title: Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines.
abstract: BACKGROUND
  Cell penetrating peptides (CPPs) have the ability to translocate through cell membranes with high efficiency and therefore can introduce biological agents with pharmaceutical properties into the cell. Transportan (TP) and its shorter analog transportan 10 (TP10) are among the best studied CPPs, however, their effects on viability of and cargo introduction into colorectal cancer (CRC) cells have yet not been investigated. The aim of our study was to evaluate the cytotoxic effects of TP and TP10 on representative CRC lines and the efficiency of protein (streptavidin) and siRNA cargo delivery by TP-biotinylated derivatives (TP-biot).
  
  
  METHODS
  HT29 (early stage CRC model) and HCT116 (metastatic CRC model) cell lines were incubated with TP, TP10, TP-biot1, TP-biot13 and TP10-biot1. The effects of studied CPPs on cell viability and cell cycle were assessed by MTT and annexin V assays. The uptake of streptavidin-FITC complex into cells was determined by flow cytometry and fluorescence microscopy, with the inhibition of cellular vesicle trafficking by brefeldin A. The efficiency of siRNA for SASH1 gene delivery was measured by quantitative PCR (qPCR).
  
  
  RESULTS
  Since up to 10 µM concentrations of each CPP showed no significant cytotoxic effect, the concentrations of 0.5-5 µM were used for further analyses. Within this concentration range none of the studied CPPs affected cell viability and cell cycle. The efficient and endocytosis-independent introduction of streptavidin-FITC complex into cells was observed for TP10-biot1 and TP-biot1 with the cytoplasmic location of the fluorescent cargo; decreased SASH1 mRNA level was noticed with the use of siRNA and analyzed CPPs.
  
  
  CONCLUSIONS
  We conclude that TP, TP10 and their biotinylated derivatives can be used as efficient delivery vehicles of small and large cargoes into CRC cells.
authors.affiliation: Department of Histology, Medical University of Gdansk, Gdansk, Poland. pwierzb@gumed.edu.pl.
authors.forename: Piotr M
authors.initials: PM
authors.lastname: Wierzbicki
authors.forename: Marzena
authors.initials: M
authors.lastname: Kogut-Wierzbicka
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Ruczynski
authors.forename: Kamila
authors.initials: K
authors.lastname: Siedlecka-Kroplewska
authors.forename: Lucyna
authors.initials: L
authors.lastname: Kaszubowska
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Rybarczyk
authors.forename: Magdalena
authors.initials: M
authors.lastname: Alenowicz
authors.forename: Piotr
authors.initials: P
authors.lastname: Rekowski
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Kmiec
mesh_terms: D001681:Biological Assay; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000633:toxicity*; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000378:metabolism; D019004:Galanin / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D018014:Gene Transfer Techniques / Q000592:standards*; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007928:Lethal Dose 50; D034741:RNA, Small Interfering / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014862:Wasp Venoms / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*
chemical_list: D057846:Cell-Penetrating Peptides; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C474743:SASH1 protein, human; D025521:Tumor Suppressor Proteins; D014862:Wasp Venoms; C110068:transportan; C586078:transportan-10; D019004:Galanin