Daniele Mercatelli

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.

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.

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.

Protein synthesis inhibition activity by strawberry tissue protein extracts during plant life cycle and under biotic and abiotic stresses.

Ribosome-inactivating proteins (RIPs), enzymes that are widely distributed in the plant kingdom, inhibit protein synthesis by depurinating rRNA and many other polynucleotidic substrates. Although RIPs show antiviral, antifungal, and insecticidal activities, their biological and physiological roles are not completely understood. Additionally, it has been described that RIP expression is augmented under stressful conditions. In this study, we evaluated protein synthesis inhibition activity in partially purified basic proteins (hereafter referred to as RIP activity) from tissue extracts of Fragaria × ananassa (strawberry) cultivars with low (Dora) and high (Record) tolerance to root pathogens and fructification stress. Association between the presence of RIP activity and the crop management (organic or integrated soil), growth stage (quiescence, flowering, and fructification), and exogenous stress (drought) were investigated. RIP activity was found in every tissue tested (roots, rhizomes, leaves, buds, flowers, and fruits) and under each tested condition. However, significant differences in RIP distribution were observed depending on the soil and growth stage, and an increase in RIP activity was found in the leaves of drought-stressed plants. These results suggest that RIP expression and activity could represent a response mechanism against biotic and abiotic stresses and could be a useful tool in selecting stress-resistant strawberry genotypes.

Quantitative imaging techniques for the assessment of osteoporosis and sarcopenia

Bone and muscle are two deeply interconnected organs and a strong relationship between them exists in their development and maintenance. The peak of both bone and muscle mass is achieved in early adulthood, followed by a progressive decline after the age of 40. The increase in life expectancy in developed countries resulted in an increase of degenerative diseases affecting the musculoskeletal system. Osteoporosis and sarcopenia represent a major cause of morbidity and mortality in the elderly population and are associated with a significant increase in healthcare costs. Several imaging techniques are currently available for the non-invasive investigation of bone and muscle mass and quality. Conventional radiology, dual energy X-ray absorptiometry (DXA), computed tomography (CT), magnetic resonance imaging (MRI) and ultrasound often play a complementary role in the study of osteoporosis and sarcopenia, depicting different aspects of the same pathology. This paper presents the different imaging modalities currently used for the investigation of bone and muscle mass and quality in osteoporosis and sarcopenia with special emphasis on the clinical applications and limitations of each technique and with the intent to provide interesting insights into recent advances in the field of conventional imaging, novel high-resolution techniques and fracture risk.

Correlation between DXA and laboratory parameters in normal weight, overweight and obese patients

OBJECTIVE The aim of this study was to review the existence and types of correlations between body composition densitometric parameters and laboratory values associated to cardiometabolic risk. METHODS We retrospectively analyzed data from 316 individuals in the weight range from normality to super-obesity, submitted to total body dual-energy x-ray absorptiometry (DXA) scans and routine biochemistry at S.Orsola-Malpighi Hospital from June 2010 to March 2014. The study included 182 women, 45.8 ± 13.4 y of age, with a body mass index (BMI) of 31.5 (± 11) kg/m2 (group F) and 134 men, 45.4 ± 13.6 y of age, with a BMI of 27.6 (± 7.8) kg/m2 (group M). All patients underwent whole-body scan (Lunar iDXA, GE Healthcare, Madison, WI, USA) and laboratory analysis (blood fasting glucose, total cholesterol, high-density lipoprotein cholesterol, tricylglycerides [TGs], aspartate aminotransferase, and alanine aminotransferase). Correlation between laboratory values and total body and regional fat mass (including visceral adipose tissue [VAT] and subcutaneous adipose tissue in the android region), and lean mass parameters were analyzed with linear and stepwise regressions analysis (significance limit, P < 0.05). Receiver operating characteristic curves were performed to assess the accuracy of the best-fit DXA parameter (VAT) to identify at least one laboratory risk factor. RESULTS In both groups, BMI and densitometric parameters showed a linear correlation with fasting blood glucose and TG levels and an inverse correlation with high-density lipoprotein cholesterol (P < 0.05), whereas no correlation was observed with total cholesterol levels. The only densitometric parameter retained in the final model of stepwise multiple regression was VAT for fasting blood glucose (group F: β = 0.4627, P < 0.0001; group M: β = 0.6221, P < 0.0001) and TG levels (group F: β = 0.4931, P < 0.0001; group M: β = 0.1990, P < 0.0261) independently of BMI. The optimal cutoff points of VAT to identify the presence of at least one laboratory risk factor were >1395 g and >1479 cm3 for men and >1281 g and >1357 cm3 for women. CONCLUSIONS DXA analysis of VAT is associated with selected laboratory parameters used for the evaluation of cardiometabolic risk and could be per se a helpful parameter in the assessment of clinical risk.

Torticollis After a Somersault: A Case of Grisel’s Syndrome

A three-year-old boy attended the emergency department with acute neck pain and torticollis after a somersault. The child performed a forward somersault keeping both palms and the top of his head in touch with the ground while pushing forward to roll, causing a mechanical compressive stress to upper cervical vertebrae. Clinical examination revealed extreme neck stiffness and head fixed in maximum left rotation with some degrees of flexion (‘cockrobin’position). Conventional radiography suggested atlanto-axial joint dislocation (Fig. 1). Computed Tomography (CT) of the cervical spine with C1-C2 three-dimensional reconstruction revealed atlanto-axial rotatory subluxation (Fig. 1) without fracture. The child was at that time affected by pharyngitis and tonsillitis with cervical lymphadenopathies, conditions confirmed by Magnetic Resonance Imaging (Fig. 2). These findings oriented the diagnosis of Grisel’s syndrome. The child was successfully treated with cervical traction together with medical therapy (anti-inflammatories and antibiotics). Grisel’s syndrome consists of atlanto-axial rotatory subluxation or fixation, secondary to an inflammatory process of the adjacent tissues such as pharyngitis with retropharyngeal abscess, or local surgery (usually adenotonsillectomy), with secondary ligamentous laxity [1]. The condition occurs primarily in children after minor trauma, sharp movements or without any traumatic event [2].

Immunoconjugates for Osteosarcoma Therapy: Preclinical Experiences and Future Perspectives

Osteosarcoma (OS) is an aggressive osteoid-producing tumor of mesenchymal origin, which represents the most common primary bone malignancy. It is characterized by a complex and frequently uncertain etiology. The current standard care for high-grade OS treatment is neoadjuvant chemotherapy, followed by surgery and post-operative chemotherapy. In order to ameliorate survival rates of patients, new therapeutic approaches have been evaluated, mainly immunotherapy with antibody-drug conjugates or immunoconjugates. These molecules consist of a carrier (frequently an antibody) joined by a linker to a toxic moiety (drug, radionuclide, or toxin). Although several clinical trials with immunoconjugates have been conducted, mainly in hematological tumors, their potential as therapeutic agents is relatively under-explored in many types of cancer. In this review, we report the immunoconjugates directed against OS surface antigens, considering the in vitro and in vivo studies. To date, several attempts have been made in preclinical settings, reporting encouraging results and demonstrating the validity of the idea. The clinical experience with glembatumumab vedotin may provide new insights into the real efficacy of antibody-drug conjugates for OS therapy, possibly giving more information about patient selection. Moreover, new opportunities could arise from the ongoing clinical trials in OS patients with unconjugated antibodies that could represent future candidates as carrier moieties of immunoconjugates.