SERPINB3 inibisce il poro di transizione della permeabilità mitocondriale attraverso la regolazione della produzione di ROS mitocondriali e incrementa la resistenza ai trattamenti chemioterapici

Resistance to chemotherapeutic agents is well known in patients with hepatocarcinoma. Inhibition of the mitochondrial permeability transition pore (PTP) is a crucial step in tumor cell resistance to apoptosis induced by anticancer drugs. Since SERPINB3 is overexpressed in hepatocellular carcinoma and has an anti-apoptotic activity, aim of the study was to assess the role of this serpin on PTP modulation during treatment with chemotherapeutic agents. HepG2 cells stably transfected with SERPINB3 where assayed for cell death induced by Cisplatin, Doxorubicin, Etoposide and 5-Fluorine Uracil. Reactive Oxygen Species (ROS) were detected with dichlorofluorescein. Threshold of PTP opening was evaluated by CRC assay and Complex I activity was determined by spectrophotometric assay. After cell death induction by Cisplatin and Doxorubicin, HepG2 cells expressing SERPINB3 showed a significant increase in viability compared to controls, while there was no difference in cell death after Etoposide and 5-FU treatments. The addition of the antioxidant N-acetyl cysteine abrogated cell death induction by Cisplatin and Doxorubicin, suggesting that SERPINB3 protects from death through an antioxidant activity. Since Cisplatin and Doxorubicin induce mitochondrial oxidative stress and favor PTP opening, cells were treated with the PTP inducer EM20-25 that acts at mitochondrial level. In presence of SERPINB3 the effect of EM20-25 resulted in PTP opening inhibition and decreased ROS formation. Subcellular localization analysis revealed that a fraction of SERPINB3 was located in mitochondria and that it increased after death-promoting treatments. Mitochondrial SERPINB3 was found associated to respiratory chain Complex I by immunoprecipitation experiments. In vitro analysis confirmed the inhibition by SERPINB3 of Complex I activity, known as one of the main sources of mitochondrial ROS. In conclusion, SERPINB3 acts at mitochondrial level protecting cells from chemotherapeutic-induced oxidative stress and consequent cell death. This antioxidant function could represent a relevant advantage for a transformed cell to balance its ROS equilibrium and for SB3-expressing cells exposed to anticancer treatments

Gold(III)-pyrrolidinedithiocarbamato Derivatives as Antineoplastic Agents

Transition metals offer many possibilities in developing potent chemotherapeutic agents. They are endowed with a variety of oxidation states, allowing for the selection of their coordination numbers and geometries via the choice of proper ligands, leading to the tuning of their final biological properties. We report here on the synthesis, physico-chemical characterization, and solution behavior of two gold(III) pyrrolidinedithiocarbamates (PDT), namely [AuIIIBr2(PDT)] and [AuIIICl2(PDT)]. We found that the bromide derivative was more effective than the chloride one in inducing cell death for several cancer cell lines. [AuIIIBr2(PDT)] elicited oxidative stress with effects on the permeability transition pore, a mitochondrial channel whose opening leads to cell death. More efficient antineoplastic strategies are required for the widespread burden that is cancer. In line with this, our results indicate that [AuIIIBr2(PDT)] is a promising antineoplastic agent that targets cellular components with crucial functions for the survival of tumor cells

Clinical and molecular characterization of a cohort of patients with novel nucleotide alterations of the Dystrophin gene detected by direct sequencing

<p>Abstract</p> <p>Background</p> <p>Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements.</p> <p>Methods</p> <p>We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in <it>DMD </it>gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis.</p> <p>Results</p> <p>We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the <it>DMD </it>gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65.</p> <p>Conclusion</p> <p>The analysis of our patients' sample, carrying point mutations or complex rearrangements in <it>DMD </it>gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.</p

SERPINB3 inibisce il poro di transizione della permeabilità mitocondriale attraverso la regolazione della produzione di ROS mitocondriali e incrementa la resistenza ai trattamenti chemioterapici

Resistance to chemotherapeutic agents is well known in patients with hepatocarcinoma. Inhibition of the mitochondrial permeability transition pore (PTP) is a crucial step in tumor cell resistance to apoptosis induced by anticancer drugs. Since SERPINB3 is overexpressed in hepatocellular carcinoma and has an anti-apoptotic activity, aim of the study was to assess the role of this serpin on PTP modulation during treatment with chemotherapeutic agents. HepG2 cells stably transfected with SERPINB3 where assayed for cell death induced by Cisplatin, Doxorubicin, Etoposide and 5-Fluorine Uracil. Reactive Oxygen Species (ROS) were detected with dichlorofluorescein. Threshold of PTP opening was evaluated by CRC assay and Complex I activity was determined by spectrophotometric assay. After cell death induction by Cisplatin and Doxorubicin, HepG2 cells expressing SERPINB3 showed a significant increase in viability compared to controls, while there was no difference in cell death after Etoposide and 5-FU treatments. The addition of the antioxidant N-acetyl cysteine abrogated cell death induction by Cisplatin and Doxorubicin, suggesting that SERPINB3 protects from death through an antioxidant activity. Since Cisplatin and Doxorubicin induce mitochondrial oxidative stress and favor PTP opening, cells were treated with the PTP inducer EM20-25 that acts at mitochondrial level. In presence of SERPINB3 the effect of EM20-25 resulted in PTP opening inhibition and decreased ROS formation. Subcellular localization analysis revealed that a fraction of SERPINB3 was located in mitochondria and that it increased after death-promoting treatments. Mitochondrial SERPINB3 was found associated to respiratory chain Complex I by immunoprecipitation experiments. In vitro analysis confirmed the inhibition by SERPINB3 of Complex I activity, known as one of the main sources of mitochondrial ROS. In conclusion, SERPINB3 acts at mitochondrial level protecting cells from chemotherapeutic-induced oxidative stress and consequent cell death. This antioxidant function could represent a relevant advantage for a transformed cell to balance its ROS equilibrium and for SB3-expressing cells exposed to anticancer treatments.L'epatocarcinoma (HCC) è la quinta forma di cancro più diffusa nel mondo ed è caratterizzato da un'alta proliferazione cellulare e da un'alta resistenza ai trattamenti chemioterapici. Negli ultimi anni l'associazione tra la ov-serpina SERPINB3 (precedentemente conosciuta anche come SCCA1) e la progressione della patologia epatica e lo sviluppo di HCC si è fatta sempre più stretta, portando ad ipotizzare che questa proteina possa giocare un ruolo importante nella biologia del tumore epatico. SERPINB3 è in grado di inibire la morte cellulare in sistemi cellulari di tipo epiteliale, ma non si conosce ancora il suo esatto meccanismo d'azione molecolare né la sua funzione nel fegato. Lo scopo dello studio è stato verificare il ruolo di SERPINB3 nella morte cellulare indotta da trattamenti chemioterapici in un modelli epatici sperimentali e di indagarne l'azione a livello molecolare. Esprimendo SERPINB3 in una linea di epatoma (HepG2), si è messo in evidenza come la presenza della proteina riduce la morte cellulare dopo trattamento con Cisplatino e Doxorubicina, ma non dopo trattamento con Etoposide o 5-FluoroUracile. Esperimenti successivi hanno documentato come la resistenza alla morte sia associata ad un diminuito stress ossidativo, definito come riduzione dei livelli di ROS, nelle cellule che esprimono SERPINB3. E' stato dimostrato che la diminuzione dei livelli di ROS si associa alla presenza di SERPINB3 a livello mitocondriale. Utilizzando l'EM20-25, un composto che fa parte della famiglia dei chemioterapici BH3-mimetici, è stato confermato che la protezione viene conferita a livello mitocondriale in quanto viene diminuita l'attività del Complesso I della catena respiratoria con conseguente diminuzione dei livelli di ROS. Questo meccanismo molecolare è stato confermato mediante la documentazione dell'inibizione del Poro di Transizione di Permeabilità mitocondriale da parte di SERPINB3, che è considerato un regolatore chiave dell'innesco della morte cellulare. Dato che SERPINB3 non è presente nel fegato sano, ma la sua espressione progressivamente aumenta in corso di infiammazione cronica e di cirrosi, la sua azione antiossidante, inizialmente protettiva, potrebbe contribuire alla sopravvivenza di cellule trasformate e allo sviluppo di tumore. Nell'ambito dell'epatocarcinoma SERPINB3 potrebbe contribuire alla resistenza ai trattamenti chemioterapici, nota caratteristica di questa forma tumorale epatica

Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep

none4noThe crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.openCiscato, Francesco; Chiara, Federica; Filadi, Riccardo; Rasola, AndreaCiscato, Francesco; Chiara, Federica; Filadi, Riccardo; Rasola, Andre

Retroactive insulation system for highly effective thermal control in an induction heating DSS furnace for Si-ingot casting

none6noneDughiero F.; Forzan M.; Ciscato D.; Giusto F.; Spagnolo A.; Doni ADughiero, Fabrizio; Forzan, Michele; Ciscato, Dario; Giusto, Francesco; Spagnolo, A.; Doni, Angel