Biological processes involved in the antitumor effect of CIGB-552
Since CIGB-552 is a synthetic peptide that possess modified aminoacids (D aminoacids), which cannot be translated inside cells, we decided to use L-2 peptide instead, for gene expression studies. L-2 represents the primary sequence that has been modified in order to generate a more stable peptide, the CIGB-552. Subtractive hybridization (SSH) analysis on laryngeal tumor Hep-2 cells showed that L-2 peptide treatment impacts the expression of genes related to biological processes and pathways involved in cancer such as: DNA repair, mitosis, and angiogenesis (Vallespi et al., 2010). Furthermore, a yeast two-hybrid study of L-2 and a pull-down technique identified COMMD1 protein as a target of CIGB-552. The results of two hybrid and pull-down experiments indicate that the interaction between peptides and COMMD1 is specific and the strength of this interaction may be relevant for the antitumor effect of the peptides (Fernandez Masso et al., 2013). This interaction was recently confirmed in live cells measuring the in situ COMMD1 expression level after treatment with CIGB-552 (Astrada et al., 2018). We found that in MCF-7 and NCI-H460 cells the peptide accumulated COMMD1 in the cytoplasm. In addition, we identified the interaction between CIGB-552 and COMMD1 as a co-localization of both proteins at early endosome using confocal microscopy approach. This result was corroborated by ELISA assay (Astrada et al., 2016). Such finding is very interesting to the mechanism of CIGB-552, because define a specific intracellular target for the peptide and the initial cellular compartment were this interaction occurs.
To identify other proteins that interact with CIGB-552, two chemical proteomic approaches were then conducted using PBS-soluble proteins derived from Hep-2 cells (Rodriguez-Ulloa et al., 2015). A total of 161 proteins constitute the identified potential target profile of CIGB-552. Biological processes related to carbohydrate metabolism, protein modification, and cell cycle, are significantly represented on this dataset. Interestingly, such biological processes are also represented in the transcriptomic profile regulated by L-2 peptide in Hep-2 tumor cells. Functional subnetworks which are perturbed by CIGB-552 include anti-apoptosis and negative regulation of cell cycle; extracellular structure organization and response to hypoxia. Positive regulation of NF-𝜅B transcription factor activity is disrupted by the CIGB-552 target profile essentially at two network nodes: RelA and TRAF6 (Rodriguez-Ulloa et al., 2015). On the other hand, we study the proteins modulated by treatment with CIGB-552 in HT-29 cells using subcellular protein and peptide fractionation by chemical proteomic approach. In particular, we explored the nuclear proteome of HT-29 cells at five hours of treatment with CIGB-552, identifying 68 differentially modulated proteins, 49 of which localize to the nucleus (Nunez de Villavicencio-Diaz et al., 2015). The differentially modulated proteins were analyzed following a system biology approach. Results pointed to a modulation of apoptosis, oxidative stress, NF-κB activation, inflammatory signaling and cell adhesion and motility. These results demonstrated that even in different cell lines (HT-29, Hep-2) the CIGB-552 antitumor effect is exerted by modulating similar biological pathways.
According to proteomic and genomic data, oxidative stress and apoptosis are the main biological processes modulated by CIGB-552 in tumor cells (Figure 3). This makes sense, considering the crucial role of COMMD1 in both cellular functions. COMMD1 impairs the antioxidant superoxide dismutase 1 (SOD1) activity by reducing the expression levels of active SOD1 homodimers, late in the posttranslational maturation process of this enzyme (Vonk, Wijmenga, Berger, van de Sluis, & Klomp, 2010). Furthermore, COMMD1 is also involved in apoptotic cell death, mainly due to negative regulation of NF-κB (Thoms et al., 2010). Consequently, the treatment with CIGB-552 must be able to induce oxidative damage and apoptosis in cancer cells. In fact, this was demonstrated in human lung cancer cells in presence of the peptide, which increased the levels of protein and lipid peroxidation as a sign of oxidative stress damage (Fernandez Masso et al., 2013). In addition, the downregulation of COMMD1 in these cells abrogated the negative effect of CIGB-552 on SOD1 activity, demonstrating the contribution of COMMD1 to this process. In the same way, the peptide activated the apoptotic pathway in human lung cancer cells trough the modulation of the Bax/Bcl-2 protein ratio and the cleavage of caspase 3 and PARP (Fernandez Masso et al., 2013).