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Penetratins 35
In a more recent report, Chikh et al. have shown that the addition of liposomes
stabilizes AntpHD-Cw3 and protects it against degradation. 41 Association with liposomes
is spontaneous regardless of lipid composition; about 50% of the tethered
peptide can also be exchanged from these liposomes. Moreover, this association
with liposomes confers a partial resistance against protease degradation and does
not alter the capacity of the recombinant peptide to be internalized in macrophages
and dendritic cells and to prime T-cell response in vivo through the MHC-I pathway.
2.4.3 VECTORIZATION WITH PENETRATIN PEPTIDE, IN VITRO STUDIES
2.4.3.1 Oligonucleotide and Oligopeptide Delivery
In this section we shall only consider the in vitro delivery of oligopeptides and
oligonucleotides. A specific section is devoted to full-length proteins and to in vivo
approaches. Two main domains of application (cell death and cell cycle and signal
transduction) will be developed.
2.4.3.1.1 Cell Death and Cell Cycle Regulation
Several pathways lead to apoptosis in PC12 cells (nitric oxide (NO), withdrawal of
trophic factors). Incubation of PC12 cells with an antisense oligonucleotide against
superoxyde dismutase 1 (SOD-1) mRNA efficiently decreases SOD-1 activity (50
to 60%) and promotes apoptotic cell death specifically induced by accumulation of
NO. 42 A 100-fold increase in antisense efficiency on both effects was observed after
linkage to penetratin-1 by a disulfide bridge. 36 When linked to penetratin, oligonucleotide
internalization became insensitive to the presence of serum during incubation.
In the same experimental model, incubation of PC12 cells with a penetratinlinked
oligopeptide which mimics the catalytic site of interleukin 1β-converting
enzyme (ICE)-like proteases protected cells from apoptosis. 43 Equivalent effects
were obtained with the permeant competitive ICE inhibitor ZVAD-FMK, but at
much higher concentrations (200-fold). Two different penetratin-linked cargoes
(SOD antisense, ICE oligopeptide) have been used simultaneously without interference.
Penetratin peptides have been used to interfere with the cell cycle, in particular
to antagonize the interaction between cyclins and cyclin-dependent kinases (CDK).
Cell cycle inhibitors, also tumor suppressors, are grouped in two families: the
p21 Cip1/Waf1 family (including p21, p27, and p57) and the p16 family (including
p15 INK4b , p16 INK4b , p18 INK4c , and p19 INK4d ). 44 P16 CDK2/INK4A is deleted or mutated
in a large number of human cancers and its overexpression blocks transition through
the G1/S phase of the cell cycle. P16 binding blocks CDK 4,CDK 6 activity and
pRb phosphorylation. Peptides derived from the p16 binding domain to CDK4,
CDK6 and internalized after penetratin linkage mimicked the p16 inhibitory action
on CDK4 and CDK6 and prevented pRb phosphorylation and cell cycle progression.
45,46
Inhibition of pRb phosphorylation in pancreatic cancer cells devoid of endogenous
p16 activity was also obtained by Fujimoto et al. 47 Indeed, they restored a p16
function using the same peptide as Fahraeus et al. 45,46 Consequently, the growth of
cancer cells was inhibited through arrest in G 1. Another study with p16-derived