Role of sialic acids on lactoferrin anticancer activity
Role of sialic acids on lactoferrin anticancer activity
- Prof. Antimo Cutone;
- Prof. Giovanni Musci;
- Prof.ssa Eleonora Sgambati;
- Prof.ssa Sabrina Di Bartolomeo.
Objectives and activities
Lactoferrin (Lf), a cationic iron-binding glycoprotein of the innate immune defense, is constituted by 689 amino acid residues and folds into two distinct lobes (N- and C-lobes), each bearing a high affinity iron-binding site. Since human (hLf) and bovine (bLf) lactoferrins show high sequence homology and share identical multifunctionality, most studies have been carried out using commercial bLf.
With regard to glycosylation, bLf contains five N-glycosylation sites, four of which (Asn233, Asn368, Asn476, and Asn545) are invariably glycated, with two oligomannosidic type and three biantennary N-acetyllactosamine type glycans, partially fucosylated and sialylated. The sialic acid (Sia), usually present in a ratio of about three residues per molecule, could affect the functionality and bioavailability of Lf. Due to its structural features, including iron binding and a high cationic charge, Lf exerts a plethora of functions, such as antibacterial, antiviral, anti-inflammatory, antioxidant, and anticancer activities.
Lf is highly bioavailable after oral administration, showing high selectivity toward cancer cells and a wide range of molecular targets that control tumor proliferation, survival, migration, invasion, and metastasization (1). In this context, the mechanistic aspects responsible for the high selectivity of Lf for cancer cells, which might involve a primary interaction with peculiar cancer cell surface receptors, have not yet been elucidated. In this respect, most cancerous cells present a higher content of Sia when compared to normal cells. In particular, it has been observed that sialylation promotes glioblastoma (GBM) growth, sustaining cell stemness and invasion (2).
Moreover, suppression of sialylation increases sensitivity of GBM cells to chemotherapeutic drugs.
The aim of the present project is to investigate the role of Sia, both linked to the glycoprotein Lf and expressed on the surface of GBM cells, in mediating the selective targeting of Lf toward tumor cells and allowing the glycoprotein to exert its anti-cancer activity.
- Cutone A, et al. Cell Signal. 2020
- Gc S, et al. JCI Insight. 2022
Methodologies to be used to achieve the objectives and timetable of the activities
In order to evaluate the role of the Sia present on the glycan chains of bLf, a desialylated version of the glycoprotein will be produced by treatment with neuraminidase, an enzyme able to specifically cleave and release Sia.
After digestion, bLf will be purified by cation exchange chromatography, which allows the selective binding of the glycoprotein and the release of neuraminidase (pI around 6). The integrity of the desialylated protein will be assessed by SDS-PAGE and Coomassie Blue staining and the desialylation will be checked by a lectin-based Western blot. Desialylated bLf will then be tested for cell and nuclear targeting as well as for its anticancer activity.
To preliminarily evaluate the content of different types of Sias on the surface of cancer cells, lectin histochemistry and immunohistochemistry analysis will be carried out.
Lectin histochemistry will be used to identify monomeric forms of Sia linked α2-3 to galactose (Gal) and α2-6 to Gal or N-acetyl-D-galactosamine (GalNAc). Immunohistochemistry will allow us to identify Sia polymeric form, the so- called PolySia.
To disclose the contribution of Sias to the ability of bLf to selectively target cancer cells, different glioblastoma cell lines, already available in the lab, will be treated with neuraminidase. In detail, GBM established cell lines (e.g. U87MG, GL15) and primary cells from patients will be employed and compared with an astroglial commercial non-tumoral cell line, as control. GBM cells, untreated or treated with neuraminidase, will be incubated with native and desialylated bLf at different concentrations (ranging from 10 to 500 μg/ml) for 4h, 24h and 48h.
First, the evaluation of bLf internalization, and nuclear targeting, will be carried out after 4 and 24h of treatment both by Western blot on cytosolic and nuclear fractions and by immunofluorescence. Since our previous experiments and literature data have shown an anticancer activity of bLf in several cancer models, including GBM, we will perform proliferation and migration assays in bLf-treated GBM cells. Cell count with Thoma chamber or MTS assay will be employed to analyze proliferation and scratch/invasion tests will be used for evaluating the invasion capabilities of cells.
Finally, we will evaluate the apoptotic response of GBM cells to the drug Temozolomide in combination with bLF and the putative role of sialylation in this context. To analyze the apoptotic process, both microscopy assays and Western blot experiments will be performed.
Several molecular mechanisms underlying Lf anti-cancer ability have been unveiled, including the modulation of cell cycle, promotion of apoptosis, hindering of migration and invasiveness, as well as immunomodulation. Except for the indirect immunomodulatory pathway, the other mechanisms require the direct recognition of and selection between cancerous and normal cells by Lf. In this respect, most cancerous cells present a high content of glycoconjugates such as proteoglycans, glycoproteins and glycolipids, some of which are rich in Sias, all well-known interactors for Lfs. This crude recognition could be the base of the Lf anti-cancer specificity and selectivity. Based on this evidence, through both the production of a desialylated form of Lf and the desialylation of GBM cell lines, our study will disclose the contribution of Sia on the cell surface in the anti-cancer activity of Lf.
The desialylation of bLf could impact the cell internalization and nuclear translocation of the glycoprotein, thus possibly modifying its downstream biological activities. On the other hand, desialylated bLf could show a normal intracellular transport, whereas presenting an impaired anti-cancer activity. The desialylation of GBM cells will demonstrate the actual mechanistic role of different types of Sias in the high selectivity of Lf towards cancer cells.