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).