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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Jun 21;177(16):3625–3634. doi: 10.1111/bph.15132

Antitumour peptide based on a protein derived from the horseshoe crab: CIGB‐552 a promising candidate for cancer therapy

Brizaida Oliva Arguelles 1, Mario Riera‐Romo 2,✉, Maribel Guerra Vallespi 1
PMCID: PMC7393197  PMID: 32436254

Abstract

Peptide‐based cancer therapy has been of great interest due to the unique advantages of peptides, such as their low MW, the ability to specifically target tumour cells, easily available and low toxicity in normal tissues. Therefore, identifying and synthesizing novel peptides could provide a promising option for cancer patients. The antitumour second generation peptide, CIGB‐552 has been developed as a candidate for cancer therapy. Proteomic and genomic studies have identified the intracellular protein COMMD1 as the specific target of CIGB‐552. This peptide penetrates to the inside tumour cells to induce the proteasomal degradation of RelA, causing the termination of NF‐κB signalling. The antitumour activity of CIGB‐552 has been validated in vitro in different human cancer cell lines, as well as in vivo in syngeneic and xenograft tumour mouse models and in dogs with different types of cancers. The aim of this review is to present and discuss the experimental data obtained on the action of CIGB‐552, including its mechanism of action and its therapeutic potential in human chronic diseases. This peptide is already in phase I clinical trials as antineoplastic drug and has also possible application for other inflammatory and metabolic conditions.

Abbreviation

MDR

multidrug resistance

COMMD1

Copper Metabolism Mur 1 Domain containing protein 1

CIGB

Centre for Genetic Engineering and Biotechnology

LALF

Limulus anti‐LPS factor

PARP

Poly ADP Ribose Polymerase

CPPs

cell‐penetrating peptides

SSH

subtractive hybridization

TRAF6

TNF receptor associated factor 6

Bcl‐2

B‐cell lymphoma 2

Bax

Bcl‐2 associated protein X

BRCA1

breast cancer type 1 susceptibility protein

HIF‐1

hypoxia inducible factor 1

NSAID

non‐steroidal anti‐inflammatory drugs

CFTR

cystic fibrosis transmembrane conductance regulator

1. INTRODUCTION

Cancer is a multifactorial and complex disease, which presents different clinical outcomes depending on the affected tissue and the genetic background of the patient (Luo, Solimini, & Elledge, 2009). Tumour cells express a variety of molecular targets involved in cancer progression and exhibit a deregulation in normal growth, proliferation and survival, among other vital functions (Hanahan & Weinberg, 2011). Because of its complexity, the huge variety of molecular targets are involved and the high variability in the therapeutic response, cancer has become a major health problem worldwide and is the one the diseases that is the most difficult to treat in the 21st century.

Commonly, traditional chemotherapeutical drugs target tumour cells by disrupting essential cell components, such as DNA, RNA or proteins (Huang et al., 2014). However, chemotherapy is inefficient and is highly toxic because it does not specifically target tumour cells, thus causing many side/unwanted effects (Amit & Hochberg, 2010). Additionally, multidrug resistance (MDR) is one of the main reasons by which chemotherapy fails to cure patients (Huang et al., 2014). Because of these limitations therapeutic strategies based on peptides are receiving increased attention.

There are several advantages of peptides, such as their small size, easy synthesis and modification, tumour penetrating ability and having a good biocompatibility (Wu et al., 2014). A growing number of studies have indicated that peptides may be beneficial for drug discovery and development. Peptides offer minimal immunogenicity, excellent tissue penetrability, low‐cost manufacturability and they are also relatively easy to modify to enhance in vivo stability and biological activity, such properties which make them ideal candidates for cancer treatment (Yavari, Mahjub, Saidijam, Raigani, & Soleimani, 2018).

Peptides have also been demonstrated to play a role in cancer therapy, including in early diagnosis, as prognostic predictors and directly in the treatment of cancer patients. Unlike other therapies, peptides seem to be more effective due to their specificity. Recently, some peptide‐based treatments against cancer, such as peptide vaccines have attracted increased attention. Anticancer activity of different peptides is attributed to a variety of mechanisms that restrict tumour growth (Borghouts, Kunz, & Groner, 2005).

CIGB‐552 is a synthetic peptide “first‐in‐class” that increases the level of the intracellular protein COMMD1 (Copper Metabolism Mur 1 Domain containing protein 1) and inhibits the anti‐apoptotic genes regulated by NF‐κB. CIGB‐552 leads to the selective degradation of RelA, a NF‐κB subunit, and induces apoptosis in multiple types of tumour cells in the absence of toxicity to normal cells. In addition, CIGB‐552 inhibits the transcriptional activity of NF‐κB induced by TNF‐α and IL‐1β in human colon cancer cells. The mechanism of action of CIGB‐552 involves a new‐targeted anticancer therapy to regulate oncogenic‐inflammatory activity of NF‐κB in cancer cells and as such having a specificity of action. This novel peptide has a potential application against solid tumours and inflammation‐associated cancers, including colorectal, breast and lung cancer, lymphomas and others.

2. ORIGIN OF CIGB‐552 PEPTIDE

In the Centre for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba, Vallespi, Colas, Garay, Reyes, and Arana (2004) obtained an antimicrobial peptide against septic shock designed from the region 31–52 of the Limulus anti‐LPS factor (LALF), a protein derived from the Horseshoe crab Limulus polyphemus. The peptide CIGB‐550 (LALF31–52) demonstrated cell‐penetrating capacity due to its net positive charge and amphipathic structure by alternating positive/hydrophobic basic residues. It showed the capacity to bind, neutralize bacterial LPS and block the inflammatory response mediated by LPS. Vallespi et al. (2003) demonstrated anti‐inflammatory and immunomodulatory properties for CIGB‐550 but also showed an antiviral effect for this peptide, mediated by IFN‐γ and IFN‐α. It has been reported that antimicrobial peptides exhibit a broad spectrum of cytotoxic activity against cancer cells (Hoskin & Ramamoorthy, 2008) and CIGB‐550 is not an exception. The ability to inhibit biological functions associated with heparin, such as anticoagulation, angiogenesis and tumour cell proliferation was attributed to this peptide, although there are no supporting experimental data exists for this. In order to study the structure–function relationship of LALF31–52 and its connection with the biological properties attributed to the peptide, a synthetic library was generated by alanine scanning (Vallespi et al., 2010). The resulting peptides were evaluated by LPS‐binding ability, antitumour effect, penetration capacity in live cells and immunostimulatory activity. These results lead to the development of L‐2, a peptide optimized for anticancer activity. L‐2 has lost its LPS‐binding capacity, however it is a powerful cytotoxic agent against murine and human tumour cell lines (Vallespi et al., 2010). One of the weaknesses of peptides as drugs is their short t 1/2 and fast elimination (Fosgerau & Hoffmann, 2015). By modifications of the primary structure of L‐2, we have developed a second‐generation peptide as anticancer drug for evaluation in clinical trials named CIGB‐552 (Figure 1). CIGB‐552 maintains the cell‐penetrating capacity and shows a higher antitumour effect similar to CIGB‐550 and L‐2.

FIGURE 1.

FIGURE 1

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Process of generation of antitumor peptide CIGB‐552. The “second‐generation” peptide CIGB‐552 was obtained by chemical modifications of L‐2, an antitumor peptide developed from an alanine library from CIGB‐550, the LPS‐binding region of the LALF protein. The image illustrates the incorporation of D‐amino acids and N‐terminal modifications in the peptide L‐2 to obtain CIGB‐552. The properties acquired by each peptide are showed inside colour boxes

3. CIGB‐552 HAS ANTITUMOUR EFFECTS IN VITRO AND IN VIVO

The studies of anticancer activity of CIGB‐552 were performed using in vitro culture systems as well as clinically relevant in vivo models, as is shown in Figure 2. We began with the demonstration of a selective antiproliferative activity in several cancer cell lines. We demonstrated that CIGB‐552 has powerful antiproliferative and cytotoxic effects compared to its precursor peptide L‐2. In this assays, we evaluated the effect of the peptide on non‐tumour cell lines were the IC50 was higher with respect to tumour cells. These differences suggest a selectivity of this peptide for malignant cells (Fernandez Masso et al., 2013). The IC50 values observed in tumour cell lines suggest a tumour type‐dependent pattern of sensitivity. Recently, we demonstrated that this difference is caused by the internalization mechanism for the peptide (Astrada et al., 2016).

FIGURE 2.

FIGURE 2

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Preclinical summary of CIGB‐552 as novel antitumor peptide. CIGB‐552 has been evaluated in vitro in cytotoxicity assays, protein–protein interaction (PPI) studies, where COMMD1 was identified as its main molecular target, intracellular effects including the modulation of NF‐κB activity, proteomic studies, and fluorescence microscopy/cell penetration assays. In vivo, the pharmacokinetic profile of the peptide was obtained in BALB/c mice, and its antitumor properties were validated in various animal models including CT‐26 syngeneic and HT‐29 xenograft mice models and pet dogs with spontaneous cancer

Since cell death often occurs as a consequence of a cell cycle blockade, we checked whether the cytotoxicity of L‐2 and CIGB‐552 was mediated by an alteration in the cell division cycle. Analyses by flow cytometry of tumour cells treated with L‐2 showed the absence of the G2/M peak and the accumulation of cells in S phase (Vallespi et al., 2010). In the same way, CIGB‐552 affects cell cycle progression in the lung cancer cell line NCI‐H460 increasing the SubG0 phase, which is clearly indicative of cell death induction. This effect was compared between wild type and COMMD1‐knockdown NCI‐H460 cells, corroborating the main role of COMMD1 accumulation in the antitumour mechanism of the peptide (Fernandez Masso et al., 2013). We also evaluated the effect of CIGB‐552 in combination with cisplatin on cell cycle and apoptosis‐related proteins in NCI‐H460 cells. We demonstrated that the combination of CIGB‐552 and cisplatin induces cell cycle arrest and cell death, potentiating the activation of caspase 3, caspase 8 and caspase 9. The activation of caspase 3 and the cleavage of the poly ADP ribose polymerase (PARP) is promoted by both pathways, but mostly and firstly by the intrinsic pathway. This result confirms that the combination of CIGB‐552 with cisplatin significantly increases apoptosis through the activation of the caspases pathway in the NCI‐H460 cell line, confirming the synergic interaction of both drugs (unpublished data). This is in accordance with recent reports of novel anticancer peptides, which are also selectively cytotoxic, targeting intracellular proteins, inducing apoptosis or causing necrotic cell death by membrane disruption (Chen et al., 2020; Emelianova et al., 2018; Huang et al., 2020). That is the case of ChMAP‐28, an antimicrobial peptide isolated from leukocytes of the goat Capra hircus, which like CIGB‐552 has a natural origin with minor modifications and is a potent cytotoxic compound to a variety of tumour cells, mainly leukaemia, but inducing necrotic cell death. Interestingly, ChMAP‐28 is also selective to cancer cells and is non‐haemolytic, properties that confer it therapeutic potential (Emelianova et al., 2018). In the same way, p28, a peptide derived from the copper‐containing protein Azurin from Pseudomonas aeruginosa, displays high and selective anticancer activity inducing apoptosis and cell cycle arrest, targeting the p53 and the receptor TK pathways. This peptide has demonstrated tolerability and antineoplastic activity in two phase I clinical trials in adult and paediatric patients (Huang et al., 2020).

The in vivo therapy of solid tumours was a subsequent phase in the proof of concept of CIGB‐552 as an anticancer drug (Figure 2). In our institute, we developed a murine model of tumour CT‐26. This model was used to study the administration route and the dosage regimen for the treatment. Firstly, we demonstrated that the s.c. administration of CIGB‐552 led to a significant reduction of tumour growth compared to the group treated with saline solution, this route of administration being more effective that the i.p. After 2 weeks of treatment, the tumour volume had decreased and this action was observed until the end of the study (Vallespi et al., 2014). Therefore, this in vivo study demonstrated that s.c. administration of CIGB‐552 was able to inhibit 50% of the tumour growth when the peptide was given once a week over a 2 week period. In collaboration with the EPO‐Berlin Institute, we further evaluated the effect of four s.c. injections of CIGB‐552 in a xenograft model of human colon cancer HT‐29. In this mouse model, we monitored changes in body weight as an indicator of tolerability. As in the syngeneic CT‐26 model, the peptide showed a significant reduction of tumour volume compared with the control group. For all mice in the CIGB‐552 treated group their body weight remained stable over the period of the study, indicating safety and tolerability of CIGB‐552. This study also included a group treated with oxaliplatin, a standard drug for the therapy of colorectal cancer. Oxaliplatin induced a significant inhibition of tumour growth, but this effect was accompanied by a severe loss of body weight and two deaths, probably due related to its high toxicity (Vallespi et al., 2014).

The mechanisms involved in the reduction of tumour growth in response to CIGB‐552 were evaluated in both murine syngeneic and human xenograft models. In vivo, the groups treated with CIGB‐552 exhibit induction of apoptosis in the tumour and a decrease in microvessels density (Vallespi et al., 2014). The resistance to programmed cell death and the sustained angiogenesis are included in a group of properties called hallmarks of cancer, a set of functional adaptions acquired during the multistep development of human tumours. Thus, the antitumour mechanism of the peptide CIGB‐552 is associated with proteins and signalling pathways that trigger apoptosis inhibiting the proliferation and inducing the reduction of blood vessels formation.

Monolayers of tumour cells cultivated in vitro and mouse xenografts implanted with these cells have been the standard toolkit for cancer biologists for decades. However, the need for better and more clinically predictive models of human cancer is required. Spontaneous cancer in pet dogs is considered an attractive model to study the efficacy of drug candidates for cancer therapy (Rowell, McCarthy, & Alvarez, 2011). Cancers in affective animals such as pet dogs are characterized by histological features similar to human cancer,tumour growth over long periods, inter‐individual and intra‐tumour heterogeneity, the development of recurrent or resistant diseases and metastasis to relevant distal sites (Hawai et al., 2013). Based on that, we evaluated the safety, tolerability and antineoplastic effect of CIGB‐552 in tumour‐presenting dogs. In this study, we kept the same dosage regimen and administration route used in mouse models. The treatment with CIGB‐552 had no negative effects on total leukocytes number and did not affect the levels of liver transaminases or haemoglobin during the study (Vallespi et al., 2017). Interestingt, the white blood count returned to normal levels after completing the study, which sugges that treatment with the peptide does not induce leukopenia, a common problem for chemotherapy regimens. The dogs enrolled into the study, which presented different types of spontaneous cancer including cutaneous and splenic lymphoma, p.o. carcinoma and malignant melanoma, showed a tumour regression (>50%), demonstrating a stabilization of the disease and the potential of CIGB‐552 to be useful in a variety of malignancies (Vallespi et al., 2017).

Altogether, these preclinical results validate the peptide CIGB‐552 as a potent antiproliferative and antitumour drug, which mechanism is related to the inhibition of various hallmarks of cancer. In addition, the s.c. administration of CIGB‐552 is safe and effective as an anticancer treatment, at least at preclinical level.

4. CIGB‐552 EXHIBITS AN INTRACELLULAR MECHANISM BASED ON NF‐κB INHIBITION

4.1. Cell‐penetrating capacity

The cellular uptake mechanism of cell‐penetrating peptides (CPPs) is an essential piece for the optimization of in vivo therapeutic applications. Cell‐penetrating peptides have been shown 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 of these mechanism, different internalization patterns are also observed inside the cells. Endocytosis produces a punctuated pattern which is the 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. 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 analysed the internalization of fluorescent labelled CIGB‐552 in these cell lines by confocal microscopy. The intracellular uptake of the peptide was observed in the cytoplasm after 10 min and mainly shows punctuate staining close to the nuclei (Fernandez Masso et al., 2013; Vallespi et al., 2014). Expression of two different proteins Rab5A and fibrillarin were analysed. 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 and 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. Co‐localization was found between CIGB‐552 and these two markers, indicating that this peptide is effectively able to enter cells through these two different mechanisms (Astrada et al., 2016).

However, CIGB‐552 incorporation efficiency and contribution of each of each of these mechanisms 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 cell lines studied (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, through endocytosis as the 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 its cytotoxic effects. This mechanism has been observed for other novel antitumour peptides. For example, the hybrid peptide kla‐TAT, evaluated in the human lung cancer cell line A549, enters the cell by clatrin‐mediated endocytosis or by membrane disruption, inducing apoptosis and compromising mitochondrial membrane integrity (Chen et al., 2020).

4.2. Biological processes involved in the antitumour effect of CIGB‐552

Since CIGB‐552 is a synthetic peptide that possesses modified amino acids (D‐amino acids), 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, CIGB‐552. Subtractive hybridization (SSH) analysis on laryngeal tumour 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 antitumour 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 caused the accumulation COMMD1 in the cytoplasm. In addition, using confocal microscopy approach we identified the interaction between CIGB‐552 and COMMD1 as both proteins were found to be co‐localized at early endosome. This result was corroborated by ELISA (Astrada et al., 2016). Such a finding is very interesting in understanding the mechanism of action of CIGB‐552, because we can identify a specific intracellular target for the peptide and also the initial cellular compartment were this interaction occurs.

To identify other proteins that might 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 were identified as potential targets for CIGB‐552. They were from biological pathways mainly related to carbohydrate metabolism, protein modification and the cell cycle. Interestingly, such biological pathways are also represented in the transcriptomic profile regulated by L‐2 peptide in Hep‐2 tumour cells. Functional subnetworks, which are affected by CIGB‐552 include anti‐apoptosis and negative regulation of cell cycle, extracellular structure organization and responses 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 (TNF receptor associated factor 6) (Rodriguez‐Ulloa et al., 2015). On the other hand, we also studied the proteins modulated by treatment with CIGB‐552 in HT‐29 cells using subcellular protein and peptide fractionation using a chemical proteomic approach. In particular, we explored the nuclear proteome of HT‐29 cells at 5 h after treatment with CIGB‐552, identifying 68 differentially modulated proteins, 49 of which were localize to the nucleus (Nunez de Villavicencio‐Diaz et al., 2015). These differentially modulated proteins were analysed using a system biological approach. Results pointed to the modulation of apoptosis, oxidative stress, NF‐κB activation, inflammatory signalling and cell adhesion and motility. These results demonstrated that even in different cell lines (HT‐29 and Hep‐2), the CIGB‐552 exerts its antitumour effect 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 tumour cells (Figure 3). This makes sense, considering the crucial role of COMMD1 in both these cellular functions. COMMD1 impairs the antioxidant SOD1 activity by reducing the expression levels of active SOD1 homodimers, late in post‐translational 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 the 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 down‐regulation of COMMD1 in these cells abolished the negative effect of CIGB‐552 on SOD1 activity, demonstrating the contribution of COMMD1 to this process. In the same way, CIGB‐552 activated the apoptotic pathway in human lung cancer cells decreasing B‐cell lymphoma 2 (Bcl‐2) and increasing Bcl‐2 associated protein X (Bax) protein levels and inducing the cleavage of Caspase 3 and PARP (Fernandez Masso et al., 2013).

FIGURE 3.

FIGURE 3

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Model of molecular mechanism of CIGB‐552. CIGB‐552 penetrates the cell by transduction and/or endocytosis, it interacts with COMMD1 stabilizing it, preventing its ubiquitination and proteasomal degradation, and inducing its nuclear translocation. Once in the nucleus, COMMD1 inhibits the NF‐κB transcriptional activity leading eventually to a process of apoptosis. In the cytosol, the complex COMMD1–CIGB‐552 activates the apoptotic pathway through oxidative stress, the modulation of Bax/Bcl‐2 ratio, and the activation of Caspase 8 and Caspase 9. Red arrows indicate inhibition, and green arrows indicate activation. PS, phosphatidylserine; C, cytochrome C

4.3. Copper Metabolism Mur 1 Domain containing protein 1 (COMMD1) and NF‐κB activity

The COMMD protein family is highly conserved among multicellular eukaryotic organisms. COMMD1 is the best characterized member of the family and is conserved among vertebrates (Burstein et al., 2005). This protein represents a pleiotropic factor involved in the regulation of many cellular and physiological processes that include oxidative stress, protein aggregation, protein trafficking, NF‐κB‐mediated transcription and oncogenesis (Bartuzi et al., 2016; Phillips‐Krawczak et al., 2015; Vonk et al., 2010). The potential of COMMD1 in cancer therapy is becoming a focus of attention as this protein offers a way to modulate crucial events in oncogenesis and even produces ROS that contribute to the apoptosis of tumour cells, in a safe and specific manner (Riera‐Romo, 2018).

The proteomics and genomics approach indicated that COMMD1 represents a molecular target of the peptide CIGB‐552. The development of the interactome of CIGB‐552, integrating the data obtained by genomics and proteomics studies, suggests a direct connection with the NF‐κB pathway, in a COMMD1‐dependent manner. According to these findings, the cellular expression of COMMD1 in whole‐cell lysates of human cancer cells of different histological origin was determined using Western blot analysis. These experiments revealed an increase in the levels of endogenous COMMD1 after 5 h of treatment with the peptide (Fernandez Masso et al., 2013). This effect on COMMD1 was not accompanied by significant changes in mRNA expression of the protein, suggesting a post‐transcriptional effect of CIGB‐552 on COMMD1 levels (Fernandez Masso et al., 2013). It is known that nuclear localization of the protein COMMD1 accelerates the ubiquitination and degradation of the RelA subunit of NF‐κB and decreases the activation of antiapoptotic genes (Maine, Mao, Komarck, & Burstein, 2007; Thoms et al., 2010). In this sense, we demonstrated that in the response to CIGB‐552, COMMD1 localizes into the nucleus, a fact that is related with increasing amounts of ubiquitinated RelA and apoptosis induction. This effect was abrogated decreasing the levels of COMMD1 by interference RNA gene silencing method (knockdown), indicating the functional role of this protein in the antitumour activity of CIGB‐552 (Fernandez Masso et al., 2013).

As a downstream event, the transcriptional activity of NF‐κB was evaluated in the reporter cell line HT‐29‐NF‐κB‐hrGFP E5, in which the NFKB gene is linked to the GFP gene. The peptide CIGB‐552 inhibited the NF‐κB activity in these cells in the presence or absence of proinflammatory cytokines such as TNF‐α and IL‐1β. Besides, CIGB‐552 reduces the levels of IL‐8 in cell culture supernatants confirming the inhibition of NF‐κB activity (Nunez de Villavicencio‐Diaz et al., 2015).

5. ROLE OF THE PRIMARY STRUCTURE IN THE ANTITUMOUR MECHANISM OF CIGB‐552

Biostability of peptides in blood and serum is an important issue for development of these molecules as clinical drugs. Besides, the establishment of the main sequence necessary for the observed antitumour activity of CIGB‐552 this was an important question that our group needed to address. In vitro degradation of peptides in serum and plasma is considered the primary methodology for studying the degradation pattern of peptides (Werle & Bernkop‐Schnurcch, 2006). For that reason, our group examined the in vitro metabolic stability of CIGB‐552 in serum during 120 min. A typical serine‐proteases degradation pattern was suggested for this peptide sequence, with potential cleavage sites at alanine 2, lysine 1 and lysine 16, resulting in a series of metabolites (Vallespi et al., 2014). The main metabolites of CIGB‐552, termed as peptides 1a, 3 and 5, showed variations in size and MW (Table 1), but no great differences regarding hydrophobicity, hydrophilicity and isoelectric point. However, the analysis of the cytotoxic effect, cell‐penetrating capacity, antitumour mechanism, apoptosis induction and interaction with its intracellular target, COMMD1, demonstrated a loss of antitumour capacity for the metabolites compared to CIGB‐552 (Figure 4). These findings suggest the importance of C‐terminal amino acidic residues (Lys18, Phe19, and Trp20) in the antitumour activity of CIGB‐552 (Astrada et al., 2016). The role of these residues in the peptide–membrane interaction was analysed by molecular dynamic simulation (Astrada et al., 2016). CIGB‐552 and the 17 amino acid metabolites (peptide 5) have a conserved structural motif. Two tryptophan residues and a tyrosine create a hydrophobic cluster that brings together the C‐terminal carboxylate moieties and one arginine residue, forming a stable salt bridge interaction that leads to a looped conformation. Indeed, tryptophan–tryptophan and arginine–C‐terminal carboxylate distances in both systems display very stable interactions.

TABLE 1.

Description of CIGB‐552 and its main metabolites

Name Sequence MW
CIGB‐552 HARIKPTFRRLKWKYKGKFW 2,647.51
5 IKPTFRRLKWKYKGKFW 2,283.06
1a IKPTFRRLKWKYKG 1,821.45
3 IKPTFRRLKW 1,344.81
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FIGURE 4.

FIGURE 4

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CIGB‐552 and its main metabolites. The analysis of CIGB‐552 stability in serum and its characterization by ESI‐MS (electrospray ionization MS) identified the main metabolites of CIGB‐552 (peptides 1a, 3, and 5). The evaluation of in vitro properties of these metabolites demonstrated that amino acids of C‐terminal ending are essential for antitumor activity. The black arrows indicate the potential cleavage sites in the sequence of CIGB‐552. The MW of each metabolite is represented in the figure, and their properties are summarized in colour boxes. The colour scale indicates the nature of the effect

Altogether, these results demonstrate that the primary structure of CIGB‐552 is considered the minimum active sequence needed to produce a potent antitumour effect in cell and animal models. In this sense, the protection of CIGB‐552 from proteolytic degradation is one of the most important challenges that face this peptide in its development as an anticancer drug. However, it has demonstrated a good stability, rapid bio‐distribution and tumour bio‐accumulation in vivo after s.c. injection, an administration route that may help to avoid or minimize serum protolithic degradation. This was corroborated at clinical level in the phase I study of CIGB‐552 in humans, where the s.c. injection of the peptide resulted in a good therapeutic response with no adverse reactions (unpublished results). The development of CIGB‐552 as an s.c. anticancer drug is an attractive idea for a less invasive therapeutic intervention with a better patient management.

6. CONCLUSIONS AND FUTURE PERSPECTIVES

CIGB‐552 is a “first‐in class” antitumour peptide developed by a series of modifications from a structural motif present in the antimicrobial protein LALF. This peptide in capable of penetrate inside the cell to interact with different target proteins. COMMD1 has been identified as one of them. This pleotropic protein regulates the stability and activity of several intracellular, endosomal and transmembrane proteins, regulating a variety of biological processes. It has been described to participate in the termination of NF‐κB signalling and in the negative regulation of SOD1, among other molecular events. CIGB‐552 acting through COMMD1, is able of induce oxidative stress and trigger apoptosis in cancer cells being effective, safe and tolerable in different animal models of cancer including mice and dogs. All these results, point out CIGB‐552 as a promising candidate to cancer therapy. In fact, this peptide drug is already in clinical trials and has concluded recently the phase I study, which demonstrated an overall positive effect in patients with no toxicity or side effects.

In spite of that, the potential of CIGB‐552 is still under evaluation. Recent studies have revealed new findings about COMMD1 that could expand the therapeutic applications of this peptide (Table 2). Fedoseienko et al. (2016) demonstrated that nuclear COMMD1 decreased protein expression of the DNA repair gene breast cancer type 1 susceptibility protein (BRAC1) and the apoptosis inhibitor BCL2 in ovarian cells A‐2780, conferring sensitivity to cisplatin. In addition, they demonstrated through tissue micro‐arrays that nuclear expression of COMMD1 is associated with an improved response to chemotherapy. This information supports the idea of the use of CIGB‐552 in combined therapy with other chemotherapeutic drugs, to improve antitumour effect and reduce toxicity. In the same way, Yeh et al. examined the association of COMMD1 with stemness. The down‐regulation of COMMD1 amplifies stemness‐associated property of cancer cells (Yeh et al., 2016), suggesting that CIGB‐552 could target cancer steam cells, which are involved in tumour re‐emergence, metastasis and multiple drug resistance. Furthermore, COMMD1 has been reported as a negative regulator of the hypoxia inducible factor 1 (HIF‐1), which is implicated in tumour angiogenesis and metastasis (van de Sluis et al., 2009; van de Sluis et al., 2010). Thus, this could be another cancer‐associated process modulated by CIGB‐552. Interestingly, in accordance with this hypothesis, the peptide reduced microvessel density in vivo in our HT‐29 xenograft mouse model.

TABLE 2.

Prospective therapeutic applications of CIGB‐552

Disease/condition Potential use Modulated pathway Target cell/tissue
Cancer Antineoplastic NF‐κB, apoptosis Solid tumours
Metastasis Antiangiogenic, antimetastatic HIF‐1, stemness Cancer stem cells Metastatic cells
Colitis, IBD, CD Anti‐inflammatory NF‐κB Gastrointestinal tract
Atherosclerosis Anti‐lipidemic LDL recycling Hepatocytes
Cystic fibrosis CFTR modulator CFTR activity/expression Epithelial cells
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Abbreviations: CD, Cronh's disease; IBD, inflammatory bowel disease. HIF‐1 hypoxia inducible factor 1 CFTR, cystic fibrosis transmembrane conductance regulator.

CIGB‐552 differentially modulates genes and proteins involved in key signalling pathways of cancer cells such as NF‐κB, hypoxia, apoptosis and inflammation. The recovery of COMMD1 levels by CIGB‐552 negatively regulates the NF‐κB activity in the absence or presence of inflammatory cytokines. Therefore, the molecular mechanism described for CIGB‐552 validate its pharmacological use in chronic diseases such as human cancer, by inhibiting inflammation, tumour angiogenesis and stemness process or even in certain inflammatory conditions such as, colitis and colitis‐related cancer, inflammatory bowel disease and Crohn's disease among others. Furthermore, CIGB‐552 could be used in combination regimens with non‐steroidal anti‐inflammatory drugs (NSAID), antineoplastic agents, and other target therapies. Substantial evidence indicates that aspirin and related NSAIDs have potential as chemo‐preventive/therapeutic agents (Drew, Cao, & Chan, 2016; Patrignani & Patrono, 2016). However, these drugs cannot be universally recommended for prevention purposes due to their potential side‐effect profiles. Stark et al. have demonstrated that aspirin inhibits the transcriptional activity of NF‐κB through stabilization of COMMD1 in colon cancer cells (O'Hara et al., 2014; Thoms et al., 2010). The intervention of CIGB‐552 in combination with aspirin could mediate an improved effect and could reduce the clinical dose of both drugs in cancer therapy. The constitutive activation of NF‐κB is one of the resistance mechanisms to antineoplastic treatments. The inhibition of NF‐κB activity by CIGB‐552 could be an attractive strategy to sensitize tumours to antineoplastic agents, reducing the doses of drugs and providing a better life quality for patients.

Increasing evidence is pointing out COMMD1 as an attractive molecular target involved in a variety of human disorders. CIGB‐552 as a biotechnological product that accumulates this protein and potentiates its intracellular effect represents an interesting candidate not only for cancer therapy but also for other metabolic diseases in which COMMD1 is implicated. For example, the research group of Fedoseienko et al. has also studied the role of COMMD1 in atherosclerosis. The localization of COMMD1 in the endosome is critical for LDL receptor recycling and cholesterol levels in mice (Bartuzi et al., 2016). Likewise, Drevillon et al. (2011) demonstrate that COMMD1 modulates the activity and surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, an activity that is down‐regulated in cystic fibrosis patients. In line with this, CIGB‐552 could be a therapeutical or therapeutic alternative to the treatment of atherosclerosis and cystic fibrosis, two human disorders that do not possess a current effective treatment. The molecular evidence and experimental data discussed throughout this review demonstrate that the peptide CIGB‐552 is an interesting, effective and versatile drug that could be developed as a novel anti‐inflammatory and anticancer drug, which could also be effective for different human chronic conditions.

6.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Christopoulos et al., 2019; Alexander, Cidlowski et al., 2019).

CONFLICT OF INTEREST

The authors declare no conflict of interests.

Oliva Arguelles B, Riera‐Romo M, Guerra Vallespi M. Antitumour peptide based on a protein derived from the horseshoe crab: CIGB‐552 a promising candidate for cancer therapy. Br J Pharmacol. 2020;177:3625–3634. 10.1111/bph.15132

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