Gender Influence on XOR Activities and Related Pathologies: A Narrative Review.

Taking into account the patient's gender is the first step towards more precise and egalitarian medicine. The gender-related divergences observed in purine catabolism and their pathological consequences are good examples of gender medicine differences. Uric acid is produced by the activity of xanthine oxidoreductase (XOR). The serum levels of both XOR activity and uric acid differ physiologically between the genders, being higher in men than in women. Their higher levels have been associated with gout and hypertension, as well as with vascular, cardiac, renal, and metabolic diseases. The present review analyzes the gender-related differences in these pathological conditions in relation to increases in the serum levels of XOR and/or uric acid and the opportunity for gender-driven pharmacological treatment.

New Insights on Saporin Resistance to Chemical Derivatization with Heterobifunctional Reagents.

Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.

Heterophyllin: A New Adenia Toxic Lectin with Peculiar Biological Properties.

Ribosome-inactivating proteins (RIPs) are plant toxins that were identified for their ability to irreversibly damage ribosomes, thereby causing arrest of protein synthesis and induction of cell death. The RIPs purified from Adenia plants are the most potent ones. Here, we describe a novel toxic lectin from Adenia heterophylla caudex, which has been named heterophyllin. Heterophyllin shows the enzymatic and lectin properties of type 2 RIPs. Interestingly, in immunoreactivity experiments, heterophyllin poorly cross-reacts with sera against all other tested RIPs. The cytotoxic effects and death pathways triggered by heterophyllin were investigated in three human-derived cell lines: NB100, T24, and MCF7, and compared to ricin, the most known and studied type 2 RIP. Heterophyllin was able to completely abolish cell viability at nM concentration. A strong induction of apoptosis, but not necrosis, and the involvement of oxidative stress and necroptosis were observed in all the tested cell lines. Therefore, the enzymatic, immunological, and biological activities of heterophyllin make it an interesting molecule, worthy of further in-depth analysis to verify its possible pharmacological application.

Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

Anti-PD-L1 immunoconjugates for cancer therapy: Are available antibodies good carriers for toxic payload delivering?

Immune checkpoint mechanisms are important molecular cell systems that maintain tolerance toward autoantigens in order to prevent immunity-mediated accidental damage. It is well known that cancer cells may exploit these molecular and cellular mechanisms to escape recognition and elimination by immune cells. Programmed cell death protein-1 (PD-1) and its natural ligand programmed cell death ligand-1 (PD-L1) form the PD-L1/PD-1 axis, a well-known immune checkpoint mechanism, which is considered an interesting target in cancer immunotherapy. In fact, the expression of PD-L1 was found in various solid malignancies and the overactivation of PD-L1/PD-1 axis results in a poor patient survival rate. Breaking PD-L1/PD-1 axis, by blocking either the cancer side or the immune side of the axis, is currently used as anti-cancer strategy to re-establish a tumor-specific immune response. For this purpose, several blocking antibodies are now available. To date, three anti-PD-L1 antibodies have been approved by the FDA, namely atezolizumab, durvalumab and avelumab. The main advantages of anti-PD-L1 antibodies arise from the overexpression of PD-L1 antigen by a high number of tumor cells, also deriving from different tissues; this makes anti-PD-L1 antibodies potential pan-specific anti-cancer molecules. Despite the good results reported in clinical trials with anti-PD-L1 antibodies, there is a significant number of patients that do not respond to the therapy. In fact, it should be considered that, in some neoplastic patients, reduced or absent infiltration of cytotoxic T cells and natural killer cells in the tumor microenvironment or presence of other immunosuppressive molecules make immunotherapy with anti-PD-L1 blocking antibodies less effective. A strategy to improve the efficacy of antibodies is to use them as carriers for toxic payloads (toxins, drugs, enzymes, radionuclides, etc.) to form immunoconjugates. Several immunoconjugates have been already approved by FDA for treatment of malignancies. In this review, we focused on PD-L1 targeting antibodies utilized as carrier to construct immunoconjugates for the potential elimination of neoplastic cells, expressing PD-L1. A complete examination of the literature regarding anti-PD-L1 immunoconjugates is here reported, describing the results obtained in vitro and in vivo. The real potential of anti-PD-L1 antibodies as carriers for toxic payload delivery is considered and extensively discussed.

Chronic kidney disease: Which role for xanthine oxidoreductase activity and products?

The present review explores the role of xanthine oxidoreductase (XOR) in the development and progression of chronic kidney disease (CKD). Human XOR is a multi-level regulated enzyme, which has many physiological functions, but that is also implicated in several pathological processes. The main XOR activities are the purine catabolism, which generates uric acid, and the regulation of cell redox state and cell signaling, through the production of reactive oxygen species. XOR dysregulation may lead to hyperuricemia and oxidative stress, which could have a pathogenic role in the initial phases of CKD, by promoting cell injury, hypertension, chronic inflammation and metabolic derangements. Hypertension is common in CKD patients and many mechanisms inducing it (upregulation of renin-angiotensin-aldosterone system, endothelial dysfunction and atherosclerosis) may be influenced by XOR products. High XOR activity and hyperuricemia are also risk factors for obesity, insulin resistance, type 2 diabetes and metabolic syndrome that are frequent CKD causes. Moreover, CKD is common in patients with gout, which is characterized by hyperuricemia, and in patients with cardiovascular diseases, which are associated with hypertension, endothelial dysfunction and atherosclerosis. Although hyperuricemia is undoubtedly related to CKD, controversial findings have been hitherto reported in patients treated with urate-lowering therapies.

Toxin and Immunotoxin Based Therapeutic Approaches.

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Sequence, Structure, and Binding Site Analysis of Kirkiin in Comparison with Ricin and Other Type 2 RIPs.

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

Xanthine oxidoreductase: A leading actor in cardiovascular disease drama.

Cardiovascular diseases (CVD) are the leading cause of global mortality and their pathogenesis lies mainly in the atherosclerotic process. There are close connections linking oxidative stress and inflammation to endothelial dysfunction, atherosclerosis and, consequently, to CVD. This review focuses on the role of xanthine oxidoreductase (XOR) and its products on the development of chronic inflammation and oxidative stress, responsible for atheromatous plaque formation. Evidence is reported that an excessive level of XOR products favors inflammatory response and plaque development, thereby promoting major cardiovascular risk factors. Also, the relationship between hyperuricemia and hypertension as well as between XOR activity and CVD is confirmed. In spite of the increasing number of clinical studies investigating the output of cardiovascular patients treated with urate-lowering therapies (including uricosuric drugs, XOR inhibitors and recombinant uricase) the results are still uncertain. The inhibition of XOR activity appears more promising than just the control of uricemia level in preventing cardiovascular events, possibly because it also reduces the intracellular accumulation of urate, as well as the production of reactive oxygen species. However, XOR inhibition also reduces the availability of the multifaced mediator nitric oxide and, at present, can be recommended only in hyperuricemic patients.

Antibody Conjugates for Sarcoma Therapy: How Far along Are We?

Sarcomas are one of the most difficult type of cancer to manage and treat because of their extremely heterogeneous molecular and morphological features. Despite the progress made over the years in the establishment of standard protocols for high and low grading/staging sarcoma patients, mostly with chemotherapy and/or radiotherapy, 50% of treated patients experience relapse episodes. Because of this, in the last 20 years, new therapeutic approaches for sarcoma treatment have been evaluated in preclinical and clinical studies. Among them, antibody-based therapies have been the most studied. Immunoconjugates consist of a carrier portion, frequently represented by an antibody, linked to a toxic moiety, i.e., a drug, toxin, or radionuclide. While the efficacy of immunoconjugates is well demonstrated in the therapy of hematological tumors and more recently also of epithelial ones, their potential as therapeutic agents against sarcomas is still not completely explored. In this paper, we summarize the results obtained with immunoconjugates targeting sarcoma surface antigens, considering both preclinical and clinical studies. To date, the encouraging results obtained in preclinical studies allowed nine immunoconjugates to enter clinical trials, demonstrating the validity of immunotherapy as a promising pharmacological tool also for sarcoma therapy.

Xanthine oxidoreductase: One enzyme for multiple physiological tasks.

Human xanthine oxidoreductase (XOR) is a multiple-level regulated enzyme, resulting from a complicated evolutionary process that assigned it many physiological roles. The main XOR activities are: (i) xanthine dehydrogenase (XDH) activity that performs the last two steps of purine catabolism, from hypoxanthine to uric acid; (ii) xanthine oxidase (XO) activity that, besides purine catabolism, produces reactive oxygen species (ROS); (iii) nitrite reductase activity that generates nitric oxide, contributing to vasodilation and regulation of blood pressure; (iv) NADH oxidase activity that produces ROS. All these XOR activities contribute also to metabolize various endogenous and exogenous compounds, including some drugs. About XOR products, it should be considered that (i) uric acid is not only a proinflammatory agent, but also a fundamental antioxidant molecule in serum and (ii) XOR-derived ROS are essential to the inflammatory defensive response. Although XOR has been the object of a large number of studies, most of them were focused on the pathological consequences of its activity and there is not a clear and schematic picture of XOR physiological roles. In this review, we try to fill this gap, reporting and graphically schematizing the main roles of XOR and its products.

Kirkiin: A New Toxic Type 2 Ribosome-Inactivating Protein from the Caudex of Adenia kirkii.

Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.

Pro-Aging Effects of Xanthine Oxidoreductase Products.

The senescence process is the result of a series of factors that start from the genetic constitution interacting with epigenetic modifications induced by endogenous and environmental causes and that lead to a progressive deterioration at the cellular and functional levels. One of the main causes of aging is oxidative stress deriving from the imbalance between the production of reactive oxygen (ROS) and nitrogen (RNS) species and their scavenging through antioxidants. Xanthine oxidoreductase (XOR) activities produce uric acid, as well as reactive oxygen and nitrogen species, which all may be relevant to such equilibrium. This review analyzes XOR activity through in vitro experiments, animal studies and clinical reports, which highlight the pro-aging effects of XOR products. However, XOR activity contributes to a regular level of ROS and RNS, which appears essential for the proper functioning of many physiological pathways. This discourages the use of therapies with XOR inhibitors, unless symptomatic hyperuricemia is present.

Primary Sequence and 3D Structure Prediction of the Plant Toxin Stenodactylin.

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.

Early Response to the Plant Toxin Stenodactylin in Acute Myeloid Leukemia Cells Involves Inflammatory and Apoptotic Signaling.

Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Adenia stenodactyla Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt's lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.

Insights into penicillin-induced Chlamydia trachomatis persistence.

Chlamydia persistence is a viable, but non-cultivable, growth stage, resulting in a long-term relationship with the infected host cell. In vitro, this condition can be induced by different stressor agents, including beta-lactam antibiotics, as penicillin. The aim of this study was to get new insights into the interactions between Chlamydia trachomatis (serovars D and L2) and the epithelial host cells (HeLa) during persistence condition. In particular, we evaluated the following aspects, by comparing the normal chlamydial development cycle with penicillin-induced persistence: (i) cell survival/death, (ii) externalization of phosphatidylserine, (iii) caspase 1 and caspase 3/7 activation, and (iv) reactive oxygen species (ROS) production by the infected cells. At 72 h post-infection, the cytotoxic effect displayed by CT was completely abolished for both serovars and for all levels of multiplicity of infection only in the cells with aberrant CT inclusions. At the same time, CT was able to switch off the exposure of the lipid phosphatidylserine on the surface of epithelial cells and to strongly inhibit the activation of caspase 1 and caspase 3/7 only in penicillin-treated cells. Forty-eight hours post-infection, CT elicited a significant ROS expression both in case of a normal cycle and in case of persistence. However, serovar L and penicillin-free infection activated a higher ROS production compared to serovar D and to penicillin-induced persistence, respectively. In conclusion, we added knowledge to the cellular dynamics taking place during chlamydial persistence, demonstrating that CT creates a suitable niche to survive, switching off signals able to activate phagocytes/leukocytes recruitment. Nevertheless, persistent CT elicits ROS production by the infected cells, potentially contributing to the onset of chronic inflammation and tissue damages.

Ricin: An Ancient Story for a Timeless Plant Toxin.

The castor plant (Ricinus communis L.) has been known since time immemorial in traditional medicine in the pharmacopeia of Mediterranean and eastern ancient cultures. Moreover, it is still used in folk medicine worldwide. Castor bean has been mainly recommended as anti-inflammatory, anthelmintic, anti-bacterial, laxative, abortifacient, for wounds, ulcers, and many other indications. Many cases of human intoxication occurred accidentally or voluntarily with the ingestion of castor seeds or derivatives. Ricinus toxicity depends on several molecules, among them the most important is ricin, a protein belonging to the family of ribosome-inactivating proteins. Ricin is the most studied of this category of proteins and it is also known to the general public, having been used for several biocrimes. This manuscript intends to give the reader an overview of ricin, focusing on the historical path to the current knowledge on this protein. The main steps of ricin research are here reported, with particular regard to its enzymatic activity, structure, and cytotoxicity. Moreover, we discuss ricin toxicity for animals and humans, as well as the relation between bioterrorism and ricin and its impact on environmental toxicity. Ricin has also been used to develop immunotoxins for the elimination of unwanted cells, mainly cancer cells; some of these immunoconjugates gave promising results in clinical trials but also showed critical limitation.

Survival and death of intestinal cells infected by Chlamydia trachomatis.

The sexually transmitted pathogen Chlamydia trachomatis (CT) is able to replicate and survive in human intestinal epithelial cells, being the gastro-intestinal tract a suitable site of residence for this microorganism. In this context, no detailed information about the mechanisms of cell death in intestinal cell lines after a chlamydial infection is available. The aim of this study was to compare the effect of two different CT serovars (D and L2) on the survival/death of different intestinal cell lines (Caco-2 and COLO-205), using endocervical cells (HeLa) as a reference model of genital infection. Seventy two hours after chlamydial infection at different multiplicity of infection (MOI) levels, the viability of HeLa, Caco-2 and COLO 205 cells was evaluated through dose-response experiments by means of a MTS-based assay. To get deeper insights in the mechanisms of cell death induced by CT, cell viability was assessed in presence of different inhibitors (i.e. pan-caspase inhibitor Z-VAD, necroptosis inhibitor Necrostatin-1, hydrogen peroxide scavenger catalase, caspase-1 inhibitor Ac-YVAD-cmk). Moreover, the activation of effector caspases and the presence of cellular apoptotic/necrotic changes were evaluated at different time points after CT infection. Our results demonstrated that, for both chlamydial serovars, intestinal cell lines are more resistant to CT-induced cell death compared to HeLa, thus representing a suitable 'niche' for chlamydial residence and replication. In literature, apoptosis has been widely described to be the main cell death mechanism elicited by chlamydia infection. However, our data demonstrate that necroptosis plays a relevant role, proceeding in parallel with apoptosis. The protective effect of catalase suggests the involvement of oxidative stress in triggering both cell death pathways. Moreover, we demonstrated that caspase-1 is involved in CT-induced cell death, potentially contributing to host inflammatory response and tissue damage. Cells infected by L2 serovar displayed a higher activation of effector caspases compared to cells infected with serovar D, suggesting a serovar-specific activation of apoptotic pathways and potentially explaining the greater virulence of L serovars. Finally, we found that Chlamydia elicits the early externalization of phosphatidylserine on the external leaflet of plasma membrane independently of caspase activation.

Hexadecenoic Fatty Acid Positional Isomers and De Novo PUFA Synthesis in Colon Cancer Cells.

Palmitic acid metabolism involves delta-9 and delta-6 desaturase enzymes forming palmitoleic acid (9cis-16:1; n-7 series) and sapienic acid (6cis-16:1; n-10 series), respectively. The corresponding biological consequences and lipidomic research on these positional monounsaturated fatty acid (MUFA) isomers are under development. Furthermore, sapienic acid can bring to the de novo synthesis of the n-10 polyunsaturated fatty acid (PUFA) sebaleic acid (5cis,8cis-18:2), but such transformations in cancer cells are not known. The model of Caco-2 cell line was used to monitor sapienic acid supplementation (150 and 300 µM) and provide evidence of the formation of n-10 fatty acids as well as their incorporation at levels of membrane phospholipids and triglycerides. Comparison with palmitoleic and palmitic acids evidenced that lipid remodelling was influenced by the type of fatty acid and positional isomer, with an increase of 8cis-18:1, n-10 PUFA and a decrease of saturated fats in case of sapienic acid. Cholesteryl esters were formed only in cases with sapienic acid. Sapienic acid was the less toxic among the tested fatty acids, showing the highest EC50s and inducing death only in 75% of cells at the highest concentration tested. Two-photon fluorescent microscopy with Laurdan as a fluorescent dye provided information on membrane fluidity, highlighting that sapienic acid increases the distribution of fluid regions, probably connected with the formation of 8cis-18:1 and the n-10 PUFA in cell lipidome. Our results bring evidence for MUFA positional isomers and de novo PUFA synthesis for developing lipidomic analysis and cancer research.