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.