siRNA-cell-penetrating peptides complexes as a combinatorial therapy against chronic myeloid leukemia using BV173 cell line as model

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• 作者：Joã ; o Miguel Freire^a ; ¹ ; Inê ; s Rego de Figueiredo^a ; ¹ ; Javier Valle^b ; Ana Salomé ; Veiga^a ; David Andreu^b ; Francisco J. Enguita^a ; ^{fenguita@medicina.ulisboa.pt} ; Miguel A.R.B. Castanho^a ; ^{macastanho@medicina.ulisboa.pt}
• 关键词：Chronic myeloid leukemia ; Bcr-Abl ; siRNA ; Dengue virus capsid protein ; Cell-penetrating peptide ; Bioportide ; Microarray
• 刊名：Journal of Controlled Release
• 出版年：2017
• 出版时间：10 January 2017
• 年：2017
• 卷：245
• 期：Complete
• 页码：127-136
• 全文大小：1797 K
• 卷排序：245

文摘

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 (48 h to 168 h) 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 120 h. 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.