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ABSTRACT: In mammalian cells, adaptation to hypertonic conditions leads to the activation of an array of early (cell shrinkage, regulatory volume increase) and late (accumulation of compatible osmolytes) responses and increased level of HSPs (heat shock proteins). Protein synthesis is strongly inhibited few minutes after the hypertonic challenge as demonstrated in whole cells and as reproduced under controlled conditions in cell-free systems. Different mechanisms known to mediate the accumulation of HSP70, such as mRNA transcription and stabilization, require fully active protein synthesis. We show that the 5'-untranslated region of HSP70 messenger drives an hypertonicity-resistant translation (up to 0.425 osm/kg of water), whereas cap-dependent protein synthesis is almost totally blocked under the same conditions. The results, obtained in cell-free systems and in whole cells, might help to explain why HSP70 is accumulated in cells when total protein synthesis is impaired. We also observed that translation initiated by a viral IRES (from Cricket paralysis virus) is highly efficient in cells exposed to hyperosmolarity, suggesting that the resistance to hypertonic conditions is a more general feature of cap-independent translation. The described mechanism may also play a role in the control of translation of other messengers encoding for proteins involved in the adaptation to hypertonicity.
Biochemical and Biophysical Research Communications 01/2013; · 2.48 Impact Factor
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Elena Galvani,
Elisa Giovannetti,
Francesca Saccani,
Andrea Cavazzoni,
Leticia G Leon,
Henk Dekker,
Roberta Alfieri,
Caterina Carmi,
Marco Mor,
Andrea Ardizzoni, Pier Giorgio Petronini,
Godefridus J Peters
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ABSTRACT: Overcoming the emergence of acquired resistance to clinically approved epidermal growth factor receptor (EGFR) inhibitors is a major challenge in the treatment of advanced non-small cell lung cancer (NSCLC). The aim of this study was to investigate the effects of a series of novel compounds affecting viability of NSCLC NCI-H1975 cells (carrying the EGFR T790M mutation). The inhibition of the autophosphorylation of EGFR occurred at nanomolar concentrations and both UPR1282 and UPR1268 caused a significant induction of apoptosis. Targeting of EGFR and downstream pathways was confirmed by a peptide substrate array, which highlighted the inhibition of other kinases involved in NSCLC cell aggressive behavior. Accordingly, the drugs inhibited migration (about 30% vs. control), which could be, in part, explained also by the increase of E-cadherin expression. Additionally, we observed a contraction of the volume of H1975 spheroids, associated with the reduction of the cancer stem-like cell hallmark CD133. The activity of UPR1282 was retained in H1975 xenograft models where it determined tumor shrinkage (P < .05) and resulted well tolerated compared to canertinib. Of note, the kinase activity profile of UPR1282 on xenograft tumor tissues showed overlapping results with respect to the activity in H1975 cells, unraveling the inhibition of kinases involved in pivotal proliferation and invasive signaling pathways. In conclusion, UPR1282 and UPR1268 are effective against various processes involved in malignancy transformation and progression and may be promising compounds for the future treatment of gefitinib-resistant NSCLCs.
Neoplasia (New York, N.Y.) 01/2013; 15(1):61-72. · 5.48 Impact Factor
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Maurizio Brigotti,
Valentina Arfilli,
Domenica Carnicelli,
Laura Rocchi,
Cinzia Calcabrini,
Francesca Ricci,
Pasqualepaolo Pagliaro,
Pier Luigi Tazzari,
Roberta R Alfieri, Pier Giorgio Petronini,
Piero Sestili
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ABSTRACT: Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05-0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.
Toxins. 01/2013; 5(2):431-44.
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Andrea Cavazzoni,
Roberta R Alfieri,
Daniele Cretella,
Francesca Saccani,
Luca Ampollini,
Maricla Galetti,
Federico Quaini,
Gallia Graiani,
Denise Madeddu,
Paola Mozzoni,
Elena Galvani,
Silvia La Monica,
Mara Bonelli,
Claudia Fumarola,
Antonio Mutti,
Paolo Carbognani,
Marcello Tiseo,
Elisabetta Barocelli, Pier Giorgio Petronini,
Andrea Ardizzoni
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ABSTRACT: BACKGROUND: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. RESULTS: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. CONCLUSION: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.
Molecular Cancer 12/2012; 11(1):91. · 3.99 Impact Factor
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Elena Galvani,
Roberta Alfieri,
Elisa Giovannetti,
Andrea Cavazzoni,
Silvia La Monica,
Maricla Galetti,
Claudia Fumarola,
Mara Bonelli,
Marco Mor,
Marcello Tiseo,
Godefridus J Peters, Pier Giorgio Petronini,
Andrea Ardizzoni
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ABSTRACT: Conventional chemotherapeutic regimens have reached an efficacy plateau against most solid tumors and deal with significant toxicity. Recently, the goal of the oncologic research to improve outcome and reduce treatment-related side-effects has led to the development of novel anticancer treatments targeting specific proteins or genes involved in cancer growth and progression. In particular, the tyrosine-kinase inhibitors (TKIs) gefitinib and erlotinib targeting the epidermal growth factor receptor (EGFR) have been approved for the treatment of non-small-cell lung cancer (NSCLC). Their clinical activity has been related to different clinical and biological parameters, such as the presence of activating mutations in the kinase domain of the target. Disappointingly, their clinical efficacy is limited by the development of resistance which is caused in more than 50% of the cases by the emergence of a secondary point-mutation (T790M) in the ATP-binding cleft of EGFR. Several novel EGFR inhibitors, able to covalently bind the target and prolong its inactivation, have been developed with the aim to overcome such resistance and are evaluated in ongoing clinical studies. However, not all clinical outcomes, including tolerability, are explained, and the identification/validation of novel biomarkers of sensitivity or resistance to such agents is a viable area of research to improve their clinical use. This review summarizes the current knowledge on the functional role of activating mutations of EGFR, pivotal primary/acquired resistance mechanisms as well as clinical data of small molecule EGFR-TKIs, and discusses the future of such therapeutic approach in NSCLC.
Current pharmaceutical design 09/2012; · 4.41 Impact Factor
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ABSTRACT: Irreversible inhibitors provide potent and selective inhibition of tyrosine kinase enzymes. Use of such inhibitors has proved promising in overcoming the tumor resistance encountered with reversible tyrosine kinase inhibitors. Irreversible inhibitors inactivate their protein target through covalent interaction with a nucleophilic cysteine residue within the nucleotide binding pocket of the kinase domain. Different irreversible tyrosin kinase inhibitors directed against epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor tyrosine kinase (FGFR) have been developed and some of them have been employed clinically as anticancer agents. This review focuses on recent preclinical and clinical progress with currently available irreversible tyrosine kinase inhibitors. The chemical structures of the candidates, structure-activity relationships, biological activities and results of current clinical investigations are described.
Biochemical pharmacology 08/2012; · 4.25 Impact Factor
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Andrea Cavazzoni,
Mara A Bonelli,
Claudia Fumarola,
Silvia La Monica,
Kinda Airoud,
Ramona Bertoni,
Roberta R Alfieri,
Maricla Galetti,
Stefano Tramonti,
Elena Galvani,
Adrian L Harris,
Lesley-Ann Martin,
Daniele Andreis,
Alberto Bottini,
Daniele Generali, Pier Giorgio Petronini
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ABSTRACT: Development of resistance to endocrine therapy is a clinical issue in estrogen receptor (ER)-positive breast cancer. Here we show that persistent activation of AKT/mTOR signaling is crucial to the acquisition of letrozole resistance in cell clones generated from MCF-7/AROM-1 aromatase-expressing breast cancer cells after prolonged letrozole exposure. ERα plays a marginal role in this context. As a proof of concept, the association between PI3K/AKT/mTOR signaling and insensitivity to endocrine therapies was confirmed in breast cancer patients who developed early letrozole resistance in neoadjuvant setting. In addition our results suggest that, regardless of the mechanism mediating the activation of AKT/mTOR pathway, either RAD001 or NVP-BEZ235 treatment may represent a promising strategy to overcome acquired resistance to letrozole in breast cancers dependent on AKT/mTOR signaling.
Cancer letters 04/2012; 323(1):77-87. · 4.86 Impact Factor
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Caterina Carmi,
Elena Galvani,
Federica Vacondio,
Silvia Rivara,
Alessio Lodola,
Simonetta Russo,
Stefania Aiello,
Fabrizio Bordi,
Gabriele Costantino,
Andrea Cavazzoni,
Roberta R Alfieri,
Andrea Ardizzoni, Pier Giorgio Petronini,
Marco Mor
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ABSTRACT: Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.
Journal of Medicinal Chemistry 03/2012; 55(5):2251-64. · 4.80 Impact Factor
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Roberta R Alfieri,
Maricla Galetti,
Stefano Tramonti,
Roberta Andreoli,
Paola Mozzoni,
Andrea Cavazzoni,
Mara Bonelli,
Claudia Fumarola,
Silvia La Monica,
Elena Galvani,
Giuseppe De Palma,
Antonio Mutti,
Marco Mor,
Marcello Tiseo,
Ettore Mari,
Andrea Ardizzoni, Pier Giorgio Petronini
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ABSTRACT: Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment.The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness.
In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation.
Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC cells lacking activating mutations. On the other hand, in metabolizing cells, the inhibition of CYP1A1 might lead to increased local exposure to the active drug and thus increase gefitinib potency.
Molecular Cancer 11/2011; 10:143. · 3.99 Impact Factor
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Mara A Bonelli,
Claudia Fumarola,
Roberta R Alfieri,
Silvia La Monica,
Andrea Cavazzoni,
Maricla Galetti,
Rita Gatti,
Silvana Belletti,
Adrian L Harris,
Stephen B Fox,
Dean B Evans,
Mitch Dowsett,
Lesley-Ann Martin,
Alberto Bottini,
Daniele Generali, Pier Giorgio Petronini
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ABSTRACT: Estrogens induce breast tumor cell proliferation by directly regulating gene expression via the estrogen receptor (ER) transcriptional activity and by affecting growth factor signaling pathways such as mitogen-activated protein kinase (MAPK) and AKT/mammalian target of rapamycin Complex1 (mTORC1) cascades. In this study we demonstrated the preclinical therapeutic efficacy of combining the aromatase inhibitor letrozole with the multi-kinase inhibitor sorafenib in aromatase-expressing breast cancer cell lines. Treatment with letrozole reduced testosterone-driven cell proliferation, by inhibiting the synthesis of estrogens. Sorafenib inhibited cell proliferation in a concentration-dependent manner; this effect was not dependent on sorafenib-mediated inhibition of Raf1, but involved the down-regulation of mTORC1 and its targets p70S6K and 4E-binding protein 1 (4E-BP1). At concentrations of 5-10 μM the growth-inhibitory effect of sorafenib was associated with the induction of apoptosis, as indicated by release of cytochrome c and Apoptosis-Inducing Factor into the cytosol, activation of caspase-9 and caspase-7, and PARP-1 cleavage. Combination of letrozole and sorafenib produced a synergistic inhibition of cell proliferation associated with an enhanced accumulation of cells in the G(0)/G(1) phase of the cell cycle and with a down-regulation of the cell cycle regulatory proteins c-myc, cyclin D1, and phospho-Rb. In addition, longer experiments (12 weeks) demonstrated that sorafenib may be effective in preventing the acquisition of resistance towards letrozole. Together, these results indicate that combination of letrozole and sorafenib might constitute a promising approach to the treatment of hormone-dependent breast cancer.
Breast Cancer Research and Treatment 11/2010; 124(1):79-88. · 4.43 Impact Factor
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Maurizio Brigotti,
Pier Luigi Tazzari,
Elisa Ravanelli,
Domenica Carnicelli,
Stefania Barbieri,
Laura Rocchi,
Valentina Arfilli,
Gaia Scavia,
Francesca Ricci,
Andrea Bontadini,
Roberta R Alfieri, Pier Giorgio Petronini,
Carmine Pecoraro,
Alberto E Tozzi,
Alfredo Caprioli
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ABSTRACT: The endothelial damage induced by Stx represents the main pathogenic event in the HUS associated with STEC infections in humans. Stx, released in the gut by bacteria, enter the bloodstream and are targeted to renal endothelia. The role of PMN as a toxin carrier has been the object of controversy. In this paper, we confirm the binding of Stx1 to PMN, also showing its degranulating effects on full-loaded leukocytes, and support the carrier role of PMN by using a two-chamber transmigration device, in which PMN, loaded in vitro with different amounts of Stx1, transmigrated through confluent monolayers of endothelial cells, mimicking the toxin-induced renal endothelial injury. Stx1 was transferred during PMN transmigration, impairing protein synthesis and triggering production of proinflammatory cytokines in endothelial cells. PMN, carrying low toxin amounts, induced the release of high levels of cytokines in viable endothelial cells, whereas cytokine production was blocked in cells challenged with PMN fully loaded with Stx as a result of an almost total impairment of translation and of the activation of the apoptotic program. In agreement with previous unexplained observations in animal models, the results obtained with our experimental setting suggest that a self-amplifying circle triggered by low doses of toxin may lead to the production of proinflammatory mediators of renal damage in HUS.
Journal of leukocyte biology 04/2010; 88(1):201-10. · 4.99 Impact Factor
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Maricla Galetti,
Roberta R Alfieri,
Andrea Cavazzoni,
Silvia La Monica,
Mara Bonelli,
Claudia Fumarola,
Paola Mozzoni,
Giuseppe De Palma,
Roberta Andreoli,
Antonio Mutti,
Marco Mor,
Marcello Tiseo,
Andrea Ardizzoni, Pier Giorgio Petronini
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ABSTRACT: Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation.
Biochemical pharmacology 04/2010; 80(2):179-87. · 4.25 Impact Factor
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Caterina Carmi,
Andrea Cavazzoni,
Stefano Vezzosi,
Fabrizio Bordi,
Federica Vacondio,
Claudia Silva,
Silvia Rivara,
Alessio Lodola,
Roberta R Alfieri,
Silvia La Monica,
Maricla Galetti,
Andrea Ardizzoni, Pier Giorgio Petronini,
Marco Mor
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ABSTRACT: Irreversible EGFR inhibitors can circumvent acquired resistance to first-generation reversible, ATP-competitive inhibitors in the treatment of non-small-cell lung cancer. They contain both a driver group, which assures target recognition, and a warhead, generally an acrylamide or propargylamide fragment that binds covalently to Cys797 within the kinase domain of EGFR. We performed a systematic exploration of the role for the warhead group, introducing different cysteine-trapping fragments at position 6 of a traditional 4-anilinoquinazoline scaffold. We found that different reactive groups, including epoxyamides (compounds 3-6) and phenoxyacetamides (compounds 7-9), were able to irreversibly inhibit EGFR. In particular, at significant lower concentrations than gefitinib (1), (2R,3R)-N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(piperidin-1-ylmethyl)oxirane-2-carboxamide (6) inhibited EGFR autophosphorylation and downstream signaling pathways, suppressed proliferation, and induced apoptosis in gefitinib-resistant NSCLC H1975 cells, harboring the T790M mutation in EGFR.
Journal of Medicinal Chemistry 02/2010; 53(5):2038-50. · 4.80 Impact Factor
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ABSTRACT: Fragile histidine triad (FHIT) is a tumor suppressor gene whose allelic loss is associated to a number of human cancers. FHIT protein acts as a diadenosine oligophosphate hydrolase, but its tumor suppressive activity appears as independent from its enzymatic activity. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) can induce apoptosis in the FHIT-negative non-small lung cancer cell line Calu-1. We generated four FHIT-inducible Calu-1 cell clones and demonstrated that FHIT expression was able to protect cells from TRAIL-induced apoptosis, without affecting TRAIL-receptors surface expression. FHIT-specific small interference RNA transfection of SV40-immortalized normal bronchial BEAS cells that show levels of FHIT protein comparable to those of normal bronchial cells, resulted in a significant increase of TRAIL-induced apoptosis. Of note, suramin-mediated inhibition of FHIT enzymatic activity also enhanced TRAIL-induced apoptosis. We conclude that FHIT expression in lung cancer cells is protective from TRAIL-induced apoptosis. Our data suggest that FHIT exerts this protective effect downstream TRAIL-receptors and likely requires its dinucleoside-triphosphate hydrolase activity. As TRAIL represents in the near future a good candidate for death ligands-based anticancer therapy, its potential therapeutic use should be envisaged as preliminary to molecular genetics interventions or drug-induced epigenetic modulations aimed to restoring FHIT gene expression levels in non-small cells lung tumors.
Journal of Cellular Physiology 06/2009; 220(2):492-8. · 3.87 Impact Factor
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Silvia La Monica,
Maricla Galetti,
Roberta R Alfieri,
Andrea Cavazzoni,
Andrea Ardizzoni,
Marcello Tiseo,
Marzia Capelletti,
Matteo Goldoni,
Sara Tagliaferri,
Antonio Mutti,
Claudia Fumarola,
Mara Bonelli,
Daniele Generali, Pier Giorgio Petronini
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ABSTRACT: The epidermal growth factor receptor (EGFR) is a validated target for therapy in non-small cell lung cancer (NSCLC). Most patients, however, either do not benefit or develop resistance to specific inhibitors of the EGFR tyrosine kinase activity, such as gefitinib or erlotinib. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation and survival pathways and has been associated with resistance to EGFR tyrosine kinase inhibitors. In this study, we assessed the effects of combining the mTOR inhibitor everolimus (RAD001) with gefitinib on a panel of NSCLC cell lines characterized by gefitinib resistance and able to maintain S6K phosphorylation after gefitinib treatment. Everolimus plus gefitinib induced a significant decrease in the activation of MAPK and mTOR signaling pathways downstream of EGFR and resulted in a growth-inhibitory effect rather than in an enhancement of cell death. A synergistic effect was observed in those cell lines characterized by high proliferative index and low doubling time. These data suggest that treatment with everolimus and gefitinib might be of value in the treatment of selected NSCLC patients that exhibit high tumor proliferative activity.
Biochemical pharmacology 06/2009; 78(5):460-8. · 4.25 Impact Factor
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Valentina Zuliani,
Caterina Carmi,
Mirko Rivara,
Marco Fantini,
Alessio Lodola,
Federica Vacondio,
Fabrizio Bordi,
Pier Vincenzo Plazzi,
Andrea Cavazzoni,
Maricla Galetti,
Roberta R Alfieri, Pier Giorgio Petronini,
Marco Mor
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ABSTRACT: Benzylidene hydantoins have been recently reported as a new class of EGFR inhibitors. We describe here a simple and efficient methodology for the parallel solution-phase synthesis of a library of 5-benzylidene hydantoins, which were evaluated for antiproliferative activity on the human lung adenocarcinoma A549 cell line. Various substituents at positions 1, 3 and 5 on the hydantoin nucleus were examined. In the presence of a 5-benzylidene group and of a lipophilic substituent at position 1, most of the tested compounds inhibited cell proliferation at a concentration of 20 microM. Compound 7 (UPR1024), bearing 1-phenethyl and (E)-5-p-OH-benzylidene substituents, was found to be the most active derivative of the series. It inhibited EGFR autophosphorylation and induced DNA damage in A549 cells. Compound 7 and other synthesized 5-benzylidene hydantoin derivatives increased p53 levels, suggesting that the dual mechanism of action was a common feature shared by compound 7 and other member of the series.
European journal of medicinal chemistry 03/2009; 44(9):3471-9. · 3.27 Impact Factor
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Marcello Tiseo,
Marzia Capelletti,
Giuseppe De Palma,
Vittorio Franciosi,
Andrea Cavazzoni,
Paola Mozzoni,
Roberta R Alfieri,
Matteo Goldoni,
Maricla Galetti,
Beatrice Bortesi,
Cecilia Bozzetti,
Maura Loprevite,
Luca Boni,
Roberta Camisa,
Guido Rindi, Pier Giorgio Petronini,
Andrea Ardizzoni
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ABSTRACT: Epidermal growth factor receptor (EGFR) gene intron 1 contains a polymorphic single sequence dinucleotide repeat (CA)n whose length has been found to inversely correlate with transcriptional activity. This study was designed to assess the role of (CA)n polymorphism in predicting the outcome of gefitinib treatment in advanced non-small cell lung cancer (NSCLC).
Blood and tumor tissue from 58 patients with advanced NSCLC submitted to gefitinib were collected. EGFR intron 1 gene polymorphism, along with EGFR gene mutation, gene copy number and immunohistochemistry expression were determined. Moreover, a panel of lung cancer cell lines characterized for EGFR intron 1 polymorphism was also studied.
EGFR intron 1 polymorphism showed a statistically significant correlation with the gefitinib response (response rate 25 versus 0%, for patients with a (CA)16 and with a (CA)else genotype, respectively; p = 0.044). Patients with a (CA)16 genotype had a longer survival compared with those with a (CA)else genotype (11.4 versus 4.8 months, respectively; p = 0.037). In addition, cell lines lacking the (CA)16 allele showed a statistically significant higher IC50 compared with cell lines bearing at least one (CA)16 allele (p = 0.003).
This study supports a potential role of EGFR intron 1 polymorphism in predicting the outcome of gefitinib treatment in advanced NSCLC.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 11/2008; 3(10):1104-11. · 4.55 Impact Factor
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Andrea Cavazzoni,
Roberta R Alfieri,
Caterina Carmi,
Valentina Zuliani,
Maricla Galetti,
Claudia Fumarola,
Raffaele Frazzi,
Mara Bonelli,
Fabrizio Bordi,
Alessio Lodola,
Marco Mor, Pier Giorgio Petronini
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ABSTRACT: In this study, we examined the mechanism of action of the novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor 5-benzylidene-hydantoin UPR1024, whose structure was designed to interact at the ATP-binding site of EGFR. The compound had antiproliferative and proapoptotic effects when tested on the non-small cell lung cancer cell line A549. The growth inhibitory effect was associated with an accumulation of the cells in the S phase of the cell cycle. Moreover, UPR1024 induced significant level of DNA strand breaks associated with increased expression of p53 and p21(WAF1) proteins, suggesting an additive mechanism of action. The presence of wild-type p53 improved the drug efficacy, although the effect was also detectable in p53 null cells. We also noted apoptotic cell death after treatment with UPR1024 at concentrations above 10 mumol/L for >24 h, with involvement of both the extrinsic and intrinsic pathways. The present data show that UPR1024 may be considered a combi-molecule capable of both blocking EGFR tyrosine kinase activity and inducing genomic DNA damage. UPR1024 or its derivatives might serve as a basis for development of drugs for the treatment of lung cancer in patients resistant to classic tyrosine kinase inhibitors.
Molecular Cancer Therapeutics 03/2008; 7(2):361-70. · 5.23 Impact Factor
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ABSTRACT: Treatment of human endothelial cells with Shiga toxin 1 and 2 leads to the upregulation of genes encoding proinflammatory molecules involved in the pathogenesis of hemolytic-uremic syndrome. The paradoxical effect of inhibitors of mRNA translation, such as Shiga toxins, that at the same time induce protein expression was investigated by studying the relationship between their enzymatic activity (abstraction of adenine from nucleic acids) and the induction of interleukin-8 and granulocyte-macrophage colony-stimulating factor in human endothelial cells. As a positive control, the fungal toxin alpha-sarcin, acting on the same rRNA sequence targeted by Shiga toxins with a different mechanism (RNase activity), was used. The three toxins caused ribosomal lesions that, in turn, induced the activation of p38 stress kinase with kinetics that paralleled the inhibition of translation. Alpha-sarcin was devoid of activity on DNA. Shiga toxin 2 targeted nuclear DNA with more rapid kinetics than did Shiga toxin 1. Since the fungal ribotoxin was fully effective in the induction of proinflammatory proteins, we conclude that damage to ribosomes is indispensable and sufficient to activate protein expression via induction of the stress-kinase cascade. However, gene upregulation events induced by Shiga toxin 2 were much more efficient than those triggered by Shiga toxin 1, although the two toxins impaired translation to the same extent and had overlapping time courses of stress kinase activation. Regulations independent of the ribotoxic stress were assumed to operate in intoxicated cells. We hypothesized that the two bacterial toxins recognize different DNA sequences inducing different regulating effects on gene expression.
Infection and Immunity 06/2007; 75(5):2201-7. · 4.16 Impact Factor
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ABSTRACT: Cellular responses induced by stress are essential for the survival of cells under adverse conditions. These responses, resulting in cell adaptation to the stress, are accomplished by a variety of processes at the molecular level. After an alteration in homeostatic conditions, intracellular signalling processes link the sensing mechanism to adaptive or compensatory changes in gene expression. The ability of cells to adapt to hyperosmotic stress involves early responses in which ions move across cell membranes and late responses characterized by increased synthesis of either membrane transporters essential for uptake of organic osmolytes or of enzymes involved in their synthesis. The goal of these responses is to return the cell to its normal size and maintain cellular homeostasis. The enhanced synthesis of molecular chaperones, such as heat shock proteins, is another important component of the adaptive process that contributes to cell survival. Some responses are common to different stresses, whereas others are specific. In the first part of the review, we illustrate the characteristic and specific features of adaptive response to hypertonicity; we then describe similarities to and differences from other cellular stresses, such as genotoxic agents, nutrient starvation and heat shock.
Pflügers Archiv - European Journal of Physiology 06/2007; 454(2):173-85. · 4.46 Impact Factor
