Letizia Polito

CTLA‐4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction

CTLA‐4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA‐4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA‐4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast‐like cultures). However, by reverse transcriptase‐PCR, CTLA‐4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA‐4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA‐4‐expressing tumor lines with recombinant forms of the CTLA‐4‐ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase‐8 and caspase‐3. The level of apoptosis was reduced by soluble CTLA‐4 and by anti‐CTLA‐4 scFvs antibodies. The novel finding that CTLA‐4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.

Pathophysiology of circulating xanthine oxidoreductase: new emerging roles for a multi-tasking enzyme.

The enzyme xanthine oxidoreductase (XOR) catalyses the last step of purine degradation in the highest uricotelic primates as a rate-limiting enzyme in nucleic acid catabolism. Although XOR has been studied for more than a century, this enzyme continues to arouse interest because its involvement in many pathological conditions is not completely known. XOR is highly evolutionarily conserved; moreover, its activity is very versatile and tuneable at multiple-levels and generates both oxidant and anti-oxidant products. This review covers the basic information on XOR biology that is essential to understand its enzymatic role in human pathophysiology and provides a comprehensive catalogue of the experimental and human pathologies associated with increased serum XOR levels. The production of radical species by XOR oxidase activity has been intensively studied and evaluated in recent decades in conjunction with the cytotoxic consequences and tissue injuries of various pathological conditions. More recently, a role has emerged for the activity of endothelium-bound enzymes in inducing the vascular response to oxidative stress, which includes the regulation of pro-inflammatory and pro-thrombotic activities of endothelial cells. The possible physiological functions of circulating XOR and the products of its enzyme activity are presented here together with their implications in cardiovascular and metabolic diseases.

Xanthine oxidoreductase in atherosclerosis pathogenesis: Not only oxidative stress

Endothelial xanthine oxidoreductase (XOR) together with NAD(P)H oxidase and nitric oxide (NO) synthase plays a physiologic role in inflammatory signalling, the regulation of NO production and vascular function. The oxidative stress generated by these enzymes may induce endothelial dysfunction, leading to atherosclerosis, cardiovascular diseases and metabolic syndrome. XOR activity creates both oxidant and anti-oxidant products that are implicated in the development of hypertension, smoking vascular injury, dyslipidemia and diabetes, which are the main risk factors of atherosclerosis. In particular, uric acid may have a protective as well as a detrimental role in vascular alterations, thus justifying the multi-directional effects of XOR inhibition. Moreover, XOR products are associated with cell differentiation, leading to adipogenesis and foam cell formation, as well as to the production of monocyte chemoattractant protein-1 from arterial smooth muscle cells, after proliferation and migration. The role of XOR in adipogenesis is also connected with insulin resistance and obesity, two main features of type 2 diabetes.

Immunotoxins and other conjugates containing saporin-s6 for cancer therapy.

Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface.

Saporin-S6: A Useful Tool in Cancer Therapy

Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials.

Saporin induces multiple death pathways in lymphoma cells with different intensity and timing as compared to ricin.

Ribosome-inactivating protein (RIP)-containing immunotoxins are currently used in clinical trials as anti-tumour drugs, in particular against haematological malignancies. In cell killing-based therapies it is important to identify the death pathways induced by the cytotoxic agent. The purpose of this work was to compare the pathways of cell death induced by the RIP saporin with those carried out by ricin in the L540 human Hodgkins lymphoma-derived cell line. Protein synthesis inhibition, activation of caspases, DNA fragmentation and loss of viability have been evaluated. The two toxins triggered a similar DNA fragmentation and cell death, at concentrations giving the same level of cell protein synthesis inhibition, although the inhibitory effect of ricin on protein synthesis was more rapid than that of saporin. Moreover, the intrinsic apoptotic pathway was equally activated by both toxins, whilst ricin activated the extrinsic caspase pathway and the effector caspase-3/7 more efficiently than saporin. The complete inhibition of caspases by Z-VAD was only partially effective in cell rescue which appeared to be time limited. Necrostatin-1, a new inhibitor of non-apoptotic death, rescued cells from death by RIPs, although the effect was also partial and temporary. Despite the high RIP doses used no necrosis was detectable by Annexin V/Propidium Iodide (PI) test. These results suggest that more than one death mechanism was elicited by both ricin and saporin, however, with different timing and strength. The perspective of modulating cell death of neoplastic lymphocytes through different pathways could add new opportunities to reduce side effects and develop combined synergic immuno-chemotherapy.

Xanthine Oxidoreductase-Derived Reactive Species: Physiological and Pathological Effects

Xanthine oxidoreductase (XOR) is the enzyme that catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid and is widely distributed among species. In addition to this housekeeping function, mammalian XOR is a physiological source of superoxide ion, hydrogen peroxide, and nitric oxide, which can function as second messengers in the activation of various pathways. This review intends to address the physiological and pathological roles of XOR-derived oxidant molecules. The cytocidal action of XOR products has been claimed in relation to tissue damage, in particular damage induced by hypoxia and ischemia. Attempts to exploit this activity to eliminate unwanted cells via the construction of conjugates have also been reported. Moreover, different aspects of XOR activity related to phlogosis, endothelial activation, leukocyte activation, and vascular tone regulation, have been taken into consideration. Finally, the positive and negative outcomes concerning cancer pathology have been analyzed because XOR products may induce mutagenesis, cell proliferation, and tumor progression, but they are also associated with apoptosis and cell differentiation. In conclusion, XOR activity generates free radicals and other oxidant reactive species that may result in either harmful or beneficial outcomes.

Sequence determination of lychnin, a type 1 ribosome-inactivating protein from Lychnis chalcedonica seeds.

Abstract The complete amino acid sequence of lychnin, a type 1 ribosome-inactivating protein (RIP) isolated from Lychnis chalcedonica seeds, has been determined by automated Edman degradation and ESI-QTOF mass spectrometry. Lychnin consists of 234 amino acid residues with a molecular mass of 26 131.14 Da. All amino acid residues involved in the formation of the RIP active site (Tyr69, Tyr119, Glu170, Arg173 and Trp203) are fully conserved. Furthermore, a fast MALDI-TOF experiment showed that two out of three cysteinyl residues (Cys32 and Cys115) form a disulfide bridge, while Cys214 is in the thiol form, which makes it suitable for linking carrier molecules to generate immunotoxins and other conjugates.

Hepatotoxicity of ricin, saporin or a saporin immunotoxin: xanthine oxidase activity in rat liver and blood serum

Male Wistar rats each received an i.p. injection of the ribosome-inactivating proteins ricin or saporin, or a Ber-H2 (anti-CD30)-saporin immunotoxin at a dose corresponding to three times the LD50 calculated for mice. Animals were killed 24, 48 or 72 h after treatment. Histological examination showed hepatic necrosis in all treated animals, although the sinusoidal lining was affected only in ricin-poisoned rats. The activities of xanthine dehydrogenase (D-form) and oxidase (O form) were determined spectrophotometrically in liver and serum samples. In ricin-treated animals the liver enzyme was progressively converted from the D- to the O-form, which accounted for more than 60% of total activity after 48 h of poisoning, whilst no change in the xanthine oxidase activity was found in the serum. In the liver of rats treated with free or Ber-H2-conjugated saporin, the D-form was more than 75%, as in normal animals. In the same animals the serum xanthine oxidase activity was up to three-fold control values. The determination of serum xanthine oxidase may prove helpful in the evaluation of liver damage in patients treated with immunotoxins. It may become a diagnostic tool for the differential diagnosis of liver diseases.

Apoptosis and necroptosis induced by stenodactylin in neuroblastoma cells can be completely prevented through caspase inhibition plus catalase or necrostatin-1

BACKGROUND Stenodactylin is a highly toxic plant lectin purified from the caudex of Adenia stenodactyla, with molecular structure, intracellular routing and enzyme activity similar to those of ricin, a well-known type 2 ribosome-inactivating protein. However, in contrast with ricin, stenodactylin is retrogradely transported not only in peripheral nerves but also in the central nervous system. PURPOSE Stenodactylin properties make it a potential candidate for application in neurobiology and in experimental therapies against cancer. Thus, it is necessary to better clarify the toxic activity of this compound. STUDY DESIGN We investigated the mechanism of stenodactylin-induced cell death in the neuroblastoma-derived cell line, NB100, evaluating the implications of different death pathways and the involvement of oxidative stress. METHODS Stenodactylin cytotoxicity was determined by evaluating protein synthesis and other viability parameters. Cell death pathways and oxidative stress were analysed through flow cytometry and microscopy. Inhibitors of apoptosis, oxidative stress and necroptosis were tested to evaluate their protective effect against stenodactylin cytotoxicity. RESULTS Stenodactylin efficiently blocked protein synthesis and reduced the viability of neuroblastoma cells at an extremely low concentration and over a short time (1 pM, 24 h). Stenodactylin induced the strong and rapid activation of apoptosis and the production of free radicals. Here, for the first time, a complete and long lasting protection from the lethal effect induced by a toxic type 2 ribosome-inactivating protein has been obtained by combining the caspase inhibitor Z-VAD-fmk, to either the hydrogen peroxide scavenger catalase or the necroptotic inhibitor necrostatin-1. CONCLUSION In respect to stenodactylin cytotoxicity, our results: (i) confirm the high toxicity to nervous cells, (ii) indicate that multiple cell death pathways can be induced, (iii) show that apoptosis is the main death pathway, (iv) demonstrate the involvement of necroptosis and (v) oxidative stress.