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38 Cell-Penetrating Peptides: Processes and Applications
of two inhibitory PNAs on the catalytic activity of human telomerase. Telomerase
function in normal cells is to maintain chromosome integrity, but several reports
raise the question of telomerase implication in cell immortality and cancer. From a
therapeutic point of view, it is of interest to block telomerase activity in transformed
cells. To add telomeric repeats to the end of chromosomes, telomerase copies a small
RNA sequence complementary to DNA. The strategy used here was to internalize
two PNAs, an 11mer and a 13mer designed to cover the 5′-proximal region of the
RNA and thus to inhibit human telomerase activity. The 13mer PNA coupled to
penetratin through a disulfide-bound down-regulated telomerase activity in intact
live cells, but the doubling time of the treated cells was only slightly decreased and
telomeres were not shortened. 65
A more successful use of PNAs linked to penetratin-1 was the antagonizing of
the biological activity of galanin, a peptide neuromediator involved in pain transduction
(see the section devoted to in vivo use of penetratin-derived peptides).
2.4.3.3 Delivery of Entire Proteins
AntpHD and penetratin are able to deliver large molecular weight protein into live
cells. Among them are homeoproteins like Engrailed, HoxA-5, HoxB-4, HoxC-8,
Pax 6, HoxC-4, Emx2, and Otx2 68-70 (unpublished results).
A good example of the successful delivery of entire proteins fused to penetratin-1
is the study of the replicative senescence phenomenon. Genetically programmed
senescence is induced when cells approach their limit of population doubling. Interestingly,
p16 INK4b RNA and protein levels rise as cells enter senescence, but there
was no evidence that p16 accumulation drives senescence. Using penetratin-1 to
internalize p16 INK4b into human diploid fibroblasts, Kato et al. have shown that
p16 INK4b , but not a functionally compromised variant, provokes G1 arrest. 71 This
arrest is certainly due to inhibition of pRb phosphorylation and the arrested cells
display a senescent phenotype, strongly suggesting that p16 INK4b is implicated in
replicative senescence.
2.4.3.4 Chemical Drug Delivery
Doxorubicin, an anthracyclin, is a commonly used anticancer drug. Anthracyclins
are among the most active antitumor compounds. Not only do they, as intercalating
agents, induce DNA damage but they also bind cell surface acidic phospholipids,
like cardiolipids, and induce necrosis and apoptosis via the sphingomyelin–ceramide
pathway. However, anthracyclin treatments often lead to multidrug-resistant tumors
through an enhanced expression of a multidrug-resistance gene (mdr). This gene
encodes a 170-kD protein called the P-glycoprotein (Pgp) that actively transports
several cytotoxic agents out of the cells. 72
To overcome this problem, Mazel et al. have covalently linked doxorubicin to
the penetratin N terminus and tested this conjugate in human erythroleukemia K562
cells. 73 Doxo-penetratin was about 20-fold more effective than doxorubicin alone to
kill K562 doxorubicin-resistant cells. This suggests that the mode of internalization
of penetratin, very likely through inverted micelles, allows the conjugate to escape
Pgp activity.