The Impact of Spermidine on C2C12 Myoblasts Proliferation, Redox Status and Polyamines Metabolism under H2O2 Exposure.

A central feature of the skeletal muscle is its ability to regenerate through the activation, by environmental signals, of satellite cells. Once activated, these cells proliferate as myoblasts, and defects in this process profoundly affect the subsequent process of regeneration. High levels of reactive oxygen species such as hydrogen peroxide (H2O2) with the consequent formation of oxidized macromolecules increase myoblasts' cell death and strongly contribute to the loss of myoblast function. Recently, particular interest has turned towards the beneficial effects on muscle of the naturally occurring polyamine spermidine (Spd). In this work, we tested the hypothesis that Spd, upon oxidative challenge, would restore the compromised myoblasts' viability and redox status. The effects of Spd in combination with aminoguanidine (Spd-AG), an inhibitor of bovine serum amine oxidase, on murine C2C12 myoblasts treated with a mild dose of H2O2 were evaluated by analyzing: (i) myoblast viability and recovery from wound scratch; (ii) redox status and (iii) polyamine (PAs) metabolism. The treatment of C2C12 myoblasts with Spd-AG increased cell number and accelerated scratch wound closure, while H2O2 exposure caused redox status imbalance and cell death. The combined treatment with Spd-AG showed an antioxidant effect on C2C12 myoblasts, partially restoring cellular total antioxidant capacity, reducing the oxidized glutathione (GSH/GSSG) ratio and increasing cell viability through a reduction in cell death. Moreover, Spd-AG administration counteracted the induction of polyamine catabolic genes and PA content decreased due to H2O2 challenges. In conclusion, our data suggest that Spd treatment has a protective role in skeletal muscle cells by restoring redox balance and promoting recovery from wound scratches, thus making myoblasts able to better cope with an oxidative insult.

Unraveling the role of male reproductive tract and haemolymph in cantharidin-exuding Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae): a comparative transcriptomics approach.

BACKGROUND: Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. RESULTS: Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. CONCLUSIONS: The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Epileptic seizures and oxidative stress in a mouse model over-expressing spermine oxidase.

Several studies have demonstrated high polyamine levels in brain diseases such as epilepsy. Epilepsy is the fourth most common neurological disorder and affects people of all ages. Excitotoxic stress has been associated with epilepsy and it is considered one of the main causes of neuronal degeneration and death. The transgenic mouse line Dach-SMOX, with CD1 background, specifically overexpressing spermine oxidase in brain cortex, has been proven to be highly susceptible to epileptic seizures and excitotoxic stress induced by kainic acid. In this study, we analysed the effect of spermine oxidase over-expression in a different epileptic model, pentylenetetrazole. Behavioural evaluations of transgenic mice compared to controls showed a higher susceptibility towards pentylentetrazole. High-performance liquid chromatography analysis of transgenic brain from treated mice revealed altered polyamine content. Immunoistochemical analysis indicated a rise of 8-oxo-7,8-dihydro-2'-deoxyguanosine, demonstrating an increase in oxidative damage, and an augmentation of system xc- as a defence mechanism. This cascade of events can be initially linked to an increase in protein kinase C alpha, as shown by Western blot. This research points out the role of spermine oxidase, as a hydrogen peroxide producer, in the oxidative stress during epilepsy. Moreover, Dach-SMOX susceptibility demonstrated by two different epileptic models strongly indicates this transgenic mouse line as a potential animal model to study epilepsy.

Emerging Role for Linear and Circular Spermine Oxidase RNAs in Skeletal Muscle Physiopathology.

Skeletal muscle atrophy is a pathological condition so far without effective treatment and poorly understood at a molecular level. Emerging evidence suggest a key role for circular RNAs (circRNA) during myogenesis and their deregulation has been reported to be associated with muscle diseases. Spermine oxidase (SMOX), a polyamine catabolic enzyme plays a critical role in muscle differentiation and the existence of a circRNA arising from SMOX gene has been recently identified. In this study, we evaluated the expression profile of circular and linear SMOX in both C2C12 differentiation and dexamethasone-induced myotubes atrophy. To validate our findings in vivo their expression levels were also tested in two murine models of amyotrophic lateral sclerosis: SOD1G93A and hFUS+/+, characterized by progressive muscle atrophy. During C2C12 differentiation, linear and circular SMOX show the same trend of expression. Interestingly, in atrophy circSMOX levels significantly increased compared to the physiological state, in both in vitro and in vivo models. Our study demonstrates that SMOX represents a new player in muscle physiopathology and provides a scientific basis for further investigation on circSMOX RNA as a possible new therapeutic target for the treatment of muscle atrophy.

Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase.

Fourteen polyamine analogues, asymmetric or symmetric substituted spermine (1-9) or methoctramine (10-14) analogues, were evaluated as potential inhibitors or substrates of two enzymes of the polyamine catabolic pathway, spermine oxidase (SMOX) and acetylpolyamine oxidase (PAOX). Compound 2 turned out to be the best substrate for PAOX, having the highest affinity and catalytic efficiency with respect to its physiological substrates. Methoctramine (10), a well-known muscarinic M2 receptor antagonist, emerged as the most potent competitive PAOX inhibitor known so far (Ki = 10 nM), endowed with very good selectivity compared with SMOX (Ki=1.2 µM vs SMOX). The efficacy of methoctramine in inhibiting PAOX activity was confirmed in the HT22 cell line. Methoctramine is a very promising tool in the design of drugs targeting the polyamine catabolism pathway, both to understand the physio-pathological role of PAOX vs SMOX and for pharmacological applications, being the polyamine pathway involved in various pathologies.

A multilocus view on Mediterranean aeolid nudibranchs (Mollusca): Systematics and cryptic diversity of Flabellinidae and Piseinotecidae.

Recent molecular studies revealed high level of endemism and numerous cryptic species within opisthobranchs, with Mediterranean taxa clearly understudied. Here we used genetic data from both mitochondrial and nuclear gene fragments as well as morphological data from taxonomically relevant characters to investigate the phylogenetic relationships and systematics of Mediterranean taxa of the Flabellinidae and Piseinotecidae families. Phylogenetic analyses based on Bayesian and Maximum-Likelihood methods indicate that Flabellinidae and Pisenotecidae taxa and species within the genera Flabellina, Calmella and Piseinotecus do not form monophyletic clades. These results are supported by our morphological analyses which allowed the re-evaluation of the triseriate radula condition in Pisenotecidae and Calmella taxa and their inclusion in the genus Flabellina as Flabellina gaditanacomb. nov. (synonym of F. confusa), Flabellina gabiniereicomb. nov. and Flabellina cavolinicomb. nov. Species delimitation and barcoding gap analyses allowed uncovering cryptic species within Flabellina gracilis (Alder and Hancock, 1844), F. trophina (Bergh, 1890), F. verrucosa (M. Sars, 1829) and F. ischitana Hirano and Thompson, 1990, the latter with an Atlantic form which is under description. This study corroborates the relevance of combining molecular and morphological data from multiple populations and species in the assessment of nudibranch diversity and classification.

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms.

Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on the thermal denaturation of SMOX and its association forms in native and reducing conditions. By combining spectroscopic methods (circular dichroism, fluorescence) and thermal methods (differential scanning calorimetry), we provide new insights into the structure, the transformation, and the stability of SMOX. While the crystal structure of this protein is not available yet, experimental results are interpreted also on the basis of a novel SMOX structural model, obtained in silico exploiting the recently solved acetylspermine oxidase crystal structure. We conclude that while at least one specific intermolecular disulfide bond links two SMOX molecules to form the homodimer, the thermal denaturation profiles can be justified by the presence of at least one intramolecular disulfide bond, which also plays a critical role in the stabilization of the overall three-dimensional SMOX structure, and in particular of its flavin adenine dinucleotide-containing active site.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

The Glu²¹6/Ser²¹8 pocket is a major determinant of spermine oxidase substrate specificity.

SMO (spermine oxidase) and APAO (acetylpolyamine oxidase) are flavoenzymes that play a critical role in the catabolism of polyamines. Polyamines are basic regulators of cell growth and proliferation and their homoeostasis is crucial for cell life since dysregulation of polyamine metabolism has been linked with cancer. In vertebrates SMO specifically catalyses the oxidation of spermine, whereas APAO displays a wider specificity, being able to oxidize both N¹-acetylspermine and N¹-acetylspermidine, but not spermine. The molecular bases of the different substrate specificity of these two enzymes have remained so far elusive. However, previous molecular modelling, site-directed mutagenesis and biochemical characterization studies of the SMO enzyme-substrate complex have identified Glu²¹6-Ser²¹8 as a putative active site hot spot responsible for SMO substrate specificity. On the basis of these analyses, the SMO double mutants E216L/S218A and E216T/S218A have been produced and characterized by CD spectroscopy and steady-state and rapid kinetics experiments. The results obtained demonstrate that mutation E216L/S218A endows SMO with N¹-acetylspermine oxidase activity, uncovering one of the structural determinants that confer the exquisite and exclusive substrate specificity of SMO for spermine. These results provide the theoretical bases for the design of specific inhibitors either for SMO or APAO.

Polyamines metabolism and breast cancer: state of the art and perspectives.

Breast cancer (BC) is a common disease that generally occurs in women over the age of 50, and the risk is especially high for women over 60 years of age. One of the major BC therapeutic problems is that tumors initially responsive to chemotherapeutic approaches can progress to more aggressive forms poorly responsive to therapies. Polyamines (PAs) are small polycationic alkylamines, naturally occurring and essential for normal cell growth and development in eukaryotes. The intracellular concentration of PA is maintained within strongly controlled contents, while a dysregulation occurs in BC cells. Polyamines facilitate the interactions of transcription factors, such as estrogen receptors with their specific response element, and are involved in the proliferation of ER-negative and highly invasive BC tumor cells. Since PA metabolism has a critical role in cell death and proliferation, it represents a potential target for intervention in BC. The goal of this study was to perform a literature search reviewing the association between PA metabolism and BC, and the current evidence supporting the BC treatment targeting PA metabolism. We here describe in vitro and in vivo models, as well as the clinical trials that have been utilized to unveil the relationship between PA metabolism and BC. Polyamine pathway is still an important target for the development of BC chemotherapy via enzyme inhibitors. Furthermore, a recent promising strategy in breast anticancer therapy is to exploit the self-regulatory nature of PA metabolism using PA analogs to affect PA homeostasis. Nowadays, antineoplastic compounds targeting the PA pathway with novel mechanisms are of great interest and high social impact for BC chemotherapy.

Inflammation, carcinogenesis and neurodegeneration studies in transgenic animal models for polyamine research.

Natural polyamines (PA) are cationic molecules affecting cell growth and proliferation. An association between increased polyamine biosynthesis and inflammation-induced carcinogenesis has been recognised. On the other hand, there are indications that inflammatory stimuli can up-regulate polyamine catabolism and that altered polyamine metabolism could affect pro- and anti-inflammatory cytokines. Since the polyamine content is strictly related to cell growth, a consistent number of evidences relate polyamine metabolism dysfunction with cancer. The increase of polyamine levels in malignant and proliferating cells attracted the interest of scientists during last decades, addressing polyamine depletion as a new strategy to inhibit carcinogenesis. Several studies suggest that PA also play an important role in neurodegeneration, but the mechanisms by which they participate in neuronal death are still unclear. Furthermore, the role of endogenous PA in normal brain functioning is yet to be elucidated. The consequences of an alteration of polyamine metabolism have also been approached in vivo with the use of transgenic animals overexpressing or devoid of some enzymes involved in polyamine metabolism. In the present work we review the experimental investigation carried out on inflammation, cancerogenesis and neurodegeneration using transgenic animals engineered as models for polyamine research.

Spermine metabolism and radiation-derived reactive oxygen species for future therapeutic implications in cancer: an additive or adaptive response.

Destruction of cells by irradiation-induced radical formation is one of the most frequent interventions in cancer therapy. An alternative to irradiation-induced radical formation is in principle drug-induced formation of radicals, and the formation of toxic metabolites by enzyme catalyzed reactions. Thus, combination therapy targeting polyamine metabolism could represent a promising strategy to fight hyper-proliferative disease. The aim of this work is to discuss and evaluate whether the presence of a DNA damage provoked by enzymatic ROS overproduction may act as an additive or adaptive response upon radiation and combination of hyperthermia with lysosomotropic compounds may improve the cytocidal effect of polyamines oxidation metabolites. Low level of X-irradiations delivers challenging dose of damage and an additive or adaptive response with the chronic damage induced by spermine oxidase overexpression depending on the deficiency of the DNA repair mechanisms. Since reactive oxygen species lead to membrane destabilization and cell death, we discuss the effects of BSAO and spermine association in multidrug resistant cells that resulted more sensitive to spermine metabolites than their wild-type counterparts, due to an increased mitochondrial activity. Since mammal spermine oxidase is differentially activated in a tissue specific manner, and cancer cells can differ in term of DNA repair capability, it could be of interest to open a scientific debate to use combinatory treatments to alter spermine metabolism and deliver differential response.

Description of two new Jujubinus species (Gastropoda: Trochidae) from the Sicily Channel, with notes on the Jujubinus curinii species complex.

Based on shell characters, two new species of the gastropod family Trochidae, Jujubinus eleonorae n. sp. and Jujubinus trilloi n. sp., from the Sicily Channel are described. Shells of the new taxa were collected in the lower infralittoral of the Skerki and Talbot Banks, respectively. The new taxa are compared with Jujubinus curinii Bogi & Campani, 2005, morphologically the most closely related species.

Unveiling the hidden players: noncoding RNAs orchestrating polyamine metabolism in disease.

Polyamines (PA) are polycations with pleiotropic functions in cellular physiology and pathology. In particular, PA have been involved in the regulation of cell homeostasis and proliferation participating in the control of fundamental processes like DNA transcription, RNA translation, protein hypusination, autophagy and modulation of ion channels. Indeed, their dysregulation has been associated to inflammation, oxidative stress, neurodegeneration and cancer progression. Accordingly, PA intracellular levels, derived from the balance between uptake, biosynthesis, and catabolism, need to be tightly regulated. Among the mechanisms that fine-tune PA metabolic enzymes, emerging findings highlight the importance of noncoding RNAs (ncRNAs). Among the ncRNAs, microRNA, long noncoding RNA and circRNA are the most studied as regulators of gene expression and mRNA metabolism and their alteration have been frequently reported in pathological conditions, such as cancer progression and brain diseases. In this review, we will discuss the role of ncRNAs in the regulation of PA genes, with a particular emphasis on the changes of this modulation observed in health disorders.

Checklist of the marine malacofauna of Culuccia Peninsula (NW Sardinia, Italy), with notes on relevant species.

Background: Culuccia is a small peninsula of about 3 km2 placed in north-western Sardinia (Italy) at the margin of the Maddalena Archipelago. The marine area surrounding this Peninsula is a Special Area of Conservation, included in the European Natura 2000 Ecological Network of protected areas, but until now, no information on biodiversity of this area is available. In 2021, a research project to study both terrestrial and marine biodiversity of Culuccia has started in order to fill this gap of knowledge. New information: This work provides the first inventory of the marine malacofauna of the coast of Culuccia. Fifteen sites were sampled seasonally for one-year by using different sampling methods and the present study shows the results from approximately 50 scientific SCUBA and free dive surveys, carried out in all main marine habitats of the studied area. In total, 259 species of molluscs were recorded along the coasts of the Culuccia Peninsula (0-25 m depth), belonging to the classes Bivalvia, Gastropoda, Polyplacophora and Scaphopoda. Amongst the four classes recorded, gastropods were the most represented (66.90%; 173 species), followed by bivalves (28.10%; 73 species), polyplacophorans (4.60%; 12 species) and scapophods (0.40%; 1 species). Notes about distribution, conservation status and ecology for some valuable species are provided, together with images of representative species, consisting mainly of in situ photographs. Additionally, the present investigation recorded the presence of four alien species, whose Mediterranean distribution was extended to north-western Sardinia.

Structure-function relationships in the evolutionary framework of spermine oxidase.

Spermine oxidase is a FAD-dependent enzyme that specifically oxidizes spermine, and plays a central role in the highly regulated catabolism of polyamines in vertebrates. The spermine oxidase substrate is specifically spermine, a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function, cellular signalling, nitric oxide synthesis and inhibition of immune responses. The oxidative products of spermine oxidase activity are spermidine, H2O2 and the aldehyde 3-aminopropanal that spontaneously turns into acrolein. In this study the reconstruction of the phylogenetic relationships among spermine oxidase proteins from different vertebrate taxa allowed to infer their molecular evolutionary history, and assisted in elucidating the conservation of structural and functional properties of this enzyme family. The amino acid residues, which have been hypothesized or demonstrated to play a pivotal role in the enzymatic activity, and substrate specificity are here analysed to obtain a comprehensive and updated view of the structure-function relationships in the evolution of spermine oxidase.

Inhibition of acetylpolyamine and spermine oxidases by the polyamine analogue chlorhexidine.

Acetylpolyamine and spermine oxidases are involved in the catabolism of polyamines. The discovery of selective inhibitors of these enzymes represents an important tool for the development of novel anti-neoplastic drugs. Here, a comparative study on acetylpolyamine and spermine oxidases inhibition by the polyamine analogue chlorhexidine is reported. Chlorhexidine is an antiseptic diamide, commonly used as a bactericidal and bacteriostatic agent. Docking simulations indicate that chlorhexidine binding to these enzymes is compatible with the stereochemical properties of both acetylpolyamine oxidase and spermine oxidase active sites. In fact, chlorhexidine is predicted to establish several polar and hydrophobic interactions with the active site residues of both enzymes, with binding energy values ranging from -7.6 to -10.6 kcal/mol. In agreement with this hypothesis, inhibition studies indicate that chlorhexidine behaves as a strong competitive inhibitor of both enzymes, values of Ki being 0.10 µM and 0.55 µM for acetylpolyamine oxidase and spermine oxidase, respectively.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma that includes fusion-positive (FP) and fusion-negative (FN) molecular subtypes. FP-RMS expresses PAX3-FOXO1 fusion protein and often shows dismal prognosis. FN-RMS shows cytogenetic abnormalities and frequently harbors RAS pathway mutations. Despite the multimodal heavy chemo and radiation therapeutic regimens, high risk metastatic/recurrent FN-RMS shows a 5-year survival less than 30% due to poor sensitivity to chemo-radiotherapy. Therefore, the identification of novel targets is needed. Polyamines (PAs) such as putrescine (PUT), spermidine (SPD) and spermine (SPM) are low-molecular-mass highly charged molecules whose intracellular levels are strictly modulated by specific enzymes. Among the latter, spermine oxidase (SMOX) regulates polyamine catabolism oxidizing SPM to SPD, which impacts cellular processes such as apoptosis and DNA damage response. Here we report that low SMOX levels are associated with a worse outcome in FN-RMS, but not in FP-RMS, patients. Consistently, SMOX expression is downregulated in FN-RMS cell lines as compared to normal myoblasts. Moreover, SMOX transcript levels are reduced FN-RMS cells differentiation, being indirectly downregulated by the muscle transcription factor MYOD. Noteworthy, forced expression of SMOX in two cell lines derived from high-risk FN-RMS: 1) reduces SPM and upregulates SPD levels; 2) induces G0/G1 cell cycle arrest followed by apoptosis; 3) impairs anchorage-independent and tumor spheroids growth; 4) inhibits cell migration; 5) increases γH2AX levels and foci formation indicative of DNA damage. In addition, forced expression of SMOX and irradiation synergize at activating ATM and DNA-PKCs, and at inducing γH2AX expression and foci formation, which suggests an enhancement in DNA damage response. Irradiated SMOX-overexpressing FN-RMS cells also show significant decrease in both colony formation capacity and spheroids growth with respect to single approaches. Thus, our results unveil a role for SMOX as inhibitor of tumorigenicity of FN-RMS cells in vitro. In conclusion, our in vitro results suggest that SMOX induction could be a potential combinatorial approach to sensitize FN-RMS to ionizing radiation and deserve further in-depth studies.