Bioactive cationic peptides as potential agents for breast cancer treatment

Abstract Breast cancer continues to affect millions of women worldwide, and the number of new cases dramatically increases every year. The physiological causes behind the disease are still not fully understood. One in every 100 cases can occur in men, and although the frequency is lower than among women, men tend to have a worse prognosis of the disease. Various therapeutic alternatives to combat the disease are available. These depend on the type and progress of the disease, and include chemotherapy, radiotherapy, surgery, and cancer immunotherapy. However, there are several well-reported side effects of these treatments that have a significant impact on life quality, and patients either relapse or are refractory to treatment. This makes it necessary to develop new therapeutic strategies. One promising initiative are bioactive peptides, which have emerged in recent years as a family of compounds with an enormous number of clinical applications due to their broad spectrum of activity. They are widely distributed in several organisms as part of their immune system. The antitumoral activity of these peptides lies in a nonspecific mechanism of action associated with their interaction with cancer cell membranes, inducing, through several routes, bilayer destabilization and cell death. This review provides an overview of the literature on the evaluation of cationic peptides as potential agents against breast cancer under different study phases. First, physicochemical characteristics such as the primary structure and charge are presented. Secondly, information about dosage, the experimental model used, and the mechanism of action proposed for the peptides are discussed.


Introduction: the need for new therapeutic options for breast cancer
Cancer is defined as a broad group of diseases characterized by uncontrolled and abnormal cell growth, which frequently invades adjacent organs or tissues and spreads into the body. The latter feature is known as metastasis and is a principal cause of death from this malignancy. Cancer is the first or second leading cause of death before the age of 70 years in 112 of 183 countries [1]. Breast cancer is the world's most commonly diagnosed malignancy, according to statistics released by the International Agency for Research on Cancer (IARC) in December 2020 [2]. It can occur in women of any age, including cases with no identifiable cancer risk factors. According to statistics from the World Health Organization (WHO), more than 2.3 million women were diagnosed with breast cancer in 2020, while there were 685000 deaths globally [3]. Male breast cancer is considered a rare disease, accounting for approx. 1% of all breast cancer cases, but, like female breast cancer, its incidence has increased over the past 25 years [4]. The breast comprises glands, including the breast lobes and breast ducts, whose function is to produce milk during the lactation period. The lobes are connected by the mammary ducts, which carry milk to the nipple. The glands and ducts of the breast are embedded in adipose tissue and connective tissue, which, together with lymphatic tissue, form the breast. The pectoral muscle, located between the ribs and the breast, acts as a retaining wall. Finally, the skin covers and protects the entire breast structure [5]. express and define in the treatment are the human epidermal growth factor receptor 2 (HER2-positive), hormone receptor-positive breast cancer, BRCA gene mutations, and triple-negative breast cancer (TNBC) [36]. Trastuzumab, pertuzumab, and margetuximab are monoclonal antibodies that bind to the HER2 protein on cancer cells, preventing the cells from growing. Therapy with HER2-targeted treatments combined with chemotherapy, has led to an improvement in the clinical outcomes of patients [37]. Targeted therapy for hormone receptor-positive breast cancer includes palbociclib, ribociclib, and abemaciclib, which block CDK4 and CDK6. In hormone receptor-positive breast cancer cells, blocking these proteins helps stop proliferation of the cells; which can delay the progression of cancer [38]. Although different types of medication are available, they have a different mechanism of action to chemotherapy drugs and frequently have side effects. Common targets in breast cancer include olaparib, talazoparib and PARP inhibitors, which have been studied in women with breast or ovarian cancers associated with deleterious germline mutations in BRCA1 and BRCA2. In terms of median progression-free survival, they have proven efficacy [39]. The cancer cells in TNBC lack estrogen and progesterone receptors and overproduce the HER2 protein. Some drugs, such as pembrolizumab and iniparib, are currently in clinical trials with promising effects in TNBC, but serious adverse events have been reported [40]. Although therapies directed at these receptors are administered to decrease their activity, there are limitations related to adverse effects. For example, in the case of endocrine therapy, significant side effects are menopause and arthropathy; while less common but potentially fatal side effects are are pulmonary embolism, endometrial cancer, and osteoporotic fracture [41]. The main limitations of monoclonal antibodies are their size and high molecular weight, which are related to their tissue penetration properties. This hinders their internalization into solid tumors [34]. Furthermore, nonspecific uptake of these molecules has been reported in parts of the endothelial reticulum system such as the liver, spleen, and bone marrow [42,43].
The complexity of cancer and the burden it represents for the health system necessitates the intervention of multiple areas of science focused on the search for new breast cancer treatment strategies. Current therapeutic options involve long treatments with numerous side effects that affect the quality of life of patients. Therefore, the search for new antiproliferative agents continues to be a priority. These compounds must be capable of eliminating cancer cells and be selective enough not to cause damage to the healthy cells of the tissue surrounding the lesions. Therefore, it is necessary to develop new therapeutic strategies based on systems that increase selectivity for use individually or synergistically with conventional breast cancer procedures. These can offer patients more selective and less cytotoxic alternatives, thus improving their quality of life.

Cationic peptides as agents against breast cancer
Several studies have shown that cancer cells develop multidrug resistance to chemotherapeutics [44][45][46][47]. Changes are induced at the cellular level that include overexpression of enzymes and drug transporters capable of reducing the concentration of chemotherapeutics in the cytoplasm, allowing the cancer cells to repair damage caused by chemotherapy [48]. To solve this problem, it is necessary to explore and evaluate new molecules that are capable of eliminating cancer cells while having low levels of cytotoxicity against the cells of the healthy tissue surrounding the lesions. A promising possibility in this respect are bioactive cationic peptides (BCPs), which have emerged indirectly as an alternative for cancer treatment. BCPs are widely distributed in nature and are produced by almost all organisms as part of the nonspecific immune system [49][50][51][52][53][54]. These molecules were initially studied as potential substitutes for antibiotics. However, they have been shown to have a broad spectrum of target organisms ranging from viruses to parasites [55][56][57][58], and have the potential to treat polymicrobial biofilms [59,60]. BCPs are small molecules composed of up to 50 amino acids, making chemical synthesis and modification relatively easy. Moreover, although they vary significantly in structure and sequence, they share some general characteristics, being amphipathic and containing a high proportion of cationic and hydrophobic residues [56,61,62]. BCPs have been classified by their sequence and structure as either anionic or cationic, and rich in cysteine forming disulfide bonds, α-helices, β-sheets, cyclic, and linear ( Figure 1) [63]. There is a wide diversity of BCPs, since their primary structures are very heterogeneous, leading to varied secondary structures. The vast majority of reported biologically active peptides are amphipathic and cationic at physiological pH, with charges from +3 to +9 [64,65].
Different mechanisms of action have been proposed to explain how bioactive peptides exert their activity, all based on complex molecular interactions. However, the biological action of all of these mechanisms primarily involves altering the membrane of the target cells [66]. Therefore, peptides have become a promising potential agent in breast cancer treatment, since they reduce the generation of resistance mechanisms by cancer cells. Chemotherapeutics must enter cancer cells to exert their action, allowing the cells to develop resistance mechanisms to combat their effect. In contrast, one of the advantages of BCPs is that they act from outside the membrane, a mechanism that cannot be compensated for by tumor cells [67,68]. The mechanism of action of BCPs is composed of several stages, the first of which is mediated by electrostatic interactions between the positively charged residues of the peptide and the negatively charged groups of the tumor membrane [69]. After that, the hydrophobic interactions between the acyl chains of lipid membranes and non-polar residues then allow the incorporation of the peptide into the bilayer through various modes including the Barrel-stave, carpet detergent, and toroidal pore modes [63,70] (Figure 2). Although the later stages are based on the peptide's ability to induce changes in the membrane, altering its structural properties and compromising its integrity, the first stage is considered fundamental in explaining the biological activity of the peptides and their potential selectivity [71]. Therefore, peptides induce instability and structural and physicochemical changes in the lipid bilayer, leading to cell death [72][73][74].
Furthermore, the higher phosphatidylserine (PS) concentration of cancer cell membranes favors electrostatic interaction between them and the peptides, unlike the membranes of normal cells that are considered neutral because they are mainly made up of zwitterionic lipids ( Figure 3) [75,76]. Additionally, malignant cells are more fluid, and have lower cholesterol content tha the normal cells. Their lower cholesterol content makes malignant cells more susceptible to cell lysis by facilitating the destabilization of the membrane [69]. Leuschner et al. (2004) studied how the cholesterol content of eukaryotic cells acts as a protective factor against the cytolytic effect of BCPs [77,78]. Finally, several authors have reported that cancer cells present microvilli or cell membrane projections [79][80][81]. This would probably increase the surface area of cancer cells compared with normal cell membranes, which could in turn lead to increased interaction with BCPs [82]. However, this theory is still not proven. All these characteristics play a fundamental role in the selectivity of BCPs for malignant cells.

Anticancer activities of BCPs
BCPs exhibit a wide range of anticancer activities. The main effects observed in various studies of the in vitro and in vivo models of breast cancer are cytotoxicity, antiproliferative activity, induction of cell death by necrosis or apoptosis, and inhibition of cell migration ( Figure 4). The results of extensive research on the activity of cationic peptides against breast cancer are summarized in Table 1. An initial experimental approach model to evaluate the biological effects of BCPs against breast cancer includes in vitro cell-based analyses. Studies using cell line cultures have advantages, including easy maintenance, reproducibility of toxicity responses, and vast commercial availability of different cell types that allow the comparison of results between different treatment groups. Consequently, several breast cancer cell       lines have been widely used for breast cancer modeling. Nevertheless, as shown in Table 1, MCF-7 and MDA-MB-231 cell lines are the most frequently employed in the associated studies [83]. Cytotoxic effect on MCF-7 or MDA-MB-231 cancer cell lines has been reported for the cationic peptides Bovine lactoferricin [84], its Bovine variant lactoferricin 6 [85], pBmje [86], Magainin II [87], the Lysine-substituted VmCT1 analogs [88], IW13 [89], Peptoide 1 [90], Pseudhymenochirin-1Pa and Pseudhymenochirin-2Pa [91]. Moreover, cell proliferation assays revealed that Kale antifungal peptide impaired the proliferation of MCF-7 cells. In addition, ERα17p peptide decreased the number of colonies formed by different cancer cells, indicative of an antiproliferative effect [92]. However, the authors of these studies concluded that the peptides had a dose-dependent cytotoxic or antiproliferative activity without thoroughly investigating the mechanism of death induction.
Other researchers evaluated the differential response to BCPs in MCF-7 and MDA-MB-231 cell lines due to their important phenotypic variations. MCF-7 is estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+). On the other hand, MDA-MB-231 is estrogen receptor-negative (ER−) and progesterone receptor-negative (PR−). In general, treatments with BCPs significantly decreased the viability of both types of cells in a dose-dependent manner, and, as is evident in IC 50 values, receptor-positive MCF-7 cells were more sensitive to peptide treatments than receptor-negative cells (MDA-MB-231) [92][93][94][95][96]. Additional evidence reported by Duffy and Sorolla [97] showed that melittin was significantly more potent against HER2-enriched breast cancer cells. Cytotoxic effect was related to the  suppression of activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells [97].
As described previously in this review, the mechanism of action of BCPs in targeting cell membranes is based on electrostatic interactions between the cationic residues on the peptide and anionic lipids on cancer cell membranes. In this respect, several authors have suggested that the mode of action is probably dependent on membrane disruption and subsequent induction of necrosis, as was reported for breast cancer cells treated with Temporina-1CEa [95], pHLIP-(KLAKLAK) 2 construct [98], Maculatin 1.1 [99], NC peptide [100], EVP50 [101], and NRC-03 NRC-07 peptides [102].
After the action of BCPs on the cell membrane, the peptides can also infiltrate intracellular spaces. Hence, the biological effects of BCPs are also associated with the targeting of other cellular structures, such as mitochondria [117][118][119], as well as interference with signaling pathways linked to apoptosis cell death [66] and cell cycle [120,121]. Many BCPs are reported to induce these cellular changes. For example, Ting et al. reported that, in MDB-MA-231 cells treated with TP4 peptide, while the induction of DNA fragmentation or caspase 3 activation after treatment was not evident, lactate dehydrogenase (LDH) increased at 3 h post-TP4 treatment in TNBC cells, indicating that this peptide induces necrotic death in TNBC cells. Furthermore, the mechanism action of TP4 showed that it binds to the mitochondria, disrupts Ca 2+ homeostasis, and ultimately induces FOSB protein to activate TNBC cell death [104]. Another study reported that VES-H8R8 peptide is cytotoxic to breast cancer cells through mitochondria depolarization, increased reactive oxygen species (ROS) production, reduced cell bioenergetics, and triggering of apoptosis G 1 cell cycle arrest [105]. Similarly, Wang et al. observed that Temporin-1CEa induces cell death, which is associated with rapid intracellular Ca 2+ leakage, collapse of mitochondrial membrane potential, and overgeneration of ROS [106]. Figure 4 summarizes all the proposed mechanisms for the BCPs. Aurein 1.2, Buforin IIb, and BMAP-28m induce apoptotic cell death, as was evidenced in MCF-7 cells, where peptides provoked PS exposure in treated cells. Additionally, Bufforin IIb activity was associated with activation of caspase-9 and cleavage of PARP [107]. Soleimani et al. reported that chimeric protein p28-NRC induces cell injury in MCF-7 and MDA-MB-231 in a dose-dependent manner, with increased expression levels of the proapoptotic genes AIF, BAX, and Caspase-3, and decreased expression of the anti-apoptotic gene Bcl-2 [108]. Similar results were published previously for [G10a]SHa-BCTP conjugate peptide, where treatment induced high DNA fragmentation, down-regulating the expression of Bcl-2, and up-regulating BAX and caspase-3 [109].
Many chemotherapeutics affect cancer cells by altering the cell cycle, generally in specific control points; indeed, some BCPs have been reported to affect the growth and division of breast cancer cells. For instance, in MDA-MB-231 cells, melitinin reduced DNA synthesis at the S phase and increased G 1 /S transition, with related low expression of mRNA and protein level of the regulator protein Cyclin D1. Similarly, LTX-315 showed increased G 1 /S transition and time-dependent alterations in the chromatin morphology of the treated cells, which is related to apoptosis [110]. FR8P and FR11P peptides induced G 2 /M phase cell arrest in MDA-MB-231 cells, linked to depolarization of mitochondrial membrane potential and activation of caspases [111].
Since metastasis is responsible for therapeutic failure, molecules that can specifically interfere in the cell migration process are helpful for cancer treatment. Various BCPs with capacity to inhibit cell migration in breast cancer cells have been reported. For example, PR39 treatment significantly inhibited 4T1 cell invasion and migration, and it was suggested that it could have a synergistic effect with Stat3 siRNA, efficiently inhibiting cellular proliferation and migration [112]. FR8P and FR11P peptides also induce a down-regulation of the P44/42 MAP kinase protein responsible for the migration of breast cancer cells [111]. Another study reported an IC 50 value of 61.5 μM for MAP-04-03, although the peptide was very effective at inhibiting the cell migration at 5 μM, with inhabitation of approx. 40% of cell migration [113].
The biological effects induced by BCPs also have been evaluated in vivo controlled environments using animal testing. Rats and mice injected with breast cancer cells are the most common model for tumors. In vivo models employing BCP treatments significantly inhibited tumor growth, as was reported for peptoid 1 [90], melittin [97], and amphipathic α-helical peptide [93]. In other reports, tumor growth reduction was linked to necrosis, for example in ERα17p [92], TP4 [104], and NRC-03 and NRC-07 peptides [102]. Further, vascularization and angiogenesis inhibition in xenograft tumors were reported after buforin IIb [107] and CDAK [96]. The co-treatment of BCPs with standard chemotherapeutics also have been evaluated. In breast cancer, LTX-315 in co-treatment with doxorubicin induced substantial local necrosis and immune-mediated changes in the tumor microenvironment, followed by complete regression in most animals treated [122]. Encouragingly, most of the in vivo studies found that BCP treatment did not have any noticeable adverse side effects. Despite several studies on the discovery or design of anticancer peptides against breast cancer, only LTX-315 is tested in clinical trials. Results of Phase I trial in eight patients with breast cancer (NCT01986426) show that intratumoral injection of LTX-315 is well tolerated. The dosing regimen used for LTX-315 induces necrosis and CD8 + T-cell infiltration into the tumor microenvironment [116].

Current status and future directions
Breast cancer continues to be one of the leading causes of women's deaths worldwide. The search for new therapies for this disease is a priority, especially in view of the very well-known side effects of traditional treatments. Although researchers have been studying the potential of BCPs for cancer treatment, there are still some critical barriers to overcome. Firstly, the selectivity of most BCPs is not sufficiently differentiated between cancer cells and normal cells, resulting in limited clinical applications. Second, the low resistance of BCPs to proteolytic cleavage is one of the aspects of peptides that has raises the most questions. It explains their short half-life and, therefore, low bioavailability in vivo [123], a limitation that avoid using peptides as pharmaceutical agents.
However, different pharmaceutical companies have made progress in evaluating and developing drugs from natural or modified peptides, demonstrating the potential use of these compounds. This potential is based on the easy modification of the sequence, net charge, hydrophobicity, amphipathicity, and therefore the peptide's secondary structure. Some of the more unique peptides have reached phase II and III clinical studies, and are intended for use topically or intravenously to treat localized and systemic infections [124]. This is the case with the peptide derived from lactoferrin hLF-1-11 (AM-Pharma), for use in the treatment of transplant-associated infections; the peptide PAC113, based on histatin 5 (PacGen) from human saliva and used for the treatment of oral candidiasis; and the peptide Mersacidin (Novacta Biosystems Ltd), derived from bacteriocin and used for the treatment of infections of Gram-positive bacteria [124]. One of the most promising peptides developed in recent years is the synthetic peptide LTX-315, a derivative of lactoferricin, known by its trade name as Oncopore™, which is active in several cancer cell lines and is in phase II clinical trials [125]. LTX-315 lyses cancer cells (necrosis) through a membrane destabilizing mechanism followed by the release of danger-associated molecular patterns (DAMPs), thereby reprogramming the tumor microenvironment while presenting low cytotoxicity against human erythrocytes [118,126]. The results using a fibrosarcoma model have shown that 80% of animals treated with LTX-315 show regression in the size of the treated tumor [115,127]. Currently, it is considered an alternative treatment for different types of cancer, but it is mainly used in melanoma. The development of this peptide was the basis for the foundation of the company Lytix Biopharma, whose objective is the pharmacological development of oncolytic peptides [128]. The next generation of peptides will be based on modifications focused on improving the cancer targeting, specificity, and efficacy of peptides, reducing their potential side effects.