open access

Vol 52, No 4 (2014)
Original paper
Submitted: 2014-11-27
Accepted: 2014-12-09
Published online: 2014-12-12
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Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Piotr M. Wierzbicki, Marzena Kogut-Wierzbicka, Jaroslaw Ruczynski, Kamila Siedlecka-Kroplewska, Lucyna Kaszubowska, Agnieszka Rybarczyk, Magdalena Alenowicz, Piotr Rekowski, Zbigniew Kmiec
DOI: 10.5603/FHC.a2014.0035
·
Pubmed: 25511292
·
Folia Histochem Cytobiol 2014;52(4):270-280.

open access

Vol 52, No 4 (2014)
ORIGINAL PAPERS
Submitted: 2014-11-27
Accepted: 2014-12-09
Published online: 2014-12-12

Abstract

Introduction. 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).

Material and 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.

Abstract

Introduction. 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).

Material and 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.
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Keywords

CPP; transportan; TP10; HT29; HCT116; SASH1; siRNA; cell cycle; apoptosis

About this article
Title

Protein and siRNA delivery by transportan and transportan 10 into colorectal cancer cell lines

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 52, No 4 (2014)

Article type

Original paper

Pages

270-280

Published online

2014-12-12

DOI

10.5603/FHC.a2014.0035

Pubmed

25511292

Bibliographic record

Folia Histochem Cytobiol 2014;52(4):270-280.

Keywords

CPP
transportan
TP10
HT29
HCT116
SASH1
siRNA
cell cycle
apoptosis

Authors

Piotr M. Wierzbicki
Marzena Kogut-Wierzbicka
Jaroslaw Ruczynski
Kamila Siedlecka-Kroplewska
Lucyna Kaszubowska
Agnieszka Rybarczyk
Magdalena Alenowicz
Piotr Rekowski
Zbigniew Kmiec

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