Studio dell’azione antiproliferativa di oxaliplatino e pemetrexed in linee cellulari di carcinoma del colon-retto: meccanismi cellulari e molecolari

Oxaliplatin is active in the treatment of colorectal cancer and its effect is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluated the pharmacological interaction between oxaliplatin and pemetrexed and established the optimal combination schedule in colorectal cancer cell lines. HT29, WiDr, SW620 and LS174T cells were treated with drugs, alone or in combination: a dose-dependent inhibition of cell growth was observed after oxaliplatin and pemetrexed exposure, while a synergistic interaction was observed mostly with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase thus rendering cells more sensitive to pemetrexed. Drug combinations increased apoptotic index with respect to single agents, and treatment with oxaliplatin and pemetrexed inhibited Akt phosphorylation. RT-PCR showed a correlation between the FPGS/(TS×DHFR×GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of genes involved in DNA repair (ERCC1 and ERCC2), TS, DHFR and GARFT after drug exposure. These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression

Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small cell lung cancer cells

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5'-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations

Evidence for novel binding sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen

The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen

Evidence for novel binding sites on the platelet glycoprotein IIb and IIIa subunits and immobilized fibrinogen

The present study was designed to examine the interaction of the purified platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa or integrin alpha IIb beta 3) and the individual subunits of the complex with immobilized fibrinogen. Although 125I-GP IIb-IIIa binding to fibrinogen immobilized on Sepharose was specific, this interaction exhibited properties distinct from those of reversible fibrinogen binding to platelets: 125I-GP IIb-IIIa binding appeared irreversible, but non-covalent, Ca(2+)-independent, and was inhibited only weakly, or not at all, by the anti-(GP IIb-IIIa) monoclonal antibodies 10E5 and 7E3 and synthetic peptides from known platelet-binding domains of fibrinogen. Reversibly dissociated GP IIb or GP IIIa subunits inhibited 125I-GP IIb-IIIa binding to immobilized fibrinogen and bound directly to the fibrinogen. However, these subunits did not bind to peptides derived from known platelet-binding domains within the fibrinogen alpha- and gamma-chains, although the GP IIb-IIIa complex did. These results show that the complexed form of full-length GP IIb and GP IIIa is required for binding to these synthetic peptides, but not necessarily for binding to immobilized fibrinogen. Thus GP IIb-IIIa can bind to immobilized fibrinogen by a distinct mechanism that appears to involve novel binding sites on each subunit of the GP IIb-IIIa complex and on fibrinogen

Effect of clopidogrel discontinuation at 1 year after drug eluting stent placement on soluble CD40L, P-selectin and C-reactive protein levels: DECADES (Discontinuation Effect of Clopidogrel After Drug Eluting Stent): a multicenter, open-label study

Antiplatelet therapy with clopidogrel has been shown to reduce major adverse cardiac events in acute coronary syndromes and after percutaneous interventions. This effect is not only due to its anti-platelet effect but also possibly due to an anti-inflammatory effect. The effect of clopidogrel cessation after one year of therapy on markers of inflammation has been investigated in diabetics and showed an increase in platelet aggregation as well as hsCRP and surface P-selectin levels. This was an exploratory multicenter prospective open-label single arm study of 98 non-diabetic patients who had received one or more drug eluting stents and were coming to the end of their 12 months course of clopidogrel therapy. The effect of clopidogrel cessation on expression of biomarkers: sCD40L, soluble P-selectin and hsCRP was measured right before clopidogrel cessation (day 0), and subsequently at 1, 2, 3 and 4 weeks after drug withdrawal. A median increase in sCD40L expression from 224 to 324.5 pg/ml was observed between baseline and 4 weeks after clopidogrel cessation, which corresponded to a 39% mean percent change based on an ANCOVA model (P < 0.001). Over the 4 weeks observation period the change in sCD40L expression correlated weakly with soluble P-selectin levels (at 4 weeks Spearman’s correlation coefficient = 0.32; P = 0.0024). Increase in P-selectin expression from baseline was statistically significant at week 1 and 2. Conversely, hsCRP level decreased by 21% at 1 week (P = 0.008) and was still reduced by 18% by 4 weeks (P = 0.062). The change in sCD40L expression appeared to vary with the type of drug eluting stent. Patients treated with drug eluting stents at 1 year after implantation display significant increase in sCD40L and decrease in hsCRP after clopidogrel cessation. Further studies should elucidate if this increase in sCD40L levels reflects solely the removal of the inhibitory effects of clopidogrel on platelet activity or rather an increase in pro-inflammatory state. The latter hypothesis may be less likely given decrease in hsCRP levels. Randomized studies are urgently needed to establish potential link of clopidogrel discontinuation and vascular outcomes

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

The present study was performed to investigate the capability of gemcitabine and pemetrexed to synergistically interact with respect to cytotoxicity and apoptosis in T24 and J82 bladder cancer cells, and to establish a correlation between drug activity and gene expression of selected genes in tumour samples. The interaction between gemcitabine and pemetrexed was synergistic; indeed, pemetrexed favoured gemcitabine cytotoxicity by increasing cellular population in S-phase, reducing Akt phosphorylation as well as by inducing the expression of a major gemcitabine uptake system, the human equilibrative nucleoside transporter-1 (hENT1), and the key activating enzyme deoxycytidine kinase (dCK) in both cell lines. Bladder tumour specimens showed an heterogeneous gene expression pattern and patients with higher levels of dCK and hENT1 had better response. Moreover, human nucleoside concentrative transporter-1 was detectable only in 3/12 patients, two of whom presented a complete response to gemcitabine. These data provide evidence that the chemotherapeutic activity of the combination of gemcitabine and pemetrexed is synergistic against bladder cancer cells in vitro and that the assessment of the expression of genes involved in gemcitabine uptake and activation might be a possible determinant of bladder cancer response and may represent a new tool for treatment optimization

Preclinical emergence of vandetanib as a potent antitumour agent in mesothelioma: molecular mechanisms underlying its synergistic interaction with pemetrexed and carboplatin

BACKGROUND: Although pemetrexed, a potent thymidylate synthase (TS) inhibitor, enhances the cytoytoxic effect of platinum compounds against malignant pleural mesothelioma (MPM), novel combinations with effective targeted therapies are warranted. To this end, the current study evaluates new targeted agents and their pharmacological interaction with carboplatin-pemetrexed in human MPM cell lines. METHODS: We treated H2052, H2452, H28 and MSTO-211H cells with carboplatin, pemetrexed and targeted compounds (gefitinib, erlotinib, sorafenib, vandetanib, enzastaurin and ZM447439) and evaluated the modulation of pivotal pathways in drug activity and cancer cell proliferation. RESULTS: Vandetanib emerged as the compound with the most potent cytotoxic activity, which interacted synergistically with carboplatin and pemetrexed. Drug combinations blocked Akt phosphorylation and increased apoptosis. Vandetanib significantly downregulated epidermal growth factor receptor (EGFR)/Erk/Akt phosphorylation as well as E2F-1 mRNA and TS mRNA/protein levels. Moreover, pemetrexed decreased Akt phosphorylation and expression of DNA repair genes. Finally, most MPM samples displayed detectable levels of EGFR and TS, the variability of which could be used for patients' stratification in future trials with vandetanib-pemetrexed-carboplatin combination. CONCLUSION: Vandetanib markedly enhances pemetrexed-carboplatin activity against human MPM cells. Induction of apoptosis, modulation of EGFR/Akt/Erk phosphorylation and expression of key determinants for pemetrexed and carboplatin activity contribute to this synergistic interaction, and, together with the expression of these determinants in MPM samples, warrant further clinical investigation

Progettazione e sintesi di derivati eterociclici azotati come nuovi inibitori di aldeide deidrogenasi.

Nel mio lavoro di tesi sperimentale mi sono dedicata allo studio dell’enzima aldeide deidrogenasi isoforma 1A3 (ALDH1A3) ed alla progettazione e alla sintesi di nuovi derivati eterociclici capaci di inibirlo. La classe delle aldeidi deidrogenasi (ALDH) presenta 19 isoforme enzimatiche che prevalentemente catalizzano l’ossidazione di aldeidi esogene ed endogene nel corrispondente acido carbossilico, in una reazione NAD(P)+ dipendente. Grazie a questa attività funzionale le aldeide deidrogenasi esercitano un’azione detossificante, rimuovendo composti elettrofili altamente reattivi dalle riconosciute attività citotossica, mutagena e carcinogenica. Inoltre, proteggono nei confronti dello stress ossidativo, contribuendo a rifornire la cellula di coenzina NAD(P)H ridotto, e completano il metabolismo degli alcoli trasformando i loro prodotti di ossidazione. Ad esempio, nel caso dell’etanolo assunto con l’alimentazione, l’acetaldeide prodotta da questo per intervento di alcol deidrogenasi viene definitivamente ossidata ad acido acetico da ALDH. [1] Grazie alla loro azione ossidante, le ALDH sono gli enzimi primari coinvolti nella biosintesi dell'acido retinoico (RA, retinoic acid), prodotto a partire dal retinale, a sua volta metabolita della vitamina A, e comprendente le forme tutto-trans (ATRA, all-trans retinoic acid), più potente biologicamente, ma anche 9-cis (9-cis-RA) e 13-cis (13-cis-RA). RA è un metabolita chiave capace di regolare la trascrizione di numerosi geni target con conseguenti effetti proliferativi che promuovono la crescita e la differenziazione cellulare; inoltre svolge un ruolo importante nella biosintesi di molecole che regolano l'omeostasi. La biosintesi di RA è catalizzata in particolare dalla sottofamiglia di enzimi ALDH1A, comprendente le isoforme ALDH1A1, 1A2 e 1A3. Queste sono espresse soprattutto nei tessuti embrionali e nelle cellule staminali, una particolare popolazione cellulare caratterizzata dalla capacità di proliferare, auto-rinnovarsi e differenziarsi. [2] L’elevata espressione di ALDH1A1, 1A2 e 1A3 nelle cellule staminali di tipo tumorale (CSC, cancer stem cells) ha reso queste isoforme enzimatiche non solo un marker utile per identificare la presenza delle CSC in un tessuto tumorale eterogeneo, [3] ma anche un target privilegiato per lo sviluppo di approcci terapeutici antitumorali nuovi ed efficaci. In particolare, vista la dimostrata iper-espressione della isoforma ALDH1A3 nelle CSC di glioblastoma, composti in grado di inibire l’attività catalitica di questo enzima sono divenuti oggetto di studio come nuovi candidati farmaci per la cura di questa particolare forma di tumore che, ad oggi, ancora manca di un trattamento efficace. Il primo derivato eterociclico descritto come capace di inibire ALDH1A3 ed efficace in un modello murino di glioblastoma è stato il composto denominato GA11, a nucleo imidazo[1,2-a] piridinico. GA11 si lega all’entrata del sito catalitico di ALDH1A3 impedendo parzialmente l’ingresso del substrato, bloccando così l’attività catalitica dell’enzima. [4] Mantenendo lo stesso nucleo eterociclico, abbiamo ottimizzato il GA11 sintetizzando nuovi derivati 2,6-difenil sostituiti che presentino gruppi in grado di permettere una maggiore interazione con il sito catalitico della proteina ed al contempo presentino una solubilità migliore rispetto ai composti fatti in precedenza (Figura 1). Inoltre, abbiamo aggiunto un ulteriore sostituente aromatico nella posizione 8 del nucleo, allo scopo consentire ai composti di inserirsi con maggiore efficacia nel tunnel lipofilico che delinea il sito catalitico dell’enzima (Figura 1). Infine, abbiamo progettato e sintetizzato nuovi derivati 2-aminopiridinici 3,5-disostituiti come analoghi strutturali aperti del nucleo imidazo[1,2-a]piridinico, per rendere la struttura più mobile e maggiormente adattabile al sito attivo dell’enzima. BIBLIOGRAFIA: [1] R. Januchowski, K. Wojtowicz, e M. Zabel, «The role of aldehyde dehydrogenase (ALDH) in cancer drug resistance», Biomed. Pharmacother., vol. 67, n. 7, pagg. 669–680, set. 2013, doi: 10.1016/j.biopha.2013.04.005. [2] G. Vassalli, «Aldehyde Dehydrogenases: Not Just Markers, but Functional Regulators of Stem Cells», Stem Cells Int., vol. 2019, pagg. 1–15, gen. 2019, doi: 10.1155/2019/3904645. [3] L. Zhou et al., «Identification of cancer-type specific expression patterns for active aldehyde dehydrogenase (ALDH) isoforms in ALDEFLUOR assay», Cell Biol. Toxicol., vol. 35, n. 2, pagg. 161–177, apr. 2019, doi: 10.1007/s10565-018-9444-y. [4] P. Cheng et al., «FOXD1–ALDH1A3 Signaling Is a Determinant for the Self-Renewal and Tumorigenicity of Mesenchymal Glioma Stem Cells», Cancer Res., vol. 76, n. 24, pagg. 7219–7230, dic. 2016, doi: 10.1158/0008-5472.CAN-15-2860. [5] L. Quattrini et al., «Progress in the Field of Aldehyde Dehydrogenase Inhibitors: Novel Imidazo[1,2- a ]pyridines against the 1A Family», ACS Med. Chem. Lett., vol. 11, n. 5, pagg. 963–970, mag. 2020, doi: 10.1021/acsmedchemlett.9b00686

Plan de negocios: planta recicladora de neumáticos

Este plan de negocios evalúa los canales de comercialización, la factibilidad técnica, económica y financiera de construir una Planta de Reciclado de Neumáticos Fuera de Uso por medio de trituración mecánica, basándose y tomando como punto de inicio el trabajo denominado Estudio de Factibilidad de una Planta de Reciclado de Neumáticos, Autor: Mario MarcosEsta tesis en PDF no tiene permisos por parte del autor para ser reproducida. Puedes venir a consultarla a la Biblioteca Di Tella pero recuerda que no podrás copiarla, ni grabarla en ningún dispositivo, ni enviarla, ni imprimirla. La consulta se hace solo bajo reserva escribiendo a [email protected] eres el autor de la tesis y quieres dar tu autorización para la reproducción, puedes ponerte en contacto con [email protected]