Filippo Minutolo

Publications (65) View all

• Conference Proceeding: Targeting metabolic reprogramming in hypoxic models of pancreatic cancer: Preclinical emergence of novel LDH inhibitors and molecular mechanisms underlying their synergistic interaction with gemcitabine

  Amir Avan, Rocco Sciarrillo, Carlotta Granchi, Niccola Funel, Richard J. Honeywell, Marco Macchia, Filippo Minutolo, Godefridus J. Peters, Elisa Giovannetti
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  ABSTRACT: Background/Aim: Pancreatic ductal adenocarcinoma (PDAC) is an extremely scirrhous and hypoxic tumor. Intra-tumoral hypoxia is a driving force in pancreatic cancer growth and metastasis, implicated as a major cause of drug resistance to conventional chemotherapy and radiotherapy. Another major consequence effect of intra-tumoral hypoxia is cell metabolic switch, which is required for tumor proliferation under low oxygen and nutrient. Since the muscle-isoform of lactate-dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic-glycolysis, ensuring a sufficient energy supply even in hypoxic environment, we investigated the molecular mechanisms underlying the pharmacological interaction of a series of novel LDH-A inhibitors, PI-FLY21 and PI-FLY124, with gemcitabine in PDAC. Methods: In vitro studies were performed in 7 pancreatic cancer cell lines and 7 primary cultures (PP), characterized by differential expression of LDH-A and HIF-1α. Cytotoxicity was evaluated with sulforhodamine-B assay, whereas LDH-A modulation was investigated by RT-PCR, Western-blot and enzymatic-activity-assay, using also specific siRNA. Cell cycle perturbation and apoptosis were studied with flow-cytometry, while pharmacological interaction with gemcitabine was investigated with the combination index (CI) method. Total cytosolic adenosine and phosphorylated deoxynucleosides were evaluated using Liquid chromatography-mass spectrometry (LC-MS/MS). Furthermore, we examined if LDH-A inhibition modulated invasiveness, expression of cancer-stem-cell (CSC) markers and EMT phenotype, in adherent-cells and spheroids. All these experiments were performed in both normoxic and hypoxic (1% O2) conditions. Results: The mRNA expression of LDH-A and HIF-1α was detected by quantitative-RT-PCR in all the cells, showing that LDH-A had a large range of mRNA levels (from 27 to 338 arbitrary unit (a.u.) in Capan-1 and PP109 cells, respectively), and correlated with HIF-1α expression. LDH-A expression significantly increased in PANC-1, PP78 and PP109 under hypoxic condition, as detected by qRT-PCR, and Western-blotting, while it was specifically reduced by siRNA-anti-LDH-A. The antiproliferative activity of gemcitabine was significantly reduced in hypoxic conditions, most probably through reduction of the active phosphorylated gemcitabine metabolites, as detected by LC-MS/MS. Conversely, the novel LDH-A-inhibitors proved to be particularly effective under hypoxia, with IC50s ranging from 0.1 to 0.8 μM, and synergistically enhanced the antiproliferative activity of gemcitabine in PANC-1 and PP78 (CI values <0.8). PI-FLY21 and PI-FLY124 significantly decreased LDH activity in PANC-1 and PP78, also in comparison with siRNA-anti-LDH-A. In addition, these compounds caused a slight cell cycle arrest in the G1-S boundary, while the drug combination reduced the percentages of cells in the G2/M phase (e.g. in PANC-1 from 21 to 10% under hypoxia, p<0.05) and significantly enhanced apoptosis induction. PI-FLY compounds reduced cell migration, the volume of PDAC spheroids growing in CSC-selective-medium under hypoxia, and the expression of the CSC markers CD133 and CD44. Conclusions: These data provide evidence that LDH-A is a viable target in pancreatic cancer, and novel LDH-A-inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool for optimizing chemotherapy in hypoxic pancreatic tumors.
  American Association for Cancer Research; Pancreatic Cancer: Progress and Challenges, Lake Tahoe, NV, USA; 06/2012
• Article: Study of apoptosis induction and deoxycytidine kinase/cytidine deaminase modulation in the synergistic interaction of a novel ceramide analog and gemcitabine in pancreatic cancer cells.

  E Giovannetti, L G Leon, S Bertini, M Macchia, F Minutolo, N Funel, C Alecci, F Giancola, R Danesi, G J Peters
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  ABSTRACT: This study investigated the interaction between the novel ceramide analog AL6 and gemcitabine in MIA PaCa-2 and PANC-1 pancreatic cancer cell lines, harboring different polymorphic variants of the gemcitabine catabolism enzyme cytidine deaminase (CDA). AL6 dose-dependently inhibited cell growth, induced apoptosis and synergistically enhanced the cytotoxic activity of gemcitabine. Moreover, it triggered apoptosis, which was significantly enhanced by the combination, and increased the ratio between gene expression of the activating enzyme deoxycytidine kinase (dCK) and CDA, potentially favoring gemcitabine activity. In conclusion, AL6 displays synergistic cytotoxic activity, enhances apoptosis, and favorably modulates enzymes involved in gemcitabine metabolism, supporting future investigation of this combination in pancreatic cancer.
  Nucleosides Nucleotides &amp Nucleic Acids 06/2010; 29(4-6):419-26. · 0.90 Impact Factor
• Article: Inhibitors of lactate dehydrogenase isoforms and their therapeutic potentials.

  C Granchi, S Bertini, M Macchia, F Minutolo
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  ABSTRACT: In many different species, lactate dehydrogenase (LDH) constitutes a major checkpoint of anaerobic glycolysis, by catalyzing the reduction of pyruvate into lactate. This enzyme has recently received a great deal of attention since it may constitute a valid therapeutic target for diseases so different as malaria and cancer. In fact, the isoform expressed by Plasmodium falciparum (pfLDH) is a key enzyme for energy generation of malarial parasites. These species mostly depend on anaerobic glycolysis for energy production, since they lack a citric acid cycle for ATP formation. Therefore, inhibitors of pfLDH would potentially cause mortality of P. falciparum and, to this purpose, several small organic molecules have been recently designed and developed with the aim of blocking this new potential antimalarial chemotherapeutic target. Moreover, most invasive tumour phenotypes show a metabolic switch (Warburg effect) from oxidative phosphorylation to an increased anaerobic glycolysis, by promoting an upregulation of the human isoform-5 of lactate dehydrogenase (hLDH-5 or LDH-A), which is normally present in muscles and in the liver. Hence, inhibition of hLDH-5 may constitute an efficient way to interfere with tumour growth and invasiveness. This review provides an overview of the LDH inhibitors that have been developed up to now, an analysis of their possible isoform-selectivity, and their therapeutic potentials.
  Current Medicinal Chemistry 01/2010; 17(7):672-97. · 4.86 Impact Factor
• Article: Salicylaldoxime derivatives as new leads for the development of carbonic anhydrase inhibitors.

  Tiziano Tuccinardi, Simone Bertini, Carlotta Granchi, Gabriella Ortore, Marco Macchia, Filippo Minutolo, Adriano Martinelli, Claudiu T Supuran
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  ABSTRACT: New compounds containing a novel zinc binding group (salicylaldoxime system) were identified as effective inhibitors of carbonic anhydrases (CAs). This structural motif seems to bind the catalytic zinc ion of CAs, revealing itself as a new valid alternative to the sulfonamide group. Computational procedures were used to investigate the binding mode of this class of compounds, within the active site of CAII. This study suggests that the salicylaldoxime moiety binds the zinc ion through the oxime oxygen atom that also forms an H-bond with T199. The results herein obtained will allow the development of new CA-inhibitors bearing the salicylaldoxime moiety.
  Bioorganic & medicinal chemistry 09/2012; · 2.82 Impact Factor
• Article: Curaxins: a new family of non-genotoxic multitargeted anticancer agents.

  Valeria Di Bussolo, Filippo Minutolo
  ChemMedChem 12/2011; 6(12):2133-6. · 3.15 Impact Factor