title: siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model.
abstract: Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by a single gene mutation, a reciprocal translocation that originates the Bcr-Abl gene with constitutive tyrosine kinase activity. As a monogenic disease, it is an optimum target for RNA silencing therapy. We developed a siRNA-based therapeutic approach in which the siRNA is delivered by pepM or pepR, two cell-penetrating peptides (CPPs) derived from the dengue virus capsid protein. These peptides have a dual role: siRNA delivery into cells and direct action as bioportides, i.e. intracellularly bioactive CPPs, targetting cancer-related signaling processes. Both pepM and pepR penetrate the positive Bcr-Abl+Cell Line (BV173). Five in silico designed anti-Bcr-Abl siRNA were selected for in vitro analysis after thorough screening. The Bcr-Abl downregulation kinetics (48h to 168h) was followed by quantitative PCR. The bioportide action of the peptide vectors was evaluated by genome-wide microarray analysis and further validated by testing BV173 cell cycle and cell proliferation monitoring different genes involved in housekeeping/cell stress (RPL13A, HPRT1), cell proliferation (ki67), cell apoptosis (Caspase 3 and Caspase 9) and cell cycle steps (CDK2, CCDN2, CDKN1A). Assays with a commercial transfection agent were carried out for comparison purposes. Maximal Bcr-Abl gene knockdown was observed for one of the siRNA when delivered by pepM at 120h. Both pepM and pepR showed downregulation effects on proliferative CML-related signaling pathways having direct impact on BV173 cell cycle and proliferation, thus reinforcing the siRNA effect by acting as anticancer molecules. With this work we show the therapeutic potential of a CPP shuttle that combines intrinsic anticancer properties with the ability to deliver functional siRNA into CML cell models. By such combination, the pepM-siRNA conjugates lowered Bcr-Abl gene expression levels more extensively than conventional siRNA delivery technologies and perturbed leukemogenic cell homeostasis, hence revealing their potential as novel alternative scaffolds for CML therapy.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.
authors.forename: João Miguel
authors.initials: JM
authors.lastname: Freire
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.
authors.forename: Inês
authors.initials: I
authors.lastname: Rego de Figueiredo
authors.affiliation: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, E-08003 Barcelona, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Valle
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal.
authors.forename: Ana Salomé
authors.initials: AS
authors.lastname: Veiga
authors.affiliation: Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, E-08003 Barcelona, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Andreu
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. Electronic address: fenguita@medicina.ulisboa.pt.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Enguita
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. Electronic address: macastanho@medicina.ulisboa.pt.
authors.forename: Miguel A R B
authors.initials: MA
authors.lastname: Castanho
mesh_terms: D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000008:administration & dosage*; D003131:Combined Modality Therapy; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D055785:Gene Knockdown Techniques; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000628:therapy*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage*
chemical_list: C581634:BCR-ABL1 fusion protein, human; D057846:Cell-Penetrating Peptides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016044:Fusion Proteins, bcr-abl
keywords: Bcr-Abl; Bioportide; Cell-penetrating peptide; Chronic myeloid leukemia; Dengue virus capsid protein; Microarray; siRNA