N-acetylglucosamine is an essential amino sugar moiety for protein glycosylation [41]. Glycosylation, a posttranslational protein modification, plays a crucial biological role in many physiological and pathological events [42]. In studies, N-glycan changes have been detected during breast cancer progression [43]. In particular, numerous studies have focused on the dysregulated glycosylation involved in the development and progression of breast cancer [44, 45]. Previous studies concluded that control of glycosylation could be a practical treatment approach for cancer progression [46]. Therefore, regulation of glycosylation is an essential process for breast cancer and many types of cancer [46, 47].
Glucosamine plays an essential role in cancer treatment. Daily glucosamine injection has reduced cell mass and large haemorrhagic areas in Sarcoma 37 tumors in mice [48]. Although glucosamine treatment did not result in complete tumor regression, approximately double the survival time of treated mice has been reported [49]. Additionally, in a study investigating the protective effect of glucosamine, a linkage was observed between the use of glucosamine and a lower risk of lung and colorectal cancer [50]). It is known that O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible posttranslational modification of serine/threonine residues and that the O-linked N-acetyl-glucosamine transferase enzyme mediates O-GlcNAsylation of various proteins involved in cervical cancer tumorigenesis [51]. Alteration of protein glycosylation has been correlated with tumorigenesis, but the effect of regulation of glycosylation on tumor development remains unclear [8].
Our study focused on the immune system modulator effect of D-GlcNAc as a candidate supplement compound against breast cancer. An administrative dose was determined using the machine learning method (Table 1), which was later applied in vitro to MCF-7 and 4T1 cell lines, as the most common breast cancer cell lines with in vivo to xenograft murine model. In vitro, results demonstrated that D-GlcNAc inhibits cell proliferation by promoting apoptosis in MCF-7 and 4T1 cell lines in a concentration-dependent manner and following our findings, in another study, overexpressed MGAT3 in breast cancer MDA-MB-231 cells which leads to bisecting of N-GlcNAc, which causes suppression on the EGFR/Erk signalling and reduced migratory ability, cell proliferation, and clonal formation [45].
Our in vitro results showed promoted apoptosis and Fas expression in MCF-7 and 4T1 cell lines depending on the increasing concentrations of D-GlcNAc, and accordingly suppressed proliferation in cancer cells. Furthermore, the data have been consistent with the hypothesis of our studies using the machine learning method, which has indicated 2 mM D-GlcNAc as the cut-off level for apoptosis, Fas expression, and proliferation rate were confirmed with in vitro results. Therefore, we have used 2 mM D-GlcNAc to treat the xenograft murine model.
In a previous study, abnormal glycosylation correlated with N-glycan alterations in breast cancer cells compared to normal cells [52]. In addition, GlcNAc and glucosamine (GlcN) have been used as contrast agents to detect breast tumors, by targeting tumor cells using magnetic resonance imaging [53]. Afterwards, a study closely related to our findings indicated that GlcNAc sensitizes non-small cell lung cancer cells (NSCLC) via TRAIL-induced apoptosis, which can be a novel effective agent for TRAIL-mediated NSCLC-targeted therapy [4]. Since the tumor growth and metastatic spread of 4T1 cells in BALB/c mice closely mimic human breast cancer, we have preferred applying the 4T1 mouse model. The D-GlcNAc was administered on the 1st day or 18th day after inoculation of 4T1 cells to evaluate the pre-tumoral and post-tumoral effect of D-GlcNAc. The in vivo results demonstrated that D-GlcNAc could reduce tumor size, mitosis, and angiogenesis in the post-treatment group, which could indicate the tumour-targeted effect of D-GlcNAc.
Sialylations mediate signaling, immunological responses, and cell-cell interactions in normal cells. However, abnormalities in sialylations can occur due to mutated genes, altered gene expressions, or defective enzymes involved in metabolism, including sialyltransferases and sialidases. They affect sialic acid metabolism and normal sialylation of glycoproteins and glycolipids associated with diseases. Sialic acid is a biomarker of several diseases, including cancer [54]. Sialylation generally increases in tumor cells and is incorporated with N-glycans and O-glycans. N-glycan sialylation could serve as a regulatory mechanism for various receptor tyrosine kinases, as shown by following the activation of MET and RON receptors by α2-3Sia.
Contrary to the effect of galectins, death receptor ligands, and chemotherapeutic drugs inducing apoptosis, increase α2-6Sia on N-glycans because ST6GAL1 up-regulation in cancer cells enhances integrin-mediated cell motility and protects cells against apoptosis [7]. Here, we have assumed a reversible effect of exogenously administered D-GlcNAc that removes cells' protection against apoptosis by binding on HER2 protein (Fig. 6, Fig. 7). Furthermore, in a previous study, HER2 showed an inhibitory role in the cGAS–STING-Mediated immune response, which causes DNA damage [55]. Our presented data with molecular docking and dynamics analysis have proved that the binding of D-GlcNAc to HER2 could be associated with preventing DNA damage (Fig. 6, Fig. 7). In vitro assays and murine model studies have supported our hypothesis according to the induced apoptosis in D-GlcNAc-administered cancer cell lines (Fig. 4, Fig. 5).
The predicted interferences related to the induction of tumor progression involving β1–4 branched tetra-antennary N-glycan formation by MGAT4 expression enhances lattice formation via galectin binding to poly-N-acetyllactosamines [7]. The exogenously D-GlcNAc administration could result in the displacement of Galactin, and D-GlcNAc's binding on HER2 could revert tumor progression into the recession phase due to apoptosis and cell death.
The cancer cells spread throughout the body and show high metastasizing, which can avoid detection and neutralization by the immune system. This case relies on mimicking the glycosylation patterns of healthy immune cells, which is the reason for self-signalling and avoiding the immune system attack. Sialylation converts the surface of cancer cells to prime ligands for sialic acid-binding immunoglobulin-type lectins (Siglecs), similar to the immune cells' surface architecture [56, 57]. Bounding to sialylated glycans produces Siglecs promotion due to immunosuppressive signalling and protects the tumour cell [58–60]. Therefore, natural killer (NK)-cell-mediated tumor cell death was inhibited by interactions between NK-expressed Siglec-7 or Siglec-9 and sialylated glycans (Siglec ligands), which leads to the hiding of tumor cells from immune cells [61, 62]. In previous studies, monoclonal antibodies targeting Siglec-7 and Siglec-9 have been suggested to neutralize Siglec–Siglec ligand interactions [63]. This study showed that the binding of Siglec-9-expressing macrophages plays a role in the induction of a tumour-associated macrophage phenotype that has primed the myeloid cells to release factors that promote disease progression (such as interleukin 6 and macrophage colony-stimulating factor) that are based on soluble mucin-1 and mucin-1 expressed on T47D breast cancer cells [63, 64]. Multiple myeloma cells are possible, which bind to Siglec-7 and Siglec-9 and avoid NK cells. Still, once the sialic acid residues treat with neuraminidase or inhibited by sialylation using the sialyltransferase inhibitor 3Fax-Neu5Ac34, NK cells can kill multiple myeloma cells [65].
The cancer cells hide from immune defence cells by mimicking the sialic acid-binding immunoglobulin(Ig)-like lectins, which are the members of the immunoglobulin superfamily, and act like immune regulatory receptors on the transmembrane cell surface. Consequently, sialic acid-binding sites activate or inhibit the immune response [66]. In the insights of previous findings related to sialic acid-binding Ig-like lectin mechanisms, our study showed that D-GlcNAc could compensate for lectin, previously desired in the sialic acid-binding Ig lectin mechanism. We suggested that the cancer cells could be deceived and directed to apoptosis by the presence of D-GlcNAc. As parameters related to the inhibition of cancerogenic cells, we have observed a significant decrease in metastasis and an increase in apoptosis and Fas expression on D-GlcNAc treated cells (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5). We have also observed a veinal decrease and recession of metastasis in mice murine model studies, which could be associated with the positive effect of D-GlcNAc.
Alternatively, factor H limiting activation and modulation of tumour cells helps for metastasis and suppresses antitumour immunity by inhibition of Siglecs. Some tumour cells express de-N-acetyl (deNAc) gangliosides, which protect tumour cells from apoptosis and activate the epidermal growth factor receptor (EGFR). Mucins could prevent intercellular interactions and inhibit cadherins and integrins from functioning, resulting in apoptosis. Hence, they might block and mask recognition by major histocompatibility complex molecules [7]. Mucin secretion results from the loss of normal topology and polarisation of epithelial cells in cancer due to impairment of O-GalNAc molecule structure. Tumour cells attack tissues and the bloodstream when they have mucin on their cell surfaces. O-GlcNAc levels are altered in many cancers, which serve as a nutrient sensor to regulate signalling, transcription, mitochondrial activity, and cytoskeletal functions [67, 68]. Since this case activates EGF or IGF-1 signalling, we assumed it to have a reversible effect of D-GlcNAc, leading breast cancer to apoptosis and Fas expression (Fig. 2, Fig. 3).
O-GlcNAcylation influences cancer cell metabolism due to changes in the stability or activity of transcription factors and kinases, which the administration of D-GlcNAc could modulate. In recent studies, the findings on targeting glycan-dependent molecular interactions have attracted researchers' attention to enhance the immune system. The sialoglycan–Siglec interaction displays another critical point of view of the immune checkpoint. A function-blocker and modulator compound introduced to replace Siglecs, such as lectin precursor GlcNAc, could be suggested [69, 70]. Therefore, D-GlcNAc could be considered and tested in clinical studies on account of its potential effect by combining with other approved methods and anticancer drugs developed to block tumour cells via administration of sialic acid mimetics or the targeted delivery of sialidases to tumour cell surfaces [69].
Our findings reveal a major role of D-GlcNAc, which is necessary for activating the immune system resulting in early apoptosis and recession in metastasis that we have observed in our mice murine model. O-GlcNAc transferase enzyme (OGT) functions on UDP-GlcNAc as a nuclear and cytoplasmic metabolism substrate. This interaction increases in multiple cancers, and a decrease in OGT level blocks tumour growth. Previous studies have reported MannaC as an inhibitor of sialic acid in cancer cells. We have predicted the impairment in the mechanism related to converting UDP-GlcNAc to Mannac. Therefore, it does not work as a sialic acid inhibitor, preventing apoptosis and Fas expression. However, D-GlcNAc could not be utilized by cancer cells and decrease the transferase enzyme, which could reduce metastasis and veining as we have observed in mice murine model studies [70–72]. As reported in a previous study, the viral agents' energy consumption, the predicted reason for breast cancer, and HCMV infection did not increase intracellular UDP-sugar metabolite pools. However, this process induced UDP-sugar biosynthesis depending on UDP-glucose and the biosynthetic flux of UDP-GlcNAc. This study suggested the role of sugar metabolism, which induced peculiar to HCMV infection [73]. Consistent with this finding, Pan et al. [74] showed upregulation of UDP-glucose dehydrogenase gene expression via ERK and PI3K/Akt pathway due to EBV latent membrane protein 2A detected in nasopharyngeal carcinoma. These findings demonstrate a linkage between virus infection and cancer formation depending on glycosylation pathways in the cell. As published in our previous studies, the stimulative effect of D-GlcNAc can potentially increase the defence capacity of the immunological response to SARS-CoV-2 [9, 10]. Moreover, N-acetyl-D-glucosamine-coated poly(amidoamine) structures have also induced upregulation of antibody formation in the rat recombinant cells [75], which could be a predicted response as expected in the human cell.
In accordance with our study, Denning et al. [76] showed that ROS induces Fas expression, which causes apoptosis in intestinal epithelial cells. In D-GlcNAc treated cells, we have observed higher Fas expression than untreated cells. ROS-induced Fas expression could be the reason for oxidative stress exposure to breast cancer due to D-GlcNAc administration. Moreover, oxidative stress causes starvation on cell growth, which activates Fas expression leading to apoptosis [77]. In our study, the higher Fas expression in D-GlcNAc treated cells and the increased apoptotic rate could be suggested triggering of apoptotic pathways and enhancing Necrosis on cancer cells depending on D-GlcNAc administration (Fig. 2, Fig. 3, Fig. 4, Fig. 5).