humans. Healthy volunteers consumed microencapsulated OLWPE for 4 weeks, in a food matrix [one portion (30 g) of a meat product]. High-fat-fed rats developed a metabolic dysfunction, with increased LDL and insulin levels and decreased HDL; this syndrome was significantly impaired when treated with OLWPE. Treated rats had increased total plasma antioxidant capacity, while several phenolic compounds were detected in their blood. These findings were also verified in humans that consumed OLWPE, daily, for 4 weeks. Interestingly, in individuals with elements of cardio-metabolic risk, OLWPE consumption resulted in reduced glucose, insulin, total cholesterol, LDL and oxLDL levels. Our data clearly show that OLWPE can improve glucose and lipid profile, indicating its possible use in the design of functional food and/or therapeutic interventions.

Accumulating evidence during the last decades revealed that androgen can exert membrane initiated actions that involve signaling via specific kinases and the modulation of significant cellular processes, important for prostate cancer cell growth and metastasis. Results of the present work clearly show that androgens can specifically act at the membrane level via the GPCR oxoeicosanoid receptor 1 (OXER1) in prostate cancer cells. In fact, OXER1 expression parallels that of membrane androgen binding in prostate cancer cell lines and tumor specimens, while in silico docking simulation of OXER1 showed that testosterone could bind to OXER1 within the same grove as 5-OxoETE, the natural ligand of OXER1. Interestingly, testosterone antagonizes the effects of 5-oxoETE on specific signaling pathways and rapid effects such as actin cytoskeleton reorganization that ultimately can modulate cell migration and metastasis. These findings verify that membrane-acting androgens exert specific effects through an antagonistic interaction with OXER1. Additionally, this interaction between androgen and OXER1, which is an arachidonic acid metabolite receptor expressed in prostate cancer, provides a novel link between steroid and lipid actions and renders OXER1 as new player in the disease. These findings should be taken into account in the design of novel therapeutic approaches in prostate cancer.

The stilbene resveratrol exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines, through diverse mechanisms, including antioxidant effects, enzyme, growth factor and hormone receptor binding, and nucleic acid direct or indirect interactions. Although resveratrol accumulates in the liver, its effect on hepatocellular carcinoma has not been extensively studied. We have used the human hepatocyte-derived cancer cell line HepG2 to address the possible action of resveratrol on cell growth and to examine some possible mechanisms of action. Our results indicate that the stilbene inhibits potently cell proliferation, reduces the production of reactive oxygen species and induces apoptosis, through cell cycle arrest in G1 and G2/M phases. Furthermore it modulates the NO/NOS system, by increasing iNOS and eNOS expression, NOS activity and NO production. Inhibition of NOS enzymes attenuates its antiproliferative effect. These data could be of value in possible prevention or adjuvant treatment of hepatocellular carcinoma, through an increased consumption of resveratrol-rich foods and beverages.

Background: Oxidative stress may play a critical role in the pathogenesis and development of obesity-associated co-morbidities. Reactive oxygen and nitrogen species are produced as a consequence of normal aerobic metabolism and removed and/or inactivated in vivo by both endogenous (uric acid, bilirubin, thiols) and diet-derived (exogenous) antioxidants. The purpose of this study is to measure the total plasma antioxidant capacity (TAC), as well as the corrected TAC (cTAC, an index of exogenous provided antioxidants) in morbidly obese patients before and after surgical weight reduction. Methods: 16 morbidly obese (5 male and 11 female) candidates for surgical intervention, median age 34 (range 22-56) years, median weight 128 (range 96-186) kg, median excess weight 62 (range 28-115) kg and median BMI 44.4 (range 33.7-60.1) kg/m2 were evaluated before and 6 months after implantation of an intragastric balloon. 15 healthy blood donors (4 male and 11 female) on a normal diet, median age 35 (range 21-52) years, median weight 64.3 (range 46-78) kg and median BMI 24.2 (range 23.7-25.2) kg/m2 were also evaluated. Blood samples for routine clinical chemistry, TAC and cTAC determination were drawn, and weight and BMI calculation were performed once in the control group, and in the morbidly obese patients (MO) before and 6 months after the balloon implantation. Results: 6 months after balloon placement, weight and BMI of the MO patients were statistically significantly reduced from the preoperative values (P

Hazardous chemicals escape to the environment by a number of natural and/or anthropogenic activities and may cause adverse effects on human health and the environment. Increased combustion of fossil fuels in the last century is responsible for the progressive change in the atmospheric composition. Air pollutants, such as carbon monoxide (CO), sulfur dioxide (SO 2 ), nitrogen oxides (NOx), volatile organic compounds (VOCs), ozone (O 3 ), heavy metals, and respirable particulate matter (PM2.5 and PM10), differ in their chemical composition, reaction properties, emission, time of disintegration and ability to diffuse in long or short distances. Air pollution has both acute and chronic effects on human health, affecting a number of different systems and organs. It ranges from minor upper respiratory irritation to chronic respiratory and heart disease, lung cancer, acute respiratory infections in children and chronic bronchitis in adults, aggravating pre-existing heart and lung disease, or asthmatic attacks. In addition, short- and long-term exposures have also been linked with premature mortality and reduced life expectancy. These effects of air pollutants on human health and their mechanism of action are briefly discussed.

Opioids, acting via G-protein coupled membrane receptors, induce analgesia. However their role is not limited to their anti-nociceptive action. They are found in several peripheral tissues acting as negative regulators of cellular processes. Even though that is not fully elucidated, it becomes obvious that opioids exert their effects in close relation to other neuropeptides such as somatostatin. Hepatocellular carcinoma is one tumor, among others, which secrete bioactive peptides while somatostatin analogs exert an inhibitory effect. We have used the human hepatocyte-derived cancer cell line HepG2, in order to examine the effect of opioids on cell growth and their possible mode of action. Our results show that the opioid ethylketocyclazocine (EKC) inhibits cell proliferation and induces apoptosis. This inhibitory effect is not exerted via opioids receptors since it was not reversed by the opioid antagonist diprenorphine and functional opioid receptors were not found on HepG2 cells. On the contrary, we show that EKC binds to somatostatin receptors, and activates a PTP signalling cascade. In this respect, the interaction of opioids with somatostatin receptors on hepatocellular carcinoma cells, and the fact that they are widely used for pain control, may provide some additional clues for the discrepancies during treatment with somatostatin analogues.

Genomic signaling mechanisms require a relatively long time to get into action and represent the main way through which steroid hormones affect target cells. In addition, steroids may rapidly activate cellular functions by non-genomic signaling mechanisms involving membrane sites. Understanding in depth the molecular mechanisms of the non-genomic action represents an important frontier for developing new and more selective pharmacologic tools for endocrine therapies. In the present study, we report that membrane-impermeable testosterone-bovine serum albumin (BSA) acts synergistically with paclitaxel in modifying actin and tubulin cytoskeleton dynamics in LNCaP (androgen sensitive) and DU-145 (androgen insensitive) human prostate cancer cell lines. In addition, coincubation of either cell line with testosterone-BSA and paclitaxel induced inhibition of cell proliferation and apoptosis. Finally, in vivo experiments in LNCaP and DU-145 tumor xenografts in nude mice showed that both agents decrease tumor mass, whereas testosterone-BSA enhances the effect of paclitaxel. Our findings suggest that chronic activation of membrane androgen receptors in vitro and in vivo facilitates and sustains for a longer time the antitumoral action of cytoskeletal acting agents. [Mol Cancer Ther 2006;5(5):1342–51]

Experimental and epidemiological studies indicate that antioxidant food polyphenols could have antimitotic activities, interfering with cancer initiation, progression or mortality. Circulating polyphenols are far lower than the nominal value in foods. In the rare studies dealing with polyphenol bioavailability, it was noted that their active concentrations in the blood are M or pM). Our results indicate that cell growth was decreased, with cells being arrested at the S phase of the cycle. In addition, we provide evidence of a bimodal modulation of the NO/NOS system, affecting its activity and transcription. We show that modulation of this system is sufficient to explain polyphenol action on this cell line. This result suggests a potential importance of wine ingestion and possibly the consumption of other polyphenol-rich dietary foods and drinks in the control of breast cancer cell growth.

The present work reports a new mode of action of the naturally occurring flavanols catechin and epicatechin and their dimers B2 and B5, in the breast cancer T47D cell line, namely, their interaction with membrane androgen receptors. We show that monomeric and dimeric flavanols are complete (B2) or partial displacers of radiolabeled testosterone bound on T47D membranes, with affinities ranging from 1.7 (B5) to 82.2 nM (B2). In addition, they trigger the phosphorylation of the same signaling molecules (FAK, PI3K) as testosterone-BSA, minutes after binding to membrane receptors, leading to actin cytoskeleton polymerization and redistribution, with formation of filopodia and lamellipodia. The PI3K inhibitor wortmannin reverts the effect of polyphenols and testosterone-BSA, providing additional evidence about activation of a similar signaling cascade. Incubation of T47D cells for more than 2 h with polyphenols or testosterone-BSA induces apoptosis, which follows the same time-dependent pattern. We conclude that flavanols (monomers or dimers) are agonists of membrane androgen receptors and could be used as testosterone–protein conjugates for the management of tumors, in which, application of testosterone-BSA induces regression, providing additional data about the mechanism of their antiproliferative action.

Nongenomic androgen actions imply mechanisms different from the classical intracellular androgen receptor (iAR) activation. We have recently reported the identification of a membrane androgen receptor (mAR) on LNCaP human prostate cancer cells, mediating testosterone signal transduction within minutes. In the present study we provide evidence that activation of mAR by nonpermeable, BSA-coupled testosterone results in 1) inhibition of LNCaP cell growth (with a 50% inhibitory concentration of 5.08 nM, similar to the affinity of testosterone for membrane sites); 2) induction in LNCaP cells of both apoptosis and the proapoptotic Fas protein; and 3) a significant decrease in migration, adhesion, and invasion of iAR-negative DU145 human prostate cancer cells. These actions persisted in the presence of antiandrogen flutamide or after decreasing the content of iAR in LNCaP cells by iAR antisense oligonucleotides. Testosterone-BSA was also effective in inducing apoptosis of DU145 human prostate cancer cells, negative for iAR, but expressing mAR sites. In LNCaP cell-inoculated nude mice, treatment with testosterone-BSA (4.8 mg/kg body weight) for 1 month resulted in a 60% reduction of tumor size compared with that in control animals receiving only BSA, an effect that was not affected by the antiandrogen flutamide. Our findings suggest that activators of mAR may represent a new class of antitumoral agents of prostate cancer.

Opioids and somatostatin mediate their cellular effects through specific membrane receptors. Three major receptor classes (delta, mu and kappa) were identified for opioids, while for somatostatin, five different receptor classes (SSTR1-5) have been cloned. Through the interaction with their receptors, opioids and somatostatin exert their effects on cell growth, proliferation, differentiation and secretion. Specific actions of each receptor type have been reported, to be implicated in one or more of the cell functions referred above but have been mainly correlated with cell growth control. In several systems the effect of either neuropeptide is the reverse, inducing cell growth rather than antiproliferative and proapoptotic signals. In recent years, a growing number of reports indicate a possible interaction between opioid and somatostatin system. This could occur at the receptor level, through a cross-interaction of either neuropeptide with either receptor type, or receptor hetero-dimerization, and at a post-receptor level, via interaction with specific signaling molecules. These interactions provide new directions for the identification of specific molecules acting at the receptor and post-receptor level, mimicking the effects of both categories of agents.

Background Oxidative stress may play a critical role in the vascular disease of end stage renal failure and hemodialysis patients. Studies, analyzing either discrete analytes and antioxidant substances, or the integrated total antioxidant activity of human plasma during hemodialysis, give contradictory results. Methods Recently, we have introduced a new automated method for the determination of Total Antioxidant Capacity (TAC) of human plasma. We have serially measured TAC and corrected TAC (cTAC: after subtraction of the interactions due to endogenous uric acid, bilirubin and albumin) in 10 patients before the onset of the dialysis session, 10 min, 30 min, 1 h, 2 h and 3 h into the procedure and after completion of the session. Results Our results indicate that TAC decreases, reaching minimum levels at 2 h. However, corrected TAC increases with t1/2 of about 30 min. We then repeated the measurements in 65 patients undergoing dialysis with different filters (36 patients with ethylene vinyl alcohol copolymer resin filter -Eval-, 23 patients with two polysulfone filters -10 with F6 and 13 with PSN140-, and 6 patients with hemophan filters). Three specimens were collected (0, 30, 240 min). The results of this second group confirm our initial results, while no significant difference was observed using either filter. Conclusions Our results are discussed under the point of view of possible mechanisms of modification of endogenous antioxidants, and the interaction of lipid- and water-soluble antioxidants.

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors, which modify the actin cytoskeleton and increase the secretion of prostate-specific antigen (PSA) within minutes. Membrane steroid receptors are, indeed, a newly identified element of steroid action that is different from the classical intracellular sites. In the present work, using a nonpermeable analog of testosterone (testosterone-BSA), we investigated the signaling pathway that is triggered by the membrane testosterone receptors' activation and leads to actin cytoskeleton reorganization. We report that exposure of cells to testosterone-BSA resulted in phosphorylation of focal adhesion kinase (FAK), the association of FAK with the phosphatidylinositol-3 (PI-3) kinase, and the subsequent activation of the latter as well as the activation of the small guanosine triphosphatases Cdc42/Rac1. Pretreatment of cells with the specific PI-3 kinase inhibitor wortmannin abolished both the activation of the small guanosine triphosphatases and the alterations of actin cytoskeleton, whereas it did not affect the phosphorylation of FAK. These findings indicate that PI-3 kinase is activated downstream of FAK and upstream of Cdc42/Rac1, which subsequently regulate the actin organization. Moreover, wortmannin diminished the secretion of PSA, implying that the signaling events described above are responsible for the testosterone-BSA-induced PSA secretion. Our results are discussed under the prism of a possible implication of these membrane receptors in prostate cancer chemotherapy.

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short-time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non-internalizable analog testosterone-BSA increase rapidly the release of prostate-specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non-internalizable fluorescent analog testosterone-BSA-FITC, on whole cells. Binding on these sites is time- and concentration-dependent and specific for testosterone, presenting a KD of 10.9 nM and a number of 144 sites/mg protein (approximately 13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

Introduction The oncoprotective role of food-derived polyphenol antioxidants has been described but the implicated mechanisms are not yet clear. In addition to polyphenols, phenolic acids, found at high concentrations in a number of plants, possess antioxidant action. The main phenolic acids found in foods are derivatives of 4-hydroxybenzoic acid and 4-hydroxycinnamic acid.

In addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types. Receptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells. Our work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.

AIM: The balance between oxidants and antioxidants can play an important role in the initiation and development of liver diseases. Recently, we have described a new automated method for the determination of total antioxidant capacity (TAC) in human serum and plasma. METHODS: We measured TAC and corrected TAC (CTAC -abstraction of interactions due to endogenous uric acid, bilirubin and albumin) in 52 patients with chronic liver diseases (41 patients with primary biliary cirrhosis (PBC), 10 patients with chronic hepatitis C and 13 patients with viral HCV cirrhosis) as well as in 10 healthy controls. In 23 PBC patients measurement were also done 6 mo after treatment with ursodeoxycholic acid (UDCA). The TAC assay was based on a modification of the crocin bleaching assay. The results were correlated with routine laboratory measurements and the histological stage of PBC. RESULTS: There were no significant differences in TAC between the various groups. However, CTAC was consi-derably increased in the PBC group compared to controls and cirrhotics. Analysis of these patients according to disease stages showed that this increase was an early phenomenon observed only in stages I and II compared to controls, cirrhotics and patients with chronic hepatitis C). After 6 mo of treatment with UDCA, levels of CTAC decreased to those similar to that of controls. CONCLUSION: Patients in the early stages of PBC present with high levels of corrected total antioxidant capacity and this maybe related to the pathophysiology of the disease. UDCA treatment restores the levels of CTAC to control levels.

Background Antioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product. Methods In the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay), based on the crocin bleaching (oxidation) method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L), and performed using an end point measurement. Results The TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS) test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM), while a diet rich in antioxidants more than doubled this value. Conclusions The proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and/or exogenous metabolites. The antioxidant capacity of plasma thus calculated can be used as a useful indicator of the antioxidant value of foods and beverages in the daily diet.