Massimo Bortolotti

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.

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.

Xanthine oxidoreductase in cancer: more than a differentiation marker

Human xanthine oxidoreductase (XOR) catalyzes the last two steps of purine catabolism and is present in two interconvertible forms, which may utilize O2 or NAD+ as electron acceptors. In addition to uric acid, XOR products may comprise reactive oxygen and nitrogen species that have many biologic effects, including inflammation, endothelial dysfunction, and cytotoxicity, as well as mutagenesis and induction of proliferation. XOR is strictly modulated at the transcriptional and post‐translational levels, and its expression and activity are highly variable in cancer. Xanthine oxidoreductase (XOR) expression has been negatively associated with a high malignity grade and a worse prognosis in neoplasms of the breast, liver, gastrointestinal tract, and kidney, which normally express a high level of XOR protein. However, the level of XOR expression may be associated with a worse outcome in cancer of low XOR‐expressing cells, in relation to the inflammatory response elicited through the tissue damage induced by tumor growth. Xanthine oxidoreductase (XOR) has been implicated in the process of oncogenesis either directly because it is able to catalyze the metabolic activation of carcinogenic substances or indirectly through the action of XOR‐derived reactive oxygen and nitrogen species. The role of uric acid is characterized by both oxidant and antioxidant action; thus, it is still debatable whether control of uricemia may be helpful to improve the outcomes of tumor illness.

Plant Toxin-Based Immunotoxins for Cancer Therapy: A Short Overview

Immunotoxins are chimeric proteins obtained by linking a toxin to either an intact antibody or an antibody fragment. Conjugation can be obtained by chemical or genetic engineering, where the latter yields recombinant conjugates. An essential requirement is that the target molecule recognized by the antibody is confined to the cell population to be deleted, or at least that it is not present on stem cells or other cell types essential for the organism’s survival. Hundreds of different studies have demonstrated the potential for applying immunotoxins to many models in pre-clinical studies and in clinical trials. Immunotoxins can be theoretically used to eliminate any unwanted cell responsible for a pathological condition. The best results have been obtained in cancer therapy, especially in hematological malignancies. Among plant toxins, the most frequently employed to generate immunotoxins are ribosome-inactivating proteins, the most common being ricin. This review summarizes the various approaches and results obtained in the last four decades by researchers in the field of plant toxin-based immunotoxins for cancer therapy.

ATG-saporin-S6 immunotoxin: a new potent and selective drug to eliminate activated lymphocytes and lymphoma cells

Anti‐thymocyte globulins (ATG) are currently used to prevent graft‐versus‐host disease in haematopoietic stem cell transplants from alternative donors and to treat and prevent acute organ rejection after transplantation. Many recent studies have demonstrated that ATG can also be beneficial in patients with myeloma, lymphoma, leukaemia and myelodysplastic syndrome. This study showed, for the first time, that the cytotoxic effect of ATG can been enhanced by conjugation with saporin‐S6, which is one of the most stable and active type‐1 ribosome‐inactivating proteins. The ATG‐saporin‐S6 immunotoxin showed a strong cytotoxic effect on five lymphoma‐ and leukaemia‐derived cell lines as well as on activated lymphocytes while sparing non‐haematological cell lines. ATG‐saporin‐S6 induced a time‐dependent activation of caspase‐3/7 in RAJI cells. The caspase inhibitor Z‐VAD‐fmk partially rescued the cells that were treated with ATG‐saporin‐S6, suggesting that multiple cell death pathways, some of which are caspase independent, play a role in ATG‐saporin‐S6 toxicity. In our experiments ATG increased the complement‐independent cytotoxicity of activated lymphocytes by a magnitude of 3–5 logs after conjugation. These findings suggest that the ATG‐saporin‐S6 immunotoxin is a promising therapeutic tool for many pathological conditions involving T lymphocytes and T and B neoplastic cells.

Two Saporin-Containing Immunotoxins Specific for CD20 and CD22 Show Different Behavior in Killing Lymphoma Cells

Immunotoxins (ITs) are hybrid proteins combining the binding specificity of antibodies with the cytocidal properties of toxins. They represent a promising approach to lymphoma therapy. The cytotoxicity of two immunotoxins obtained by chemical conjugation of the plant toxin saporin-S6 with the anti-CD20 chimeric antibody rituximab and the anti-CD22 murine antibody OM124 were evaluated on the CD20-/CD22-positive cell line Raji. Both ITs showed strong cytotoxicity for Raji cells, but the anti-CD22 IT was two logs more efficient in killing, probably because of its faster internalization. The anti-CD22 IT gave slower but greater caspase activation than the anti-CD20 IT. The cytotoxic effect of both immunotoxins can be partially prevented by either the pan-caspase inhibitor Z-VAD or the necroptosis inhibitor necrostatin-1. Oxidative stress seems to be involved in the cell killing activity of anti-CD20 IT, as demonstrated by the protective role of the H2O2 scavenger catalase, but not in that of anti-CD22 IT. Moreover, the IT toxicity can be augmented by the contemporary administration of other chemotherapeutic drugs, such as PS-341, MG-132, and fludarabine. These results contribute to the understanding of the immunotoxin mechanism of action that is required for their clinical use, either alone or in combination with other drugs.

Plants Producing Ribosome-Inactivating Proteins in Traditional Medicine.

Ribosome-inactivating proteins (RIPs) are enzymes that deadenylate nucleic acids and are broadly distributed in the plant kingdom. Many plants that contain RIPs are listed in the pharmacopoeias of folk medicine all over the world, mostly because of their toxicity. This review analyses the position occupied in traditional medicine by plants from which RIPs have been isolated. The overview starts from the antique age of the Mediterranean area with ancient Egypt, followed by the Greek and Roman classic period. Then, the ancient oriental civilizations of China and India are evaluated. More recently, Unani medicine and European folk medicine are examined. Finally, the African and American folk medicines are taken into consideration. In conclusion, a list of RIP-expressing plants, which have been used in folk medicine, is provided with the geographical distribution and the prescriptions that are recommended by traditional healers. Some final considerations are provided on the present utilization of such herbal treatments, both in developing and developed countries, often in the absence of scientific validation. The most promising prospect for the medicinal use of RIP-expressing plants is the conjugation of purified RIPs to antibodies that recognise tumour antigens for cancer therapy.