Anticancer potential of lactoferrin: effects, drug synergy and molecular interactions

Abstract

Cancer treatment is among today’s most active and challenging research fields. In recent years, significant progress has been made in developing new cancer therapies, including nutraceuticals and natural compounds with anticancer properties. Lactoferrin, a glycoprotein present in mammals, is of significant interest due to its pleiotropic behavior, demonstrating a broad spectrum of biological activities such as antimicrobial, antioxidant, anti-inflammatory, immunomodulatory, and anticancer effects. In this review, we examine the current knowledge of Lf’s role in cancer. In addition, it exhibits a synergistic effect along with conventional drugs, potentially enhancing their efficacy and, at the same time, reducing the side effects associated with most traditional therapies. However, it is essential to consider the precise molecular mechanism by which Lf exerts its antitumor activity. Searching interactions with several molecules can provide insight into this mechanism. Additionally, finding lactoferrin receptors can improve the strategies for the specific release of the conjugates. For all these reasons, Lactoferrin becomes a potential therapeutic agent that should be examined in depth.

Introduction

Cancer is becoming one of the primary cause of death worldwide; in 2022, roughly 20 million new cases were diagnosed, and almost 10 million people died because of this pathology, it is estimated that by 2045, this figure will increase to 16 million deaths (Bray et al. 2024). Developing non-toxic or natural-derived products against cancer and improving therapies have become among the most researched fields (Chhikara and Parang 2022). Lactoferrin (Lf), a cationic glycoprotein from milk, has been of great interest as a potential therapeutic agent for many health problems due to its multifaceted nature of biological properties, including its activity against cancer; it can reduce proliferation, survival, migration, and metastasis of cancer cells through the modulation of genes involved in the cell cycle and apoptosis (Kaczyńska et al. 2023). These functions can be linked to its capacity for iron binding, glycosylated structure, local microenvironment, interactions with cellular receptors, and ability to disrupt the cytoskeleton, altering microfilament architecture (Cao et al. 2023; Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021). However, detailed molecular mechanisms are still unknown.

One of the limitations of the treatments is the delivery of the drug and the traditional drugs cannot recognize normal cells from cancerous cells. Lf has emerged as a promising molecule for developing nanocarriers and improving the current therapies (Ramezani et al. 2023). This review will examine the role of Lf in cancer inhibition, explore the more recent advances in drug-Lf conjugated nanosystems, and the importance of searching for receptors of Lf to achieve maximum efficacy within the targeted delivery system. The aim is to provide a detailed overview of Lf’s relevance in anticancer therapy.

Lactoferrin and cancer

Lf is a non-hematic iron-binding glycoprotein found for the first time in bovine milk in 1939 (Sorensen and Sorensen 1939) and subsequently isolated from human milk in 1960 (Johanson 1960). Lf is present in exocrine fluids, including saliva, tears, bile, and sweat, and mainly in milk and colostrum of mammalians, reaching concentrations of 2 and 7 g/L, respectively. Another source of Lf is secondary granules of neutrophils, which are released into the blood during an infectious or inflammatory process. Thus, Lf is a host defense protein (Sienkiewicz et al. 2022) Lf is composed of a single polypeptide chain comprising roughly 700 amino acids, and it has a molecular weight of 77–80 kDa with a high homology among species. Lf has been recognized as a multifunctional glycoprotein with nutraceutical properties due to its antibacterial, antiviral, antifungal, antiinflammatory, immunomodulatory, and anticancer activities (Rascón-Cruz et al. 2021).

The anticancer activity of Lf was discovered in the 1990s by Bezault et al. (1994) the administration of hLf inhibited the tumor growth of melanoma and fibrosarcoma from murine models in both cases (Bezault et al. 1994). Since then, numerous studies have been conducted on different types of cancer, both in vivo and in vitro, to comprehend the underlying mechanism of this activity. It is well known that Lf exogenous inhibits cell proliferation, angiogenesis, metastasis, and induces apoptosis in cancer cells. These activities can be carried out indirectly by modulating the immune response or directly interacting with the cell (Ramírez-Rico et al. 2022; Siqueiros-Cendón et al. 2014). Moreover, iron is a requirement for cancer cells. Lf’s capacity to chelate iron ions may be one of the mechanisms by which it induces its anti-cancer cytotoxic effects (Rascón-Cruz et al. 2021).

One of the surprising traits of Lf as an anticancer agent is its selectivity. Lf exhibits cytotoxicity on cancer cells while having any or minimum effect on normal cells at equal concentrations (Guedes et al. 2018; Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021; Olszewska et al. 2022). This selectivity could be associated to the electrostatic attraction between the positively-charged Lf and the negatively-charged membrane (Barragán-Cárdenas et al. 2020), mainly due to the union with sialic acid, glycosaminoglycans (GAGs) and proteoglycans, which are overexpressed in cancer cells (Mann et al. 1994; Nakamura et al. 2023). In addition, phosphatidylserine exposed in the outer leaflet of the membrane of non-apoptotic tumor cells has also been proposed as a target of Lf selectivity (Barragán-Cárdenas et al. 2020; Riedl et al. 2015).

This protein not only serves as an anticancer agent but also as a chemopreventive compound, which helps to prevent, delay, and reduce the risk of developing cancer, thereby decreasing the incidence and improving the potential of curative treatment of this disease. Using mouse models with induced hepatocellular carcinoma demonstrated that pre-treatment with iron-saturated bLf (Holo-bLf) prevented and reduced necrosis (associated with early stages of carcinogenesis), reactive oxygen species (ROS), and the formation of precancerous lesions (Hernández-Galdámez et al. 2024). In human beings, in a clinical trial with patients with colorectal polyps, oral-administrated bLf reduced the number and size of polyps in the early stages of their development since the removal of colorectal polyps is used as a preventive measure against cancer, bLf is potentially as a chemopreventive agent (Kozu et al. 2009).

These effects have mainly been attributed to the administration of exogenous Lf. In fact, the Lf gene (LTF) shows a decreased expression in various types of cancer (Hoedt et al. 2010). This downregulation may be due to hypermethylation of the Lf promoter, which can lead to its silencing (Yi et al. 2006). Considering this assumption and in addition to numerous studies in animal models demonstrating that the administration of Lf inhibits carcinogenesis (Aboda et al. 2020), and clinical trials in humans that have demonstrated the inhibition of tumor growth, and beneficial clinical effects in cancer patients (Kozu et al. 2009; Moastafa et al. 2014), we consider that Lf contributes to regulating cancer development. Based on these findings, statistical analyses have been conducted to predict the probability of survival in relation to Lf levels in cancer patients compared to no-cancer patients. These analyses indicated that low Lf levels are associated with decreased patient survival (Hernández-Galdámez et al. 2024). Nevertheless, other studies suggested that high levels of Lf promote cancer progression and contribute to the development of more aggressive phenotypes, particularly in the case of hormone-dependent cancers such as breast cancer (Gallo and Antonini 2024; Gopal and Das 2016). This dual role has been more closely linked to human lactoferrin (hLf), as the findings on the role of bovine lactoferrin (bLf) are less controversial and better established than hLf (Gallo and Antonini 2024). In this sense, the dosage of Lf is a key element in determining its effect. Therefore, it is necessary to conduct studies that evaluate the different concentrations and forms of administration of this bioactive component.

Effects of Lf in hallmarks of cancer

There could be grouped in hallmarks of cancer established by the similarities in all types of cancer cells regarding their cellular phenotype. These characteristics include sustaining proliferation signals, evading growth suppressors, resisting cell death (apoptosis), replicative immortality, angiogenesis, invasion, and metastasis, reprogramming cellular metabolism, suppressing immune destruction, and two emerging hallmarks: unlocking phenotypic plasticity and senescent cells (Hanahan 2022). Lf from various species, its fragments, peptides, or conjugates have been shown to contrast some of its characteristics (Fig. 1). In summary, these characteristics are described below.

Fig. 1.

Molecular mechanisms of Lf’s anticancer activity based on hallmarks of cancer. Created using Biorender (www.biorender.com)

One of the primary and most notable hallmarks of cancer is its excessive proliferation. Cancer cells deregulate growth-promoting signals, and simultaneously sustain proliferation signals, speeding up the cycle, evading steps, or staying in constant division (Hanahan and Weinberg 2000). Lf can modulate cell proliferation, exhibiting both inhibitory and enhancing effects, as demonstrated in various in vitro and in vivo models (Li et al. 2017). Lf can arrest the cell cycle at different checkpoints, such as G1/S, S, G2/M, and G0/G1, depending on the cancer cell type (Chea et al. 2018b; Cidem et al. 2024; Damiens et al. 1999; Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021). This behavior may be associated with suppressing the activity of cyclin-dependent kinases and activating p21 and p27 inhibitors of CDKs (Cidem et al. 2024; Harper et al. 1995), and increasing ERK phosphorylation which is involved in the regulation of cell cycle progression and arrest at the G0/G1 phase (Cidem et al. 2024). Lf peptides, such as the N-lobe of human Lf, have been shown to be more effective in inhibiting proliferation than the full-length Lf protein (Nakamura et al. 2023).

Lf also acts with other natural compounds, like curcumin, to enhance its antiproliferative effects unlike when used individually (Costantini et al. 2022). Moreover, Lf regulates survival and growth factors, such as p53, p21, and mdm2, and can act as a sensor of DNA damage, inducing apoptosis or cell growth arrest (Bukowska-Ośko et al. 2022; Oh et al. 2004). Additionally, Lf suppresses the expression of growth/differentiation factor 15 (GDF15), which is involved in cancer metastasis (Pan et al. 2021) and inhibits the PI3K/AKT/mTOR signaling pathway, a crucial regulator of cell proliferation, metastasis, and apoptosis, further supporting its chemopreventive and antimetastatic activities (Shi et al. 2019; Xu et al. 2024). Also, Lf prevents carcinogenesis by decreasing AKT activity (Hegazy et al. 2019).

Cancer cells can reprogram the cellular metabolism, particularly in the generation of ATP. While normal cells typically obtain ATP through oxidative phosphorylation, malignant cells have been able to generate it instead through glycolysis and lactate fermentation, primarily to evade high levels of ROS and consequently evade cell death (Bebber et al. 2020). Ferroptosis is a form of cell death initiated by the accumulation of ROS, regulated by iron. The accumulation of intracellular iron increases the availability of free iron to catalyze lipid peroxidation, leading to cellular damage and, consequently, cell death (Li et al. 2020). Lf, an iron chelator with antioxidant activity, has a dual behavior in ferroptosis depending on its iron saturation, Holo-Lf increased iron concentration, promoted ROS generation, and enhanced ferroptosis and iron-free Lf (Apo-Lf) inhibited ferroptosis (Zhang et al. 2021). This inhibition is consistent with other anticancer features, where Holo-Lf shows a more pronounced effect than those with Apo-Lf; even if these differences have not been fully understood, this may be a possible explanation. In a study examining ferroptosis regulation, three relevant genes (SCL7A11, GPX4, and ACSL4) were evaluated in HepG2 cells with and without p53 mutations under normal and hypoxic conditions (Kosim et al. 2022). The expression of SCL7A11, an inhibitor of ferroptosis, varied in each treatment. GPX4 (glutathione peroxidase) increased in both treatments under hypoxia. At the same time, ACSL4 (promotes lipid peroxidation) is upregulated only in cells with p53 mutation, which suggests an increase in ferroptosis just in Lf-treatment on the mutant-type p53 (Kosim et al. 2022).

On the other hand, angiogenesis is a biological process involving forming new blood vessels from pre-existing ones (Hanahan and Weinberg 2000). However, angiogenesis plays a crucial role in the development of cancer and tumor growth (DeCicco-Skinner et al. 2014). The vascular endothelial growth factor (VEGF) and tube formation stimulate angiogenesis; bLf downregulates the VEGFR2 receptor and VEGFA expression and has been demonstrated to decrease new blood vessels in HT29-induced tumors on mice (Li et al. 2017). Exposure to bLf or a VEGF inhibitor caused tube junctions to break in both tumor endothelial cells (TECs) and normal endothelial cells (NECs). However, bLf treatment increased tube junctions in normal endothelial cells, while the VEGF inhibitor SU668 significantly decreased it. These observations suggest bLf can differentiate between TECs and NECs (Ayuningtyas et al. 2023).

The main cause of cancer death is invasion and metastasis, a process that involves the ability of a cell to transition into a mesenchymal state, acquiring mobility and an invasive capacity that ultimately allows it to migrate and colonize other tissues (Hanahan and Weinberg 2000). The major proteins involved in epithelial-to-mesenchymal transition (EMT) include SNAIL, TWIST, and SLUG; these proteins are responsible for the repression of cadherins, which are involved in maintaining cell adhesion (Saitoh 2018). In oral and glioblastoma cells, bLf downregulates TWIST through the ERK 1/2 pathway stimulates the expression of E-cadherin, and downregulates SNAIL and vimentin (a hallmark of EMT) respectively (Chea et al. 2018b; Cutone et al. 2020). In addition, bLf inhibited the IL-6/STAT3 axis, and STAT3 possess oncogenic capabilities, promoting tumor survival (Cutone et al. 2020). In MDA-MB-231 cells, Holo-bLf inhibited 90% of the migration and secretion of MMP-2 and MMP-9, enzymes involved in breaking down the extracellular matrix (Rodriguez-Ochoa et al. 2023). In another study, the fusion of hLf with human serum albumin (hLf-HAS) promoted the antimigratory effect and downregulated MMP1 in PC-14 lung cells. However, hLf alone was unexpectedly demonstrated to induce migration and upregulated MMP-1 expression (Nopia et al. 2023). Finally, integrins regulate the activation of signaling pathways that promote cell survival (Desgrosellier and Cheresh 2010). Lf fused reduced α3 and β1 integrin (involved in tumor progression) in PC3 and DU145 cells. However, β1 reduction was not significant. These results suggest natural compounds could help maintain homeostatic balance, preventing the survival of cancer cells (Costantini et al. 2022).

Cancer’s survival is also due to its ability to evade cell death (Carneiro and El-Deiry 2020). Apoptosis is a natural process that involves the activation of enzymes that degrade the cellular components in a well-organized manner resulting in cell death (Carneiro and El-Deiry 2020; Obeng 2021). Lf persuades morphological changes in cancer cells, such as chromatin condensation, cell shrinking, and blebbing in the cell membrane, those all being apoptosis-related morphological changes (Arredondo-Beltrán et al. 2023; Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021; Pan et al. 2021).

The rhLf shows selective cytotoxicity in the triple-negative MDA-MB-231 and its lung metastatic variant MDA-MB-231-LM2-4 cell lines (Iglesias-Figueroa et al. 2019). In contrast, bLf exhibited a more significant apoptotic effect in no-metastatic breast cancer cells MCF-7 than MDA-MB-231. This difference was associated with a mutation of the p53 gene in MDA-MB-231. In particular, phosphorylated p53 at Ser15 and Ser46 increased in MCF-7 cells, while downregulation occurred for all three phosphorylation sites of p53 in MDA-MB-231 cells (Gibbons et al. 2015).

Apoptosis mediated by bLf in MDA-MB-231, prostate cancer PC-3, and osteosarcoma MG-63 cells was associated with an elevated extracellular acidification rate and vacuolar H + -ATPase (V-ATPase) expression levels and distribution, if it is localized at the plasma membrane, treatment with bLf can be used successfully in contrast with those with only intracellular V-ATPase localization (Guedes et al. 2018; Pereira et al. 2016).

In AGS and MCF-7 cells, Lf increased BAX and BAK expression apoptosis (Khalafi et al. 2020). In He-La cells, bLf upregulates the expression of p53, BAX, and active caspase-3 while downregulating BCL-2 (Shu et al. 2023). On the other hand, Lf suppressed antiapoptotic proteins like survivin, leading to apoptosis activation (Gibbons et al. 2015). Additionally, lactoferrin induced the intrinsic apoptosis pathway in liver cancer cells (HepG2 Hep3B and SK-Hep1) by suppressing BCL-2 thus decreasing the BCL-2/BAX ratio, and activating proapoptotic factors such as c-jun and cytochrome C (Cidem et al. 2024).

The same effects have been observed in in vivo models. In mice bearing EMT6 breast carcinoma the volume of the tumor was reduced associated with the regulation of the expression of BCL-2, BAX and activation of caspase 3 (Wang et al. 2011). In models of cervical cancer, BCL-2, BAX and receptor FAS are upregulated while expression of antiapoptotic Bcl-2 is decreased (Li et al. 2011b). These modifications of apoptosis-related genes were also observed in colon mucosa of azoxymethane-treated rats (Fujita et al. 2004a, 2004b).

Bovine lactoferricin (bLfcin) and bovine lactoferrampin (bLfampin) trigger apoptosis in liver cancer and leukemia cells by increasing the expression of the mitochondrial pathway (Arredondo-Beltrán et al. 2023). In AGS cells, bLfcin inhibited cell growth and increased apoptosis, activating caspases 3, 6, 7, and 9 (Pan et al. 2013). Another fragment of Lf, rtHLF4 upregulated proapoptotic proteins BAX, BAK, and p53, while suppressing antiapoptotic BCL-2 (Shu et al. 2023). It was suggested that BAK and BAX activation is due to the internalization of rtHLF and the consequently increased stress factors in the cell, triggering the activation of proapoptotic proteins (Pan et al. 2021).

One of Lf’s attributes is the ability to modulate the immune response by interacting with immune cells and enhancing their activity. Since cancer can suppress immune destruction, Lf can improve the growth, differentiation, and activation of macrophages, natural killer cells (NK), and T lymphocytes (Rascón-Cruz et al. 2021). Since Lf can bind to DNA and modulate gene expression, various signal pathways can be activated. Lf suppresses proinflammatory cytokines, including IL-1, IL-4, IL-6, and TNF-α, which maintain malignant cells (Kanwar and Kanwar 2013). Moreover, Lf downregulated the expression of growth factors and conversely upregulated the expression of receptors on the surface of the cells for the recognition of immune cells (Bukowska-Ośko et al. 2022). Myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment stimulated the migration and metastasis. In tumors of B16-F10 in mice lacking Lf, MDSCs increased, and the rate of metastasis was higher compared with mice-induced wild type, suggesting Lf can modulate the differentiation and apoptosis of MDSCs (Wei et al. 2020). Neutrophils are a crucial component of the innate system. One of its host defense mechanisms is neutrophils’ extracellular traps (NETs). NETs play a role in inflammation to combat the pathogens in the extracellular space. However, persistent inflammation can lead to an environment for the survival of cancer, facilitating the metastatic process (Akhtar et al. 2023). Lf, an integral constituent of NETs, cannot inhibit the formation of these structures. In contrast, exogenous Lf demonstrates the ability to suppress NET formation. The 25 amino acids of the N-terminal region were identified as critical for preventing the formation and release of NETs (Okubo et al. 2016). Furthermore, this activity may be related to one of the enabling hallmarks of cancer: tumor-promoting inflammation. Thus, this antiinflammatory property can help prevent the development of malignancy. Another way to exert its antiinflammatory effect is to facilitate the elimination of apoptotic cells by macrophages by inhibiting the chemotaxis of neutrophils (Bournazou et al. 2009). These features position Lf as a promising candidate for less invasive therapies, such as immunotherapies. Table 1 provides a concise summary of the key activities in hallmarks of cancer provided by experimental evidence.

Table 1.

Lactoferrin action in hallmarks of cancer. The cancer hallmarks are observed in the left column, while the effects exerted on them by Lf are shown in the right column

		Lactoferrin potential
Hallmarks of cancer	Replicative immortality, evading growth suppressors and sustaining proliferation signals	Arrest in cell cycle phase S, G2/M, G0/G1 (Chea et al. 2018a; Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021) Reduction of proliferation time and doses-dependent (Costantini et al. 2022; Nakamura et al. 2023) Reduction in tumor volume (Li et al. 2017) Regulation of PI3K/AKT/mTOR (Hegazy et al. 2019; Xu et al. 2024) Regulation of p53, p21 and mdm2 (Oh et al. 2004) Suppression of the GDF15 factor (Pan et al. 2021)
	Angiogenesis	Negative regulation of VEGR receptor (Li et al. 2017) Reduction of tube injections (Ayuningtyas et al. 2023)
	Evading cell death	Morphological changes associated with apoptosis (Iglesias-Figueroa et al. 2019; Nakamura-Bencomo et al. 2021; Pan et al. 2021) Elevation of extracellular acidification rate and expression of V-ATPase (Guedes et al. 2018; Pereira et al. 2016) Increasing gene expression of the mitochondrial pathway (Arredondo-Beltrán et al. 2023) Activation of caspases, BID, BAX, BAK, p53, FAS, p53 (Fujita et al. 2004a; Khalafi et al. 2020; Li et al. 2011b; Moradian and Mohammadzadeh 2023; Pan et al. 2013; Shu et al. 2023) Suppression of survivin (Gibbons et al. 2015)
	Reprogramming cell metabolism	Promoted ROS generation and increasing ferroptosis (Kosim et al. 2022; Zhang et al. 2021)
	Invasion, and metastasis	Antimigratory activity (Costantini et al. 2022; Nopia et al. 2023; Rodriguez-Ochoa et al. 2023) Restoring the epithelial properties (Chea et al. 2018b) Inhibition of MMP-1, MMP-2 and MMP-9 enzymes (Nopia et al. 2023; Rodriguez-Ochoa et al. 2023) Inhibition of IL-6/STAT3 axis (Cutone et al. 2020) Reduction of α3 and β1 integrins (Costantini et al. 2022)
	Suppressing immune destruction	Improving the growth, differentiation, and activation of macrophages, natural killer cells (NK), and T lymphocytes (Bukowska-Ośko et al. 2022; Wei et al. 2020) Suppressing proinflammatory cytokines (IL-1, IL-4, IL-6, and TNF-α) (Kanwar and Kanwar 2013) Upregulated the expression of receptors for the recognition of immune cells (Bukowska-Ośko et al. 2022)

Considering all these anticancer effects, it is evident that Lf shows a wide range of properties similar to those exerted by the currently used pharmacology, including the ability to inhibit tumor growth and induce apoptosis of cancer cells. Furthermore, it has been demonstrated that this compound has a low toxicity profile, making it an attractive option for developing effective and safe anticancer treatments. Emerging technologies in this context have used nanostructures loaded with Lf and Lf conjugates with commonly used chemotherapeutics, which provide a synergistic effect. This strategy has become one of the main methods for applying Lf as an anticancer agent. The following section summarizes of the most recent studies using these strategies.

Synergistic complex nano-systems of Lf

Nano-systems can be composed of individual nanoparticles or complex assemblies of diverse materials and can be designed differently.

Nanoparticles with Lf

Use of nanocarriers or nanocapsules to improve Lf stability, survival, and controlled target release. Nanoparticles (NPs) have been explored in many investigations, and different materials have been used to carry out this process. These nanomaterials include polymers, lipids, peptides, metallic nanoparticles, and carbon nanotubes (Kumari et al. 2014).

Selenium nanoparticles (SE-NPs) of Apo-Lf enhance antiproliferation and apoptotic effects on MCF-7, HepG-2, and Caco-2 cells over Lf or SE-NP’s alone (El-Fakharany et al. 2023). On the other hand, chitosan NPs with Lf exhibited a more pronounced growth inhibition effect than Lf alone in gastric cancer AGS cells. The expression of proapoptotic proteins such as Bax and Bak were dose-dependent, indicating the induction of apoptosis, an effect of Lf. In contrast, chitosan alone had no inhibitory effect on the cells (Moradian and Mohammadzadeh 2023).

Another delivery system is exosomes, which are extracellular vesicles produced by endosomes involved in carrying drugs (Liang et al. 2021). Exosomes of MDA-MB-231 loaded with bLf (ExoLf) exert an anticancer effect on MDA-MB-231 cells but not in human adipose-derived mesenchymal stem cells (MSC). The optimum concentration of bLf was lower than bLf alone, from 100 to 1 mg/mL in ExoLf. Nonetheless, the toxicity of ExoLf was predominantly necrotic phenotype (Ramezani et al. 2023).

Conjugated Lf-drug

Since Lf is a hydrophilic protein, it may be employed to enhance drug solubility and selectivity (Abdellatif et al. 2023). This design allows taking advantage of targeting (Lf coating NPs) and anti-cancer properties (active compound) of Lf to have a specific action on target cells. Lf-drug conjugates are a promising cancer strategy since functionalizing NPs with Lf enhances their bioavailability and stability, facilitating efficient and targeted cellular internalization, which can subsequently improve therapeutic efficacy (Akdaşçi et al. 2024). Indeed, drugs’ optimal efficacy is achieved when they are employed in combination. In addition, encapsulation protects them from premature degradation, allowing them to reach cancer cells intact (Kondapi 2020). The most widely used is a multifunctional design consisting of the nanoparticle, composed of some material described previously functionalized with Lf, and then simultaneously loaded with a drug.

Lf- docetaxel

Docetaxel (DTX) inhibits mitosis and cell division by arresting the cell cycle at the G2/M phase. Complex DTX-Lf has a dual effect on many types of cancers. In prostate cancer cell MAT-LyLu, the complex enters the cell through the Lf receptor and exhibits an antiproliferative effect of 2.5 times higher than DTX alone (Muj et al. 2023). In addition, in in vivo model, bioavailability, and anticancer effect were increased twice compared to DTX alone (Muj et al. 2023). These findings concord with studies of breast cancer in mice (Ehrlich ascites tumor cells), dual conjugate Lf-DTX-CST (Celastrol, which triggers apoptosis by activating caspase − 7, − 8 and − 9) suppressed tumor growth by 117.9% compared with DTX and CST alone (Abdelmoneem et al. 2021). Furthermore, it has shown suppression of NF-kB p65, TNF-α, COX-2, and Ki-67 expression levels, which are involved in survival, inflammation, and proliferation activities (Abdelmoneem et al. 2021).

The limited ability to breach the blood–brain barrier (BBB) is challenging in treating gliomas. Since Lf has been demonstrated to cross BBB through its receptor LRP-1, which is highly expressed in the cell of BBB, conjugates with Lf enhance the uptake into cells (Singh et al. 2016). Solid lipid nanoparticles-DTX conjugated reduce the IC50 values compared to DTX alone in glioblastomas U-87 cells. This behavior was attributed to an improvement in the internalization resulting from the presence of LfRs. Nanoparticles were ineffective when LfRs were saturated with free Lf (Singh et al. 2016).

Lf-temozolomide

Temozolomide (TMZ) is an alkylating agent that induces cell death by inhibiting DNA transcription (Zhang et al. 2012). TMZ is the first chemotherapy option for the treatment of malignant gliomas. However, TMZ has a poor half-life in blood, and cancer cells develop resistance to it, decreasing its efficiency quickly (Cui et al. 2023). Treatment with nanoparticles of Lf loaded with TMZ (TMZ-LfNPs) in mice leads to a considerable decrease in tumor size increased apoptosis of tumor cells and enhanced median survival (Kumari et al. 2017). In the study conducted by Mital et al., nanostructured lipid carriers (NCL) with Lf primarily as a targeted delivery system, where loaded with resveratrol and temozolomide, the conjugate increased the permeability of the BBB in ex vivo models by up to 88.5% at 24 h compared to resveratrol (25.76%) and temozolomide (31.10%) without conjugation. Moreover, the IC50 value exhibited a statistically significant improvement in the LTR-NLC treatment (Mittal et al. 2023).

On the other hand, drug efflux is a key mechanism of resistance of cancer cells to chemotherapy, cancer cells can actively pump out drugs from the intracellular environment reducing the concentration of drugs within the cells, rendering them less effective (Xu et al. 2018). This is a significant challenge in cancer treatment, as multi-drug resistance can render many chemotherapeutic agents ineffective, leading to failure treatment. Large pore silica NPs (USLP) loaded with TMZ, polyethylene glycol (PEG), and Lf significantly reduced the efflux of TMZ in human glioblastoma U-87 cells compared with TMZ alone. Moreover, the apoptosis rate was improved with USLP-TMZ particles compared to just TMZ. Lf’s presence principally enhances the delivery time (Janjua et al. 2023). To corroborate the correct delivery across the BBB, healthy mice were used to evaluate the USLP-based delivery system’s ability to accumulate in the brain, suggesting that it can permeate effectively the BBB (Janjua et al. 2023).

Lf-etoposide

The combination of rhLf-etoposide (a topoisomerase II inhibitor) inhibited the growth of human lung adenocarcinoma A549 cells by causing arrest in the cell cycle at the G2/M phase and triggering apoptosis, as compared with etoposide alone, which showed a lower inhibition of cell growth (Olszewska et al. 2022). On the other hand, Kuo and Chen developed conjugates of PLG [poly (L-lactide-co-glycolide)] nanoparticles with etoposide, Lf, and folic acid Etoposide-Lf-FA-PLG demonstrating an increase in both permeability through the BBB and antiproliferative effect in brain-microvascular endothelial cells and U87-MG cells, respectively (Kuo and Chen 2015).

Lf-doxorubicin

Doxorubicin DOX kills cells by reducing the topoisomerase II activity, preventing cell proliferation(Janjua et al. 2021). In breast cancer 4T1 cells, the conjugated Lf-liposome-Dox was found in higher amounts in the cytoplasm than in cells treated with Liposome-Dox. Both nanosystems caused similar toxicity after 24 h. However, Lf-Liposome-Dox exhibited more cytotoxicity after 36 h of incubation. Furthermore, Lf-Liposome-Dox had higher tumor accumulation than Liposome-Dox due to active targeting in mice bearing 4T1 tumors (Zhang et al. 2019). Lactoferrin − doxorubicin − mesoporous maghemite nanoparticles (Lf-Doxo-MMNPs) designed by Sharifi et al. also demonstrated an increase in accumulation in the targeted tissue which results in a reduction of the tumor volume in breast cancer in vivo model. Moreover, the Lf-Doxo-MMNPs with the application of a magnetic field and photothermal therapy demonstrated a significant increase in antimetastatic activity, indicating their potential as a promising therapeutic approach to combat metastatic cancer (Sharifi et al. 2020). Additionally, studies suggest that the size of NPs is linked to their toxicological effect, where smaller nanoparticles (< 50 nm) tend to have a more toxic effect due to their larger specific surface area, which allows them to interact more easily with cellular components (Huang et al. 2017). It is the case of ultrasmall size (30 nm) with large pore silica nanoparticles (> 7 nm) loaded with Dox and Lf that were able to permeate the BBB and improve the efficiency of apoptosis in glioblastomas U-87 cells (Janjua et al. 2021).

Other compounds

Mesalazine (MSZ), a drug used to treat colon diseases, combined with Lf, and resveratrol was encapsulated into the hydrophobic core of LF-MSZ nanoparticles. The nanoparticles demonstrated improved cellular uptake and toxicity against HCT colon cancer cells through LfR-mediated endocytosis. When administered orally, the nanoparticles significantly reduced tumor size in a mouse colon cancer model, increasing antioxidant enzymes and stimulating apoptotic pathways (Abd Elhamid et al. 2024).

Pterostilbene (PTS) is a promising compound against breast cancer. Solid lipid nanoparticles loaded with PTS and coated with Lf and chondroitin-sulfate for targeted delivery increased the cytotoxicity and cellular uptake in MDA-MB-231 cells. Furthermore, this formulation exhibited enhanced suppression of VEGF, downregulation of cyclin D1, and upregulation of caspase-3 and BAX, compared to PTS alone (Aly et al. 2023). Finally, in another study using Lf as a bioactive compound, PLGA NPs with hyaluronic acid for targeting CD44 receptors, which are highly expressed in many types of cancer (Senbanjo and Chellaiah 2017), increased cellular uptake from 11.03% to 65.50% in lung cancer compared to the control group. Furthermore, adding polydatin, a glycosylated resveratrol derivative, increased this figure to 228.53%. (Nashaat Alnagar et al. 2024).

Lf can exert its functionalities while allowing the controlled delivery of the drug. Additionally, some methods employ NPs to transport the Lf itself, aiming to improve the transport and preserve protein’s biological characteristics (Kondapi 2020). We can determine that the effect of NPs with Lf can depend on the size, which commonly ranges between 1 and 100 nm, with the smallest being the most effective. The composition of the material and the synthesis method are also influenced, which are described in more depth in a recent review by Tran et al., (2023).

Generally, cancer treatments typically involve the simultaneous use of various therapies to enhance the therapeutic effect. Similarly, the utilization of NPs is no exception, as studies like the one presented by Sarifi et al. (2020) have demonstrated that this multifaceted approach can significantly improve the efficacy of treatments.

In addition to combining therapies, another key factor for achieving greater efficacy in NP-based treatments is incorporating diverse bioactive agents. By obtaining conjugates with different active components, a therapeutic synergy can be achieved, where each component provides the anticancer effect, the protective effect, and the targeting effect, ultimately enhancing the effectiveness and biocompatibility of the treatment. As a result, one of the future perspectives in this context is focused on exploring the impact of combining NPs with different treatments and the design of multi-component NPs to deliver Lf and multiple agents simultaneously. However, for successful clinical application, certain limitations must be addressed, such as the scaling up of functionalized NPs, their long-term systemic effect, and the optimization of the design to achieve maximum efficacy.

Finally, the last factor that could improve the anti-cancer effect of these conjugates, is the affinity of Lf for specific receptors that may be overexpressed in tumor cells. This recognition allows for actively targeting of malignant tissues, minimizing toxicity in healthy cells. However, Lf receptors have remained understudied. Some studies have sought to identify and characterize these receptors as a target to optimize the use of Lf for cancer therapies. The last section identifies Lf targets that may be crucial to improving its practical applications.

Interaction of Lf with molecular targets

The multiple functions of Lf have been associated with binding to the mammalian Lf receptor (LfR), which primarily plays an essential role in iron absorption in the intestine (Jiang and Lönnerdal 2018). The binding of Lf to its receptors facilitates its internalization. It activates multiple signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway, which is crucial for cell proliferation, differentiation, and survival. These interactions are essential for the functioning of diverse physiological processes. Additionally, Lf’s hydrophobic and cationic nature influences these interactions (Rascón-Cruz et al. 2025). Lf binds to several types of targets, such as immune cells, DNA, heparin (Van Berkel et al. 1997) lipopolysaccharide, and glycosaminoglycans (GAGs) (Mann et al. 1994). Furthermore, other molecules interact with Lf, such as osteopontin, (Yamniuk et al. 2009), ceruloplasmin (Sokolov et al. 2009), and plasminogen (Zwirzitz et al. 2018) in blood and other biological fluids, additionally others molecules also have been predicted through bioinformatics approach (Fig. 2). However, the functionality of some bindings has yet to be fully understood. We will focus on those interactions that can provide information to elucidate the mechanism of action or recognition of Lf in the cancer context.

Fig. 2.

Molecular interactions of Lf. The experimental interactions are shown in white rectangles, and interactions predicted through in silico analysis are shown in pink rectangles (DNA has been determined both in vitro and in silico). The possible processes involving these interactions are presented in light purple rectangles connected by dashed arrows. Lf modeling was created using Pymol.

GAGs

Among GAGs, the binding with heparan sulfate proteoglycans (HSPG) triggers antiproliferative effects in breast cancer cells (Damiens et al. 1998). Recently this interaction was rectified and identified as a key mechanism in the activity against the coronavirus. The binding Lf-HSPG blocks the entry of SARS-CoV-2 as the virus relies on HSPG binding to the cell surface as a key step in the infection process (Mirabelli et al. 2021). Lf also binds to chondroitin sulfate-E, a type of glycosaminoglycan involved in various neurodegenerative disorders. The interaction with GAGs is associated with the N-terminal region of Lf (Nakamura et al. 2021). GAGs are abundant in the extracellular matrix of tissues; changes in GAGs levels contribute to various cancer hallmarks, including metabolic reprogramming, persistent growth signals, immunosuppression, angiogenesis, tumor invasion, and metastasis. In fact, GAGs have been proposed as diagnostic markers for various cancers due to their overexpression in these cells (Douglah et al. 2024). These characteristics make this union beneficial by creating a selective accumulation of Lf in the tumor microenvironment. Additionally, the union inhibits the interaction of GAGs with other factors that promote tumor development, which represents an additional advantage.

LRP-1

One of the well-known Lf receptors is the low-density lipoprotein (LDL) receptor-related protein 1 (LRP-1) or CD91, which, in the cancer context, prevents the degradation or remodeling of the extracellular matrix by internalizing various proteins such as metalloproteinases, associated with metastasis (Dedieu and Langlois 2008). The binding with Lf activates the ERK1/2 pathway in fibroblasts, osteoblasts, keratinocytes, and adipocytes (Takayama et al. 2003). As described in the previous section, this receptor is of great interest as a target for developing technologies aimed at brain cancers, especially gliomas, due to its ability to mediate the transport of molecules such as Lf across the BBB. The overexpression of LRP-1 in gliomas and other brain tumors makes it a promising target for selective therapeutic interventions. By binding to LRP-1, Lf-functionalized NPs or can influence receptor-mediated endocytosis to achieve efficient cellular internalization.

INTL1

Intelectin-1 is another important Lf receptor found in the small intestine’s epithelium and plays a role in the endocytosis of Lf (Suzuki et al. 2001). For example, in Caco2 cells Lf is internalized and shows a co-localization with intelectin-1 (Akiyama et al. 2013). In a recent study, inhibition of INTL1 expression in liver cancer cells suppressed the entry of Lf and the subsequent phosphorylation of p38 MAPK, JNK, and ERK induced by Lf suggesting that INTL1 acts as Lf, receptor in HepG2 cells (Cidem et al. 2024). ITLN1 suppresses invasion in ovarian cancer by downregulating MMP-1, but conversely, ITLN1 abolishes the effect of Lf on motility and invasive potential. Interestingly, the binding of ITLN1 prevents its interaction with LRP1 which exerts antitumor activities (Au-Yeung et al. 2020). These features reiterate the diverse effects of Lf use depending on the cellular context and the type of cancer.

CXCR4

The cytokine receptor C-X-C chemokine receptor type 4 is highly expressed in various types of cancer and promotes cell proliferation and angiogenesis (Xu et al. 2015). CXCR4 is a specific receptor for stromal-derived factor-1 (SDF-1). The study by Takamaya et al. (2017) showed that bLf mimics the binding of CXCR4 to SDF-1, including receptor dimerization, tyrosine phosphorylation, and mono-ubiquitination. Furthermore, this union was capable of activating the PI3K/Akt signaling pathway, an undesired result in the context of cancer: However, it has also been demonstrated to deregulate CXCR4 negatively; this can be an advantage in cancer, as reducing the amount of the receptor could also reduce proliferation (Takayama et al. 2017). Despite this, Lf can be used as a recognition system or for blocking the CXCR4/SDF-1 axis to reduce its metastatic properties. However, additional research on this union across various cell types is needed to understand its effects fully.

Nucleolin

Nucleolin is a multifunctional protein of the nucleolus that is involved in many cell processes, such as cell survival and cell cycle (Chen and Xu 2016). Nucleolin can be translocated to the cell membrane and is overexpressed in various cancers (Carvalho et al. 2021). Nucleolin promotes the antiapoptotic mRNA expression, blocks proapoptotic factors such as FAS receptor, and suppresses FAS-ligand interaction (Wise et al. 2013). In addition, it was found that nucleolin interacts with the intracellular domain of EGFR (member of the receptor tyrosine kinase), inducing the activation of this pathway involved in proliferation and differentiation (Farin et al. 2011). Nucleolin is an Lf-binding protein, and this interaction promotes the internalization of both proteins into the cell. A small percentage of Lf is also translocated into the nucleus (Legrand et al. 2004). Lf can abolish the binding of C1QTNF4 (another ligand of nucleolin) in ex vivo monocytes and HEK293 cells, indicating competition for the same binding site. This finding further reinforces the interaction between Lf and nucleolin (Vester et al. 2021). Indeed, nucleolin has become a very promising target for drug delivery due to its overexpression in cancer cells (Thongchot et al. 2023). This fact positions this union as a potential strategy in the use of NPs.

IGBP1

Immunoglobulin binding protein 1 (IGBP1) is overexpressed in esophageal squamous cell carcinoma, predicting a poor prognosis in lung adenocarcinoma (Jiang et al. 2019; Sato et al. 2017). IGBP1 is a regulator of protein phosphatase 2A (PP2Ac), which plays a vital role in growth, differentiation, and apoptosis. The binding of IGBP1 and PPCAc exerts an antiapoptotic effect (Sato et al. 2017). Li et al. identified an interaction between IGBP1 and bLf using a protein microarray analysis. The IGBPI-bLf binding complex inhibited the binding of IGBPI to PP2Ac, inhibiting the antiapoptotic activity and thus enhancing apoptosis in PC-14 lung adenocarcinoma cells (Li et al. 2011a).

DNA

Lf can bind and degrade DNA. Two binding sites have been identified with different affinities in the N-lobe of hLf (He and Furmanski 1995; Kanyshkova et al. 1999) Lf acts as a transcription factor that activates specific DNA sequences, such as the nuclear factor-kB (NF-kB) and the consequent transactivation of the p53 gene, regulates oncogenes, and degrades free radicals (Bukowska-Ośko et al. 2022). The binding to DNA has been studied by bioinformatics, peptides from camel lactoferrin (cLf) bind DNA, especially CLFchimera (camel LFcin + camel LFampin), The residues Lys5, Lys9, Lys13, Arg16, Lys18, Arg27, Lys34, and Lys35 were the more relevant amino acids for binding (Pirkhezranian et al. 2020).

V-ATPase

V-ATPase is essential for cellular homeostasis. However, in cancer, they play a role in the acidification of the tumor microenvironment (Santos-Pereira et al. 2021b). Bioinformatics predicted an interaction between Lf and V-ATPase vía docking molecular (Santos-Pereira et al. 2021a). Results suggest that Lf binds in AB parts of the ATP hydrolysis site of V-ATPase. Thus, the ATP hydrolysis is blocked, and consequently, the whole V-ATPase is affected. Various residues of Lf were identified as necessary for this interaction: Arg3, Arg4, Arg27, Arg587, Gln666, Arg249, Arg86, Arg89, Arg652, and Arg608 (Santos-Pereira et al. 2021a). These results are aligned with those obtained experimentally. As mentioned in the section on apoptosis caused by Lf, it was associated with increased V-ATPase expression, suggesting a possible interaction, this in silico study supports these findings. Furthermore, some of the interacting residues, especially Arg3 and Arg4, have been reported as essential for their interaction with cells and other molecules in vitro, for example, in the case of the binding of Lf to Jurkat lymphoblastic T (Legrand et al. 1997) cells and human colon carcinoma HT29-18-C1 cells, where was determined that the binding sites were heparan sulfate and chondroitin sulfate GAGs (El Yazidi-Belkoura et al. 2001). Furthermore, it has been shown that through these residues, hLf interacts with heparin, bacterial lipopolysaccharide lipid A, human lysozyme, and DNA (Van Berkel et al. 1997). Finally, a recent study determined that the binding of Lf with the SARS-CoV-2 Spike protein is primarily due to its N-terminus, where these residues are found (Babulic et al. 2024).

UKK1, ATG13, and ATG101

Autophagy has become an alternative pathway for killing cancer cells. It is regulated by mTORC1 and AMPK pathways. Aizawa et al. reported that bLf activates the AMPK pathway to promote autophagy through the LRP1 receptor (Aizawa et al. 2017). In MC-F7 cells, the expression of ULK1 (initiator of autophagy pathway) was elevated compared to that of the control group when treated with Lf. Based on these results, bioinformatic studies of interaction were carried out. Lf showed to interact with ULK1 protein: the N-lobe region of lactoferrin; Gly150, Ile161, Asp162, Arg163, Asn179, Asn176, Arg186, and Ala182 interacts by hydrogen bonds with the PS domain of the ULK1 protein; His543, Arg546, Arg549, Leu550, Ggy551, and Arg553. Besides ULK1, Lf also interacts with ATG13 and ATG101 proteins, which are components of the autophagy initiation complex (Karabi et al. 2022). In a recent study, Lf’s N-lobe region interacted with the HORMA domain of the ATG101 protein (Mashhadi-Kholerdi et al. 2024). In addition, interactions with mTOR and AMPK proteins were also found in the fat and CTD domains, respectively. However, Lf did not stimulate gene expression in vitro, suggesting that the induction of autophagy, based on the in silico results, could occur through interactions with proteins related to this process (Mashhadi-Kholerdi et al. 2024).

BAK/BAX

Proapoptotic members of the intrinsic apoptosis pathway that control cytochrome C release by influencing the permeability of the mitochondrial membrane (Obeng 2021). Besides its gene-related effects, Lf interaction with BAX and BAK proteins was determined via docking molecular. As described above, in several studies, Lf has been shown to upregulate the expression of both genes, increasing the apoptotic rate. Residues in the N-lobe of lactoferrin: Glu13, Lys18, Arg20, Arg25, Arg22, Lys174, Glu176, Arg186 and Pro293 interacts with Glu90, Phr93, Argg94, Ala97, Glu146, Arg147, Leu149 and Met191 from the C-terminal and BH1 domain of BAX. On the other hand, Glu13, Trp16, Arg20, Arg21, Arg28, Pro293 from Lf interacts with Glu25, Glu105, Tyr108, Glu109, Asp160, His164 and Ser166 from BH4 and BH1 domain of BAK (Moradian and Mohammadzadeh 2023). These results are similar to previous findings, where the N-lobe of Lf interacts with both proteins, probably to its positive region containing Lys and Arg residues, which can facilitate interactions with negatively charged residues, in this case with Glu, which was found in repeated instances in the interacting residues of BAX and BAK.

HER2

The human epidermal growth factor receptor 2 is one of the main targets in breast cancer (Loibl and Gianni 2017). Camel Lf peptides were analyzed by molecular docking to search for interaction with HER2. Most of the peptides had a stable hydrogen bonding interaction with residues in the active site of HER2 (Ser728, Phe731, Lys753, Asp845, Arg849, Asn850, Arg860, Asp863, and Lys887). Notably peptide PEP66 demonstrated high stability in molecular dynamics simulations, aligning with experimental results that showed it had the highest inhibitory effect on MCF-7 breast cancer cells (Baothman et al. 2024). However, the relationship between HER2 and Lf remains limited in the literature, with some research suggesting high Lf levels may contribute to the downregulation of receptors like ERα, PR, and potentially HER2, which could lead to the development of triple-negative breast cancer phenotypes (Ha et al. 2011). Further research is essential to thoroughly elucidate the relationship in this matter.

Conclusions

Lf emerges as a promising modulator in cancer, from regulation of signaling pathways to regulation of tumoral microenvironment. This review provides an overview of the anticancer features of Lf from a perspective related to cancer hallmarks, bringing to light all the Lf approaches for integral cancer treatment. In addition, the capacity of Lf to complex with drugs highlights its potential as a versatile transporter of anticancer agents, improving the efficacy and development of new combinatorial therapies. Lf has inherent properties that will enhance drug solubility and shelf life of drugs. Its functionality is ensured when it is encapsulated or combined with other materials. The Lf structure is protected under adverse conditions and controlled and targeted release is enabled. In addition, the anticancer effect can be potentiated by combining Lf with chemotherapeutic drugs and various bioactive agents that provide complementary therapeutic characteristics. Its versatility makes Lf a multifunctional tool in the design of drug delivery systems. An efficient design of NPs should start on identifying tumor-associated antigens (TAAs) that are expressed at significantly higher levels in tumors compared to normal tissues with potential affinity for binding to Lf, as is the case of CXCR4 and Nucleolin. This selective targeting contributes to Lf”s low systemic toxicity and enhanced selectivity, making it a promising targeted therapy. This field has been understudied, yet the search for molecules / Lf receptors is imperative to develop more effective delivery systems. More research is necessary to gain a deeper understanding of new therapeutic strategies, further solidifying the role of Lf and its derivatives in cancer treatments.

Data availability

No datasets were generated or analysed during the current study.