Recent advances and applications of peptide–agent conjugates for targeting tumor cells

Abstract

Background

Cancer, being a complex disease, presents a major challenge for the scientific and medical communities. Peptide therapeutics have played a significant role in different medical practices, including cancer treatment.

Method

This review provides an overview of the current situation and potential development prospects of anticancer peptides (ACPs), with a particular focus on peptide vaccines and peptide-drug conjugates for cancer treatment.

Results

ACPs can be used directly as cytotoxic agents (molecularly targeted peptides) or can act as carriers (guiding missile) of chemotherapeutic agents and radionuclides by specifically targeting cancer cells. More than 60 natural and synthetic cationic peptides are approved in the USA and other major markets for the treatment of cancer and other diseases. Compared to traditional cancer treatments, peptides exhibit anticancer activity with high specificity and the ability to rapidly kill target cancer cells. ACP's target and kill cancer cells via different mechanisms, including membrane disruption, pore formation, induction of apoptosis, necrosis, autophagy, and regulation of the immune system. Modified peptides have been developed as carriers for drugs, vaccines, and peptide–drug conjugates, which have been evaluated in various phases of clinical trials for the treatment of different types of solid and leukemia cancer.

Conclusions

This review highlights the potential of ACPs as a promising therapeutic option for cancer treatment, particularly through the use of peptide vaccines and peptide–drug conjugates. Despite the limitations of peptides, such as poor metabolic stability and low bioavailability, modified peptides show promise in addressing these challenges.

Graphical abstract

Various mechanism of action of anticancer peptides. Modes of action against cancer cells including: inducing apoptosis by cytochrome c release, direct cell membrane lysis (necrosis), inhibiting angiogenesis, inducing autophagy-mediated cell death and immune cell regulation.

Introduction

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells, evasion of apoptosis, insensitivity to antigrowth factors, and sustained angiogenesis invasion, which is a leading cause of mortality and morbidity worldwide (Hanahan and Weinberg 2011). According to the World Health Organization (WHO) rankings in 2019, cancer is the first or second cause of death in 112 out of 183 countries (Sung et al. 2021). In 2018, cancer accounted for 9.6 million deaths and 18.1 million new diagnoses worldwide. This number is expected to increase in the future, with 27.5 million new cases of cancer diagnosed annually worldwide by 2040 (Bray et al. 2018).

In 2023, 1,958,310 new cancer diagnosed cases and 609,820 cancer deaths were registered in the USA. This number is expected to increase in the future, and by 2040, 27.5 million new cases of cancer will be diagnosed annually worldwide (Bray et al. 2018). Worldwide, female breast (2.26 million cases, 11.7%), lung (2.21 million, 11.4%), and prostate cancers (1.41 million, 7.3%) are the commonly diagnosed cancers (Ferlay et al. 2021).

Among all of the cancers, lung and breast cancers are the most common cancers, contributing 12.2% and 12.5% of the total number of new cases diagnosed in 2020 (Ferlay et al. 2021). In men, lung cancer with 15.4% of the total number of new cases diagnosed and in women, breast cancer with contributing 25.8% of the total number of new cases diagnosed are the most common cancers in worldwide (Sung et al. 2021). Lung (21.5%) and breast (15.5%) cancers are the most lethality cancers in man and women, respectively. The rate of reduction in cancer mortality has slowly accelerated from about 1% per year during the 1990s to 2% per year from 2015 through 2020. In cancer, mortality has slowly accelerated from about 1% per year during the 1990s, to 1.5% per year during the 2000s, and to 2% per year from 2015 through 2020 (Siegel et al. 2023; Sung et al. 2021).

Currently, the preferred treatments for diagnosed cancer are surgery, radiation, chemotherapy, and immunotherapy, which are designed to eliminate tumor cells by directly removing or killing them (Mahalaksmi and Nandhini 2023).

Due to drug resistance and the distribution of chemotherapy drugs throughout the entire body without specifically targeting the tumors, chemotherapeutic drugs tend to present increased risks of side effects and may therefore may be reserved for adjuvant treatment or inoperable tumors (Mansoori et al. 2017).

Although monoclonal antibodies show promising therapeutic efficacy in clinical studies, adverse effects related to safety, high production costs, immunogenicity, intracellular penetration in tumor tissues, and difficulty in removing the drug from the body due to long half-life have limited the clinical use of antibodies (Chames et al. 2009). While radiotherapy is used in patients with cancer who have not benefited from surgical treatment, this treatment method is expensive and has many complications, with a long course of treatment (Long et al. 2019).

Anticancer peptides (ACPs), as polymers composed of fewer than 45 amino acids (500–5000 Da), can kill fungi, bacteria, and tumor cells with specificity and toxicity to cancer cells (Tornesello et al. 2020). ACPs are a superior choice for tumor therapy compared to antibodies, and conventional chemotherapy due to their high specificity, high penetration, and ease of modification allows them to better inhibit tumor migration, angiogenesis, and tumor cell proliferation (Otvos 2008; Vlieghe et al. 2010).

Several anticancer peptides destroy cancer cells through necrosis and apoptosis by membrane lysis or pore formation (Hoskin and Ramamoorthy 2008). Depending on the amino acid composition, sequence length, molecular weight, isoelectric point, net charge, hydrophobicity, secondary structure, and structural orientation, the properties of peptides can be used as both a molecularly targeted drug and a guided micelle to inhibit cell proliferation or completely eradicate cells (Roudi et al. 2017).

Based on the US National Institutes of Health Clinical Trials database (https://clinicaltrials.gov/), more than 1000 peptide drugs have entered preclinical and clinical trials for targeting different types of cancer, and there are about 60 approved peptide drugs in the market generating an annual sale of more than $13 billion (Fosgerau and Hoffmann 2015; Pan et al. 2020).

The current research has the potential to provide significant value addition to the field of cancer therapy. Despite significant progress in cancer treatment, there is still a critical need for more effective and targeted treatments that can minimize side effects and improve patient outcomes. Peptide–agent conjugates represent a promising approach in this regard, as they can selectively target tumor cells while sparing healthy tissues. Recent advances in this field, such as the development of novel peptide sequences and improved conjugation strategies, have further enhanced the potential of peptide–agent conjugates for cancer therapy. This research aims to provide a comprehensive overview of the recent advances and applications of peptide–agent conjugates in targeting tumor cells, with the ultimate goal of improving cancer treatment and patient outcomes. This paper focuses on the classification, sources of bioactive peptides, mechanism of action, and conjugated ACPs, and their use in peptide–drug conjugates (PDCs) for advancing targeted cancer therapeutics. In addition, the review provides a valuable reference for the modification of ACPs and the successful development of new peptide anti-tumor drugs.

Classification of ACPs

ACPs can be classified according to various criteria such as their chemical structure and mechanism of action.

Classified according to chemical structure

According to chemical structure, ACPs are divided into four categories: α-helical, β-pleated sheets, random coil and cyclic (Xie et al. 2020a, b).

α-helical ACPs

α-helical ACPs are the most typical structure type of ACPs, with short length and simple structure (Huang et al. 2010). Although most α-helical ACPs have adequate inhibitory effects on different tumor cells, they can have cytotoxicity and side effects (Xie et al. 2020a, b). Examples of ACPs with α-helical structures that have anticancer effects on lung, glioblastoma, and breast tumor cells at low IC50 include Magainin II, Aurein, and L-K6 (Chin et al. 2017; Dennison et al. 2007; Lehmann et al. 2006).

β-pleated sheet APCs

β-pleated sheet APCs are mainly found in animals and plants and are more complex than α-helical ACPs (Marsh et al. 2007). ACPs with β-pleated sheets have good stability due to having two or more disulfide bonds (Marsh et al. 2007). Because β-pleated ACPs are less toxic to normal tissue cells, they have good prospects for future development as a promising treatment method for various cancers. Bovine lactoferrin (LfcinB), MPLfcinB6, and human neutrophil peptide (HNP-1) are ACPs with β-pleated sheet structures that have been studied for the treatment of gastric, T-leukemia, and hepatocellular carcinoma cancer cells, respectively (Gaspar et al. 2015; Hilchie et al. 2013; van der Kraan et al. 2004).

Random coil structure APCs

ACPs with random coil structure are usually rich in glycine and proline and lack a typical secondary structure (Veldhuizen et al. 2014). Alloferon and PR-35 are ACPs with a random coil structure whose effects have been investigated on various cancer cells (Bae et al. 2013; Jeon et al. 2019). Random coil ACPs have a lower inhibitory effect on tumor cells than other types. Additionally, killing effect of random coil ACPs on normal cells is worse than α-helical and β-pleated sheet APCs (Xie et al. 2020a, b).

Cyclic ACPs

Cyclic ACPs are a family of backbone-cyclized plant peptides with disulfide bonds that form cysteine knots and are more stable than linear structures (Colgrave et al. 2008). Cyclic ACPs have more cell inhibitory effects and lower toxicity than the other three types of APCs (Zhang et al. 2014a). Additionally, due to their cyclic structure, these ACPs have high resistance to proteases and can be used as a reference for the modification of natural or synthetic peptides (Xie et al. 2020a, b). Examples of cyclic ACPs with anticancer effects on prostate, melanoma, and colorectal cancer cells include Diffuse Cytide 1–3, H-10, and RA-XII (Hu et al. 2015; Wang et al. 2019; Zhang et al. 2014a).

Classified according to mechanism of action

According to mechanism of action, ACPs are divided into three categories: molecularly targeted peptides, binding peptides, and cell-stimulating peptides.

Molecularly targeted peptides

Molecularly targeted peptides specifically bind to cancer cells and directly inhibit or kill cancer cells in various stages of proliferation or carcinogenesis (Ray et al. 2018). Molecularly targeted peptides act on tumor cells through the inhibition of protein–protein interactions, angiogenesis, signal transduction pathways, proteins, enzymes, or gene expression (Kakde et al. 2011; Karagiannis and Popel 2008; Kritzer et al. 2005; Mochly-Rosen and Qvit 2010). Additionally, these APCs act as pro-apoptotic peptides that mediate significant induction of apoptosis or antagonists that bind to a known receptor in tumors (Cornelio et al. 2007;; Ellerby et al. 1999; Sotomayor et al. 2010; Walensky et al. 2004). Most molecularly targeted peptides are selective against cancer cells, but some of them target both cancerous and healthy cells (Chen et al. 2010; Jiang et al. 2014; Li et al. 2019). Pore-forming peptides, cell-penetrating peptides, and tumor-targeting peptides are the most common form of molecularly targeted peptides that kill cancer cells by inducing apoptosis or necrosis, transporting small molecules to oligonucleotides or proteins and binding to receptors on the cancer cell surface for cell internalization, respectively (Boohaker et al. 2012).

Binding peptides or guiding missile peptides

Binding peptides or guiding missile peptides are delivery carriers that transport drugs, toxins, (RNAi), and other agents into the cancer cell targets (Leite et al. 2018). These peptides help to maintain drug concentration during transport to target tumor environment and slow release of the drug in the tumor area (Dossa et al. 2018).

Cell-penetrating peptides are cationic peptides with short oligopeptides consisting of 5–30 amino acid residues with high specificity and affinity to cancer cells (Leite et al. 2018). These peptides bind to the cargoes (drug, toxin, RNAi, etc.) with either covalent or non-covalent bonds to protect the cargo from degradation and increase the stability and non-soluble drugs in tumor environments (Copolovici et al. 2014). Guiding missile peptides enhance the specific binding to cancer targets, circulating stability, controlled release, and protection from enzymatic digestion of the drug in tumor cells and the tumor environment (Conibear et al. 2020).

Cell-stimulating peptides

Peptides that stimulate immune system

Peptides can be used as immune system stimulants or hormone stimulants to control metastasis and eradicate cancer cells by activating host immune system (Darabi et al. 2014; Glover et al. 2004). Due to the low immunogenicity property of ACPs, these peptides are often combined with adjuvants to increase specific immune responses (Li et al. 2012). Examples of ACPs that have been combined with various compounds for immunotherapy against cancers due to their highly immunogenic properties include E75 peptide, melittin RADA32, and APDTR (Apostolopoulos and McKenzie 1994; Jin et al. 2018; Salem et al. 2018). Peptide vaccines such as UroRCC, IMA950, hTERT, and Hp91, which respond specifically to prostate cancer, metastatic renal cell carcinoma, glioma, prostate, and cervical cancer, have been studied in various phase I/II clinical trials (Dutoit et al. 2018; Rausch et al. 2019; Talebi et al. 2017).

Peptides that stimulating hormones

Some of the hormones have a critical the role in the growth and proliferation of cancer cells. An antagonist peptide of growth hormone-releasing hormone (GHRH) inhibits proliferation of acute myeloid leukemia (AML) cells (Jimenez et al. 2018). OB3 peptide inhibits leptin-induced ovarian cancer cells via stimulation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and estrogen receptor α-activation (Chin et al. 2017). ACPs prevent cancer cell initiation and proliferation by controlling hormone release via their receptors (Glover et al. 2004).

Peptide drug conducting (PDC)

Small organic drug molecule (chemotherapy drugs) have high toxicity and off-target effect on normal cells (Nguyen 2011). In addition, rapid renal clearance and altered bioavailability are other problems associated with chemotherapy drugs (Manzari et al. 2021; Patel et al. 2021). Fortunately, targeted drug therapies and targeted drug delivery techniques using tumor cell characteristics [including cell pH, cell glutathione (GSH) content, cell morphology, and enzymes] are emerging as powerful ways to circumvent such problems (Fan et al. 2022; Khawar et al. 2015). These targeting methods allow the drug to be selectively and effectively localized to predefined targets (such as receptors overexpressed in cancer) while limiting its access to normal cells. This approach maximizes the therapeutic index and ameliorates the systemic side effects commonly associated with cytotoxic chemotherapy (Zhang et al. 2012).

Peptides, as building blocks of proteins, are promising, effective, and safe anticancer peptide therapies and are involved in almost all biological functions. Small peptides do not induce unwanted autoimmune responses, and they have high specificity and are able to cross the cell membrane and penetrate tumors more effectively (Apostolopoulos et al. 2022a, 2022b; Bojarska et al. 2021).

Anticancer peptides, as small peptides, are specific and toxic to cancer cells (Tyagi et al. 2015). Anticancer peptides are a superior choice for tumor therapy compared to antibodies and small molecules due to their high specificity, high penetrance, and easy modification. Several anticancer peptides destroy cancer cells through necrosis and apoptosis by membrane lysis or pore formation. Depending on the amino acid composition, sequence length, isoelectric point, molecular weight, net charge, hydrophobicity, secondary structure and structural orientation, peptide properties can be used both as a molecularly targeted drug and as a conducting micelle (carrier) to inhibit cell proliferation or achieve complete eradication of cancer cells (Doti et al. 2021; Le Joncour and Laakkonen 2018).

One of the targeted methods of drug delivery to tumor tissues with minimal damage to normal cells is conjugating the drug with peptides that specifically bind to a receptor that is expressed only on cancer cells. This approach increases the concentration of the drug in tumor tissue increases and causes the death of cancer cells with minimal damage to normal and healthy cells (Kondo et al. 2021). After antibody–drug conjugates (ADCs), peptide–drug conjugates (PDCs) are the next generation of targeted drug therapy with the advantage of increased cell permeability and improved drug specificity (Fu et al. 2022). Compared with ADCs, PDC drugs have a small molecular weight, high tumor tissue penetration, large-scale synthesis using solid phase synthesis, low production cost, low immunogenicity, and relatively good pharmacokinetics (Caliceti and Veronese 2003).

A peptide–drug conjugate is a targeted therapy that functions similarly to ADCs. Peptide–drug conjugation consists of three important components: tissue-specific peptides (homing), linkers, and cytotoxic drugs (Li and Roberts 2003) (Fig. 1). All three components synergistically deliver chemotherapy agents by targeting receptors on tumor cells, extending their therapeutic effect to complement peptide therapies (Vieira et al. 2017). The peptide is an important part of the peptide–drug conjugation. Peptides facilitate the penetration of drugs into the body. In addition, peptides are ideal carrier molecules because they have a similar ability to monoclonal antibodies. They have a high affinity for receptors that are overexpressed on the surface of tumor cells without exhibiting monoclonal antibodies (mAb) toxicity (Fu et al. 2022).

Fig. 1.

Construction and necessary key properties of each part of peptide–drug conjugate

An ideal peptide for PDCs should have strong target binding affinity, efficient internalization, high stability, low immunogenicity, and a long plasma half-life (Fu et al. 2022). Homing peptides specifically target constitutive receptors that are overexpressed in tumor tissues (Ulapane et al. 2017). These peptides directly transport the loaded drug to the target cell and confine the chemotherapeutic agent outside the tissue and target (Laakkonen and Vuorinen 2010). These peptides have a higher binding affinity for target sites at nanomolar concentrations (Lingasamy and Teesalu 2021). Currently, two therapeutic PDCs are available on the market, but many more are in various phases of preclinical and clinical trial (Cooper et al. 2021). Lu-dotatate and melphalan flufenamide (melflufen, Pepaxto^®) were approved by the US Food and Drug Administration (USFDA) in 2018 and 2021 for the treatment of gastroenteropancreatic neuroendocrine and multiple myeloma cancers, respectively (Dhillon 2021; Food, Administration, approves lutetium Lu 2018).

ANG1005 is a PDCin phase III (NCT03613181) and is composed of a receptor-targeting peptide vector to the brain (Angiopep-2) with three molecules of paclitaxel (Li and Tang 2017). ANG1005 demonstrates antineoplastic potency, inhibition of human tumors, and a significant increase in the survival of glioblastoma and lung carcinoma mice (Regina et al. 2008). In addition, ANG1005 is able to penetrate the blood–brain barrier (BBB) and has higher brain uptake than paclitaxel. ANG1005 bypasses P-glycoprotein (P-gp) at the BBB in both the brain and other metastatic sites (Thomas et al. 2009). TH1902 and TH1904 are two PDCs targeting Sortilin 1 (SORT1) receptors which are overexpressed in several malignancies, including ovarian and breast cancer (Lindberg et al. 2021). TH1902 is in a phase I clinical trial (NCT04706962), and TH1904 is under preclinical investigation. They are payload with docetaxel and doxorubicin, respectively, and designed for the treatment of patients with sortilin-positive recurrent advanced solid tumors (Lindberg et al. 2021). DTX-P7 is a PDCcomposed of docetaxel (DTX) and a heptapeptide (P7) that selectively targets non-small cell lung cancer (NSCLC) (Jiang et al. 2022). This PDCspecifically binds to cell surface heat shock protein 90 (Hsp90), which is overexpressed in NSCLC cells and induces an unfolded protein response and subsequent apoptosis, killing dormant lung cancer stem cells (Jiang et al. 2022).

Overall, PDCs are next-generation targeted therapeutic agents. In the last 10 years, several advantages including selective delivery of cytotoxic payloads to target cells, enhanced tumor penetration, biodegradability, systemic toxicity, improved efficacy, reduced immunogenicity, and lower costs of synthesis have led pharmaceutical companies to develop PDCs as targeted therapeutic candidates for cancer, metabolic diseases coronavirus, metabolic diseases, and so on (Alas et al. 2020; Gong et al. 2021; Jiang et al. 2022; Kind et al. 2022; Liu et al. 2022; Long et al. 2019; Salem et al. 2018; Wang et al. 2021b; White et al. 2019).

Tumor-homing peptide like (iRGD) is a peptide sequence that was discovered through a process called in vivo phage display, which involves introducing a library of phages (viruses that infect bacteria) into a living organism and selecting those that bind to a specific target (Sioud 2019).

The iRGD peptide sequence, which stands for “internalizing RGD,” contains a sequence of amino acids that binds to the αv integrin receptors on tumor cells, as well as a C-end Rule (CendR) sequence that allows the peptide to penetrate deep into the tumor tissue. When combined with a therapeutic agent such as a chemotherapy drug, iRGD can potentially enhance the delivery of the drug to the tumor cells and improve its efficacy (González-Cruz et al. 2022). Studies have shown that iRGD can improve the delivery of a variety of therapeutic agents to tumor cells, including chemotherapy drugs, nanoparticles, and viruses (González-Cruz et al. 2022; Zoa et al. 2022). In addition, iRGD has been shown to enhance the penetration of drugs into the tumor tissue, which can be a significant challenge in cancer therapy (González-Cruz et al. 2022). One potential advantage of iRGD over other tumor-homing peptides is its ability to penetrate the blood–brain barrier (BBB), which is a protective barrier that prevents many drugs from reaching the brain. In preclinical studies, iRGD has been shown to enhance the delivery of chemotherapy drugs and nanoparticles to brain tumors, suggesting that it could be a promising approach for the treatment of brain cancer (Izci et al. 2021).

Modification of ACPs

Despite the obvious advantages of ACPs, several shortcomings, including short circulatory half-life, rapid renal clearance, and low oral bioavailability, hinder and slow down their use in clinical research and application (Di 2015; Renukuntla et al. 2013; Wu and Huang 2018). Following the synthesis of peptides with different methods, they can be modified to construct an ideal secondary structure, stabilize, and mimic to improve their biological, stability, selectivity, and solubility of the peptide drugs (Di 2015). In recent years, researchers have carried out several reconstructions and modifications to improve the cell permeability, reduce renal clearance, and enhance the chemical and proteolysis stability of ACPs, thereby improving their therapeutic properties. Cyclization, replacement of natural amino acids, substitution of non-natural amino acids, and polymer conjugation are examples of modifications that can improve peptide stability and cell permeability for economic rationality and patient compliance (Cooper et al. 2021; Deng et al. 2016; Ginn et al. 2014; Han et al. 2013).

Cyclization

The cyclization of peptides can increase proteolytic stability, cell permeability, and stabilization of the peptide secondary structure in nature (in vivo) and in the laboratory (in vitro) (Hayes et al. 2021). They are promising and of considerable interest in drug discovery and nanotechnology (Muttenthaler et al. 2021). To date, several ways have been reported for peptide cyclization. Head-to-tail (known as backbone cyclization), head-to-side chain, side chain-to-side chain, and tail-to-side chain are the main approaches for peptide cyclization (Davies 2003). Chemical methods [direct amide bond formation (Jin et al. 2016), native chemical ligation (Conibear et al. 2018), ligations relying on a C-terminal aldehyde (Adebomi et al. 2019), bio-orthogonal reactions (Soellner et al. 2006), disulfide bonds (Wilson et al. 2015)], enzymatic methods [subtiligase variants (Tang et al. 2020), sortases (Chen et al. 2016), asparaginyl endopeptidases (Chen et al. 2016), transglutaminase (Touati et al. 2011), non-ribosomal peptide synthetases (Calcott and Ackerley 2014)], protein tag for cyclization [Inteins (Scott et al. 1999), and protein tags for isopeptide bond formation (Si et al. 2016)] are common approaches for peptide cyclization which each approach having its own strengths and limitations (Hayes et al. 2021). Cyclic peptides exhibit multifold biological activities in antibiotics, cancerostatics, hormones, ion carrier systems, toxins, and antimycotics (Malesevic et al. 2004).

Replacement of natural amino acids

The sequence of amino acids ultimately determines the peptide shape and function. One way to modify ACPs is by changing the sequence of main chain amino acids. Amino acid substitution causes changes in hydrophobicity, helicity, and net charge, which can lead to changes in selectivity and function of peptides (Avrahami and Shai 2002; Chen et al. 2005). Anticancer peptide peptides usually have a positive charge and can interact with the negatively charged membrane of the cancer cells (Sok et al. 1999). Currently, changing the net charge, helicity, and hydrophobicity of peptides is being carried out as a modification approach to optimize the anti-tumor activity of ACPs (Tan et al. 2014). K7S and MEL-pep peptides are examples of modified peptides in which one or more natural amino acids in different positions are substituted with other natural amino acids to change net charge and helicity of peptides (Pouillot et al. 2012; Tan et al. 2014). Experimental results have shown that by replacing serine and lysine in K7S and substitution the 8th valine and 14th proline with lysine in melittin peptide, the net charge and helicity of peptides can be changed, and the IC50 in cervical cancer and hepatoma cells significantly decreased to 1.4 μM from 13.2 μM and 11.09 μM to 4.44 μM, respectively, thereby reducing toxicity to normal cells (Bennett et al. 2010; Pouillot et al. 2012).

Substitution of non-natural amino acids

One approach to improve the stability of the ACPs is replacement of natural amino acids with unnatural amino acids, which have physicochemical properties that are not found in natural amino acids (Venugopal et al. 2010). The main features of these amino acids include improving potential organism selectivity and potency, higher metabolic stability, and a variety of physiochemical properties (Bhonsle et al. 2013; Hicks and Russell 2012; Russell et al. 2011).

Another approach to increase the stability of ACPs is to replace L-amino acids with D-amino acids (Chen et al. 2002). Due to decreased substrate recognition and binding affinity of proteolytic enzymes in D-amino acids, the half-life of ACPs is increased (Chen et al. 2002). Octreotide and PMI are modified peptides with D-amino acids incorporated into their structure. The increased half-life from a few minutes for somatostatin to 1.5 h for octreotide and the improved stability and inhibitory activities against U87 cells with a reduced IC₅₀ value of PMI-1/4 by half compared with the original peptide are examples of modified ACPs with replacement of l-amino acids to d-amino acids (Gomes-Porras et al. 2020; Kawanishi et al. 2006). Researchers have shown that synthetic amino acids including, octahydroindole carboxylic acid (Oic) instead of proline, 1-aminocyclohexane carboxylic acid (A6c)/A5c instead of alanine, tetrahydroisoquinoline carboxylic acid (Tic) instead of phenylalanine, and 2,4-diaminobutanoic acid/2,4-diaminopropionic acid instead of lysine, have higher anti-tumor activity, decreased IC₅₀ value, and are non-toxic to normal cells compared to natural amino acids (Bana et al. 2017).

Polymer conjugation

Coupling polymer groups with free side chain groups on the molecular surface of ACPs is a classical strategy that changes the physicochemical and therapeutic properties of ACPs, improves the pharmacokinetic (PK) profile of ACPs, and sterically shields peptides from proteolytic enzymes, thereby increasing circulating half-life and bioavailability, reducing their toxicity, delaying elimination by renal clearance, and improving their selectivity (Milla et al. 2012). Polyethylene glycol (PEG) is the most frequently and highly investigated polymer used in the covalent modification of peptides (Hamley 2014; Harris and Chess 2003). PEG is a hydrophobic, semicrystalline polyether, non-antigenic, non-immunogenic, highly soluble in water, low toxic, linear polyether of ethylene glycol, and FDA approved for use in biomedical applications such as biosensing, tissue engineering, and drug delivery systems (Rahmani et al. 2022). PEG is mainly conjugated with peptides, proteins, drugs, and other macromolecules (PEGylation) to optimize the pharmacokinetics of drug substances (Caliceti and Veronese 2003; Kolate et al. 2014; Pasut and Veronese 2007). As PEG is a macromolecule (molecular weight above 1000 Da), attachment of PEG to peptides increases the molecular diameter of ACPs, thus increasing water solubility and stability, as well as reducing clearance through the kidneys, leading to an extension of their half-lives and longer circulation time (Canalle et al. 2010; Yang et al. 2004). In addition, PEGylation of peptides masked antigenicity for minimum response in the host and decreases degradation of ACPs by metabolic enzymes (Lv et al. 2021; Zhang et al. 2014b).

Temporin-1CEa is a 17-amino-acid cationic anticancer peptide, showing high antimicrobial activity against Gram-negative and Gram-positive bacteria, with broad-spectrum anticancer peptide activity that acts preferentially on cancer cells without cytotoxic effect on normal cells (Zhang et al. 2021). PEGylation of this peptide (Temporin-1CEa-LIP) is a new drug carrier system that exhibited good stability in serum and could be efficiently taken up by MCF-7 cells. In addition, Temporin-1CEa-LIP showed toxicity against MCF-7 cells as potent as Temporin-1CEa (unpegylated) (Wu et al. 2016). Although PEG modification has some limitations, the high hydrophobicity of PEG increases the solubility of peptides and improves the stability of ACPs.

In conclusion, due to some limitation of peptides for therapeutic application, such as negligible oral bioavailability and short plasma half-life, different modification approaches have been developed to alter or promote the properties of peptides.

Mechanism of ACPs

Lysis of the cell membrane

Electrostatic interactions between negatively charged cell surface of neoplastic cells and cationic amino acid within the ACPs are found to be pivotal in the membrane destabilization and cell lysis (Kordi et al. 2022; Mader and Hoskin 2006). In contrast to non-malignant cells, the outer leaflet of cancerous cells has an increased content of anionic components such as O-glycosylated mucin, sialylated glycoprotein, phosphatidylserine, glycolipid, and chondroitin sulfate proteoglycan (Kurrikoff et al. 2019). Additionally, the elevated membrane fluidity of malignant cells as a result of the difference in the cholesterol/phospholipid ratio may improve the lytic activity of ACPs by facilitating the integration of hydrophobic amino acid side chains into more fluid cell membranes. Besides, cancer cells contain higher amounts of microvilli, which increase the surface area and the opportunity for ACPs to bind to them. Interestingly, the irregularities of microvilli in shape and size have potential effects on receptor accessibility and the selective binding of ACPs to cancer cells (Agrawal et al. 2021; Campanile et al. 2023). Following the binding of ACPs to the cell membrane, it either causes transmembrane pore formation and fragmentation of the cell membrane (barrel-stave model) or aligns in parallel to the anionic cell surface, resulting in internal osmotic pressure and membrane disintegration (carpet-like fashion) (Terrettaz et al. 2003; Zhao et al. 2006). Membrane disruption could also result from the release of micellar structures from membrane areas containing high peptide densities (inverted micelle models) or by inducing membrane curvature after accumulating in a parallel orientation to the phospholipid head groups of the membrane (toroidal model) (Soon et al. 2020; Timmons and Hewage 2021). PNC-27, a 32-residue peptide, was found to promote membrane pore formation and result in tumor cell lysis (Sarafraz-Yazdi et al. 2022). This selective pore-forming effect is associated with upregulation of tumor cell surface Human double minute 2 (HDM-2) compared to normal cells (Sarafraz-Yazdi et al. 2022). It should be noticed that the anticancer peptide ability of ACPs is not only restricted to membrane disturbance. They can fight tumors via other mechanisms which will be discussed in later parts.

Apoptosis

Anticancer peptide peptides, particularly in α-helical forms, result in the disruption of the mitochondrial membrane of tumor cells and the release of cytochrome c. This leads to the oligomerization of apoptotic protease-activating factor-1 (Apaf-1), activation of caspase-9, and subsequently induction of apoptosis through the conversion of procaspase-3 to caspase-3. RScyreprocin (the recombinant product of scyreprocin) has been shown to prohibit the colony formation ability of H460 lung cancer cells in vitro and in vivo via inducing apoptosis. It could exert the production of reactive oxygen species (ROS), which further caused ER stress, Ca²⁺ secretion, and the activation of caspase-3-mediated apoptotic cascades. Moreover, treated H460 cells have revealed upregulation of pro-apoptotic proteins and low expression levels of anti-apoptotic proteins (Yang et al. 2022). The ACPs M1-8 and D-LAK-120A induce mitochondrial apoptosis by promoting a decrease in mitochondrial membrane potential and the enhancement of ROS levels, leading to caspase-mediated apoptosis of human liver cancer cells and lung cancer cells, respectively (Patil and Kunda 2022; Zeng et al. 2023). In a study by Nurdiani et al., transfection of HT-29 colon cancer cells with an anticancer peptide isolated from flathead by-products (Met1-Gly2-Pro3-Pro4-Gly5-Leu6-Ala7-Gly8-Ala9-Pro10-Gly11-Glu12-Ala13-Gly14-Arg15) induced apoptotic morphological changes and anticancer peptide activity against cancerous cells (Nurdiani et al. 2022). A similar study exhibited the anti-proliferative effect of the RF13 peptide (1RRGKGGRRVTMSF13) through triggering apoptotic genes caspase-3 and caspase-9 on human laryngeal epithelial (Hep-2) cells (Velayutham et al. 2022). ACPs could induce caspase-independent apoptosis and suppress cancer cell growth. LL-37 belongs to cathelicidins family, which, besides immune regulation, has revealed a wide variety of antimicrobial and anti-carcinogenesis activities (Engelberg and Landau 2020; Yang et al. 2020). Ren and colleagues have evaluated the cytotoxic effect of a fragment of LL-37 known as FK-16 on colon cancer cells-induced apoptosis. It was concluded that FK-16 induction of apoptosis could mediate through the activation of Tumor protein 53 (TP53) gene, which, in turn, increases the expression level of Bax and PUMA and decreases the expression of Bcl-2. Furthermore, FK-16 could enhance the nuclear expression of apoptosis-inducing factor (AIF) and endonuclease G (EndoG), which causes fragmentation of DNA and condensation of chromatin (Arnoult et al. 2003; Ren et al. 2013). Considering these results, triggering mitochondrial-mediated apoptosis is important for ACPs to exert anti-tumor effects.

Necrotic cell death

Necrosis is defined as the uncontrolled death of cells, leading to spilling of the cytoplasmic contents into extracellular space and subsequent damage (D’arcy 2019). Cancer cell necrosis could result from destabilization and disruption of cellular membranes by cationic ACPs. For instance, Rb4, a four-transmembrane domain protein derived from proteolipid protein 2 (PLP2) N-terminal, can disrupt plasma membrane integrity and nuclear condensation, leading to necrosis of murine melanoma B16F10-Nex2 cells. This mechanism was found to be a result of F-actin dynamics alterations, overexpression of two damage-associated molecular patterns (DAMPs) including calreticulin and high-mobility group protein B1 (HMGB1) (Maia et al. 2022). In a similar study, the hybrid peptide NTP-217 promoted ROS accumulation in hepatoma cells by induction of mitochondrial dysfunction, resulting in membranolysis and cytosolic components leakage (Yin et al. 2022). In addition to this, brevivin-1RL1, derived from the skin secretions of frog Rana limnocharis and composed of 24 amino acids, could inhibit A549 and HCT116 tumor cell proliferation. Compared to the control group, after transfection of cells with brevivin-1RL1, typical characteristics of necrosis including cell debris and a large amount of vacuolation were detected in the cell supernatant at an IC50 value ranging from 5 to 10 μM (Ju et al. 2021). Similar results were achieved from other ACPs, such as Smp24, isolated from the Egyptian scorpion Scorpio maurus palmatus (Guo et al. 2022), and PNC-27, a peptide containing amino acid residues 12–26 of p53 protein (Davitt et al. 2014). Collectively, necrosis-promoting peptides are assessed to be important in the disruption of the cell membrane by their lytic ability.

Inhibition of tumor angiogenesis

Tumor growth is often accompanied by promoting the formation of new blood vessels from existing ones through high expression of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), which induce tumor proliferation and metastasis (Lugano et al. 2020; Shoari et al. 2021). CIGB-300, a peptide inhibitor, limited NSCLC angiogenesis by inhibiting casein kinase 2 (CK2) and therefore decreased vascular response to angiogenesis. The IC50 value is 30 µM in NCI-H460 NSCLC cells (Pérez et al. 2023). SKACP003 peptide notably restrained migration, metastasis and angiogenesis of breast cancer cell lines (MCF-7, MDA-MB-453, and MDA-MB-231). The experimental findings revealed that this peptide plays an anti-angiogenic role by downregulating the expression of vascular endothelial growth factor (VEGF-A) genes and matrix metalloproteinase (MMP) (Selvarathinam et al. 2023). Another ACP, F4 (CNPEDCLYPVSHAHQR) derived from collagen XIX, was found to inhibit VEGF-induced pseudo-tube formation on Matrigel in a mouse melanoma model via α5β1 and αvβ3 integrin interaction, thus inhibiting tumor cell angiogenesis and migration (Oudart et al. 2021). Briefly, due to low toxicity, high specificity, and high penetrating rate of anti-angiogenic ACPs, they could be considered as new therapeutic agents for cancer patients (Shoari et al. 2021).

Autophagy

Autophagy is a process by which cells degrade and recycle their own components, not necessarily just damaged organelles. Autophagy serves many functions, including the removal of misfolded proteins, the elimination of intracellular pathogens, and the regulation of cellular metabolism (Lerksuthirat et al. 2023). In cancerous cells, autophagy plays two opposite roles by adjusting cell survival or death. In the early stage of tumorigenesis, it blocks tumor growth by eliminating damaged cargos. On the contrary, in the late stages of carcinogenesis, autophagy enhances the ability of tumor cells to adapt to metabolic stressors and therefore facilitates their proliferation and survival. Different studies have highlighted the role of ACPs in inducing autophagy-mediated cell death in cancer cells. In this regard, alpha-lactalbumin-A2 (ALA-A2) has been found to trigger autophagy in A549 lung adenocarcinoma cells (1.5-fold of untreated cells) without provoking hemolytic effects. This could be explained by disturbing the expression of heat shock protein HSP90 alpha and beta, heat shock 70 kDa protein 8, and ubiquitin-60S ribosomal protein L40 (Lerksuthirat et al. 2023). Similarly, the ACP M1-8, which is derived from Musca domestica cecropin, could repress HepG2 cell proliferation through the enhancement of autophagic LC3-II, Beclin, and TSC1 protein contents and the suppression of autophagic lysosomal fusion (Zeng et al. 2023). In contrast to these studies, Zhang et al. assessed the growth inhibitory potential of LL-37 against pancreatic cancer cells via inhibition of autophagy. They found that LL-37 could prohibit autophagy in tumor cells via activating mammalian target of rapamycin (mTOR) signaling, which led to the intracellular production of ROS, impairment of mitochondrial function, and finally, cell death. After treatment for 24 h, MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay results revealed that LL-37 at concentrations greater than 1 µM decreased the viability of cancer cells in a dose-dependent way. However, treatment for 48 and 72 h had no further inhibitory effects on cell growth. The IC50 of LL-37 in MIA PaCa-2 and PANC1 after 24 h of treatment was 11.52 and 10.17 µM, respectively. In addition, immunofluorescence confocal microscopy revealed low expression levels of LC3B after transfection with LL-37 for 24 h, leading to decreased autophagosome formation (Zhang et al. 2022). Taken together, these data suggest that ACPs-induced promotion or inhibition of autophagy could result in growth repression of cancer cells.

Regulation of the immune system

Numerous studies have illustrated the importance of the immune cells in detecting and eliminating transformed cancerous cells. Due to the importance of this issue, novel anticancer peptide strategies are designed based on immune therapeutics that can promote immune responses against tumor cells. In this regard, ACPs could enhance immunity through diverse regulatory mechanisms such as cytokines production, induction of dendritic cells (DCs) maturation, upregulating cytotoxic T cells (CD8⁺ T) cells, and reduction of regulatory T cell (T_reg) levels (Xie et al. 2020a, b). A number of ACPs have been applied in cancer therapy. PKHB1 is a thrombospondin-1 peptide mimic and is able to induce immunogenic cell death in breast cancer cells. In vivo experiments reveal that PKHB1 has stimulatory activities on intratumoral CD8⁺ T cell infiltration, and ex vivo results suggest that it induces dendritic cell maturation and T cell anti-tumor responses (Calvillo-Rodríguez et al. 2022). A 9-mer membranolytic peptide, termed LTX-315, could exert anti-tumor effects in human malignancies of diverse origins by induction of CD8⁺ and CD4⁺ T cells infiltration in the tumor area. In addition, it was able to exert a remarkable increase in the diversity and number of expanding clones of T cells (Spicer et al. 2021). LTX-401 has been shown to play significant roles in stimulating immune responses, such as enhanced production of interferon-gamma (IFN-γ) by T cells against hepatocellular carcinoma (Mauseth et al. 2019). These results suggest that ACPs could increase the immune system through immunomodulatory mechanisms and suppress the growth of tumor cells.

Peptide vaccines

Peptide vaccine strategy relies on using short peptide fragments (20–30 amino acids) to elicit protective immune responses against requested antigens. Synthetic peptides used in these vaccines deliver the specific epitopes of an antigen to the immune B and T cells (Abdelmageed et al. 2020). In terms of carcinogenesis, peptide vaccines are designed by utilizing synthetic tumor associated or specific peptides that are presented on human leukocyte antigen (HLA) molecules and detected by CD8⁺ and helper T cells (CD4⁺) receptors (Biri-Kovács et al. 2023). Peptide vaccines could also be developed to provoke immunity against diverse serovars or strains of the microorganism by formulating different noncontiguous antigenic epitopes or conserved epitopes that exist between them (Verma et al. 2018). Due to the relatively small size, peptides used in vaccines are often poorly immunogenic by themselves and hence need adjuvants (stimulants of the immune system) or efficient delivery systems to promote their efficacy (Nelde et al. 2021). The most common adjuvants were mLAPMi-R848 (Mohapatra et al. 2023), sargramostim, and imiquimod (Zelba et al. 2022), XS15 (Heitmann et al. 2022), Montanide ISA-51 (Saxena et al. 2022), and Montanide ISA-720 (Motavalli Khiavi et al. 2018). Cytokines such as interleukin-2 (IL-2), IL-4, IFN-α2b, and pegylated IFN-α were utilized in peptide vaccine clinical trials (Khong and Overwijk 2016). Regarding delivery systems, polymeric nanoparticles conjugated with antigenic peptides (Koirala et al. 2023), polyacrylate (Liu et al. 2015), and liposomes (Dai et al. 2021) have developed to boost the immunogenicity in a host. There are many peptide vaccines under progression, such as vaccines against viral infection (Heitmann et al. 2022), fungi disease (Araf et al. 2022), bacterial infections (Kumar et al. 2022), autoimmune diseases (Yu et al. 2023), and cancer (Rahman et al. 2023). Large numbers of candidate peptide vaccines are under various phase I, phase II, and phase III stages of development (Bekaii-Saab et al. 2019; Noguchi et al. 2021; Shima et al. 2019). Interestingly, phase III studies are based on vaccines indicated for the treatment of multiple types of tumors, such as prostate, glioblastoma, and breast cancer (Mittendorf et al. 2019; Narita et al. 2019; Noguchi et al. 2021). Peptide vaccines provide several benefits compared to conventional ones. They are highly specific, have low side effects, and are an inexpensive technology. In addition to stable chemical properties, they lack redundant elements with high potential for reactogenicity in the host, including toxins, lipids, and lipopolysaccharides. Moreover, incorporation of various epitopes from different antigens into peptide vaccines enhances their ability to activate multiple T cell clones. In spite of these advantages, peptide vaccines are susceptible to enzymatic degradation and may trigger unrelated immune responses. Additionally, their relatively low in vivo immunogenicity highlights the need for the development of potent adjuvants to enhance their efficacy (Tang et al. 2022).

Advantage and disadvantage of ACPs

Despite achievements made in cancer therapy, anti-tumor drugs represent systemic side effects in patients. They interfere with cancer cell proliferation by destroying them (Ward et al. 2020). However, they could also affect healthy cells. In this regard, ACPs have offered an alternative strategy against malignancies. Interestingly, several aspects make peptides an ideal tool for the development of new therapies against various diseases, including cancer. One of them is their small size, which leads to improved tissue penetration and better delivery (Grisoni et al. 2019). In addition to this, ACPs specifically attacked malignant cells through physical interaction with their negatively charged membrane components. It was revealed that peptides such as heterochiral β-peptide polymers and mastoparan have efficient effects on multidrug-resistant and slow-growing cancer cells, without causing serious damage to normal tissues (Hilchie et al. 2016; Shao et al. 2022). ACPs could also potentiate the efficacy of conventional drugs in a synergistic fashion. Cationic ACP L-K6 was found to synergistically restore the sensitivity of MCF-7/ADR tumor cells to doxorubicin via inhibiting P-glycoprotein expression (Wang et al. 2021a). In spite of multiple advantages, ACPs have several drawbacks, including the high cost of production, biological instability, relatively low oral bioavailability, susceptibility to cleavage via serum proteases combined with renal clearance, and hence a brief half-life in vivo. Furthermore, high concentrations of ACPs could be toxic and also have a risk of immunogenic effects on healthy cells (Ferrero et al. 2023). Moreover, the hydrophobicity of therapeutic peptides has been found to be responsible for their limited penetration through biological membranes and physiological barriers. In addition, ACPs tend to denature, adsorb, and aggregate in vivo, limiting their function (Haggag et al. 2018). As mentioned before, the stability of peptides is a critical issue in cancer therapeutics. Various studies have been developed to extend the plasma residence time of peptides in vitro and in vivo. These include manipulation of peptide amino acids, conjugation with immunoglobulin or albumin, post-translational modification methods in which polymers such as PEG are attached to the peptide, and carrier-mediated delivery (e.g., liposomes, nanoparticles) (Zaman et al. 2019). However, these methods must avoid any unwanted side effects. Ultimately, ACPs could be an effective treatment strategy if research addressing their disadvantages has been progressed and improved in the future.

Mitochondrial targeted peptides for cancer therapy

Cancer, as one of the most challenging clinical conditions which includes a wide range of diseases, can start because of irrepressible cell proliferation in one part or some parts of the body and destruction of cell death mechanisms (Alizadeh et al. 2014; Imanieh et al. 2014; Siegel et al. 2013). Cancer leads to imposing a heavy social and economic burden on patients and society; therefore, providing effective treatment solutions is a priority in cancer studies. Cancer treatment includes chemotherapy, endocrine therapy, radiotherapy, and surgery (Urruticoechea et al. 2010).

New treatment approaches, such as anti-angiogenesis, viral therapy, antibodies, and other small molecules, have attracted the attention of researchers in this field (Urruticoechea et al. 2010). New drugs have been discovered that target specific and distinct components of cancer cells, and therapeutic peptides are among these new drugs (Neuzil et al. 2007). Peptides are small molecules of 20–50 amino acids if these molecules are conjugated with ligand domains, they can target cancer cells more effectively and become more efficient in treatment. The use of peptides has limitations such as rapid removal from the blood due to high renal clearance and enzymatic degradation, but small size, simple synthesis, modification, ability to penetrate tumors, and biocompatibility are among the advantages of these molecules; therefore, they can be a more effective alternative target than antibodies (Neuzil et al. 2007; Sun 2013). In addition to cancer, peptides have increasing applications in the treatment of cardiovascular and diabetes diseases, and they can target diseases such as cancer, cardiovascular disease, and diabetes either directly or indirectly. Some peptides target specific organelles, such as mitochondria, and lead to the induction of cell death in cancer cells through specific mechanisms (Li and Cho 2012; Neuzil et al. 2007). The main reason for the development of mitochondrial therapeutic peptides is the difference in the function of this organelle in healthy cells and cancer cells. In the following, we will discuss mitochondria and mitochondrial peptide drugs.

As the cell’s energy engine, mitochondria play a significant role in cell survival and death. Mitochondria are bi-membranous organelles with their electron transport chain located on the inner membrane. ATP is produced with the help of the energy released by the electron transfer. This released energy has enough power to move protons from the matrix to the inter-membranous space so that the electrochemical proton gradient can act as a driving force for the backflow of protons by the ATP synthase complex (Szewczyk and Wojtczak 2002). Additionally, mitochondria play important roles in the innate immune system, cell differentiation, oxygen sensing, and calcium metabolism. Due to its various functions, this organelle could be a therapeutic target for different diseases. Mitochondrial damage can lead to the disruption of the natural cell death cycle. Programmed cell death occurs in two forms: apoptosis and necrosis. Apoptosis is a stepwise regulated form of programmed cell death, while necrosis is a passive disintegration of cells. Both forms of cell death can be regulated by a variety of pathways that can overlap (Constance and Lim 2012; Martel et al. 2012). Mitochondrial outer membrane permeabilization (MOMP) leads to release of some pro-apoptotic factors such as cytochrome c (Cyt c) that cause caspases, apoptotic-inducing factor (AIF), second mitochondria-derived activator of caspases (SMAC), endonucleases and serine protease HtrA Serine Peptidase 2 (HTRA2) activation and these events lead to the initiation of apoptosis (Martinou and Youle 2011). On the other hand, the initiation of necrosis occurs without the release of cytochrome c and with primary opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane (IMM), followed by a rapid decrease in the electrical potential difference across IMM. Therefore, ATP synthesis is disrupted, and a lot of water and solutes enter the matrix, which leads to severe swelling of mitochondria (Alizadeh et al. 2012; Jacotot et al. 1999).

According to the points explained, mitochondria is an important therapeutic target, and peptides can specifically target each of the factors mentioned above. There are four categories of therapeutic peptides according to the mechanism of action, which include peptides that affect the Bcl-2 family, elevated ROS, overloaded calcium, and voltage-dependent anion channels (VDAC) (Farsinejad et al. 2015).

First, the Bcl-2 family is known as the most important proteins regulating the intrinsic pathway of apoptosis and includes 25 pro- and anti-apoptotic members. Cell death or survival depends on the ratio of pro- and anti-apoptotics. There are four regions with high sequence similarity (BH1, BH2, BH3, and BH4) among Bcl-2 family, with some indicating an anti-apoptotic role (e.g., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl1). On the other hand, PUMA, NOXA, Bad, Bim, and Bid are pro-apoptotic members. Therefore, in general, peptide drugs developed in this category affect each of these proteins (Jabbour et al. 2009).

According to in vitro and in vivo studies, ABT-737 as a BH3-mimetic peptide exerts its anticancer effects by disrupting the Bax and Bcl-2 complex, resulting in structural changes in Bax, release of cytochrome c, activation of caspases, and finally, causing cell death (Jabbour et al. 2009). ABT-263 (Navitoclax) has the same biological characteristics as ABT-737, and due to the changes made in this peptide, its half-life has increased, and it works by interrupting Bcl-2/Bcl-xL interactions, leading to the initiation of apoptosis (Ackler et al. 2010). ABT-263 led to thrombocytopenia due to its effect on BCL-XL, which limited its use. To address this, scientists developed another drug named ABT-199, which is a modified form of ABT-263 that specifically affects Bcl-2 (Souers et al. 2013). Another drug in this group is Obatoclax or GX15-070, which leads to apoptosis by inhibiting MCL-1 and Bcl-xL and can exert its anticancer effects alone or in combination with other drugs (Nguyen et al. 2007). The last drug in this group is TLSGA-FELSRDK (TLS), which has been investigated for its therapeutic role in ovarian cancer. It exerts its effects through different mechanisms, such as anti-proliferative effects, modulation of abnormal pathways in cancer, and targeting molecules that play a direct role in apoptotic pathways, such as the Bcl-2 family (Manion and Hockenbery 2003).

The second class of mitochondrial peptide drugs was developed to deal with oxidative stress. ROS are mostly produced by the mitochondrial electron transport chain and can change in different physiological and pathological conditions. ROS production plays an important role in the release of cytochrome c. ROS elevation can lead to mitochondrial dysfunction and subsequent activation of caspases, thereby initiating the pathways of cell death. Therefore, peptides have been developed with therapeutic goals. From this group, the GO-203 peptide has entered the clinical trial phase (Manion and Hockenbery 2003). The effectiveness of GO-203 depends on the inhibition of the MUC1 C-terminal domain, a polymorphic epithelial mucin protein, and it causes a reduction of ROS (Manion and Hockenbery 2003).

Since calcium is involved in most of the body’s vital processes, peptides of the last group affect calcium-dependent pathways that are important in cell survival and death. According to previous research (Carafoli 2002; Norberg et al. 2008), Ca²⁺ is related to the regulation of pro-apoptotic proteins, and mitochondrial Ca²⁺ overloading results in the loss of mitochondrial membrane potential and the release of caspase cofactors. It also activates a mechanism of cell death that involves the opening of mPTP and subsequent MOMP (Norberg et al. 2008). Mitochondrial-targeting domain (MTD) peptide causes an increase in cytosolic calcium concentration and ultimately calcium leakage from mPTP and induces necrosis in cancer cells (Seo et al. 2009).

The last class of mitochondrial peptides is those involved in VDAC-associated mitochondrial pathways. VDAC is a group of pore-forming proteins that act as key regulators of mitochondrial metabolite flux and apoptosis. Therefore, it is related to the connection between the cytosol and the mitochondrion. VDAC activities can be modulated by the Bcl-2 family, and their effects are controversial. In general, peptides of this category cause loss of potential of the mitochondrial membrane, release of mitochondrial cytochrome c, nuclear condensation, membrane blebbing, DNA fragmentation, and decreased cellular ATP levels (Gustafsson and Gottlieb 2007). Based on the explanation given above, the development of mitochondrial peptides with a therapeutic purpose can lead to the provision of effective treatments for all types of cancers.

Clinical trials and FDA-approved ACPs

A number of peptide drugs are in various phases of clinical trials. Nerofe is a novel human hormone-peptide with anticancer activity and is in clinical phase 1. This drug is an apoptotic factor that consists of 14 amino acids and induces apoptosis in tumor cells. This apoptotic factor is administered intravenously and is in the clinical trial stage in leukemia. Research on other types of cancers, such as renal cell carcinoma, metastatic ovarian cancer, triple-negative breast cancer, metastatic colorectal carcinoma, and metastatic solid tumors, is ongoing (Sandler et al. 2010).

A peptide-based vaccine (Universal cancer peptide-based vaccine or UCPVax) is in phase 1 and 2 clinical trials that induce a CD4⁺ T-helper 1 response directed against telomerase. In trials, UCPVax was immunogenic and effective in patients with non-small cell lung cancer (NSCLC) (Adotévi et al. 2023).

Another peptide-based vaccine is in phase 1 and 2 clinical trials for prostate cancer. This vaccine targets a small GTPase (Ras homolog gene family member C or RhoC) that is overexpressed in advanced solid cancers, metastases, and cancer stem cells. Results showed that it leads to long-lasting T cell immunity in the majority of the patients (Schuhmacher et al. 2020).

For the treatment of NSCLC, a vaccine based on synthetic peptide (epitope derived from indoleamine 2, 3 dioxygenase) is in phase 1 clinical trial, which was well tolerated and did not cause severe toxicity (Iversen et al. 2014). More than 239 proteins and peptides have been approved by the US FDA for clinical usage and more than 20 of these are peptides that are used in cancer treatment (Usmani et al. 2017). In general, the number of drugs that received FDA approval in 2022 was about 50, as in 2020 and 2021 (de la Torre and Albericio 2022, 2023). Some ACPs such as Kyprolis (FDA-approval 2012), for treating multiple myeloma through inhibiting proteasome and decreasing cellular proliferation, lead to increased apoptosis in tumor cells (Pan et al. 2020). SomaKit TOC (FDA-approval 2016) acts as an anticancer peptide by binding to the somatostatin subtype 2 receptor that is overexpressed in malignant cells and controls the progression of neuroendocrine cancer (Pan et al. 2020).

Lutathera (FDA-approval 2018; NDA: 208,700) reduces free radicals produced by cancer cells by binding to the somatostatin receptor-2 (SSRT2) in malignant somatostatin receptor-positive tumors. And finally, Gallium Ga 68 DOTATOC (FDA-approval 2019; NDA: 210828) reduces somatostatin subtype 2, which is overexpressed in cancer cells (Pan et al. 2020) (Table 1).

Table 1.

In vivo and in vitro studies of ACPs peptides

Peptides	Source	Tested model	Targets	Cell type	Result	Mechanism	References
RK1	Buthus Occitanus tunetanus	In vitro [U87 (Glioblastoma) and IGR39 (Melanoma)]	It has direct effects on the ion channels (such as K+, Na+, Ca2+)	U87 (Glioblastoma) and IGR39 (Melanoma) cell lines	Cell proliferation, migration and angiogenesis of Glioblastoma and Melanoma cells decreased by using RK1	Inhibiting cell proliferation, migration and angiogenesis	Khamessi et al. (2018)
EpCAM peptide-CTLs	Cancer stem-like cells	In vitro (cell line)	Cancer stem-like cells	Breast adenocarcinoma cell line MCF-7 and the human hepatoma cell line HepG2	Enhancing effective immune responses and inhibiting tumor cells growth	High level expression of tumor-associated antigen and MHC in cancer stem cells leads to mature DCs production, T cell stimulation and generating potent CTLs	Choi et al. (2018)
Lck-486 peptide	Tyrosine-protein kinase Lck	In vitro (cell line) and in vivo (female balb/cCrlCrlj mice)	DCs	Murine rectal cancer cell line, colon 26 (H-2Ka)	Tumor cell growth inhibiting	DCs maturation was induced and suppression of tumor-infiltrating T cells, including T regulatory cells in vivo using result in tumor cell growth suppression	Matsueda et al. (2018)
Bombinin-BO1	Bombina orientalis	In vitro (cell line)	Cancerous cell membrane	Hepatoma cell lines (Hep G2/SK-HEP-1/Huh7)	It inhibits tumor cell proliferation	Selectively kill cancer cells as their hemolysis concentrations on human red cells were 52.5 and 40.3 μM	Peng et al. (2018)
Bombinin H-BO1	Bombina orientalis	In vitro (cell line)	Cancerous cell membrane	Hepatoma cell lines (Hep G2/SK-HEP-1/Huh7)	It inhibits tumor cell proliferation	Selectively kill cancer cells as their hemolysis concentrations on human red cells were 52.5 and 40.3 μM	Peng et al. (2018)
Peptide 327	A specific Eps8/EGFR inhibitor	In vitro (cell line)	Interfering with EGFR signaling by disrupting Eps8/EGFR complex formation	A549, Colo320, HepG2, SW620, HT-29, PC-3, and K562 cells	Enhance Immune response against tumor cells	It shows anti-tumor effects such as up regulation of IL-2, TNF-α, granzyme B and perforin	Xie et al. (2018)
AAP-H	Anthopleura anjunae (Sea anemone)	In vitro (cell line)	Mitochondria-mediated apoptotic pathway	Prostate cancer DU-145 cell line	Increment in mitochondrial potential and apoptosis	Apoptotic induction through pro-apoptotic proteins such as Bax, cytochrome-C, caspase-3, and caspase-9	Wu et al. (2018)

AAP-H Anthopleura anjunae anti-tumor peptide, DCs dendritic cells, TNF-α Tumor necrosis factor-alpha, EGFR Epidermal growth factor receptor, Eps8 Epidermal pathway substrate 8 gene, MHC Major histocompatibility complex, CTLs Cytotoxic T lymphocytes, IL-2 Interleukin-2, and AAP-H Anti-tumor peptide

Conclusion

ACPs are unique class of pharmaceutical compounds and are a broad spectrum of anticancer activity with characteristics including the specificity for cancer cells and ability to kill target cells rapidly. In recent years, research about ACPs due to selectivity, fewer side effects, and more effectiveness to kill cancer cells in comparison with small molecules and biologics has been given much attention. Unlike biologics, synthesis of ACPs is cheap and can decrease immunogenicity with enhanced tumor penetration. Drawbacks of peptides such as low oral bioavailability, rapid renal clearance rate and short half-life as a consequence of their relatively small size (5000 Da) which may severely hamper and slow down their use in clinical research and application. These limitations can be addressed through various chemical and physical modifications including replacement of natural amino acids, Substitution of non-natural amino acids, cholesterol modification, PEG modification cyclization, peptide stapling, and formulations. ACPs destroy cancer cells through different mechanisms including lysis of the cell membrane, apoptosis, necrotic cell death, inhibition of tumor angiogenesis, autophagy, and regulation of the immune system. A number of ACPs such as cancer vaccines, peptide drug conducting, and anti-angiogenic peptides are currently in preclinical and clinical trials (Table 2). In addition, by 2020, more than 20 ACPs have been approved by FDA and European Medicines Agency (EMA) (Table 3).

Table 2.

ACPs in clinical trials

(Source: https://www.clinicaltrials.gov/)

Peptide	Type of cancer	Result	Phase	NCT number
MUC1 peptide vaccine, poly-ICLC, MUC1 peptide-poly-ICLC adjuvant vaccine	Breast cancer	It shows positive anti-MUC1 antibody response	Early phase	NCT00986609
HER‑2/neu peptide vaccine	Breast cancer	Enhances Immune response by producing peptide-specific interferon-γ T cell and peptide-specific IL-5	Early phase 1	NCT01729884
GAA/TT-peptide vaccine and poly-ICLC	Astrocytoma, oligoastrocytoma, glioma	Enhances Immune response through Induction of GAA-specific T cell response	Early phase 1	NCT00795457
Peptide vaccine + poly-ICLC	Astrocytoma, oligoastrocytoma, glioma	Enhances Immune response by infiltration of GAA‑specific T cells	Early phase 1	NCT00795457
Gag:267–274 peptide vaccine	Melanoma	Results in vaccine peptide-specific CTL response	Early phase 1	NCT01748747
HPV16 E7 peptide-pulsed autologous DCs	Cervical cancer	Improving immune response by induction of T cell in late stage of cervical cancer	Phase 1	NCT00155766
NY-ESO-1b peptide plus CpG 7909 and Montanide ISA-5	Cancer, neoplasm	Boosting NY-ESO-1 hormonal and cellular immunity	Phase 1	NCT00199836
Anti-angiogenic peptide vaccine	Hepatocellular carcinoma	Induction of peptide specific CTL response	Phase 1	NCT01266707
RNF43-721	Colorectal cancer	In vitro specific CTL response was observed	Phase 1	NCT00641615
LY6K, VEGFR1, VEGFR2	Esophageal cancer	Peptide causes improvement in immune response such as LY6K, VEGFR1/2 specific T cells	Phase 1	NCT00561275
HLA-A*0201 or HLA-A*0206-restricted URLC10 peptides	Non-small cell lung cancer	Enhancement in immune response consist of specific CTL, antigen cascade, regulatory T cells, cancer antigens and HLA levels	Phase 1	NCT01069640
MAGE-3.A1 peptide and CpG 7909	Malignant melanoma	This peptide cause CTL response	Phase 1/Phase 2	NCT00145145
VEGFR1-1084, VEGFR2-169	Pancreatic cancer	Inducing peptide specific CTL response	Phase 1/Phase 2	NCT00655785
HER-2/neu peptide vaccine	Breast cancer	Enhancing immune response by stimulating of T cells	Phase 1/Phase 2	NCT01729884
HLA-A*2402 or A*0201 restricted peptide	Solid tumors	It causes variety immune response such as inducing CTL, antigen cascade and regulatory T cells	Phase 1/Phase 2	NCT01949688
Modified CEA peptide	Pancreatic adenocarcinoma	It causes T cell response	Phase 1/Phase 2	NCT00203892
Synthetic human papillomavirus 16 E6 peptide	Cervical cancer	Improve immune response to HPV	Phase 2	NCT00002916
WT1 126–134 peptide	Acute myeloid leukemia	Using this peptide result in T cell immune response	Phase 2	NCT00153582
G250 peptide	Metastatic renal cell carcinoma	Peptide specific CTL response	Phase 2	NCT00203866
Melanoma helper peptide vaccine, multi-epitope melanoma peptide vaccine	Melanoma	Specific CTL response and inducing T-helper cell response	Phase 2	NCT00071981
PR1 leukemia peptide vaccine	Leukemia	Effectively improving immune response	Phase 3	NCT00454168
Degarelix (LHRH antagonist)	Prostatic neoplasms	Blocking GnRH	Phase 4	NCT02475057

CTL Cytotoxic T cell lymphocytes, GnRH, Gonadotropin-releasing hormone, MUC1 Mucin factor receptor, MAGE-3 Melanoma-associated antigen 3, RNF Ring finger protein, NY-ESO New York esophageal squamous cell carcinoma, carcinoembryonic antigen, HPV Human papillomavirus, HER Human epidermal growth factor receptor, VEGFR Vascular endothelial growth

Table 3.

List of ACPs approved by FDA and EMA

Peptide	NDA/BLA	Indications	Year, FDA-approved	Features	Category	References
Ixazomib	NDA: 208,462	Multiple myeloma	2015	N-Acylated, C-boronic acid dipeptide	Free peptide	Gentile et al. (2015)
KYPROLIS (CARFILZOMIB)	NDA: 202,714	Multiple myeloma	2012	Modified tetrapeptidyl epoxide	Free peptide	Herndon et al. (2013)
VANTAS (HISTRELIN ACETATE)	NDA: 021732	Prostate cancer	2004	GnRH )Gonadotropin-releasing hormone agonist	Free peptide	Adis R&D Profile (2005)
Romidepsin	NDA: 208,574	T cell lymphoma	2009	A bicyclic depsipeptide with HDI	Free peptide	Whittaker et al. (2010)
Thymalfasin	Not FDA Approved	Hepatocellular carcinoma	2002	A chemically synthesized version of thymosin alpha 1	Free peptide	Gish et al. (2009)
Mifamurtide	Not FDA Approved	Osteosarcoma	2009	Liposomal muramyl tripeptide phosphatidyl ethanolamine	Free peptide	Meyers (2009)
KIMMTRAK (Tebentafusp)	BLA: 761,228	Melanoma	2022	A gp100 peptide-HLA-directed CD3 T cell engager	Free peptide	Dhillon (2022)
Lutathera (LUTETIUM LU 177 DOTATATE)	NDA: 208,700	Neuroendocrine tumors	2018	177Lu chelated by DOTA bound to Tyr3-octreotate	PDC	Das et al. (2019)
GALLIUM GA 68 DOTATOC	NDA: 210,828	Neuroendocrine tumors	2019	68 Ga chelated by DOTA bound to Tyr3-octreotide	PDC	Hennrich, Benešová (2020)
Melphalan flufenamide (Melflufen)	NDA: 214,383	Multiple myeloma	2021	Targets aminopeptidases. Rapidly releases alkylating agent into cancer cells	PDC	Mateos et al. (2020)
PADCEV® (Enfortumab Vedotin-Ejfv)	BLA: 761,137	Cancers expressing Nectin-4	2019	ADC with a synthetic analog of the marine natural peptide dolastatin 10	ADC	Raedler
Polivy® (Polatuzumab Vedotin-Piiq)	BLA: 761,121	Diffuse large B cell lymphoma	2019	ADC with a synthetic analog of dolastatin 10 (5-residue peptide alcohol)	ADC	Deeks (2019)
Padcev™ (Enfortumab vedotin-ejfv)	BLA: 761,137	Urothelial cancers	2019	Containing a human monoclonal antibody AGS-22 targeting the cell adhesion molecule nectin-4 and conjugated to the cytotoxic agent MMAE, via a proprietary enzyme-cleavable linker AGS-22CE	ADC	Chang et al. (2021)
TIVDAK (Tisotumab vedotin-TFTV)	BLA: 761,208	Recurrent or metastatic cervical cancer	2021	ADC with antibody moiety is conjugated to monomethyl MMAE via a valine citrulline linker	ADC	Markham (2021)
Zynlonta™) Loncastuximab tesirine-lpyl)	BLA: 761,196	Relapsed or refractory large B cell lymphoma	2021	a CD-19-directed antibody and alkylation agent conjugate	ADC	Lee (2021)

BLA biologic license application, GnRH gonadotropin-releasing hormone, HDI histone deacetylase inhibitor, ADC antibody–drug conjugates, MMAE monomethyl auristatin E, NDA new drug application

In conclusion, with the rapid developments in bioinformatics, proteomics, modification strategies, and peptide libraries, ACPs have emerged as novel promising anticancer peptide drugs or vaccines to decrease emerging cases and mortality rates of cancers in the future.

Abbreviations

ACPs

Anticancer peptides

PDCs

Peptide–drug conjugates

HNP-1

Human neutrophil peptide

LfcinB

Lactoferrin

GHRH

Growth hormone-releasing hormone

STAT3

Signal transducer and activator of transcription 3

GSH

Glutathione

ADCs

Antibody–drug conjugates

mAb

Monoclonal antibody

USFDA

US Food and Drug Administration

BBB

Blood–brain barrier

SORT1

Sortilin 1

DTX

Docetaxel

NSCLC

Non-small cell lung cancer

HDM-2

Human double minute 2

Apaf-1

Apoptotic protease-activating factor-1

ROS

Reactive oxygen species

TP53

Tumor protein 53

AIF

Apoptosis-inducing factor

EndoG

Endonuclease G

PLP2

Proteolipid protein 2

DAMPs

Damage-associated molecular patterns

HMGB1

High-mobility group protein B1

VEGF

Vascular endothelial growth factor

PDGF

Platelet-derived growth factor

FGF

Fibroblast growth factor

CK2

Casein kinase 2

MMP

Matrix metalloproteinase

VEGF-A

Vascular endothelial growth factor

ALA-A2

Alpha-lactalbumin-A2

IC50

Half maximal inhibitory concentration

DCs

Dendritic cells

Treg

Regulatory T cell

IFN-γ

Interferon-gamma

HLA

Human leukocyte antigen

IL-2

Interleukin-2

PEG

Polyethylene glycol

MOMP

Mitochondrial outer membrane permeabilization

Cyt c

Cytochrome c

AIF

Apoptotic-inducing factor

mPTP

Mitochondrial permeability transition pore

IMM

Inner mitochondria membrane

VDAC

Voltage-dependent anion channels

MTD

Mitochondrial-targeting domain

UCPVax

Universal cancer peptide-based vaccine

RhoC

Ras homolog gene family member C

SSRT2

Somatostatin receptor-2

Author contributions

VAP presented the idea. YN prepared the figures. The article was jointly written by VAP, KHRJ, MSh, YN, and AT under the supervision of AT, VR, and JF. AT drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The authors declare that no funds or grants were received during the preparation of this manuscript.

Data accessibility

This article does not contain any additional data.

Declarations

Conflict of interest

All authors declare that they have no competing interest in this article.