title: In vitro and in vivo activity of a novel locked nucleic acid (LNA)-inhibitor-miR-221 against multiple myeloma cells.
abstract: OBJECTIVE
  The miR-221/222 cluster is upregulated in malignant plasma cells from multiple myeloma (MM) patients harboring the t(4;14) translocation. We previously reported that silencing of miR-221/222 by an antisense oligonucleotide induces anti-MM activity and upregulates canonical miR-221/222 targets. The in vivo anti-tumor activity occurred when miR-221/222 inhibitors were delivered directly into MM xenografts. The aim of the present study was to evaluate the anti-MM activity of a novel phosphorothioate modified backbone 13-mer locked nucleic acid (LNA)-Inhibitor-miR-221 (LNA-i-miR-221) specifically designed for systemic delivery.
  
  
  METHODS
  In vitro anti-MM activity of LNA-i-miR-221 was evaluated by cell proliferation and BrdU uptake assays. In vivo studies were performed with non-obese diabetic/severe combined immunodeficient (NOD.SCID) mice bearing t(4;14) MM xenografts, which were intraperitoneally or intravenously treated with naked LNA-i-miR-221. RNA extracts from retrieved tumors were analyzed for miR-221 levels and modulation of canonical targets expression. H&E staining and immunohistochemistry were performed on retrieved tumors and mouse vital organs.
  
  
  RESULTS
  In vitro, LNA-i-miR-221 exerted strong antagonistic activity against miR-221 and induced upregulation of the endogenous target p27Kip1. It had a marked anti-proliferative effect on t(4;14)-translocated MM cells but not on MM cells not carrying the translocation and not overexpressing miR-221. In vivo, systemic treatment with LNA-i-miR-221 triggered significant anti-tumor activity against t(4;14) MM xenografts; it also induced miR-221 downregulation, upregulated p27Kip1 and reduced Ki-67. No behavioral changes or organ-related toxicity were observed in mice as a consequence of treatments.
  
  
  CONCLUSIONS
  LNA-i-miR-221 is a highly stable, effective agent against t(4;14) MM cells, and is suitable for systemic use. These data provide the rationale for the clinical development of LNA-i-miR-221 for the treatment of MM.
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy ; T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Di Martino
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Gullà
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Gallo Cantafio
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Altomare
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Nicola
authors.initials: N
authors.lastname: Amodio
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Leone
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Morelli
authors.affiliation: Pathology Unit, "Giovanni Paolo II" Hospital, Lamezia Terme, Catanzaro, Italy.
authors.forename: Santo Giovanni
authors.initials: SG
authors.lastname: Lio
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Caracciolo
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy ; T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Rossi
authors.affiliation: Exiqon A/S, Vedbaek, Denmark.
authors.forename: Niels M
authors.initials: NM
authors.lastname: Frandsen
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy ; T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy.
authors.forename: Pierosandro
authors.initials: P
authors.lastname: Tagliaferri
authors.affiliation: Department of Experimental and Clinical Medicine, Magna Graecia University and Medical Oncology Unit, Salvatore Venuta University Campus, Catanzaro, Italy ; T. Campanella Cancer Center, Salvatore Venuta University Campus, Catanzaro, Italy ; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, Pennsylvania, United States of America.
authors.forename: Pierfrancesco
authors.initials: P
authors.lastname: Tassone
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000494:pharmacology* / Q000627:therapeutic use; D058990:Molecular Targeted Therapy; D009101:Multiple Myeloma / Q000188:drug therapy* / Q000235:genetics; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D050760:Cyclin-Dependent Kinase Inhibitor p27; D035683:MicroRNAs; C000592539:LNA-i-miR-221; C525709:MIR221, human