Cell penetrating Capacity
The cellular uptake mechanism of cell penetrating peptides (CPPs) is an
essential piece of optimization of appropriate strategies for in
vivo therapeutic applications. CPPs have been described to enter cells
either by the classical endocytic pathway or through an
energy-independent mechanism referred to as transduction
(Heitz, Morris, & Divita, 2009). In
spite of the requirements needed for each mechanism, different
internalization patterns are also observed inside the cells. Endocytosis
produces a punctuated pattern consequence of peptide internalization
into endocytic vesicles (Ter-Avetisyan et
al., 2009). Meanwhile, transduction by direct membrane penetration
results in fully loaded cells displaying a homogeneous distribution
throughout the cytoplasm and nucleoplasm
(Tunnemann et al., 2008).
The precursor peptides CIGB-550 and L-2 have the capacity to penetrate
the cell, so we studied the internalization kinetic of CIGB-552 after
modifications in its primary structure. Both endocytosis and
transduction are involved in CIGB-552 internalization in the three cell
lines evaluated, MCF-7 (human breast adenocarcinoma), NCI-H460 (human
large cell lung cancer carcinoma) and HT-29 (human colorectal
adenocarcinoma). We analyzed the internalization of fluorescent labeled
CIGB-552 in these cell lines by confocal microscopy. The intracellular
uptake of the peptide was observed in the cytoplasm after 10 minutes and
shows mainly punctuate staining close to the nuclei
(Fernandez Masso et al., 2013;
Vallespi et al., 2014). Expression of two
different proteins Rab5A and Fibrillarin were analyzed. Rab5A is a small
GTPase, which has been found to be expressed in early endosomes
(Bucci et al., 1992), whereas Fibrillarin,
which possesses methyltransferase activity, is a commonly used marker of
active nucleoli (Rodriguez-Corona, Sobol,
Rodriguez-Zapata, Hozak, & Castano, 2015). Therefore, the use of these
two proteins, allowed us to define CIGB-552 localization with respect to
both endosomes and nucleoli (Figure 3). Co-localization was found among
CIGB-552 and these two markers, indicating that this peptide is
effectively able to enter cells through different mechanisms
(Astrada et al., 2016).
However, CIGB-552 incorporation efficiency and contribution of each
mechanism is cell-line dependent. Astrada et al. showed that
cell-penetrating capacity varies among the three cell lines studied.
H460 displayed the highest internalization levels, while MCF-7 presented
the lowest internalization capacity. Moreover, CIGB-552 used both
endocytosis and transduction as internalization mechanisms, although the
contributions of each mechanism varied among the studied cell lines
(Astrada, Fernandez Masso, Vallespi, &
Bollati-Fogolin, 2018). Altogether, our results suggest that NCI-H460
sensitivity could be explained by its high internalization capacity of
CIGB-552, by using endocytosis as a preferred mechanism, which in turn
could facilitate the interaction between the peptide and target proteins
resident in the endosomal compartment. Endocytosis constitutes the most
effective way of transporting the CIGB-552 peptide inside cells, thus
promoting higher sensitivity towards peptide cytotoxic effects.