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36 Cell-Penetrating Peptides: Processes and Applications
peptides also demonstrated that p16 inhibits α vβ 3 integrin-mediated cell spreading
on vitronectin. 48
Penetratin vectors have also been directed against downstream targets of pRb.
Members of E2F transcription factors family are positive effectors of the cell cycle
and downstream targets of pRb. Unphosphorylated pRb is able to sequester E2F,
converting it from a transcriptional activator to a trancriptional repressor. Consequently,
disruption of the pRb/E2F complex can lead to oncogenesis. Among proteins
regulating E2F, cyclin A/CDK2 complexes neutralize E2F DNA-binding. Chen et al.
have designed peptides corresponding to the cyclin A/CDK2-binding site on E2F. 49
Coupled to penetratin, these peptides were able to kill transformed cells in which
E2F was already deregulated by pRb inactivation. It is noteworthy that the same
team has recently identified about 12 peptides able to antagonize in vitro E2F-1 and
cyclin A activity. 50 The potential anticancer effect of cell-permeant versions of the
peptides (coupled to penetratin) is presently tested on tumor cells.
The tumor suppressor SSeCKS (sarc-suppressed C kinase substrate) also blocks
the G1/S transition through an interaction with cyclin D1. Internalization with
penetratin of the SSeCKS sequence responsible for this interaction demonstrated
that SSeCKS anchors cyclin D1 in the cytoplasm, thus giving a molecular basis to
G1/S arrest induced by SSeCKS. 51
Another study with penetratin-coupled antagonizing peptides was conducted to
evaluate the role of the translation initiation factor eIF4E in cellular transformation. 52
Over-expression of eIF4E is oncogenic and found in a number of human cancers;
eIF4E activity can be blocked by eIF4E binding proteins (4E-BPs). Peptides after
eIF4E-binding motifs found in 4E-Bps and introduced into cells designed bound
eIF4E and induced massive apoptosis, thus revealing an involvement of this protein
in the control of cell death not related to its known role in mRNA translation.
The tumor suppressor protein p53 is frequently targeted by oncoproteins in
transformed cells. This is typically the case for adenovirus early region 1B protein
(E1B), which binds and inactivates p53 through its sequestration in the cytoplasm.
Penetratin-coupled peptides corresponding to the p53-binding site of E1B disrupted
p53-E1B interaction allowing p53 nuclear addressing and restoring cell cycle arrest. 53
Chemotherapy or radiation therapy activates p53-mediated processes in sensitive
tissues. Thus p53 may be an appropriate target to reduce damages made to normal
tissues during cancer therapies. Using a selection of genetic suppressor elements
(GSE) from a library of randomly fragmented p53 cDNA, Mittelman and Gudkov
have isolated p53-derived antagonizing peptides. 54 One of them, GSE56, was fused
to penetratin and the fusion protein was shown to attenuate p53-mediated transactivation
and reduce anticancer treatment side effects. 55
Transcription factor c-Myc is a positive effector of cell proliferation; the misregulation
of its activity can lead to cell transformation. To counteract its activity,
Giorello et al. have developed penetratin-coupled inhibitory peptides. 56 These peptides
were efficiently internalized in Myc-transformed human breast cancer cells and
suppressed their transformation characteristics. Efficiency was improved by synthesizing
their retro-inverso forms; these new peptides were more potent (5 to 10 times)
and more stable (30 to 35 times) than their natural counterparts. 57