Introduction

Proteins are one of the main nutrients for the requirement of humans and possess functions of promoting growth and development, strengthening immunity, and being involved in metabolism (Kitada et al., 2019). Some special proteins also have particular abilities, such as anti-tumor, immunomodulation, anti-oxidant, and anti-thrombosis, and are termed bioactive proteins (Auestad and Layman, 2021). Proteins are hydrolyzed to polypeptides. These polypeptides commonly contain 2–30 amino acids and have multiple functions determined by their amino acid sequence, being called bioactive polypeptides (Chakrabarti et al., 2018; Chelliah et al., 2021). They have good health effects such as lowering blood pressure, antibacterial activity, anti-tumor, and anti-inflammation. It has been found that bioactive proteins/polypeptides have remarkable anti-tumor effects. For example, fungal immunomodulatory proteins (FIPs) are a kind of bioactive proteins from fungi such as Ganoderma lucidum and Flammulina velutipes (Li et al., 2010a, 2011a), and have inhibitory effects against multiple cancer cells such as gastric, liver and breast cancer cells (Li et al., 2011b, 2019b).

Cancer is a major health problem in the world. Breast cancer is the most commonly diagnosed cancer with 2.26 million new cases worldwide in 2020 (Ferlay et al., 2021). Despite some clinical success, high incidence of recurrence and metastasis still lead to high mortality in breast cancer patients (Akram et al., 2017). Some chemotherapeutic drugs with high doses have serious side effects and drug resistance in the therapy of breast cancer (Al-Mahmood et al., 2018). Thus, it is of great importance to discover and identify more effective agents with fewer side effects (Hu et al., 2019). Natural products are a precious resource for developing and discovering antitumor drugs due to multi-targeting efficacy, no toxicity, and no drug resistance (Hashem et al., 2022; Ullah et al., 2022). Moreover, it has been demonstrated that naturally bioactive products possess abilities in the prevention and treatment of breast cancer (Bak et al., 2016). This review aims to discuss the potential of bioactive proteins/polypeptides in the prevention and treatment of breast cancer.

Acquirement of bioactive proteins/polypeptides

Proteins and polypeptides are the material basis of life. Bioactive proteins/polypeptides are special proteins and polypeptides with particular physiological functions. Bioactive proteins/polypeptides originate mainly from meat, milk, cereal, fish, seaweeds, vegetables, and fungi (Kaur et al., 2021). The most common methods to produce proteins/polypeptides include enzyme hydrolysis and microbial fermentation (Daliri et al., 2017). Besides, a PepSAVI-MS (statistically-guided bioactive peptides prioritized via mass spectrometry) pipeline is developed for bioactive peptide discovery (Kirkpatrick et al., 2017). Using this pipeline, some bioactive peptides are successfully identified from biological species such as Enterococcus faecalis (Kirkpatrick et al., 2018b), Ustilago maydis (Kirkpatrick et al., 2018a), Viola odorata (Parsley et al., 2018) and Amaranthus tricolor (Moyer et al., 2019).

Mechanism of breast cancer suppression by bioactive proteins/polypeptides

Bioactive proteins/polypeptides have potential medicinal values, especially anti-tumor effects (Li et al., 2011b; Wu et al., 2014). Some bioactive proteins/polypeptides from plants, animals, and microbes can inhibit breast cancer cell growth (Tables 1 and 2) based on mechanisms of migration and invasion inhibition, apoptosis induction, and cell cycle arrest (Fig. 1).

Table 1 Anti-tumor and other activities of bioactive proteins/polypeptides in the cell models of human breast cancer
Full size table
Table 2 Anti-tumor activities of bioactive proteins/polypeptides in the animal models of breast cancer
Full size table
Fig. 1
figure 1

Possible anti-tumor mechanisms of bioactive proteins/polypeptides against breast cancer. Akt, PKB protein kinase B; Bad Bcl-xL/Bcl-2-associated death promoter; Bak Bcl-2 antagonist killer 1; Bax Bcl-2-associated X protein; Bcl-2 B cell lymphoma 2; Bcl-xL B-cell lymphoma extra-large; Bid BH3-interacting domain death agonist; CCR10 chemokine receptor type 10; CD147 cluster of differentiation 147; CD151 cluster of differentiation 151; CD31, PECAM-1 platelet endothelial cell adhesion molecule-1; CHOP C/EBP homologous protein; COX-2 cyclooxygenase 2; ECM extracellular matrix; EGFR epidermal growth factor receptor; ER endoplasmic reticulum; ERK extracellular signal-regulated kinases; FAK focal adhesion kinase; FoxO3a forkhead box O3a; GSK3β glycogen synthase kinase 3-β; IL-6 interleukin 6; JAK2 Janus protein tyrosine kinase 2; MAPK mitogen-activated protein kinase; MEK mitogen-activated protein kinase kinase; MMPs matrix metalloproteinases; PI3K phosphatidylinositol 3-kinase; PTEN phosphatase and tensin homolog; Ras rat sarcoma virus; ROS reactive oxygen species; Src proto-oncogene tyrosine-protein kinase Src; STAT3 signal transducer and activator of transcription 3; tBid truncated Bid

Full size image

Inhibition of migration and invasion

Cancer cells are capable of migration and invasion to spread within tissues (Chambers et al., 2002). Mechanisms of the migration include extracellular matrix (ECM) degradation, which needs corresponding enzymes, such as matrix metalloproteinases (MMPs) (Alaseem et al., 2019). MMPs are zinc-dependent endopeptidases and contribute to the pathogenesis of various diseases (Bassiouni et al., 2021). Platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31) is a 130-kDa highly glycosylated transmembrane member of the Ig superfamily (Caligiuri, 2020) and mediates cancer metastasis by activating integrins (Zhang et al., 2018). Tetraspanins CD151, a transmembrane 4 superfamily protein, regulate the epidermal growth factor receptor (EGFR)/focal adhesion kinase (FAK) signaling pathway by affecting integrins (Zhu et al., 2021). Integrins comprise a family of 24 heterodimeric receptors formed by 18 α-subunits and eight β-subunits (Desgrosellier and Cheresh, 2010). Distinct integrin heterodimers are in specific cancer cells. For example, α6β4 and ανβ3 are expressed in breast cancer. Integrins bind to ECM proteins and activate the FAK and proto-oncogene tyrosine-protein kinase Src (Src) family kinase (SFK) signaling, then impinging on the rat sarcoma virus (Ras)/extracellular signal-regulated kinases (ERK) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways (Cooper and Giancotti, 2019). The pathways contribute to the regulation of nuclear factor-kappaB (NF-κB) activity (Mali et al., 2018; Mao et al., 2019), promoting the expressions of MMP-2 (Tao et al., 2018), -7 (Liu et al., 2017a) and -9 (Tan et al., 2022). Iturin A is a lipopeptide consisting of a hydrophilic peptide moiety linked to a hydrophobic fatty acid chain and is purified from a marine bacterium Bacillus megaterium (Dey et al., 2015). Zhang et al. isolated water-soluble polypeptides from a traditional Chinese pharmaceutical and functional food Pilose antler, named PAWPs (Zheng et al., 2020). In xenograft models, both iturin A and PAWPs reduce CD31 expression, leading to metastasis inhibition (Dey et al., 2015; Zheng et al., 2020). An important characteristic of iturin A is amphiphilic. Zhao et al. found that the hydrophilic peptide (NYNQPNS) showed no effect on HepG2 cells proliferation, suggesting that the fatty acid chain plays an important role in its activity (Zhao et al., 2021). Using the FireDock web server and immunoprecipitation assay, it is found that a marine natural metabolite Cyclo (l-Leucyl-l-Prolyl) peptide (CLP) from Streptomyces mangrovisoli has a strong interaction with CD151 and reduces CD151 expression (Kgk et al., 2020). Melittin is the main component of bee venom (Raghuraman and Chattopadhyay, 2007), and inhibits the activation of FAK and MMP-9 by blocking the PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway (Jeong et al., 2014). Melittin consists of 26 amino acids (GIGAVLKVLTTGLPALISWIKRKRQQ) with a hydrophobic N-terminal and a hydrophilic C-terminal (Raghuraman and Chattopadhyay, 2007). Melittin with the total + 6 charges at physiological pH binds to negatively charged membrane surface and then disturbs the membrane integrity, ultimately leading to cell lysis (Jamasbi et al., 2016), and is also considered as a promising candidate for cancer chemotherapy (Schweizer, 2009). Lunasin, a 43-amino acid peptide isolated from soybean seed (Galvez and de Lumen, 1999), is divided into four regions: N-terminus, central part, RGD motif and C-terminus (Jones and Srivastava, 2014). The full sequence of lunasin exerts cytotoxicity to MDA-MB-231 cells through inhibition of H3 and H4 acetylation (Hernandez-Ledesma et al., 2011), while the RGD motif involves in cancer metastasis (Nieberler et al., 2017). It can also inhibit the phosphorylation of FAK, Src, Akt, and ERK, suppress the nucleus translocation of NF-κB, and reduce the activity and expression of MMP-2 and -9, resulting in the inhibition of invasion of MCF-7 and MDA-MB-231 cells (Jiang et al., 2016). This mechanism might involve that lunasin interacts with integrins, leading to the suppression of the signaling axis (Vuyyuri et al., 2018). Besides, an earthworm fibrinolytic protein (EFP) isolated from Eisenia fetida inhibits FAK expression and the migration of MCF-7 cells (Chen, 2014).

A glycoprotein cluster of differentiation 147 (CD147) is an MMP inducer and as well as promotes interleukin (IL)-6 production via NF-κB (Dana et al., 2021). IL-6 primarily activates Janus protein tyrosine kinase (JAK) 1 and JAK2 to drive signal transduction, including the activity of signal transducer and activator of transcription (STAT), mitogen-activated protein kinase (MAPK), and PI3K-Akt (Kang et al., 2020). The activated STAT3 increases the expression of MMP-2, -7, and -9 (Banerjee and Resat, 2016; Cao et al., 2022). Melittin downregulates CD147 and MMP-9, leading to inhibiting MCF-7 cell invasion (Wang et al., 2017). Ilamycins are a series of cyclic peptides and are isolated from the deep South China Sea-derived S. atratus SCSIO ZH16 and engineered mutant strains (Ma et al., 2017). Ilamycin C can inhibit the invasion and migration of MDA-MB-231 cells via decreasing MMP-2 and MMP-9 by suppressing IL-6-induced STAT3 phosphorylation (Xie et al., 2019).

Chemokine receptor type 10 (CCR10) is a member of the chemokine receptor subfamily and promotes cancer cell invasion and migration through the ERK and PI3K/Akt signaling pathway with the regulation of MMPs (Lin et al., 2017; Liu et al., 2021). A modified linear peptide brintonamide D from samples of intertidal cyanobacterial mats can be used as a CCR10 antagonist to reduce the proliferation and migration of MDA-MB-231 cells (Al-Awadhi et al., 2018). Brintonamide D forms hydrogen bonds with Arg247, Arg322 and Arg345 of CCR10 and the aromatic ring on the modified side of brintonamide D has π–π interaction with residue Tyr227 in the middle of the CCR10 β-sheet.

Inducing apoptosis

Apoptosis is a kind of regulated cell death process (Carneiro and El-Deiry, 2020). Cancer cells are capable of evading apoptosis (Brown and Attardi, 2005). Reactive oxygen species (ROS) increase the levels of phosphatase and tensin homolog (PTEN), as a tumor suppressor gene, negatively regulating PI3K/Akt pathway (Wang et al., 2020, 2022), thereby inducing caspases-dependent apoptosis through itself, forkhead box O3a (FoxO3a) (Yan et al., 2020), and glycogen synthase kinase 3-β (GSK3β) (Guo et al., 2020). Members of the caspase family participate in the initiation and execution of apoptosis (Boice and Bouchier-Hayes, 2020). In MCF-7 and MDA-MB-231 cells, roe protein hydrolysate prepared from defatted Alburnus tarichi roe powder causes the significant production of intracellular ROS, and significantly increases the expressions of caspase-3, -7, -8, and -9, inducing apoptosis (Berkoz et al., 2020). In MDA-MB-231 cells, lunasin does not exhibit the effect of apoptosis, but it promoted aspirin-induced apoptosis (Hsieh et al., 2010). While, in MCF-7 cells, lunasin upregulates PTEN promoter activity, increases PTEN transcript and protein levels, and enhances nuclear PTEN localization, leading to cellular apoptosis (Pabona et al., 2013). A small molecular oligopeptide, sea cucumber intestinal peptide (SCIP) with being rich in hydrophobic amino acids (Ala, Val, Leu, Gly, Phe, and Met) and branched-chain amino acids (Val, Ile, and Leu), is extracted from sea cucumber intestines and promotes the apoptosis of MCF-7 cells through the inactivation of PI3K/Akt signaling pathway with elevating the expression of cleaved caspase-9 and -3 (Wei et al., 2021). But the detailed mechanism remains unclear. In MDA-MB-231 and MCF-7 cells, the apoptotic effect of iturin A is attributed to the Akt-mediated FoxO3a and GSK3β (Dey et al., 2015). Although the activation of caspases is a feature of apoptosis, HN-1, a naturally occurring host defense peptide identified from Amolops hainanensis, induces caspase-independent apoptosis (Qiao et al., 2019).

Cyclooxygenase 2 (COX-2) is an inducible enzyme compared with constitutive COX-1 and is often overexpressed in breast cancer with poor survival (Ristimaki et al., 2002). COX-2 binds with and inactivates p53 (Feng et al., 2019), which regulates apoptotic genes directly and indirectly (Mihara et al., 2003; Hemann and Lowe, 2006). B cell lymphoma 2 (Bcl-2) gene family, including pro-apoptotic members such as Bcl-2-associated X protein (Bax), Bcl-2 antagonist killer 1 (Bak), Bcl-2-related ovarian killer (Bok), Bcl-2-interacting mediator of cell death (Bim), BH3-interacting domain death agonist (Bid), B-cell lymphoma extra-large (Bcl-xL)/Bcl-2-associated death promoter (Bad), and p53 upregulated modulator of apoptosis (Puma), and anti-apoptotic members such as Bcl-2, Bcl-xL, Bcl-2-like protein 2 (Bcl-w), and mantle cell lymphoma 1 (Mcl-1), has a significant role in regulating apoptosis (Ashkenazi et al., 2017). The activation of caspases is mainly regulated by the Bcl-2 family (Tzifi et al., 2012). It has been found that in MCF-7 cells, the roe protein hydrolysate downregulates COX-2 level (Berkoz et al., 2020). In MCF-7 and MDA-MB-231 cells, tuna cooking juice hydrolysate by protease XXIII (PA) with > 2.5 kDa ultrafiltration fraction (PAH2.5) (Hung et al., 2014), CPe-III derived from chickpea albumin hydrolysate (Xue et al., 2015), and AtMP1 and AtMP2 identified from Anabas testudineus antimicrobial peptides (Najm et al., 2021) significantly increase the expressions of p53 and Bax, decrease the level of Bcl-2 which could be overwhelmed by Bax (Yin et al., 1997), and upregulate caspases levels. HN-1 also activates p53 and induces a p53-dependent increase of Bax/Bcl-2 ratio in xenograft tumors (Qiao et al., 2019). Molecular docking analysis showed that Arg1, Gln2, Ala6, Ala8, and Gln9 of CPe-III combine the DNA binding domain of p53 protein (Thr102, Leu111, Asn131, Gln144, Asp228, and Asn268) by hydrogen bonds, resulting in the induction of p53 expression (Xue et al., 2015). Najm et al. found that hydrogen bonds formed between AtMP1/AtMP2 and p53, Bax, Bcl-2, or caspases (Najm et al., 2021). Balsamin is a type I ribosome-inactivating protein purified from Momordica balsamina (Kaur et al., 2012). It increases the expressions of Bax, Bid, and Bad, reduces the levels of Bcl-2 and Bcl-xL, and increases the activities of caspase-3 and -8 in MCF-7 and BT549 cells (Ajji et al., 2017). Halilectin-3 containing three subunits is isolated from Haliclona caerulea (Carneiro et al., 2013), and induces MCF-7 apoptosis with a decrease of Bcl-2 and an increase of caspase-9 (do Nascimento-Neto et al., 2018). Both the α-chain and β-chain of halilectin-3 have N-glycosylation sites with affinity to N-acetylgalactosamine (GalNAc) (Carneiro et al., 2013), thereby recognizing abnormal expressing GalNAc-containing antigen on MCF-7 cells (do Nascimento-Neto et al., 2018). α-Lactalbumin, β-lactoglobulin, and lactoferrin, nutritional components in milk and dairy products, downregulate Bcl-2 and upregulate Bax, leading to increasing caspase-3 in MDA-LM2 cells (Li et al., 2019a). Melittin increases the levels of Bax, caspase-3, and -8 in MDA-MB-231 cells (Daniluk et al., 2019; Mir Hassani et al., 2021). A cytotoxic protein fraction F5 is isolated and is mainly composed of 97.29% serine protease encoded by GME4347_g (Yap et al., 2018). The F5 induces the increases of Bax, Bid, and cleaved Bid, and the decrease of Bcl-2, leading to the upregulation of caspase-8 and -9 activities.

C/EBP homologous protein (CHOP) is a pro-apoptotic endoplasmic reticulum (ER) stress marker, which can be regulated by ROS-mediated ER stress (Zhu et al., 2022), subsequently regulating Bcl-2/Bax (Li et al., 2019c; Liu et al., 2020). WSPF is a novel protein fraction isolated from Withania somnifera roots and induces apoptosis of MDA-MB-231 cells through the production of extensive ROS, leading to reducing Bcl-2 expression, increasing Bax expression, and elevating cleaved caspase-3 expression (Dar et al., 2019). Ilamycin E is another cyclic peptide (Ma et al., 2017). It activates ER stress, increases CHOP, and downregulates Bcl-2, which promotes apoptosis in HCC1937 and MDA-MB-468 cells (Zhou et al., 2019).

Bcl-2 can also be regulated by STAT3 (Liu et al., 2017b). Ilamycin C promotes Bax/Bcl-2-related caspase-dependent apoptosis through IL-6/JAK2/STAT3 as well (Xie et al., 2019).

Cell cycle arrest

The cell cycle is a tightly regulated process including Gap 1 (G1), DNA-synthesis (S), Gap 2 (G2), and mitosis (M) phases. The core cell-cycle proteins are frequently dysregulated in human cancers, and targeting these proteins seems to represent an effective strategy for inhibiting tumors (Suski et al., 2021). Mitogenic signals upregulate cyclin D, which binds and activate cyclin‐dependent kinase (CDK) 4 or CDK6 to drive the progression from the G0 or G1 into the S phase. Degradation of cyclin D is a promising targeted therapy for the cancer cell cycle (Caudron-Herger and Diederichs, 2021; Chaikovsky et al., 2021; Maiani et al., 2021; Simoneschi et al., 2021). p21 and p27 are capable of inhibiting cyclin/CDK complexes comprising CDK1 or 2 and cyclin A; CDK1 and CDK2 can be activated by cyclin A which has a critical role in the S and G2-M phase (Suski et al., 2021; Garcia-Osta et al., 2022). Ilamycin E decreases Cyclin D1, and increases p21 and p27 levels, thereby inducing G1/S cell cycle arrest in HCC1937 and MDA-MB-468 cells (Zhou et al., 2019). PAH2.5 increases p21 and p27 protein expression and decreases cyclin A expression, which induces cell cycle arrest in the S phase in MCF-7 cells (Hung et al., 2014).

Conclusions and perspectives

Breast cancer is the leading cause of cancer death among women (Ghoncheh et al., 2016). It is a hormone-dependent tumor (Russnes et al., 2017), and its targeted therapies include human epidermal growth factor receptor 2-targeted agents and endocrine therapy (Turashvili and Brogi, 2017). However, drug resistance is still a major challenge in the treatment of breast cancer (Karami Fath et al., 2022). Bioactive proteins/polypeptides exhibit a great potential against cancers, including breast cancer. Identification of novel bioactive proteins/polypeptides and development of novel functions of the existing bioactive proteins/polypeptides are necessary. Generally, protein is digested and absorbed in the form of amino acids from diet, although proteins with medicinal value still have bioactivity (Lee et al., 2018). Treatments of iturins (Zhao et al., 2018), FIP-gmi (Hsin et al., 2020) and PAWPs (Zheng et al., 2020) by gavage still exhibit bioactivity without side effects. On the other hand, unfortunately, some proteins/polypeptides such as melittin exhibits extensive hemolysis and cytotoxicity, which may limit its application in clinical practice (Askari et al., 2021). Thus, the pharmacokinetics and pharmacodynamics of bioactive proteins/polypeptides have been well studied further. At the same time, it is critical to modify the proteins/polypeptides to enhance their effects (Berdan et al., 2021). Several analogues of galaxamide which is isolated from Galaxaura filamentosa are synthesized (Xu et al., 2008; Lunagariya et al., 2017). These analogues exhibit greater excellent toxicity toward breast cancer cells. Whey protein isolates modified with rosmarinic acid at alkaline conditions exhibits enhanced antioxidative capacity (Ali et al., 2018). The covalent complex of soy protein isolates and epigallocatechin gallate has higher thermal stability and oxidation resistance and a polyphenol-protective effect (Zhou et al., 2020a). A peptide drug conjugate named TAMpepK, consisting of melittin and a pro-apoptotic peptide, targets M2-like tumor-associated macrophages, thereby inhibiting breast cancer metastasis in the mouse model (Lee et al., 2022). In the previous study, we found that N-glycosylation significantly improves the functional properties of FIP-glu (Li et al., 2021a). Besides, combination regimen-based therapies are promising strategies (Li et al., 2021b). For example, Co-treatment of melittin and hormone therapeutic drugs (Yen et al., 2022), drugs with anti-tumor potential (Duarte et al., 2022), agents (Shaw et al., 2019), and miRNAs (Motiei et al., 2021) reveals synergistic effects in breast cancer cells. Bioactive proteins/polypeptides are suitable for gene therapy, a promising strategy for cancer treatment (Zhou et al., 2020b). The anti-tumor research of bioactive proteins/polypeptides is mainly performed in cell and animal models. However, relevant clinical studies are rare (Table 3). Although the anti-tumor mechanisms of bioactive proteins/polypeptides have been actively studied, there are still problems in the production, administration and regulation of bioactive proteins/polypeptides (Chakrabarti et al., 2018). Therefore, further studies are needed to evaluate the physiological efficacy of these bioactive proteins/polypeptides in human clinical studies. To summarize, bioactive proteins/polypeptides have impressive anti-tumor effects against breast cancer. It is suggested that bioactive proteins/polypeptides with great potential are promising agents for the treatment of breast cancer.

Table 3 Clinical research of bioactive proteins/polypeptides
Full size table