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Domenico D'ARCA

Professore Associato
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze sede ex-Sc. Biomediche

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2023 - Applying optical tweezers technology to reveal Thymidylate Synthase interactions with its consensus mRNA [Abstract in Atti di Convegno]
Malpezzi, Giulia; Viader, Xvaier; Zaltron, Annamaria; Neri, Benedetta; di Felice, Giulia; Tagliazucchi, Lorenzo; Aiello, Daniele; Moschella, MARIA GAETANA; Costi, Maria Paola; D'Arca, Domenico; Cecconi, Ciro

2023 - Development of a novel class of YAP:TEAD interface-3 molecular disruptors to treat colorectal carcinoma [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Aiello, Daniele; Zappaterra, Dana; Elisi, GIAN MARCO; Venturelli, Alberto; Moschella, MARIA GAETANA; D'Arca, Domenico; Marverti, Gaetano; Lopresti, Ludovica; Pozzi, Cecilia; Pacifico, Salvatore; Guerrini, Remo; Ponterini, Glauco; Costi, Maria Paola

2023 - Exploiting the Optical Tweezer technology to reveal thymidylate synthase interactions with its consensus mRNA [Abstract in Atti di Convegno]
Malpezzi, Giulia; Viander, Xavier; Zaltron, Annamaria; Neri, Benedetta; Di Felice, Giulia; Tagliazucchi, Lorenzo; Aiello, Daniele; Moschella, MARIA GAETANA; Costi, Maria Paola; D'Arca, Domenico; Cecconi, Ciro

2023 - Lead optimization of human thymidylate synthase dimer disrupters: from computational studies to evaluation of their biological profiles [Abstract in Atti di Convegno]
Aiello, Daniele; Malpezzi, Giulia; Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; Giulia, Saporito; Venturelli, Alberto; Gluaco, Ponterini; D'Arca, Domenico; Marverti, Gaetano; Federico, Falchi; Costi, Maria Paola

2023 - Serum Mass Spectrometry Proteomics and Protein Set Identification in Response to FOLFOX-4 in Drug-Resistant Ovarian Carcinoma [Articolo su rivista]
D'Arca, Domenico; Severi, Leda; Ferrari, Stefania; Dozza, Luca; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Pagani, Lisa; Tagliazucchi, Lorenzo; Villani, Marco; D'Addese, Gianluca; Piga, Isabella; Conteduca, Vincenza; Rossi, Lorena; Gurioli, Giorgia; De Giorgi, Ugo; Losi, Lorena; Costi, Maria Paola

2023 - Targeting the Interplay of Independent Cellular Pathways and Immunity: A Challenge in Cancer Immunotherapy [Articolo su rivista]
Lauriola, Angela; Davalli, Pierpaola; Marverti, Gaetano; Santi, Spartaco; Caporali, Andrea; D’Arca, Domenico

mmunotherapy is a cancer treatment that exploits the capacity of the body’s immune system to prevent, control, and remove cancer. Immunotherapy has revolutionized cancer treatment and significantly improved patient outcomes for several tumor types. However, most patients have not benefited from such therapies yet. Within the field of cancer immunotherapy, an expansion of the combination strategy that targets independent cellular pathways that can work synergistically is predicted. Here, we review some consequences of tumor cell death and increased immune system engagement in the modulation of oxidative stress and ubiquitin ligase pathways. We also indicate combinations of cancer immunotherapies and immunomodulatory targets. Additionally, we discuss imaging techniques, which are crucial for monitoring tumor responses during treatment and the immunotherapy side effects. Finally, the major outstanding questions are also presented, and directions for future research are described.

2023 - design, synthesis and biological evaluation of novel YAP:TEAD complex disruptors for thetreatment of colorectal cancer [Relazione in Atti di Convegno]
Tagliazucchi, Lorenzo; Venturelli, Alberto; Malpezzi, Giulia; Zappaterra, Dana; Aiello, Daniele; Elisi, GIAN MARCO; Mor, Marco; Moschella, MARIA GAETANA; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2022 - A toolbox of biophysical and analytical assays helps to confirm the activity of novel hTS dimer disrupters (Ddis) with anticancer properties [Poster]
Tagliazucchi, Lorenzo; Venturelli, Alberto; Malpezzi, Giulia; Moschella, MARIA GAETANA; Aiello, Daniele; Lopresti, Ludovica; Pozzi, Cecilia; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2022 - Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth [Articolo su rivista]
Costantino, Luca; Ferrari, Stefania; Santucci, Matteo; MH Salo-Ahen, Outi; Carosati, Emanuele; Franchini, Silvia; Lauriola, Angela; Pozzi, Cecilia; Trande, Matteo; Gozzi, Gaia; Saxena, Puneet; Cannazza, Giuseppe; Losi, Lorena; Cardinale, Daniela; Venturelli, Alberto; Quotadamo, Antonio; Linciano, Pasquale; Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; Guerrini, Remo; Pacifico, Salvatore; Luciani, Rosaria; Genovese, Filippo; Henrich, Stefan; Alboni, Silvia; Santarem, Nuno; CORDEIRO DA SILVA, Anabela; Giovannetti, Elisa; J Peters, Godefridus; Pinton, Paolo; Rimessi, Alessandro; Cruciani, Gabriele; M Stroud, Robert; C Wade, Rebecca; Mangani, Stefano; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2022 - Development of a sensitive biochemical tool to assess the expression levels of recombinant ectopic proteins in in vitro engineered cellular systems [Abstract in Atti di Convegno]
Moschella, Mg; Marverti, G; Cassanelli, Valentina; Venuti, Federica; Tagliazucchi, L; Costi, Mp; D’Arca., D

2022 - Identification of a Quinone Derivative as a YAP/TEAD Activity Modulator from a Repurposing Library [Articolo su rivista]
Lauriola, A.; Uliassi, E.; Santucci, M.; Bolognesi, M. L.; Mor, M.; Scalvini, L.; Elisi, G. M.; Gozzi, G.; Tagliazucchi, L.; Marverti, G.; Ferrari, S.; Losi, L.; D'Arca, D.; Costi, M. P.

The transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional co-activator with PDZ-binding motif) are the major downstream effectors in the Hippo pathway and are involved in cancer progression through modulation of the activity of TEAD (transcriptional enhanced associate domain) transcription factors. To exploit the advantages of drug repurposing in the search of new drugs, we developed a similar approach for the identification of new hits interfering with TEAD target gene expression. In our study, a 27member in-house library was assembled, characterized, and screened for its cancer cell growth inhibition effect. In a secondary luciferase-based assay, only seven compounds confirmed their specific involvement in TEAD activity. IA5 bearing a p-quinoid structure reduced the cytoplasmic level of phosphorylated YAP and the YAP–TEAD complex transcriptional activity and reduced cancer cell growth. IA5 is a promising hit compound for TEAD activity modulator development.

2022 - LC-MS serum proteomics reveals a panel of proteins prognostic of positive responsiveness to bevacizumab therapy in late-stages ovarian cancer patients [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; D'Addese, Gianluca; Califano, Daniela; Arenare, Laura; Mezzanzanico, Delia; Chinello, Clizia; Magni, Fulvio; Villani, Marco; Losi, Lorena; Marverti, Gaetano; D'Arca, Domenico; Chiodini, Paolo; Pignata, Sandro; Costi, Maria Paola

2022 - Lead-optimization process and characterization of the dissociative effect applied to new dimer disrupters of human Thymidylate Synthase [Abstract in Atti di Convegno]
Aiello, Daniele; Ascione, Cristian; Tagliazucchi, Lorenzo; Malpezzi, Giulia; Cassanelli, Valentina; Ponterini, Glauco; D'Arca, Domenico; Marverti, Gaetano; Moschella, MARIA GAETANA; Falchi, Federico; Venturelli, Alberto; Costi, Maria Paola

2022 - Targeting the dimer-monomer equilibrium of thymidylate synthase, to accelerate protein degradation and cancer cell growth inhibition [Abstract in Atti di Convegno]
Costi, Maria Paola; Venturelli, A; Ponterini, G; Pozzi, C; Wade, Rebecca; Giovannetti, E; Rimessi, A; Tagliazucchi, L; Moschella, M; Marverti, G; D’Arca, D

2022 - Telomere Dysfunction Is Associated with Altered {DNA} Organization in Trichoplein/Tchp/Mitostatin ({TpMs}) Depleted Cells [Articolo su rivista]
Lauriola, Angela; Davalli, Pierpaola; Marverti, Gaetano; Caporali, Andrea; Mai, Sabine; D'Arca, Domenico

Abstract: Recently, we highlighted a novel role for the protein Trichoplein/TCHP/Mitostatin (TpMs), both as mitotic checkpoint regulator and guardian of chromosomal stability. TpMs-depleted cells show numerical and structural chromosome alterations that lead to genomic instability. This condition is a major driving force in malignant transformation as it allows for the cells acquiring new functional capabilities to proliferate and disseminate. Here, the effect of TpMs depletion was investigated in different TpMs-depleted cell lines by means of 3D imaging and 3D Structured illumination Microscopy. We show that TpMs depletion causes alterations in the 3D architecture of telomeres in colon cancer HCT116 cells. These findings are consistent with chromosome alterations that lead to genomic instability. Furthermore, TpMs depletion changes the spatial arrangement of chromosomes and other nuclear components. Modified nuclear architecture and organization potentially induce variations that precede the onset of genomic instability and are considered as markers of malignant transformation. Our present observations connect the tumor suppression ability of TpMs with its novel functions in maintaining the proper chromosomal segregation as well as the proper telomere and nuclear architecture. Further investigations will investigate the connection between alterations in telomeres and nuclear architecture with the progression of human tumors with the aim of developing personalized therapeutic interventions.

2021 - Cellular uptake and metabolic degradation of a conjugate of folic acid with an anticancer peptide targeting the human Thymidylate synthase enzyme [Relazione in Atti di Convegno]
Tagliazucchi, Lorenzo; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

2021 - Designing selective Cys-ligands to unpair the binding of the Human Transcription Enhancer Associated Domain 4 (hTEAD-4) with its modulators to halt cancer cell growth [Poster]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Pozzi, Cecilia; Lopresti, Ludovica; Venturelli, Alberto; D'Arca, Domenico; Marverti, Gaetano; Cecconi, Ciro; Ponterini, Glauco; Costi, Maria Paola

The Hippo Signalling cascade is an emerging target in tumour suppression regulation, neoplastic hypertrophy, and regenerative medicine. The pathway is activated by circulating anti-proliferative signals which leads to the phosphorylation of Yes Associated Protein (hYAP1) on Ser127/381, thus 14-3-3σ mediated cytosolic retention. Genetic alterations or exogenous factor may cause YAP nuclear migration and association to TEAD1-4 (Transcription Enhancer Associated Domain), triggering up-regulation of anti-apoptotic genes [1]. hTEAD is an enhancer that activates the nuclear transcription of genes as EMT’s, EGFR and cyclins, and promotes the synthesis of survivin, tyrosine kinase HER3, and mitochondrial Bcl-xL involved in cell proliferation. TEAD binds a palmitic (palm) or myristic (myr) acids, tethered at Cys367 pocket, however its biological role is still not well known. hTEAD isoform-4 is the most represented of its family in solid tumours and its overexpression or mutation leads to cancer development and metastasis. Recent studies have considered hTEAD a promising target for anticancer drugs. Its inhibition strategy includes the disruption/prevention of YAP1:TEAD4 complex formation [2]. With the aim to develop a specific cysteine-directed inhibition strategy, we studied Cys on the protein surface and investigated their reactivity. Hence, our studies focus on characterizing the recombinant hTEAD4-ybd (aa217-434) surface though the analysis of the reactivity of its four Cys thiols (Cys310, Cys335, Cys367, Cys410), all close to YAP binding area. First, myr-Cys-367 was investigated to confirm the auto-myristoilation of the E. coli recombinant hTEAD4 through RP-chromatography on UHPLC-Orbitrap Q-Ex (ThermoFisher™) by multicharged TIC deconvolution, and the total myr-TEAD was assessed around 25%. Myristate position was confirmed by FASP protein tryptic hydrolysis and tandem-MS peptide analysis. We studied hTEAD binding of a small disulphides and thiols library with different chemical properties through the exposed cysteines residues in presence of different concentration of reducing agent [3]. Top8 DDA (HCD)-MS/MS scan on the tryptic peptides suggested the ligands’ high selectivity towards Cys335. Cys367 was never found conjugated, even in the non-Myr fraction, hinting the low accessibility to the lipid pocket. The number of surface reactive Cys was confirmed by a reverse-titration of the protein against increasing amount of thiophenol; excess of unreacted thiophenol was measured by HPLC-UV-ELSD (Agilent™ 1260), suggesting a 1:1 stoichiometry. We confirmed hTEAD-ybd ligand ratio by fluoresceine labelling with absorption and fluorescence differential spectroscopy. The ongoing work engages the screening of a larger compound library to study YAP:TEAD interaction with a ligand displacement assay of labelled TEAD to a rhodamine-tagged peptidomimetic probe to achieve structural information of the heterodimer interface and to start a hit-optimization programme. REFERENCES [1] Santucci M, Vignudelli T, et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment. J Med Chem. 2015 Jun 25;58(12):4857-73. [2] Elisi G.M, Santucci M, et al. Repurposing of Drugs Targeting YAP-TEAD Functions. Cancers 2018, 10, 329. [3] Malpezzi G MSc Degree Thesis, Solvent exposure, and reactivity of the cysteines of Transcription Enhancer Associate Domain (TEAD), a potential anticancer target, 2021. University of Pavia – University of Modena and Reggio Emilia.

2021 - Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting [Articolo su rivista]
Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; D'Arca, Domenico; Pacifico, Salvatore; Guerrini, Remo; Spyrakis, Francesca; Gozzi, Gaia; Lauriola, Angela; Santucci, Matteo; Cannazza, Giuseppe; Tagliazucchi, Lorenzo; Cazzato, Addolorata Stefania; Losi, Lorena; Ferrari, Stefania; Ponterini, Glauco; Costi, Maria P

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor alpha (FRalpha) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRalpha selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.

2021 - Structural bases for the synergistic inhibition of human thymidylate synthase and ovarian cancer cell growth by drug combinations [Articolo su rivista]
Pozzi, C.; Santucci, M.; Marverti, G.; D'arca, D.; Tagliazucchi, L.; Ferrari, S.; Gozzi, G.; Losi, L.; Tassone, G.; Mangani, S.; Ponterini, G.; Costi, M. P.

2021 - Structural insight into YAP-TEAD4 protein-protein interactions as target for cancer treatment [Abstract in Atti di Convegno]
Lopresti, Ludovica; Pozzi, Cecilia; Tagliazucchi, L; D’Arca, D; Marverti, G; Costi, Mp; Mangani, Stefano

2020 - A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells [Articolo su rivista]
Marverti, Gaetano; Gozzi, Gaia; Maretti, Eleonora; Lauriola, Angela; Severi, Leda; Sacchetti, Francesca; Losi, Lorena; Pacifico, Salvatore; Ferrari, Stefania; Ponterini, Glauco; Leo, Eliana Grazia; Costi, Maria Paola; D'Arca, Domenico

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

2020 - Depletion of Trichoplein (TpMs) Causes Chromosome Mis-Segregation, DNA Damage and Chromosome Instability in Cancer Cells [Articolo su rivista]
Lauriola, Angela; Martello, Andrea; Fantini, Sebastian; Marverti, Gaetano; Zanocco-Marani, Tommaso; Davalli, Pierpaola; Guardavaccaro, Daniele; Mai, Sabine; Caporali, Andrea; D’Arca, Domenico

Mitotic perturbations frequently lead to chromosome mis-segregation that generates genome instability, thereby triggering tumor onset and/or progression. Error-free mitosis depends on fidelity-monitoring systems that ensure the temporal and spatial coordination of chromosome segregation. Recent investigations are focused on mitotic DNA damage response (DDR) and chromosome mis-segregations with the aim of developing more efficient anti-cancer therapies. We previously demonstrated that trichoplein keratin filament binding protein (TpMs) exhibits hallmarks of a tumor suppressor gene in cancer-derived cells and human tumors. Here, we show that silencing of TpMs expression results in chromosome mis-segregation, DNA damage and chromosomal instability. TpMs interacts with Mad2, and TpMs depletion results in decreased levels of Mad2 and Cyclin B1 proteins. All the genetic alterations observed are consistent with both defective activation of the spindle assembly checkpoint and mitotic progression. Thus, low levels of TpMs found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

2020 - Trichoplein binds PCM1 and controls endothelial cell function by regulating autophagy [Articolo su rivista]
Martello, Andrea; Lauriola, Angela; Mellis, David; Parish, Elisa; Dawson, John C; Imrie, Lisa; Vidmar, Martina; Gammoh, Noor; Mitić, Tijana; Brittan, Mairi; Mills, Nicholas L; Carragher, Neil O; D'Arca, Domenico; Caporali, Andrea

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome-dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP-depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF-κB inhibition. Moreover, Tchp knock-out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy-mediated mechanism.

2019 - Cyclic Peptides Acting as Allosteric Inhibitors of Human Thymidylate Synthase and Cancer Cell Growth [Articolo su rivista]
Pacifico, Salvatore; Santucci, Matteo; Luciani, Rosaria; Saxena, Puneet; Linciano, Pasquale; Ponterini, Glauco; Lauriola, Angela; D'Arca, Domenico; Marverti, Gaetano; Guerrini, Remo; Costi, Maria Paola

Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness.

2019 - Involvement of epigenetic modification of TERT promoter in response to all-trans retinoic acid in ovarian cancer cell lines [Articolo su rivista]
Losi, Lorena; Lauriola, Angela; Tazzioli, Erica; Gozzi, Gaia; Scurani, Letizia; D'Arca, Domenico; Jean, Benhattar

Background: All-trans retinoic acid (ATRA) is currently being used to treat hematological malignancies, given the ability to inhibit cell proliferation. This effect seems to be related to epigenetic changes of the TERT (Telomerase Reverse Transcriptase) promoter. When hypomethylated, ATRA-inducible TERT repressors can bind the promoter, repressing transcription of TERT, the rate-limiting component of telomerase. Ovarian carcinomas are heterogeneous tumors characterized by several aberrantly methylated genes among which is TERT. We recently found a hypomethylation of TERT promoter in about one third of serous carcinoma, the most lethal histotype. Our aim was to investigate the potential role of ATRA as an anticancer drug in a sub-group of ovarian carcinoma where the TERT promoter was hypomethylated. Methods: The potential antiproliferative and cytotoxic effect of ATRA was investigated in seven serous ovarian carcinoma and one teratocarcinoma cell lines and the results were compared to the methylation status of their TERT promoter. Results: The serous ovarian carcinoma cell line OVCAR3, harboring a hypomethylated TERT promoter, was the best and fastest responder. PA1 and SKOV3, two cell lines with an intermediate methylated promoter, revealed a weaker and delayed response. On the contrary, the other 5 cell lines with a highly methylated promoter did not respond to ATRA, indicative of ATRA-resistant cells. Conclusions: Our results demonstrate an inverse correlation between the methylation level of TERT promoter and ATRA efficacy in ovarian carcinoma cell lines. Although these results are preliminary, ATRA treatment could become a new powerful, personalized therapy in serous ovarian carcinoma patients, but only in those with tumors harboring a hypomethylated TERT promoter.

2019 - The 1,10-phenanthroline ligand enhances the antiproliferative activity of dna-intercalating thiourea-pd(Ii) and-pt(ii) complexes against cisplatin-sensitive and-resistant human ovarian cancer cell lines [Articolo su rivista]
Marverti, G.; Gozzi, G.; Lauriola, A.; Ponterini, G.; Belluti, S.; Imbriano, C.; Costi, M. P.; D’Arca, D.

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four dierent thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative ecacy. This paralleled both a higher intracellular drug accumulation and a more ecient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.

2018 - Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth [Articolo su rivista]
Saxena, Puneet; Severi, Leda; Santucci, Matteo; Taddia, Laura; Ferrari, Stefania; Luciani, Rosaria; Marverti, Gaetano; Marraccini, Chiara; Tondi, Donatella; Mor, Marco; Laura, Scalvini; Vitiello, Simone; Losi, Lorena; Fonda, Sergio; Pacifico, Salvatore; Guerrini, Remo; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola

LR and [D-Gln4]LR peptides bind the mono- mer−monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pron]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.

2018 - Proteomic and bioinformatic studies for the characterization of response to pemetrexed in platinum drug resistant ovarian cancer [Articolo su rivista]
Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I.; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P.

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

2018 - Repurposing of drugs targeting yap-tead functions [Articolo su rivista]
Elisi, Gian Marco; Santucci, Matteo; D’Arca, Domenico; Lauriola, Angela; Marverti, Gaetano; Losi, Lorena; Scalvini, Laura; Bolognesi, Maria Laura; Mor, Marco; Costi, Maria Paola

Drug repurposing is a fast and consolidated approach for the research of new active compounds bypassing the long streamline of the drug discovery process. Several drugs in clinical practice have been reported for modulating the major Hippo pathway’s terminal effectors, namely YAP (Yes1-associated protein), TAZ (transcriptional co-activator with PDZ-binding motif) and TEAD (transcriptional enhanced associate domains), which are directly involved in the regulation of cell growth and tissue homeostasis. Since this pathway is known to have many cross-talking phenomena with cell signaling pathways, many efforts have been made to understand its importance in oncology. Moreover, this could be relevant to obtain new molecular tools and potential therapeutic assets. In this review, we discuss the main mechanisms of action of the best-known compounds, clinically approved or investigational drugs, able to cross-talk and modulate the Hippo pathway, as an attractive strategy for the discovery of new potential lead compounds.

2018 - Targeting Oxidatively Induced DNA Damage Response in Cancer: Opportunities for Novel Cancer Therapies [Articolo su rivista]
Davalli, Pierpaola; Marverti, Gaetano; Lauriola, Angela; D’Arca, Domenico

Cancer is a death cause in economically developed countries that results growing also in developing countries. Improved outcome through targeted interventions faces the scarce selectivity of the therapies and the development of resistance to them that compromise the therapeutic effects. Genomic instability is a typical cancer hallmark due to DNA damage by genetic mutations, reactive oxygen and nitrogen species, ionizing radiation, and chemotherapeutic agents. DNA lesions can induce and/or support various diseases, including cancer. The DNA damage response (DDR) is a crucial signaling-transduction network that promotes cell cycle arrest or cell death to repair DNA lesions. DDR dysregulation favors tumor growth as downregulated or defective DDR generates genomic instability, while upregulated DDR may confer treatment resistance. Redox homeostasis deeply and capillary affects DDR as ROS activate/inhibit proteins and enzymes integral to DDR both in healthy and cancer cells, although by different routes. DDR regulation through modulating ROS homeostasis is under investigation as anticancer opportunity, also in combination with other treatments since ROS affect DDR differently in the patients during cancer development and treatment. Here, we highlight ROS-sensitive proteins whose regulation in oxidatively induced DDR might allow for selective strategies against cancer that are better tailored to the patients.

2018 - pH-Promoted Release of a Novel Anti-Tumour Peptide by “Stealth” Liposomes: Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells [Articolo su rivista]
Sacchetti, Francesca; Marverti, Gaetano; D’Arca, Domenico; Severi, Leda; Maretti, Eleonora; Iannuccelli, Valentina; Pacifico, Salvatore; Ponterini, Glauco; Costi, Maria Paola; Leo, Eliana

Purpose: To evaluate the potential effects of PEGylated pH-sensitive liposomes on the intracellular activity of a new peptide recently characterized as a novel inhibitor of the human thymidylate synthase (hTS) over-expressed in many drug-resistant human cancer cell lines. Methods: Peptide-loaded pH-sensitive PEGylated (PpHL) and non-PEGylated liposomes (nPpHL) were carefully characterized and delivered to cis-platinum resistant ovarian cancer C13* cells; the influence of the PpHL on the drug intracellular activity was investigated by the Western Blot analysis of proteins involved in the pathway affected by hTS inhibition. Results: Although PpHL and nPpHL showed different sizes, surface hydrophilicities and serum stabilities, both carriers entrapped the drug efficiently and stably demonstrating a pH dependent release; moreover, the different behavior against J774 macrophage cells confirmed the ability of PEGylation in protecting liposomes from the reticuloendothelial system. Comparable effects were instead observed against C13* cells and biochemical data by immunoblot analysis indicated that PEGylated pH-sensitive liposomes do not modify the proteomic profile of the cells, fully preserving the activity of the biomolecule. Conclusion: PpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.

2017 - Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones? [Articolo su rivista]
Sacchetti, Francesca; D'Arca, Domenico; Genovese, Filippo; Pacifico, Salvatore; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia

Context: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. Objective: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. Materials and methods For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. Results and discussion Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. Conclusions Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.

2017 - The key role of Mitostatin in the maintenance of genome stability [Abstract in Rivista]
Lauriola, Angela; Caporali, Andrea; Mai, Sabine; D'Arca, Domenico

Genomic instability is a characteristic of most cancers and it refers to an increased tendency of alterations in the genome during the life cycle of cells. The fidelity of DNA replication is highly ensured by different checkpoints; the activation of spindle checkpoints prevents cells from premature entry into mitosis, avoiding incorrect chromosome segregation and aneuploidy, a typical feature of many cancers. Mitostatin, a novel protein, endowed with tumor suppressor activity, has been reported to bind centrosomal proteins Odf2 and ninein, and its depletion causes an alteration of the anchorage of microtubules to the centrosome. Since functional defects of centrosomes are associated to mitotic failure, Mitostatin may have a key role in guarding the fidelity of mitosis in cells. Here we show that the depletion of Mitostatin in cancer cells, synchronized by aphidicolin (G1/S) block and released into nocodazole-containing medium, leads to mitotic slippage and adaptation to the spindle checkpoint (SAC) in the presence of a spindle inhibitor. Concomitantly, Mitostatin depletion promotes the early degradation of Mad2 and cyclin B1. Since the activated spindle checkpoint delays cell exit from mitosis by preventing cyclin B1 proteolysis, the cyclin B1 early degradation leads to mitotic checkpoint escape and resulting chromosome instability. In this study, we report for the first time that the depletion of Mitostatin induces an increase of numerical and structural chromosomal aberrations compared to control cells. These aberrations include aneuploidy, the formation of triradials and broken chromosomes. Taken together, our observations suggest that Mitostatin plays a critical role in guarding the fidelity of mitosis, enabling the optimal activation of the spindle checkpoint (SAC). Thus, low levels of Mitostatin found in certain human tumors may contribute to cellular transformation by promoting genomic instability.

2016 - Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology [Articolo su rivista]
Ponterini, Glauco; Martello, Andrea; Pavesi, Giorgia; Lauriola, Angela; Luciani, Rosaria; Santucci, Matteo; Pela', Michela; Gozzi, Gaia; Pacifico, Salvatore; Guerrini, Remo; Marverti, Gaetano; Costi, Maria Paola; D'Arca, Domenico

Demonstrating a candidate drug' s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

2016 - ROS, Cell Senescence, and Novel Molecular Mechanisms in Aging and Age-Related Diseases [Articolo su rivista]
Davalli, Pierpaola; Mitic, T; Caporali, A; Lauriola, Angela; D'Arca, Domenico

The aging process worsens the human body functions at multiple levels, thus causing its gradual decrease to resist stress, damage, and disease. Besides changes in gene expression and metabolic control, the aging rate has been associated with the production of high levels of Reactive Oxygen Species (ROS) and/or Reactive Nitrosative Species (RNS). Specific increases of ROS level have been demonstrated as potentially critical for induction and maintenance of cell senescence process. Causal connection between ROS, aging, age-related pathologies, and cell senescence is studied intensely. Senescent cells have been proposed as a target for interventions to delay the aging and its related diseases or to improve the diseases treatment. Therapeutic interventions towards senescent cells might allow restoring the health and curing the diseases that share basal processes, rather than curing each disease in separate and symptomatic way. Here, we review observations on ROS ability of inducing cell senescence through novel mechanisms that underpin aging processes. Particular emphasis is addressed to the novel mechanisms of ROS involvement in epigenetic regulation of cell senescence and aging, with the aim to individuate specific pathways, which might promote healthy lifespan and improve aging.

2015 - ANTICANCER DRUGS [Brevetto]
Costantino, Luca; Costi, Maria Paola; Ponterini, Glauco; Marverti, Gaetano; Franchini, Silvia; Tondi, Donatella; D'Arca, Domenico; Ferrari, Stefania; Luciani, Rosaria; Venturelli, Alberto; Sammak, Susan; Lauriola, Angela; Gozzi, Gaia

Composti destabilizzanti l’omodimero timidilato sintasi

2015 - Enhanced anti-hyperproliferative activity of human thymidylate synthase inhibitor peptide by solid lipid nanoparticle delivery [Articolo su rivista]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pela', Michela; Pinetti, Diego; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia

Recently, octapeptide LSCQLYQR (LRp), reducing growth of cis-platinum (cDDP) resistant ovarian carcinoma cells by inhibiting the monomer–monomer interface of the human enzyme thymidylate synthase, has been identified. As the peptide is not able to cross the cell membrane it requires an appropriate delivery system. In this work the application of SLNs, biocompatible and efficient tools for the intracellular drug transport, applied especially for lipophilic drugs, was exploited for the delivery of the hydrophilic peptide LRp. SLNs formulated in the absence/presence of small amount of squalene showed dimensions below 150 nm, negative zeta potential and good stability to the freeze-drying process. Even though the particles formulated with squalene exhibited a less ordered crystal lattice and a lower surface hydrophobicity, a rapid drug release from these nanocarriers occurred as a result of the relevant expulsion of the drug from the lipid core during lipid crystallization. On the contrary, SLNs formulated in the absence of squalene were able to incorporate more stably the peptide showing considerable cytotoxic effect on cDDP resistant C13* ovarian carcinoma cell line at concentration 50 times lower than that used previously with a marketed delivery system. From the cell cycle analysis by the propidium iodide test in SLNs-peptide treated cancer cells an increase of apoptosis percentage was observed, indicating that SLNs were able to carry efficiently the peptide until its enzymatic target.

2015 - Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance [Articolo su rivista]
Taddia, Laura; D'Arca, Domenico; Ferrari, Stefania; Marraccini, Chiara; Severi, Leda; Ponterini, Glauco; Assaraf, Yahuda G.; Marverti, Gaetano; Costi, Maria Paola

Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemo-resistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug esistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.

2015 - pH sensitive PEGylated Liposomes delivering active hydrophilic peptide with anticancer activity: in vitro study on cDDP-resistant ovarian cell line [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pinetti, Diego; Genovese, Filippo; Maretti, Eleonora; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia

Thymidylate synthase (TS) can be considered a very interesting molecular target for the therapy of the ovarian cancer.. Recently, specific octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), have been identified. Similarly to the majority of peptides, they cannot cross the cell membrane and require a delivery system for transport into the cells and pH sensitive liposomes, destabilizing at mildly acidic pH, are considered efficient tools for delivering water-soluble drugs into the cell cytoplasm. In the present study in order to attain the peptide triggering in the cells promoting endosomal escape, stealth pH-sensitive liposomes were developed and characterized. Results suggested that pH sensitive liposomes seemed suitable carriers for the encapsulation of small hydrophilic molecules like peptides. The appreciable difference in cytotoxicity between loaded and unloaded liposomes demonstrated that the peptide, whose activity is held in the cytoplasm, was triggered in the proper biological site.

2014 - Mass Spectrometric/Bioinformatic Identification of a Protein Subset That Characterizes the Cellular Activity of Anticancer Peptides [Articolo su rivista]
Genovese, Filippo; A., Gualandi; L., Taddia; Marverti, Gaetano; S., Pirondi; C., Marraccini; P., Perco; M., Pelà; R., Guerrini; M. R., Amoroso; F., Esposito; A., Martello; Ponterini, Glauco; D'Arca, Domenico; Costi, Maria Paola

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [D-Gln4]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides’ cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.

2014 - Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth [Articolo su rivista]
M., Pelà; Saxena, Puneet; Luciani, Rosaria; Santucci, Matteo; Ferrari, Stefania; Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; Pirondi, Silvia; Genovese, Filippo; S., Salvadori; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola; R., Guerrini

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The D-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Costi, Maria Paola; Marverti, Gaetano; Pirondi, Silvia; Martello, Andrea; D'Arca, Domenico; Pela', Michela; Guerrini, R.; Marraccini, Chiara

Objectives The over-expression of thymidylate synthase (TS) and of the other folate cycle enzymes, is one of the mechanisms of resistance to cisplatin (cDDP) encountered in most of resistant human ovarian cancer cell lines, accounting for the more efficient DNA repair and synthesis. Oligopeptides were designed to inhibit TS activity by interfering with its dimerization. Among these, the LR octapeptide showed cell growth inhibitory activity against two cisplatin-sensitive human ovarian cancer cell lines. To improve the intracellular delivery of LR, we designed a bioconjugate with folic acid (FA-LR), which enters cell by exploiting the folate receptor alpha (FRα)-mediated endocytosis. Methods -Cell lines. The human ovarian cancer cell lines OAW28, COV504, IGROV-1, TOV112D, 2008, C13*, A2780 and A2780/CP. -Real Time PCR of FRα mRNA . -Flow cytometric analysis of FRα cell surface expression -Folic acid surface binding studies. -Uptake studies Results Real Time PCR, western blot analysis and folic acid surface binding assay indicate that IGROV-1 and OAW28 cells show high expression levels of FRα, while TOV112D, 2008 and 2008/C13* almost don't express FRα on their cell surface. The folate bioconjugate FA-LR blocked competitively the binding of [3H]Folic acid to FR and consequently its cellular uptake. FA-LR is detected in the cell and its stability evaluated. Conclusions The chemical modification of the folate with the LR drug motif only minimally altered the intrinsic affinity the biocongiugate for FR and suggest that the pteroate-peptide conjugate exploits FR as a substrate for its internalization. Cytotoxicity of the bioconjugate will be presented.

2012 - Anticancer activity of green tea polyphenols in prostate gland [Articolo su rivista]
Davalli, Pierpaola; F., Rizzi; A., Caporali; D., Pellacani; S., Davoli; S., Bettuzzi; M., Brausi; D'Arca, Domenico

Numerous evidences from prevention studies in humans, support the existence of an association between green tea polyphenols consumption and a reduced cancer risk. Prostate cancer is one of the most frequently diagnosed male neoplasia in the Western countries, which is in agreement with this gland being particularly vulnerable to oxidative stress processes, often associated with tumorigenesis. Tea polyphenols have been extensively studied in cell culture and animal models where they inhibited tumor onset and progression. Prostate cancer appears a suitable target for primary prevention care, since it grows slowly, before symptoms arise, thus offering a relatively long time period for therapeutic interventions. It is, in fact, usually diagnosed in men 50-year-old or older, when even a modest delay in progression of the disease could significantly improve the patients quality of life. Although epidemiological studies have not yet yielded conclusive results on the chemopreventive and anticancer effect of tea polyphenols, there is an increasing trend to employ these substances as conservative management for patients diagnosed with less advanced prostate cancer. Here, we intend to review the most recent observations relating tea polyphenols to human prostate cancer risk, in an attempt to outline better their potential employment for preventing prostate cancer.

2011 - Mitostatin Is Down-Regulated in Human Prostate Cancer and Suppresses the Invasive Phenotype of Prostate Cancer Cells [Articolo su rivista]
M., Fassan; D'Arca, Domenico; J., Letko; A., Vecchione; M. P., Gardiman; P., Mccue; B., Wildemore; M., Rugge; D., Shupp Byrne; L. G., Gomella; A., Morrione; R. V., Iozzo; R., Baffa

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in nude mice. Conversely, targeting endogenous Mitostatin by siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells enhanced the malignant phenotype in both cell lines. In agreement of these anti-oncogenic roles, we discovered that Mitostatin was absent in similar to 35% (n = 124) of prostate tumor samples and its overall reduction was associated with advanced cancer stages. Collectively, our findings indicate that MITOSTATIN may acts as a tumor suppressor gene in prostate cancer and provide a novel cellular and molecular mechanism to be further exploited and deciphered in our understanding of prostate cancer progression.

2010 - Huwe1 ubiquitin ligase is essential to synchronize neuronal and glial differentiation in the developing cerebellum [Articolo su rivista]
D'Arca, Domenico; X., Zhao; W., Xu; Ramirez Martinez, N. C.; A., Iavarone; A., Lasorella

We have generated a knockout mouse strain in which the gene coding for the ubiquitin ligase Huwe1 has been inactivated in cerebellar granule neuron precursors (CGNPs) and radial glia. These mice have a high rate of postnatal lethality and profound cerebellar abnormalities. The external granule layer of the cerebellum, which contains CGNPs, is expanded and displays aberrant proliferation and impaired differentiation of the progenitor cell population. The uncontrolled proliferation of the CGNPs is associated with accumulation of the N-Myc oncoprotein, a substrate of Huwe1, and consequent activation of the signaling events downstream to N-Myc. Furthermore, loss of Huwe1 in Bergmann glia leads to extensive disorganization of this cell population with layering aberrations, severe granule neuron migration defects, and persistence of ectopic clusters of granule neurons in the external granule layer. Our findings uncover an unexpected role for Huwe1 in regulating Bergmann glia differentiation and indicate that this ubiquitin ligase orchestrates the programming of the neural progenitors that give rise to neurons and glia in the cerebellum.

2009 - MITOSTATIN, a putative tumor suppressor on chromosome 12q24.1, is downregulated in human bladder and breast cancer [Articolo su rivista]
Vecchione, A; Fassan, M; Anesti, V; Morrione, A; Goldoni, S; Baldassarre, G; Byrne, D; D'Arca, Domenico; Palazzo, Jp; Lloyd, J; Scorrano, L; Gomella, Lg; Iozzo, Rv; Baffa, R.

Allelic deletions on human chromosome 12q24 are frequently reported in a variety of malignant neoplasms, indicating the presence of a tumor suppressor gene(s) in this chromosomal region. However, no reasonable candidate has been identified so far. In this study, we report the cloning and functional characterization of a novel mitochondrial protein with tumor suppressor activity, henceforth designated MITOSTATIN. Human MITOSTATIN was found within a 3.2-kb transcript, which encoded a similar to 62 kDa, ubiquitously expressed protein with little homology to any known protein. We found homozygous deletions and mutations of MITOSTATIN gene in similar to 5 and similar to 11% of various cancer-derived cells and solid tumors, respectively. When transiently overexpressed, MITOSTATIN inhibited colony formation, tumor cell growth and was proapoptotic, all features shared by established tumor suppressor genes. We discovered a specific link between MITOSTATIN overexpression and downregulation of Hsp27. Conversely, MITOSTATIN knockdown cells showed an increase in cell growth and cell survival rates. Finally, MITOSTATIN expression was significantly reduced in primary bladder and breast tumors, and its reduction was associated with advanced tumor stages. Our findings support the hypothesis that MITOSTATIN has many hallmarks of a classical tumor suppressor in solid tumors and may play an important role in cancer development and progression.

2009 - Prevention of urinary bladder cancer in the FHIT knock-out mouse with Rofecoxib, a Cox-2 inhibitor [Articolo su rivista]
D'Arca, Domenico; J., Lenoir; B., Wildemore; F., Gottardo; E., Bragantini; D., Shupp Byrne; N., Zanesi; M., Fassan; C. M., Croce; L. G., Gomella; R., Baffa

Objectives: Aberrant or increased expression of cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of many diseases, including cancer. However, the exact mechanism by which COX-2 may influence tumorigenesis has yet to be described. To investigate the chemopreventive role of a COX-2 inhibitor, rofecoxib, in the development of urinary bladder cancer, we studied the effect of this drug in heterozygous and nullizygous fragile histidine triad (FHIT) gene-deficient mice in a chemically induced carcinogenesis model. Materials and methods: Two-hundred eight mice consisting of 50 FHIT +/+, 63 FHIT +/- and 95 FHIT -/-, were divided into five treatment groups and followed up for 15 weeks. Mice were treated with freshly prepared solution of 0.1% or 0.01% N-butyl-N-(-4-hydroxybutyl)-nitrosamine (BBN) in their drinking water and rofecoxib was administered in mouse chow at 150 parts per million concentration. Mice were sacrificed, and accurate histological analysis of the bladder was performed. Results: Rofecoxib treatment significantly reduced the incidence of preneoplastic lesions/bladder tumors (P = 0.016). Comparing the incidence of neoplastic lesions in mice treated with rofecoxib and BBN (22/56, 39.3%) and mice treated only with BBN (32/57, 56.1%), a protective role of rofecoxib on the BBN tumor induction has been observed (P = 0.024). A similar result (P = 0.002) has been reached observing the incidence of mild and moderate dysplasia in mice treated with a lower concentration of BBN (8/16, 50.0% vs. 20/24, 83.3%). Moreover, as previously observed, a significant increase in neoplastic lesions in the FHIT +/- and FHIT -/- vs. TWIT +/+ mice after BBN treatment has been observed (P = 0.003). Conclusions: These findings suggest that rofecoxib provides a therapeutic defense against bladder carcinogenesis in our model and confirmed that the FHIT knock-out mouse is a suitable system to study in vivo bladder carcinogenesis. (C) 2010 Elsevier Inc. All rights reserved.

2009 - The N-Myc-DLL3 cascade is suppressed by the ubiquitin ligase Huwe1 to inhibit proliferation and promote neurogenesis in the developing brain. [Articolo su rivista]
Zhao, X; D'Arca, Domenico; Lim, Wk; Brahmachary, M; Carro, Ms; Ludwig, T; Cardo, Cc; Guillemot, F; Aldape, K; Califano, A; Iavarone, A; Lasorella, A.

Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are crucial events directing brain development. Here we show that the ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss- and gain-of-function experiments in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene in association with amplification of the N-myc locus. Our results indicate that Huwe1 balances proliferation and neurogenesis in the developing brain and that this pathway is subverted in malignant brain tumors.

Fassan, Matteo; D'Arca, Domenico; Letko, Juraj; Vecchione, Andrea; Morrione, Andrea; Gardiman, Marina; Mccue, Peter; Wildemore, Bernadette; Rugge, Massimo; Shupp Byrne, Dolores; Gomella, Leonard G; Iozzo, Renato V; Baffa, Raffaele

Mitostatin is a novel putative tumor suppressor gene at chromosome 12q24.1. The 62-kD Mitostatin protein is expressed in most normal human tissues and its immunohistochemical staining is reduced in advanced stages primary breast and bladder tumors. Mitostatin expression negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27.To determine the role of Mitostatin in prostate cancer we transfected LNCaP, PC3, and DU145 prostate cancer cells with an expression vector encoding Mitostatin -V5 fusion protein or expressing the antisense cDNA of the coding sequence of Mitostatin. In addition, we silenced endogenous Mitostatin by siRNA strategies. Mitostatin expression in primary human prostate tumors was analyzed by immunohistochemistry using three tissue arrays (AccuMax Array - A222, A223 and A302) consisted of 293 0.6-mm cores.Our results demonstrate that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells in addition to induce a reduction in cell growth, significantly inhibits migration and invasion. Moreover, Mitostatin inhibits colony formation in soft-agar in PC3 and LNCaP cells and LNCaP tumorigenicity in nude mice. Conversely, targeting endogenous Mitostatin with siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells promotes transformation in both cell lines. Mitostatin immunohistochemical staining was absent in 35.5% of prostate tumor samples and its overall reduction was significantly associated with advanced stage disease.Our findings support the hypothesis that Mitostatin acts as a bona fide tumor suppressor and suggest that further investigations of Mitostatin as a useful clinical marker for diagnosis and prognosis in prostate tumors are warranted.

2008 - Mitostatin, a novel mitochondrial protein, that acts as a tumor suppressor in prostate cancer cells [Abstract in Atti di Convegno]
M., Fassan; D'Arca, Domenico; J., Letko; A., Vecchione; A., Morrione; M., Gardiman; P., Mccue; B., Wildemore; M., Rugge; D., Shupp Byrne; L. G., Gomella; R. V., Iozzo; R., Baffa

Mitostatin is a novel putative tumor suppressor gene at chromosome 12q24.1. The 62-kD Mitostatin protein is expressed in most normal human tissues and its immunohistochemical staining is reduced in advanced stages primary breast and bladder tumors. Mitostatin expression negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27.To determine the role of Mitostatin in prostate cancer we transfected LNCaP, PC3, and DU145 prostate cancer cells with an expression vector encoding Mitostatin -V5 fusion protein or expressing the antisense cDNA of the coding sequence of Mitostatin. In addition, we silenced endogenous Mitostatin by siRNA strategies. Mitostatin expression in primary human prostate tumors was analyzed by immunohistochemistry using three tissue arrays (AccuMax Array - A222, A223 and A302) consisted of 293 0.6-mm cores.Our results demonstrate that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells in addition to induce a reduction in cell growth, significantly inhibits migration and invasion. Moreover, Mitostatin inhibits colony formation in soft-agar in PC3 and LNCaP cells and LNCaP tumorigenicity in nude mice. Conversely, targeting endogenous Mitostatin with siRNA and anti-sense strategies in PC3 and DU145 prostate cancer cells promotes transformation in both cell lines. Mitostatin immunohistochemical staining was absent in 35.5% of prostate tumor samples and its overall reduction was significantly associated with advanced stage disease.Our findings support the hypothesis that Mitostatin acts as a bona fide tumor suppressor and suggest that further investigations of Mitostatin as a useful clinical marker for diagnosis and prognosis in prostate tumors are warranted.

2006 - Clusterin decreases oxidative stress in lung fibroblasts exposed to cigarette smoke [Articolo su rivista]
Carnevali, S; Luppi, Fabrizio; D'Arca, Domenico; Caporali, A; Ruggieri, Mp; Vettori, Mv; Caglieri, A; Astancolle, Serenella; Panico, Francesca; Davalli, Pierpaola; Mutti, A; Fabbri, Leonardo; Corti, Arnaldo

Rationale: Cigarette smoke causes injury to lung fibroblasts, partly by means of oxidative stress, and oxidative stress can lead to various lung diseases, such as chronic obstructive pulmonary disease. Clusterin is a widely distributed protein with many functions, including cellular protection in response to oxidative stress. Objectives: To determine whether clusterin is involved in the defense of the lung against cigarette smoke, we investigated the effects of cigarette smoke extract on clusterin expression and its protective effect, if any, against oxidative stress. Methods: Fibroblasts were coincubated with conditioned medium and cigarette smoke extract, and bronchial biopsy specimens obtained from nonsmokers, smokers, and ex-smokers were analyzed by immunohistochemistry. Measurements and Main Results: At concentrations of 2.5 and 5.0%, cigarette smoke extract induced oxidative stress. It also markedly increased the expression of two clusterin isoforms (60 and 76-80 kD) and the 76-80-kD isoform was secreted in the incubation medium. Coincubation of fibroblasts with conditioned medium significantly decreased the cellular oxidation caused by the cigarette smoke extract. Immunohistochemical analysis of clusterin on bronchial biopsy specimens obtained from smokers and ex-smokers showed localization of clusterin mainly in the submucosa. Conclusions: We conclude that clusterin may have a protective effect against cigarette smoke-induced oxidative stress in lung fibroblasts.

2006 - Prevention of bladder cancer in the FHIT knock-out mouse model with rofecoxib, a COX-2 inhibitor [Abstract in Rivista]
Lenoir, J; D'Arca, Domenico; Gottardo, F; Bragantini, E; Wildemore, B; Shupp Byrne, D; Zanesi, N; Croce, Cm; Gomella, Lg; Baffa, R.

INTRODUCTION and OBJECTIVE: Epidemiological studies have indicated that non-steroidal, anti-inflammatory drugs may have a role in the prevention of human cancers. Molecular based targeting of the COX-2 isoform has lead to the development of COX-2 selective inhibitors such as rofecoxib. Previous studies have shown that administration of COX-2 inhibitors, while decreasing overall morbidity do not decrease the incidence of pre-neoplastic lesions in the murine model. Our laboratory has demonstrated that mice lacking the Fhit tumor suppressor gene are more susceptible than controls to the carcinogenic effect of N-Butyl-N-(-4-hydroxybutyl)-nitrosamine (BBN). Our hypothesis is that rofecoxib will inhibit or slow the development of BBN-induced urinary bladder cancers in mice lacking the Fhit tumor suppressor gene. METHODS: 208 mice consisting of 50 Fhit +/+, 63 Fhit +/ -, and 95 Fhit -/-, were divided into five treatment groups: 1) 0.1% BBN and rofecoxib; 2) 0.01 % BBN and rofecoxib (only Fhit -/- mice); 3) rofecoxib alone; 4) 0.1% BBN alone; and 5) 0.01% BBN alone (only Fhit -/- mice). Rofecoxib was administered at the concentration of 150 ppm in mouse chow. BBN was dissolved in water. At the end of the 15th week-treatment the mice were sacrificed and their bladders collected for histological analysis. The mice fed 150 ppm rofecoxib had blood samples taken twice during the 13 weeks to verify the blood serum concentration of rofecoxib. RESULTS: The mean concentration of rofecoxib in the blood serum was 0.37-µg/mL (± 0.15). The mice that received only rofecoxib and no BBN presented normal bladders, regardless of the genotype. The association between Fhit genotype, rofecoxib treatment, presence of preneoplastic lesions and bladder tumors, was evaluated via the two sided Fisher's exact test ( of 0.05). Comparing the means of each group the pair wise Ps were as follows : Group 1 vs. Group 4 p=1 for Fhit +/+; p=0.687 for Fhit -/-; p=0.107 for Fhit +/-. Comparing Group 2 vs. Group 5 p= 0.114. The difference between the groups was not statistically significant. There was a significant increase in neoplastic lesions in the Fhit+/- and Fhit -/- vs. Fhit +/+ mice after BBN treatment. CONCLUSIONS: Our results confirmed that Fhit null mice, both Fhit +/- and Fhit -/-, are exquisitely sensitive to the carcinogenic effect of BBN and that the Fhit-KO mouse is an extremely valid model to study in vivo bladder cancer tumorigenesis. Our preliminary results suggest also that rofecoxib does not provide a therapeutic defense in our chemically induced mouse bladder cancer model

2005 - Ca2+ depletion induces nuclear clusterin, a novel effector of apoptosis in immortalized human prostate cells [Articolo su rivista]
Ae, Caccamo; Scaltriti, Maurizio; A., Caporali; D'Arca, Domenico; Corti, Arnaldo; D., Corvetta; A., Sala; S., Bettuzzi

Clusterin (CLU) is a secreted heterodimeric glycoprotein thatcan be produced almost ubiquitously in mammalians tissues.1Its gene expression is subjected to complex regulation and canchange enormously according to different stimuli.2 Cloned andidentified as the most potently induced gene in the regressingrat ventral prostate following androgen-ablation,3 CLU wasalmost simultaneously characterized and isolated by differentresearch groups working in widely divergent areas.2 CLU iscoded by a single copy gene, located on chromosome 8.4 Thegene codes for an initial precursor peptide glycosylated andcleaved into two a and b chains of 40 kDa each, held togetherby a unique five disulfide bond motif in the extracellular matureform.1 This secreted form of CLU has been suggested to act asa molecular chaperone following stress-induced injury,5clearing extracellular debris.6 However, it has been reportedthe existence of an inactive, cytoplasmic form of CLUproduced by alternative splicing that is converted by ionizingirradiation to a truncated mature nuclear isoform,7 which bindsthe Ku70/Ku80 complex in cell-free systems8 inhibiting cellgrowth and survival7 probably by a caspase-3-independentmechanism.9 Other alternative CLU isoforms, produced eitherby exon skipping10 or by post-translational modificationsactivated by apoptosis,11,12 were recently described. Thesedifferent isoforms of CLU have been suggested to beantiapoptotic6,13 or proapoptotic.7,10,12,14–16These controversial reports on the role of CLU might berelated to specific proteomic profiles that are produced bydifferent apoptotic stimuli (i.e. the general protein pattern ofCLU and the relative ratio between different CLU isoforms).This might explain why CLU has been involved in a plethora ofpathophysiological processes, including cell–cell and cell–matrix adhesion, cell differentiation, transformation, aging17,18and cancer,19 but its biological role still remains to be clearlyestablished. Reports suggesting that CLU may be a potentialtumor suppressor gene include the finding that CLU suppressesNF-kB activity and the metastatic phenotype ofneuroblastoma cells.20 We have previously reported that CLUoverexpression inhibits cell cycle progression of simian virus40(SV40)-immortalized human prostate PNT2 and PNT1Aepithelial cells.21To further assess the role of CLU in apoptotic processes wehave studied its expression pattern during the regulation ofcalcium homeostasis. Ca2þ is an important regulator ofapoptosis and cell survival.22,23,24 Both pathological increaseof Ca2þ concentration in the cytosol compartment byinophores25 and depletion of intracellular Ca2þ stores maytrigger apoptosis by disrupting intracellular architecture andallowing effectors to gain access to their substrates.24,26,27Activation of apoptotic endonucleases28 eliciting DNA cleavageand chromatin condensation has beenwell documented.29,30,31A tight buffering of intracellular Ca2þ is required for normalgrowth. In fact, apoptosis can be induced by Ca2þ mobilizationfrom intracellular pools,23,24,27 chelation of intracellular Ca2þwith 1,2-bis-(2-aminophenoxy)ethane-N,N,N1,N1-tetra-aceticacid tetra-acetoxymethyl ester (BAPTA-AM)28,29 or removalof extracellular Ca2þ.32 Intracellular Ca2þ deficiency regulatesgene expression22,26 and induces apoptosis through caspasesactivation.23,27,33 It has been shown that extracellular Ca2þdepletion reduces expression of CLU mRNA, but its proteomicprofile has not been studied.26 Thus, we decided to study theeffect of Ca2þ depletion in prostate cells and its effect on CLUprotein expression and localization.We compared the cell proliferation rate of PNT1A cellsgrown under standard culture medium (RPMI plus 10% foetalbovine serum, FBS), keratinocyte serum-free completemedium34 (KSFM, which has subphysiological concentrationsof Ca2þ, 0.1 mM), or KSFM supplemented with 1.8mM Ca2þ(Figure 1a). Under these condition, as previously reported,35KSFM

2005 - Spermidine/spermine N1-acetyltransferase transient over-expression restores sensitivity of resistant human ovarian cancer cells to N1,N12-bis(ethyl)spermine and to cisplatin [Articolo su rivista]
Marverti, Gaetano; Monti, Maria Giuseppina; Pegg, A. E.; Mccloskey, D. E.; Bettuzzi, S; Ligabue, Alessio; Caporali, A; D'Arca, Domenico; Moruzzi, Maria Stella

The limited induction of spermidine/spermine N1-acetyltransferase (SSAT) activity has been implicated as an important determinant of the reduced response, to the spermine analogue N1,N12-bis(ethyl)spermine (BESpm) by the cisplatin (cDDP)-resistant human ovarian carcinoma cell line (C13*). We checked whether under conditions of SSAT over-expression, enzyme induction and cell sensitivity to both BESpm and cDDP were restored to levels comparable with those of more responsive cDDP-sensitive 2008 cells. We transiently transfected the SSAT repressed C13* cells with two expression vectors driving human SSAT over-expression by diverse promoters, and then we analysed their responses in the absence and in the presence of BESpm. SSAT activity was promptly but briefly expressed by transfection with both pOP/SSAT and pCMV-SSAT plasmids. However, only in the presence of BESpm, did SSAT activity reach the highest levels of induction for longer times, with different time-courses for the two vectors, which paralleled the effect on cell growth. Under these conditions, growth sensitivity to BESpm of the less-responsive C13* cells was 25% reverted to cell growth inhibition displayed by 2008 cells. More interestingly, the sensitivity to cDDP cytotoxicity also increased in parallel to SSAT over-expression. BESpm induction of pCMV-SSAT-transfected cells caused a further 20-30% reduction of cell survival induced by cDDP, almost recovering the sensitivity of 2008 cells. The enhanced effectiveness of cDDP was also confirmed by the comet assay, showing an increase in the number and length of tails of damaged DNA. These findings confirm that SSAT over-expression inhibits cell growth and enhances growth sensitivity to BESpm in C13* cells, showing for the first time that restoring high inducibility of SSAT activity subverts the reduced sensitivity to cDDP of SSAT-deficient cells, making them almost indistinguishable from the responsive parental 2008 cells.

2004 - Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin [Articolo su rivista]
Ae, Caccamo; Scaltriti, Maurizio; A., Caporali; D'Arca, Domenico; F., Scorcioni; Astancolle, Serenella; M., Mangiola; S., Bettuzzi

Clusterin, ubiquitously distributed in mammalians, was cloned and identified as the most potently induced gene during rat prostate involution following androgen deprivation. Also found to be involved in many other patho-physiological processes, its biological significance is still controversial, particularly with regard to apoptosis. We previously showed that transient over-expression of clusterin blocked cell cycle progression of simian-virus-40-immortalized human prostate epithelial cell lines PNT1A and PNT2. We show in the present study that the accumulation of an intracellular 45 kDa clusterin isoform was an early event closely associated with death of PNT1A cells caused by cell detachment followed by apoptosis induction (anoikis). Cell morphological changes, decreased proliferation rate and cell cycle arrest at G(0)/G(1)-S-phase checkpoint were all strictly associated with the production and early translocation to the nucleus of a 45 kDa clusterin isoform. Later, nuclear clusterin was found accumulated in detached cells and apoptotic bodies. These results suggest that a 45 kDa isoform of clusterin, when targeted to the nucleus, can decrease cell proliferation and promotes cell -detachment- induced apoptosis, suggesting a possible major role for clusterin as an antiproliferative gene in human prostate epithelial cells.

2004 - Clusterin (SGP-2, ApoJ) expression is downregulated in low- and high-grade human prostate cancer [Articolo su rivista]
Scaltriti, Maurizio; M., Brausi; A., Amorosi; A., Caporali; D'Arca, Domenico; Astancolle, Serenella; Corti, Arnaldo; S., Bettuzzi

Clusterin is overexpressed during tissue and cell involution and downregulated in proliferating cells. Its role in cell survival, cell death and neoplastic transformation remains debated. We studied the expression and distribution of clusterin mRNA and protein in human prostate carcinoma (CaP) specimens of different degrees of malignancy. Fresh CaP specimens were obtained from 25 patients subjected to long-term androgen ablation before surgery. Clusterin expression was studied by Northern and Western analysis, in situ hybridization and immunohistochemistry, in comparison with Gas I and histone H3 mRNA (markers of cell quiescence and S phase of the cell cycle, respectively). Clusterin is downregulated in CaP in comparison with matched benign controls. In low-grade CaP, clusterin colocalized with Gas I to the stromal compartment, and in some glands, the basal lamina was heavily stained. In high-grade CaP clusterin stained the remnants of stromal matrix while histone H3 localized to cancer cells, which were very rarely clusterin positive. High clusterin expression was found in the branches of a nerve infiltrated by tumor. The periglandular clusterin expression found in low-grade CaP could result from induction of quiescence and/or apoptosis of prostatic fibroblasts lining those glands in which tumor invasion is at an initial stage, involving basal lamina. In advanced CaP, the staining of the remnants of the extracellular matrix suggests a role for clusterin in the process of dismantling the stromal organization caused by cancer progression.

2004 - The chemopreventive action of catechins in the TRAMP mouse model of prostate carcinogenesis is accompanied by clusterin over-expression [Articolo su rivista]
A., Caporali; Davalli, Pierpaola; Astancolle, Serenella; D'Arca, Domenico; M., Brausi; S., Bettuzzi; Corti, Arnaldo

Clusterin (CLU) protein is widely distributed in animal tissues and is involved in many different processes, including apoptosis and neoplastic transformation. Green tea catechins (GTC) are known to exert chemopreventive effects in many cancer models, including transgenic adenocarcinoma mouse prostate (TRAMP) mice that spontaneously develop prostate cancer (CaP). We report here that growth of SV40-immortalized human prostate epithelial cells (PNT1A) as well as tumorigenic, poorly differentiated prostate cancer cells (PC-3) was potently inhibited by EGCG, the major green tea catechin, while normal human prostate epithelial cells were not significantly affected. IC50 doses of EGCG for 24 h caused caspase cascade activation and CLU protein accumulation in both cells lines but not in normal cells, in which CLU remained undetectable. While 100% of TRAMP mice developed CaP, only 20% of those receiving 0.3% GTC in drinking water developed the neoplasm. In TRAMP mice, the CLU gene was dramatically down-regulated during onset and progression of CaP. In GTC-treated TRAMP mice in which tumor progression was chemoprevented, CLU mRNA and protein progressively accumulated in the prostate gland. CLU dropped again to undetectable levels in animals in which GTC chemoprevention failed and CaP developed. Up-regulation of histone H3 and down-regulation of growth arrest-specific gene 1 (Gas1) mRNAs in CaP-developing TRAMP mice demonstrated a high proliferation rate in tumors, while the opposite occurred in the glands of GTC chemoprevented animals. Failure of GTC chemoprevention caused induction of both histone H3 and Gas1 and down-regulation of CLU. Immunohistochemistry experiments confirmed CLU down-regulation during CaP onset and progression, and CLU sustained expression in chemoprevented TRAMP mice. A possible role for CLU as a novel tumor-suppressor gene in the prostate is thus suggested.

2003 - Clusterin gene expression is down-regulated in transformed epithelial cells but up-regulated in fibroblasts from prostate cancer [Abstract in Rivista]
Bettuzzi, S; Scaltriti, M; Caporali, A; Brausi, M; Astancolle, S; D'Arca, D; Corti, A.

Clusterin gene expression is down-regulated in transformed epithelial cells but up-regulated in fibroblasts from prostate cancer

2003 - Clusterin is down-regulated during the progression of prostate cancer in the tramp mouse model but up-regulated during chemioprevention by green tea catechins administration [Articolo su rivista]
Davalli, Pierpaola; Astancolle, Serenella; Caporali, A.; D'Arca, Domenico; Corti, Arnaldo; Brasi, M. B.

Clusterin (CLU) protein is widely distributed in animal tissues and is involved in many different processes, including apoptosis and neoplastic transformation. Green tea catechins (GTC) are known to exert chemopreventive effects in many cancer models, including transgenic adenocarcinoma mouse prostate (TRAMP) mice that spontaneously develop prostate cancer (CaP). In TRAMP mice, the CLU gene was dramatically down-regulated during onset and progression of CaP. In GTC-treated TRAMP mice in which tumor progression was chemoprevented, CLU mRNA and protein progressively accumulated in the prostate gland. CLU dropped again to undetectable levels in animals in which GTC chemoprevention failed and CaP developed.

2003 - Nuclear traslocation of a truncated clusterin isoform is associated to induction of anoikis in SV40-immortalized human prostate epithelial cells. [Articolo su rivista]
Caccamo, A. E.; Scaltriti, M.; Caporali, A.; D'Arca, Domenico; Scorcioni, F.; Candiano, G.; Mangiola, M.; Bettuzzi, S.

Clusterin gene expression is potently induced in experimental models in which apoptosis is activated, such as rat prostate involution following castration. Nevertheless, its precise physiological role has not yet been established, and both anti-apoptotic and pro-apoptotic functions have been suggested for this gene. Clusterin expression level depends on cell proliferation state, and we recently showed that its over-expression inhibited cell cycle progression of SV40-immortalized human prostate epithelial cells PNT2 and PNT1a. Here we studied clusterin expression in PNT1a cells subjected to serum-starvation with the aim of defining clusterin early molecular changes following apoptosis induction. Under serum-starvation conditions, decreased growth rate, slow rounding-up of cells, cell detachment, and formation of apoptotic bodies indicative of anoikis (detachment-induced apoptosis) were preceded by significant downregulation of 70 kDa clusterin precursor and upregulation of 45-40 kDa isoforms. On the 8th day of serum-free culturing, only the higher molecular-weight protein-band of about 45 kDa was clearly induced and accumulated in detached cells and apoptotic bodies in which PARP was activated. Anoikis was preceded by induction and transloction of a 45-kDa clusterin isoform to the nucleus. Thus, nuclear targeting of a specific 45-kDa isoform of clusterin appeared to be an early and specific molecular signal triggering anoikis-death. Considering also that clusterin is downregulated during prostate cancer onset and progression, and that its upregulation has inhibited DNA synthesis and cell cycle progression of immortalized human prostate epithelial cells, we suggest that clusterin might be a new anti-oncogene in the prostate.

2003 - Successful prediction of prostate cancer recurrence by gene profiling in combination with clinical data: a 5-year follow-up study. [Articolo su rivista]
S., Bettuzzi; M., Scaltriti; A., Caporali; M., Brausi; D'Arca, Domenico; S., Astancolle; P., Davalli; A., Corti

We show here that gene expression profiling, performed with conventional techniques and focused on a selected group of genes, when used in combination with standard clinical information, provides reliable prognostic prediction of prostate cancer (Cap). We showed previously that changes in the expression of metabolically related genes are involved in CaP progression. We then proceeded to search further for correlations between patients' gene profiling and recurrence with a 5-year follow-up study conducted on the same cohort of patients in which the molecular data were obtained. CaP prognosis was first assessed on the basis of gene expression profiling alone; then the result was compared with the prediction obtained using clinical and pathological information (Gleason score, Tumor-Node-Metastasis staging, prostate volume, or prostate-specific antigen levels at the time of diagnosis). The best result was obtained with a selected, combination of gene profiling and clinical/pathological parameters, which resulted in prediction of recurrence in 95.7% of patients.