Cell penetrating peptides (CPP) are short amino acid sequences with the potential to be used as vectors for delivering macromolecular therapeutics into cells. Five CPPs [R8, FFR8, (RXR)4, TP10 and PFV] were studied in primary human chronic lymphocytic leukaemia (CLL) cells using fluorescence-labelled CPPs. Uptake, sub-cellular localisation and toxicity were studied by confocal microscopy and flow cytometry. Two of the CPPs were selected, based on their cellular uptake and intracellular distribution characteristics, and used as delivery vectors for peptide-based NF-κB inhibitors. Four novel NF-κB inhibitory CPPs directed against p50 and p65 subunits were tested in primary CLL cells. Apoptosis was measured using AnnexinV/PI labelling and a caspase-3 activity assay by flow cytometry. Apoptosis was evident after one hour in cells treated with TP10-p50i and TP10-p65i and the LC50 of TP10-p50i and TP10-p65i was 6 μM and 10 μM respectively at 24 hours. This represents a ten-fold increase in toxicity when compared to the commercially available CPP NF-κB-inhibitors. Western blot analysis of NF-κB subunit translocation revealed NF-κB inhibition in some of the samples treated with TP10-p50i. However, the effects of the peptide varied from sample to sample. Studies using EMSA to measure NF-κB DNA binding revealed similar inconsistencies, even when CLL cells were stimulated with CD40L or CpG. Flow cytometic analysis of cell surface makers in CLL cells demonstrated that TP10-p50i did not alter the expression of CD69, a cell surface molecule regulated by NF-κB, indicating that the variations seen previously by EMSA and western blotting did not result from direct NF- κB inhibition. Although the exact mechanism of action of TP10-p50i was not determined, the cytotoxic effects observed with TP10-p50i are not likely to be related to a modulation of NF-κB activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:633585 |
| Date | January 2014 |
| Creators | Alexandre, Rosaria |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/69352/ |
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