Document Detail


siRNA and pharmacological inhibition of endocytic pathways to characterize the differential role of macropinocytosis and the actin cytoskeleton on cellular uptake of dextran and cationic cell penetrating peptides octaarginine (R8) and HIV-Tat.
MedLine Citation:
PMID:  22465675     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic macromolecules. It is widely accepted that they can enter cells directly across the plasma membrane but also gain access through endocytic pathways that are yet to be fully defined. Here we developed siRNA methods in epithelial cell lines, HeLa and A431, to inhibit endocytic pathways regulated by clathrin heavy chain, flotillin-1, caveolin-1, dynamin-2 and Pak-1. In each case, functional uptake assays were developed to characterize the requirement for these proteins, and the pathways they regulate, in the internalisation of defined endocytic probes and also the CPPs octaarginine and HIV-Tat. Peptide uptake was only inhibited in A431 cells depleted of the macropinocytosis regulator Pak-1, but experimental variables including choice of cell line, pharmacological inhibitor, macropinocytic probe and serum starvation significantly influence our ability to assess and assign this pathway as an important route for CPP uptake. Actin disruption with Cytochalasin D inhibited peptide entry in both cell lines but the effects of this agent on dextran uptake was cell line dependent, reducing uptake in HeLa cells and increasing uptake in A431 cells. This was further supported in experiments inducing actin stabilisation by Jasplakinolide, emphasising that the actin cytoskeleton can both promote and hinder endocytosis. Overall the data identify important aspects regarding the comparative mechanisms of CPP uptake and macropinocytosis, and accentuate the significant methodological challenges of studying this pathway as an endocytic portal and an entry route for drug delivery vectors.
Authors:
M Al Soraj; L He; K Peynshaert; J Cousaert; D Vercauteren; K Braeckmans; S C De Smedt; A T Jones
Related Documents :
2535745 - Isolation and characterization of hela cell lines blocked at different steps in the pol...
8021595 - Human papillomavirus (hpv) type 18 e7 protein is a short-lived steroid-inducible phosph...
3802405 - Dna alkylation repair and the induction of cell death and sister chromatid exchange in ...
184325 - Studies on the interaction between coxsackievirus a9 and hela cells. i. plaque-forming ...
214525 - The intracellular organization of actin and tubulin in cultured c-1300 mouse neuroblast...
3014785 - Bronchiolo-alveolar carcinoma with nuclear tubular inclusions. case report and electron...
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-3-24
Journal Detail:
Title:  Journal of controlled release : official journal of the Controlled Release Society     Volume:  -     ISSN:  1873-4995     ISO Abbreviation:  -     Publication Date:  2012 Mar 
Date Detail:
Created Date:  2012-4-2     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8607908     Medline TA:  J Control Release     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier B.V.
Affiliation:
Cardiff School of Pharmacy and Pharmaceutical Sciences, Redwood Building, Cardiff University, Cardiff, Wales, CF10 3NB, United Kingdom.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) am...
Next Document:  Two-layered injectable self-assembling peptide scaffold hydrogels for long-term sustained release of...