Efficient Endocytosis of the Human Lactoferrin N-Lobe Enhances Its Antiproliferative Activity against Human Cancer Cells

Abstract

Human lactoferrin (hLF) is a glycosylated globular iron-binding protein with high functional versatility that elicits anticancer, neuroprotective, and anti-inflammatory effects. Some of the diverse functions of hLF are induced after its internalization into various cells via cell surface endocytosis receptors, such as proteoglycans, which contain glycosaminoglycan (GAG) chains. We have previously demonstrated that an hLF derivative comprising the N-terminal half of hLF (referred to as the N-lobe) is internalized by intestinal enterocyte Caco-2 cells. However, the relationship between the intracellular uptake of the N-lobe and its pharmacological activity remains poorly understood. Here, we report that the N-lobe is efficiently internalized by lung cancer cells via endocytic pathways, suppressing their proliferation. Moreover, the N-lobe showed higher intracellular uptake than hLF. We found that the N-lobe was internalized into the human lung cancer cell lines PC-14 and PC-3 via clathrin- and/or caveolae-mediated endocytosis. Intracellular uptake of the N-lobe was inhibited when an equimolar concentration of chondroitin sulfate (CS)-E, a GAG subtype involved in malignant transformation and tumor metastasis, was added. The inhibitory effect of the N-lobe on PC-14 cell proliferation decreased with the addition of CS-E in a dose-dependent manner, suggesting that the CS-recognizing sequence on the N-lobe is necessary for its internalization or that the CS proteoglycan on cancer cells acts as an endocytosis receptor. These results suggest that the efficient endocytic uptake of the N-lobe is important for its antiproliferation effects on lung cancer cell lines. Thus, the N-lobe presents a promising drug candidate for cancer treatment.

INTRODUCTION

Endocytosis is a fundamental biological process in cells to internalize extracellular materials.¹⁾ Perturbation of endocytosis leads to the onset of diseases, such as cancer, neurodegenerative diseases, diabetes, and cardiovascular diseases. Clathrin- and caveolae-mediated endocytosis are the most widely characterized mechanisms.²⁾

Proteoglycans (PGs) are heterogeneous macromolecular glycoconjugates consisting of a core protein covalently attached to glycosaminoglycan (GAG) chains, such as heparan sulfate (HS) and chondroitin sulfate (CS). HSPGs and CSPGs are receptors/co-receptors for many ligands that play important roles in cellular signaling.³⁾ HSPGs act as internalizing receptors/co-receptors for cell surface attachment to promote the internalization of biopolymers and viruses.⁴⁾ In contrast, most CSPGs are secreted from cells and serve as extracellular matrix molecules widely expressed in the central nervous system. Reported cases of cell surface CS function in endocytosis are limited to low-density lipoproteins,⁵⁾ herpes simplex viruses,⁶⁾ and Clostridium difficile toxin B.⁷⁾

Lactoferrin (LF) is a GAG-binding protein secreted in body fluids, such as milk, tears, and saliva. LF exerts multiple therapeutic properties, including anticancer, neuroprotective, and anti-inflammatory effects. Human LF (hLF) is a polypeptide chain folded into two globular lobes: N- and C-lobes. The N-lobe (the N-terminal half region of hLF) has basic amino acids and regulates physiological activities through interaction with various biopolymers, such as heparin, PspA (a virulence factor from Streptococcus pneumoniae), and lipopolysaccharide.⁸⁾ We previously demonstrated that the N-lobe is internalized by intestinal enterocyte Caco-2 cells⁹⁾ and binds to CS-E, a GAG subtype involved in cancer and neurodegenerative diseases.¹⁰⁾ However, the relationship between the intracellular uptake of the N-lobe and its pharmacological activity remains poorly understood.

This study aimed to provide detailed insights into the mechanisms underlying the intracellular uptake of the N-lobe in cancer cells while elucidating its antiproliferative activity.

MATERIALS AND METHODS

Reagents

Recombinant hLF produced by Aspergillus niger was obtained from NRL Pharma, Inc. (Kawasaki, Japan). Pitstop 2 was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.), Chlorpromazine hydrochloride (CP) from Cayman Chemical (Ann Arbor, MI, U.S.A.), Methyl-β-cyclodextrin (MβCD) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), Nystatin (Nys) from Cosmo Bio Co., Ltd. (Tokyo, Japan), Dyngo-4a from TargetMol (Boston, MA, U.S.A.), and CS-E from PG Research (Tokyo, Japan).

Cell Culture

The human lung adenocarcinoma cell lines PC-14 (poorly differentiated adenocarcinoma) and PC-3 (moderately differentiated adenocarcinoma) were purchased from Immuno-Biological Laboratories Co., Ltd. (Gunma, Japan). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and maintained at 37 °C in 5% CO₂.

Expression and Purification of the N-Lobe

The expression and purification of the N-lobe (residues 1–351 in hLF) were performed as previously reported.⁹⁾ Briefly, the N-lobe was expressed into culture supernatant with the dhfr-deficient Chinese hamster ovary-derived cell line DG44 as a host cell. The N-lobe secreted into the conditioned medium was purified via cation-exchange chromatography using Macro Cap SP (Cytiva, Marlborough, MA, U.S.A.).

Immunocytochemical Analysis of Intracellular Uptake of hLF and N-Lobe in Human Cancer Cells

PC-14 and PC-3 cells were pre-cultured at 37 °C for 24 h. Afterward, the cells were treated with 0.38 µM hLF or N-lobe and incubated at 37 °C for 1 h. The cells were then washed twice with RPMI-1640 and fixed with 4% paraformaldehyde at 37 °C for 10 min. Next, the fixed cells were washed twice with phosphate buffered saline (PBS containing 137 mM NaCl, 2.7 mM KCl, 9.6 mM Na₂HPO₄, and 1.5 mM KH₂PO₄) and permeabilized with 0.5% Triton X-100 for 1 h. The cells were blocked with Blocking One (Nacalai Tesque, Kyoto, Japan) for 30 min and incubated with an anti-LF polyclonal antibody (1 : 500, A80-144A, Bethyl Laboratories, Inc., Montgomery, TX, U.S.A.) at 4 °C for 24 h. Afterward, the cells were incubated with an Alexa Fluor-546-labeled secondary anti-rabbit immunoglobulin G (IgG) antibody (1 : 200, A11010, Invitrogen, Waltham, MA, U.S.A.). Finally, the cell nuclei were counterstained with Hoechst 33258 (2 µg/mL; FUJIFILM Wako Pure Chemical Corporation) for 10 min. Cell distribution was based on the plasma membrane outline observed in a bright field, with the area inside the outline defined as the cytoplasm and the outside defined as the cell surface membrane. Cytoplasm and cell surface membrane proportions per cell were calculated with the image analysis software ImageJ based on the quantification of the respective fluorescence values. All cells were observed under a FluoView FV3000 confocal microscope (Olympus, Tokyo, Japan).

Immunocytochemical Analysis of Fluorescently Labeled N-Lobe Uptake in Human Cancer Cells Treated with Endocytosis Inhibitors

The hLF and N-lobe used in this experiment were fluorescently labeled with Alexa Fluor-488 tetrafluorophenyl esters (Invitrogen). PC-14 and PC-3 cells were treated with 0.38 µM Alexa Fluor 488-labeled N-lobe and incubated at 37 °C for 1 h. The cells were washed twice with PBS and fixed in 4% formaldehyde for 30 min. Cell nuclei were stained with 0.1 µg/mL 4′-6-diamidino-2-phenylindole (DAPI) for 10 min. The cell membranes were stained with 10 µg/mL CF594-labeled wheat germ agglutinin (WGA; BTI Biotium) for 10 min.

PC-14 and PC-3 cells were pretreated with various pharmacological inhibitors (20 µM Pitstop 2, 5 µM CP, 5 mM MβCD, 50 µM Nys, or 30 µM Dyngo-4a) for 30 min to inhibit endocytosis. The spent medium was replaced with test media containing various inhibitors and 0.38 µM Alexa Fluor 488-labeled N-lobe and then incubated at 37 °C for 1 h. The intracellular uptake of the N-lobe was calculated using the average brightness value of the fluorescently labeled N-lobe per cell area inside the outlines of the WGA-stained cells. Comparative studies between the treatment and control groups were conducted using an unpaired Student’s t-test or one-way ANOVA.

Immunoblotting Analysis of hLF and N-Lobe Uptake in Human Cancer Cells

PC-14 and PC-3 cells were treated with 0.38 µM N-lobe only or 0.38 µM N-lobe plus 0.38 µM CS-E at 37 °C for 1 h. After washing with PBS, the cells were treated with trypsin/ethylenediaminetetraacetic acid (EDTA) for 3 min. Next, the hLF and N-lobe were analyzed via Western blotting using an anti-hLF polyclonal antibody (1 : 10000, A80-144A, Bethyl Laboratories, Inc.) and HRP-conjugated anti-rabbit IgG (H + L) (1 : 10000, Promega Corporation, Madison, WI, U.S.A.). Endogenous controls were detected using an anti-γ-actin antibody (1 : 5000, FUJIFILM Wako Pure Chemical Corporation) and HRP-conjugated anti-mouse IgG (H + L) (1 : 10000, Promega Corporation). The relative band intensities were represented graphically using GraphPad Prism software version 8.4.1. (San Diego, CA, U.S.A.).

Cell Proliferation Assay

PC-14 cells were treated with 0, 1, 5, or 10 µM hLF or N-lobe and incubated at 37 °C for 72 h. The inhibitory effect of the N-lobe on cell proliferation in the presence or absence of 1 or 10 µM CS-E was evaluated. Cell counts were determined using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.). Data were analyzed using Dunnett’s test or two-way ANOVA, and p-values <0.05 were considered statistically significant.

RESULTS AND DISCUSSION

LF exerts anticancer activity via different cancer-type specific mechanisms, such as cell membrane alteration, apoptosis induction, cell cycle arrest, and metastasis inhibition.¹¹⁾ hLF is internalized in various cells, such as Caco-2 cells⁹⁾ and cytotrophoblast BeWo cells¹²⁾; however, the mechanisms underlying N-lobe internalization remain poorly understood. We first used immunocytochemical methods to investigate the cellular localization of hLF and N-lobe in the human lung adenocarcinoma cell lines PC-14 and PC-3. Both hLF and N-lobe were present on the cell surface membrane and cytoplasm of PC-14 and PC-3 cells (Fig. 1). The rate of hLF uptake was 21% in PC-14 cells (Figs. 1A, C) and 41% in PC-3 cells (Figs. 1D, F), while that of N-lobe uptake was 88% in PC-14 cells (Figs. 1B, C) and 87% in PC-3 cells (Figs. 1E, F). These results show that the intracellular uptake of the N-lobe was more efficient than that of hLF (Figs. 1C, F).

Fig. 1. N-Lobe Shows Higher Intracellular Uptake Efficiency than hLF

Representative images of the intracellular uptake (red) of hLF or N-lobe in PC-14 (A, B) and PC-3 cells (D, E). The nuclei were visualized using Hoechst staining (blue). (C, F) Graphical representation of the fluorescence intensities of the images in A, B, D, and E.

hLF is internalized into Caco-2 cells via clathrin-mediated endocytosis.¹³⁾ As shown in Fig. 1, N-lobe uptake into the cells was higher than hLF uptake; however, the underlying mechanism remains unknown. We examined temperature-dependent N-lobe uptake by the cells. The N-lobe was taken up by PC-14 and PC-3 cells at 37 °C but not at 4 °C, indicating temperature-dependent N-lobe uptake (Figs. 2A, B, F, G, K1–2).

Fig. 2. N-Lobe Uptake Occurs via Clathrin- and/or Caveolae-Mediated Endocytosis

(A–J) Representative images of N-lobe uptake by PC-14 and PC-3 cells. (K–N) Intracellular uptake of fluorescently labeled N-lobe (green) at 37, 4, and 37 °C in the presence of endocytosis inhibitors (Pitstop 2, CP, MβCD, Nys, and Dyngo-4a). The insets in A–J are high-magnification images of the boxed areas in A–J. The plasma membrane was visualized using CF594-labeled wheat germ agglutinin (WGA) (red). Nuclei were visualized using DAPI staining (blue). Quantitative results are presented as the mean ± standard deviation (S.D.) (n = 50). *** p < 0.001.

Next, we explored the mechanism underlying N-lobe uptake using endocytosis inhibitors. PC-14 and PC-3 cells were treated with Pitstop 2 or CP, specifically inhibiting clathrin-mediated endocytosis. We observed significantly decreased intracellular N-lobe uptake in the Pitstop 2 or CP-treated PC-14 and PC-3 cells compared with the PBS-treated control cells (Figs. 2C, H, L1–2). Similar results were observed for MβCD or Nys-treated PC-14 and PC-3 cells, which are inhibitors of caveolae-mediated endocytosis (Figs. 2D, I, M1–2). We also tested whether the N-lobe is internalized via dynamin-mediated endocytosis. N-lobe uptake was inhibited in the Dyngo-4a-treated PC-14 and PC-3 cells, which is an inhibitor of dynamin-mediated endocytosis (Figs. 2E, J, N1–2). These results suggest that cancer cells take up N-lobe via clathrin- and/or caveolae-mediated endocytosis. Notably, the N-lobe was present on the plasma membranes of the endocytosis inhibitor-treated cancer cells (Figs. 2C–E, H–J, insets). Hence, the intracellular uptake of the N-lobe may involve binding to molecules present on the surfaces of PC-14 and PC-3 cells.

Cell surface-covering PGs are taken up into cancer cells via clathrin- and/or caveolae- mediated endocytosis. The binding of hLF to GAG chains, which are components of proteoglycans, is involved in the intracellular uptake of hLF.⁹⁾ Furthermore, in vitro studies have identified CS-E on the surfaces of lung cancer,¹⁴⁾ ovarian cancer,¹⁵⁾ and osteosarcoma cells.¹⁶⁾ CS-E contributes to cancer cell infiltration, metastatic potential, and spheroidization.¹⁷⁾ Therefore, CS-E present on the cancer cell surface may be utilized as a cancer antigen and drug delivery target. Moreover, the N-lobe directly binds to CS-E.¹⁰⁾ However, the role of CS-E in the intracellular uptake of the N-lobe is unknown. Therefore, we verified whether the intracellular uptake of the N-lobe into cancer cells was inhibited by CS-E. The rate of intracellular N-lobe uptake decreased by up to 12% in PC-14 cells and 32% in PC-3 cells upon the addition of an equimolar concentration of CS-E (Figs. 3A–D). These findings suggest that the CS-E-recognizing sequence on the N-lobe is necessary for its internalization or that the CSPGs on cancer cells act as endocytosis receptors. The involvement of CS-E in the cellular uptake of N-lobe should be analyzed in depth using cells with reduced endogenous CS-E due to cleavage by enzymes such as chondroitinase ABC or via suppression/deletion of N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) expression.

Fig. 3. Interaction between N-Lobe and CS-E Prevents N-Lobe Uptake

(A, C) Temperature-dependent N-lobe uptake into PC-14 and PC-3 cells. CS-E was added at an equimolar concentration to the N-lobe (0.38 µM). (B, D) Graphical representations of the relative band intensities.

Finally, the antiproliferative activities of hLF and N-lobe against PC-14 cells were compared, and the effect of N-lobe activity on intracellular uptake was evaluated. hLF did not significantly inhibit PC-14 cell proliferation at concentrations below 10 µM (Fig. 4A). By contrast, the N-lobe inhibited PC-14 cell proliferation in a dose-dependent manner (Fig. 4B). However, this inhibitory effect was diminished by the addition of CS-E in a dose-dependent manner (Fig. 4C). The addition of 1 or 10 µM of CS-E did not promote the proliferation of PC-14 cells (Fig. 4C). The N-lobe dramatically shrunk PC-14 cells to alter cell morphology. This effect was attenuated by CS-E addition in a dose-dependent manner (Fig. 4D); 10 µM hLF did not alter PC-14 cell morphology (data not shown). These results suggest that the N-lobe exhibits stronger antiproliferative activity against PC-14 cells than hLF, whose biological activity is limited by its cellular uptake. On the contrary, the insets of Figs. 2A and F show that N-lobe uptake is largely localized near the nucleus. Detailed analysis of the subcellular localization of the N-lobe in further studies may provide insight into the molecular mechanism of the anti-proliferative activity of N-lobe uptake.

Fig. 4. N-Lobe Uptake Inhibits Cancer Cell Proliferation

(A, B) Antiproliferative effects of 0, 1, 5, or 10 µM hLF or N-lobe on PC-14 cells. (C) Antiproliferative effect of the N-lobe on PC-14 cells in the presence or absence of 1 or 10 µM CS-E. (D) Morphology of PC-14 cells treated with the N-lobe in the presence or absence of 1 or 10 µM CS-E. Data are presented as the mean ± S.D. (n = 3). ** p < 0.01, *** p < 0.001. n.s., not significant.

In conclusion, we found that the N-lobe was efficiently internalized by lung cancer cell lines via clathrin- and/or caveolae-mediated endocytosis and suppressed their proliferation. Further studies are necessary to clarify whether or not the N-lobe can be used as an anticancer drug in an in vivo model. Nonetheless, our findings shed light on the mechanisms underlying the anticancer drug carrier capacity of the N-lobe in cells.

Acknowledgments

We are grateful to Prof. Atsushi Sato of the Tokyo University of Technology for the useful discussions. This work was partly supported by the Sasakawa Scientific Research Grant (to M.N.).

Conflict of Interest

The authors declare no conflict of interest.

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