Ferroptosis and Iron Metabolism in Cancer Growth

Il ferro è un micronutriente essenziale per la vita, ma è anche necessario per sostenere il metabolismo accelerato delle cellule tumorali. Inoltre, l’aumento dei livelli di ferro causa stress ossidativo nelle cellule, promuovendo la perossidazione lipidica e causando morte cellulare per ferroptosi. Perciò, la doppia natura del ferro (essenziale e tossico allo stesso tempo) lo rende un promettente target terapeutico nella lotta al cancro. Considerato ciò, in questa tesi di dottorato abbiamo indagato gli effetti degli induttori di ferroptosi e della chelazione del ferro in due diversi tipi di tumore entrambi poco caratterizzati per il loro metabolismo del ferro e per il meccanismo di ferroptosi: il glioblastoma e il rabdomiosarcoma. Il glioblastoma è il tumore del sistema nervoso centrale più frequente negli adulti, ed è caratterizzato da un’estrema aggressività e da un elevato rischio di recidive, risultanti in una scarsa prognosi dei pazienti. Recentemente, cellule staminali di glioblastoma, note come glioblastoma cancer stem cells (GBM CSCs), sono state identificate come responsabili della ricaduta della malattia. Perciò, abbiamo investigato sull’efficacia degli induttori di ferroptosi nel ridurre la vitalità cellulare delle GBM CSCs. Abbiamo dimostrato che gli induttori di ferroptosi inibiscono potentemente la crescita di linee di GBM CSCs in vitro. Inoltre, abbiamo osservato che gli induttori di ferroptosi aumentano l’efficacia della temozolomide, incoraggiando ulteriori analisi sul meccanismo della ferroptosi nelle GBM CSCs. L’altra tipologia di tumore studiata nel presente lavoro di tesi, in collaborazione con il gruppo di ricerca del Prof. Fanzani (all’Università di Brescia) è il rabdomiosarcoma, un tumore dell’età pediatrica aggressivo e metastatico che si sviluppa da miociti non differenziati. Le terapie attuali contro il RMS includono l’asportazione chirurgica, seguita, dove necessario, da diverse strategie combinate di chemio- e radioterapia; ciononostante, questi trattamenti risultano spesso non curativi. Siccome i chelanti del ferro possono impedire efficacemente la crescita del RMS, abbiamo investigato gli effetti su linee cellulari di RMS del didox, inibitore della ribonucleotide reduttasi con proprietà di chelante del ferro. Abbiamo scoperto che il didox inibisce significativamente la crescita in vitro di linee cellulari di RMS rappresentative dei due sottotipi più frequenti, embrionale e alveolare, riducendo inoltre la loro motilità, la loro clonogenicità e alterando il loro metabolismo del ferro. Inoltre, abbiamo dimostrato che il didox contrasta la crescita in vivo di linee cellulari di RMS alveolare, suggerendo che l’uso di questo inibitore potrebbe essere efficace nel trattamento del rabdomiosarcoma. Inoltre, tra le varie alterazioni molecolari identificate nei RMS, l’overespressione di caveolina 1 (Cav 1) è stata correlata con un aumento della sensibilità di linee cellulari di RMS agli induttori di ferroptosi. Perciò, abbiamo investigato sul meccanismo della ferroptosi in linee cellulari di RMS embrionale overesprimenti Cav 1. Abbiamo dimostrato che queste cellule esprimono livelli alterati di geni e proteine correlati al metabolismo lipidico. Inoltre, abbiamo rilevato, nelle cellule overesprimenti Cav 1, un aumento dei livelli di espressione, sia genica sia proteica, di ACSL4, l’enzima responsabile dell’inserzione nelle membrane cellulari degli acidi grassi polinsaturi. Di conseguenza, abbiamo investigato sul ruolo di ACSL4 nella sensibilità alla ferroptosi di queste cellule. Inibendo sia la sua attività sia la sua espressione, abbiamo scoperto che ACSL4 non sembra direttamente coinvolto nell’aumentata sensibilità alla ferroptosi di queste cellule. In aggiunta, abbiamo dimostrato che la modulazione del contenuto lipidico delle cellule overesprimenti Cav 1 può alterare la loro sensibilità all’induttore di ferroptosi RSL3.Iron is an essential micronutrient for life, but it is also required to sustain the accelerated metabolism of cancer cells. On the other hand, the increased amount of this metal causes cellular oxidative stress, promoting lipid peroxidation and causing ferroptotic cell death. Thus, the double face of iron (essential and toxic at the same time) makes it a promising target to counteract tumor growth. Therefore, in this PhD thesis we investigated the effects of ferroptosis inducers and iron modulation in two different tumor types, poorly characterized for iron metabolism and ferroptosis mechanism: glioblastoma and rhabdomyosarcoma. Glioblastoma (GBM) is the most common tumor of the central nervous system in adults and is characterized by extreme aggressiveness and elevated risk of tumor recurrence, resulting in poor patient prognosis. Recently, stem like GBM cells, named glioblastoma cancer stem cells (GBM CSCs), have been identified responsible for the resistance to current therapies and tumor relapse; therefore, new therapeutic approaches are required. In this context, we investigated whether ferroptosis inducing agents could affect the viability of GBM CSCs. We demonstrated that ferroptosis inducers can potently inhibit the growth of GBM CSC lines. Moreover, we observed that, ferroptosis inducers could increase the efficacy of temozolomide, encouraging further studies for unravelling the mechanism of ferroptosis in GBM CSCs. The other type of tumor studied in the present PhD thesis, in collaboration with Prof. Fanzani’s group (University of Brescia), is rhabdomyosarcoma (RMS), an aggressive and metastatic pediatric cancer that develops from undifferentiated myocytes. Current therapies for RMS include surgical resection followed, when required, by different strategies of aggressive chemotherapy and radiotherapy; these treatments, however, are often not curative thus requiring new therapeutic tools. Recently, since we demonstrated that iron chelators can efficiently impair RMS tumor growth, we investigated the effects on RMS cells of didox, a synthetic inhibitor of the regulatory subunit of ribonucleotide reductase RRM2 with iron chelating properties. We found that didox can effectively inhibit the in vitro growth of RMS cell lines representative of the most frequent subtypes, embryonal (ERMS) and alveolar (ARMS), also altering their motility, their clonogenic capability, and their iron status. Moreover, we demonstrated that didox was able to impair the in vivo growth of ARMS cells, suggesting that the use of this inhibitor could be effective in the treatment of RMS. Additionally, among the many molecular alterations identified in RMS, the overexpression of caveolin 1 (Cav 1) has been demonstrated to correlate with an increased sensitivity of RMS cells to ferroptosis inducers. Therefore, we investigated the mechanism of ferroptosis in Cav 1 overexpressing ERMS cells, with a particular focus on the alterations on lipid metabolism in these cells. We demonstrated that RMS cells that overexpressed Cav 1 are characterized by an altered expression of various lipid metabolism related genes and proteins. Interestingly, we found that Cav 1 overexpressing cells showed increased expression of both mRNA and protein level of long chain fatty acid CoA ligase 4 (ACSL4), the enzyme responsible for the insertion in the cell membrane of polyunsaturated fatty acids (PUFAs). Thus, we investigated the role of ACSL4 in the ferroptosis sensitivity of these cells, finding that ACSL4 seems not directly involved in the increased susceptibility to ferroptosis of these cells. Moreover, we found that the modulation of lipid content of Cav 1 overexpressing cells can alter their sensitivity to ferroptosis inducer RSL3: the treatment in combination with PUFAs further decrease the cell sensitivity to RSL3, whereas the combination with MUFAs protected the cells to RSL3-induced ferroptosis

Epoxy resins composites for X-ray shielding materials additivated by coated barium sulfate with improved dispersibility

Epoxy resins additivated by barium sulfate proved to be promising low cost, easy workable and environmentally friendly alternative to lead and steel as X-ray shielding materials, but the composites tends to be stratified, with the additive accumulating in the bottom side of the sample. This sedimentation process has been, at first, studied by in situ X-ray powder diffraction, thermogravimetric techniques and X-ray tomography and then inhibited by exploiting finer barite sources, implementing a grinding procedure, combined to a surface modification of the inorganic additives. Stearic acid and sodium dodecyl sulfate were used to coat barite surface, using a liquid assisted grinding (LAG) approach. The functionalized additives resulted more compatible with the resin and their dispersion within the polymer resulted much improved. The produced composite samples were then studied by optical and electron microscopy, X-ray radiography, X-ray diffraction, thermogravimetric analysis and tensile strength test. The use of a finer additive and the grinding procedure allowed to limit the sedimentation and induced a marked hardening of the samples, with the drawback of a reduction of their plasticity. Stearic acid coating was able to eliminate sedimentation maintaining good mechanical properties

Light Weight, Easy Formable and Non-Toxic Polymer-Based Composites for Hard X-ray Shielding: A Theoretical and Experimental Study

Composite lightweight materials for X-ray shielding applications were studied and developed with the goal of replacing traditional screens made of lead and steel, with innovative materials with similar shielding properties, but lighter, more easily formed and workable, with lower impact on the environment and reduced toxicity for human health. New epoxy based composites additivated with barium sulfate and bismuth oxide were designed through simulations performed with softwares based on Geant4. Then, they were prepared and characterized using different techniques starting from digital radiography in order to test the radiopacity of the composites, in comparison with traditional materials. The lower environmental impact and toxicity of these innovative screens were quantified by Life Cycle Assessment (LCA) calculation based on the ecoinvent database, within the openLCA framework. Optimized mixtures are (i) 20% epoxy/60% bismuth oxide/20% barite, which guarantees the best performance in X-ray shielding, largely overcoming steel, but higher in costs and a weight reduction of circa 60%; (ii) 20% epoxy/40% bismuth oxide/40% barite which has slightly lower performances in shielding, but it is lighter and cheaper than the first one and (iii) the 20% epoxy/20% bismuth oxide/60% barite which is the cheapest material, still maintaining the X-ray shielding of steel. Depending on cost/efficiency request of the specific application (industrial radiography, aerospace, medical analysis), the final user can choose among the proposed solutions

The modulation of iron metabolism affects the Rhabdomyosarcoma tumor growth in vitro and in vivo

Rhabdomyosarcoma (RMS) is an aggressive rare neoplasm that derives from mesenchymal cells, which frequently develops resistance to the current therapies and the formation of metastases. Thus, new therapies are needed. The alteration of iron metabolism in cancer cells was effective in reducing the progression of many tumors but not yet investigated in RMS. Here we investigated the effect of iron modulation in RMS both in vitro and in vivo. We first characterized the most used RMS cell lines representing the most common subtypes, embryonal (ERMS, RD cells) and alveolar (ARMS, RH30 cells), for their iron metabolism, in basal condition and in response to its modulation. Then we investigated the effects of both iron overload and chelation strategies in vitro and in vivo. RMS cell lines expressed iron-related proteins, even if at lower levels compared to hepatic cell lines and they are correctly modulated in response to iron increase and deprivation. Interestingly, the treatment with different doses of ferric ammonium citrate (FAC, as iron source) and with deferiprone (DFP, as iron chelator), significantly affected the cell viability of RD and RH30. Moreover, iron supplementation (in the form of iron dextran) or iron chelation (in the form of DFP) were also effective in vivo in inhibiting the tumor mass growth both derived from RD and RH30 with iron chelation treatment the most effective one. All the data suggest that the iron modulation could be a promising approach to overcome the RMS tumor growth. The mechanism of action seems to involve the apoptotic cell death for both iron supplementation and chelation with the concomitant induction of ferroptosis in the case of iron supplementation

The Antitumor Didox Acts as an Iron Chelator in Hepatocellular Carcinoma Cells

Ribonucleotide reductase (RR) is the rate-limiting enzyme that controls the deoxynucleotide triphosphate synthesis and it is an important target of cancer treatment, since it is expressed in tumor cells in proportion to their proliferation rate, their invasiveness and poor prognosis. Didox, a derivative of hydroxyurea (HU), is one of the most potent pharmaceutical inhibitors of this enzyme, with low in vivo side effects. It inhibits the activity of the subunit RRM2 and deoxyribonucleotides (dNTPs) synthesis, and it seems to show iron-chelating activity. In the present work, we mainly investigated the iron-chelating properties of didox using the HA22T/VGH cell line, as a model of hepatocellular carcinoma (HCC). We confirmed that didox induced cell death and that this effect was suppressed by iron supplementation. Interestingly, cell treatments with didox caused changes of cellular iron content, TfR1 and ferritin levels comparable to those caused by the iron chelators, deferoxamine (DFO) and deferiprone (DFP). Chemical studies showed that didox has an affinity binding to Fe3+ comparable to that of DFO and DFP, although with slower kinetic. Structural modeling indicated that didox is a bidentated iron chelator with two theoretical possible positions for the binding and among them that with the two hydroxyls of the catechol group acting as ligands is the more likely one. The iron chelating property of didox may contribute to its antitumor activity not only blocking the formation of the tyrosil radical on Tyr122 (such as HU) on RRM2 (essential for its activity) but also sequestering the iron needed by this enzyme and to the cell proliferation

Antithrombotic strategies in the catheterization laboratory for patients with acute coronary syndromes undergoing percutaneous coronary interventions: Insights from the EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in Italian cardiac care units Registry

Aims: In the last decades, several new therapies have emerged for the treatment of acute coronary syndromes (ACS). We sought to describe real-world patterns of use of antithrombotic treatments in the catheterization laboratory for ACS patients undergoing percutaneous coronary interventions (PCI). Methods: EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in Italian cardiac care units was a nationwide, prospective registry aimed to evaluate antithrombotic strategies employed in ACS patients in Italy. Results: Over a 3-week period, a total of 2585 consecutive ACS patients have been enrolled in 203 cardiac care units across Italy. Among these patients, 1755 underwent PCI (923 with ST-elevation myocardial infarction and 832 with non-ST-elevation ACS). In the catheterization laboratory, unfractioned heparin was the most used antithrombotic drug in both ST-elevation myocardial infarction (64.7%) and non-ST-elevation ACS (77.5%) undergoing PCI and, as aspirin, bivalirudin and glycoprotein IIb/IIIa inhibitors (GPIs) more frequently employed before or during PCI compared with the postprocedural period. Any crossover of heparin therapy occurred in 36.0% of cases, whereas switching from one P2Y12 inhibitor to another occurred in 3.7% of patients. Multivariable analysis yielded several independent predictors of GPIs and of bivalirudin use in the catheterization laboratory, mainly related to clinical presentation, PCI complexity and presence of complications during the procedure. Conclusion: In our contemporary, nationwide, all-comers cohort of ACS patients undergoing PCI, antithrombotic therapies were commonly initiated before the catheterization laboratory. In the periprocedural period, the most frequently employed drugs were unfractioned heparin, leading to a high rate of crossover, followed by GPIs and bivalirudin, mainly used during complex PCI

Contemporary antithrombotic strategies in patients with acute coronary syndromes managed without revascularization: Insights fromthe EYESHOT study

Aims Patients with acute coronary syndromes (ACSs) whoare managed without coronary revascularization represent a mixed and understudied population that seems to receive suboptimal pharmacological treatment. Methods and results We assessed patterns of antithrombotic therapies employed during the hospitalization and in-hospital clinical events of medically managed patients withACS enrolled in the prospective, multicentre, nationwideEYESHOT(EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in Italian cardiac care units) registry.Among the 2585 consecutive ACS patients enrolled in EYESHOT, 783 (30.3%) did not receive any revascularization during hospital admission. Of these, 478 (61.0%) underwent coronary angiography (CA), whereas 305 (39.0%) did not. The median GRACE and CRUSADE risk scores were significantly higher among patients who did not undergo CA compared with those who did (180 vs. 145, P, 0.0001 and 50 vs. 33, P, 0.0001, respectively). Antithrombotic therapies employed during hospitalization significantly differ between patients who received CA and those who did not with unfractioned heparin and novel P2Y12 inhibitors more frequently used in the first group, and low-molecular-weight heparins and clopidogrel in the latter group. During the index hospitalization, patients who did not receive CA presented a higher incidence of ischaemic cerebrovascular events and of mortality compared with those who underwent CA (1.6 vs. 0.2%, P = 0.04 and 7.9 vs. 2.7%, P = 0.0009, respectively). Conclusion Almost one-third of ACS patients are managed without revascularization during the index hospitalization. In this population, a lower use of recommended antiplatelet therapy and worse clinical outcome were observed in those who did not undergo CA when compared with those who did