Expression of 9- O-Acetylated Sialic Acid in HPV+ Oral Squamous Cell Carcinoma Cells

Abstract

Oral squamous cell carcinoma (OSCC) is a common type of head and neck malignancy that represents a significant global health issue. Sialylations are common events in tumor transformation, proliferation, metastasis, and immune evasion. Modifications in sialylation can be detected by lectins, whose changes in OSCC have been related to grade, invasion, and metastasis. The presence of 9-O-acetylated sialic acid (Neu5,9Ac₂) in OSCC cells and its potential expression, modification, and role are unknown. This study aimed to analyze the expression of Neu5,9Ac₂ using the Macrobrachium rosenbergii lectin (MrL) that recognizes this sialic acid (Neu5Ac) residue and also compare its effect on the SCC-152 cell line (CRL-3240, ATCC) and immortalized keratinocytes (HaCaT) as a control. We observed by immunocytochemistry that SCC-152 cells expressed more Neu5,9Ac₂ compared to HaCaT cells; the specificity of MrL was confirmed after the sialidase treatment of cells in which the loss of lectin's recognition of Neu5,9Ac₂ was observed. The electrophoretic profile was similar between both cell line types; however, the Western blot showed differences in the glycoprotein patterns recognized by lectin for each cell type. MrL increased the proliferation of SCC-152 cells, as well as the integrity and morphology of the colonies. Therefore, our results suggest that Neu5,9Ac₂ glycosylated receptors could be involved in the survival and proliferation of OSCC cells, which offers a promising avenue for developing diagnostic and prognostic tools (tumor markers) against oral squamous cell carcinoma in the future.

Keywords: HaCaT; Macrobrachium rosenbergii lectin; Neu5,9Ac2; Neu5Ac; OSCC; SCC-152; glycosylation; prognostic tools.

Figure 1

The biosynthesis of Neu5Ac in mammalian cells and the acetylation of sialic acid at carbon 9. The synthesis of sialic acid in vertebrate cells begins in the cytosolic compartment. (1) Once glucose (Glc) is located intracellularly at this site, (2) it is recognized by hexokinase to produce Glucose-6-phosphate (Glc-6P), (3) and then this product is metabolized by the hexosamine pathway to obtain UDP-N-Acetyl-D-glucosamine (UDP-GlcNAc). (4) Now, by the enzymatic action of UDP-GlcNAc-2-epimerase/ManNAc kinase on the previous metabolite in the presence of a water molecule, N-Acetylmonosamine (ManNAc-) is obtained. (5) ManNAc is recognized by UDP-GlcNAc 2-epimerase/ManNAc kinase and in the presence of ATP, N-Acetyl-monosmaine-6-phosphate (ManNAc-6-phosphate) is produced, and (6) together with Phosphoenolpyruvate (PEP), both substrates are recognized by the enzyme Neu5Ac-9-phosphate synthase to obtain N-Acetylneuraminic acid 9-phosphate (Neu5A-9-phosphate). (7) Subsequently, the catalytic activity of the Neu5Ac-phosphate phosphatase on Neu5A-9-phosphate plus a water molecule allows the production of sialic acid (Neu5Ac). (8) Neu5Ac is then transported into the nucleus (9) to combine with cytidine monophosphate (CMP) from cytidine triphosphate (CTP), thus obtaining the active form of sialic acid: CMP-Neu5Ac. Once CMP-Neu5Ac is obtained, it is sent to the (10) cytoplasm and transported to (11) the rough endoplasmic reticulum (RER) for early N-glycan synthesis or (12) to the Golgi apparatus to participate as a substrate in the terminal phase of N-glycosylation and O-glycosylation. Finally, (13) acetyl groups, donated from acetyl-CoA (AcCoA), are transferred for CMP-Neu5Ac by the action of the enzyme N-acetylneuraminate (7)-9-O-acetyltransferase (CASD1) to obtain CMP-N-Acetyl-beta-neuraminate (CMP-Neu5,9Ac₂), (14) a molecule that is used in the Golgi apparatus as a substrate for the addition of terminal 9-O-acetylated sialic acid (Neu5,9Ac₂) in N-glycan and O-glycan. The image is created by the authors using http://biorender.com/ (accessed on 22 March 2025).

Figure 2

SCC-152 cells express higher levels of Neu5,9Ac₂ compared to HaCaT cells. (A) SCC-152 and HaCaT cells were cultured in 10% FBS medium for 24 h and then fixed with 4% paraformaldehide. Indirect immunofluorescence was performed with MrL-biotin and Strptavidin-Alex594, as described in the Materials and Methods Section. Magnification: 40×. MrL (red) and nucleus (Blue). (B) The histogram displays the relative quantification of the MFI of the micrographs in Figure 2A by Fiji/ImageJ software (Version 1.54p). The results are the means ± SEM of 3 independent experiments. Statistical analysis is performed using one-way-ANOVA with using the Graphpad Prism Program (version 10.4.1). **** p < 0.0001. Abbreviations: MrL—Macrobrachium rosenbergii lectin; MFI—mean fluorescence intensity.

Figure 3

MrL recognition is mediated by sialic acid. (A) SCC-152 and (B) HaCaT cells were cultured as mentioned in the Materials and Methods Section and incubated with 0.1 IU of Clostridium perfingers sialidase at different time intervals (1–4 h) at 37 °C. Indirect immunofluorescence was performed with MrL-biotin and Strptavidin-Alex594 as described in the Materials and Methods Section. Magnification: 40×. MrL (red) and nucleus (Blue). The sialic acid hydrolysis assay shows a gradual decrease in MrL localization in both cell types over time. After 2 h of incubation, changes in fluorescence intensity are observed in the localization of the lectin compared to the control, with a very slight or almost non-existent intensity at 4 h in both cell types. (C,D) The histogram displays the relative quantification of the MFI of the micrographs in (A,B), respectively, by Fiji/ImageJ software (Version 1.54p). The results are the means ± SEM of 3 independent experiments. Statistical analysis is performed using one-way-ANOVA with using the Graphpad Prism Program (version 10.4.1). **** p < 0.0001. Abbreviations: MrL—Macrobrachium rosenbergii lectin; MFI—mean fluorescence intensity; n.s.—non-significant.

Figure 4

Glycoproteins recognized by MrL in SCC-152 and HaCaT cells. (A) Cell lysates from SCC−152 and HaCaT (30 μg) are run on 10% SDS-PAGE gels under reducing conditions. The gel is stained with Coomassie Blue to observe band resolution; however, no differences are found in the electrophoretic pattern between the two cell types. (B) A Western blot of SCC-152 and HaCaT cell lysates incubated with MrL reveals that the lectin recognizes different glycoproteins. (C) Glycoproteins A (125.4 kDa) and G (25.7 kDa) are overexpressed in SCC-152 cells compared to HaCaT cells, with statistically significant differences. In contrast, glycoproteins C (70 kDa) and D (43 kDa) show significant differences and, together with glycoproteins F (32.8 kDa) and H (23.1 kDa), show a decrease in their expression in SCC-152 cells compared to HaCaT cells. The results shown are representative of at least three independent experiments using different cell preparations. A densitometric analysis of the expression levels found for each glycoprotein is shown at the right, and the data represent the means ± SEM from at least three independent assays. Statistical analysis is performed by ANOVA followed by Tukey’s post-test. ** p < 0.01; *** p < 0.001; n.s., non-significant, compared to each condition.

Figure 5

The effect of MrL on the proliferation and growth characteristics of SCC−152 and HaCaT cells. In this study, 1.5 × 105 cells are cultured in 96-well plates with culture medium supplemented with 10% FBS overnight, and then the cells are deprived of culture medium containing FBS for 24 h. The next day, MrL is added at concentrations of 1, 2.5 and 5 μg/mL for 24, 48 and 72 h. The proliferation assay with MTT is mentioned in the Materials and Methods Section. (A) The SCC-152 cell line shows a significant increase in cell proliferation at 24 h when incubated with MrL at concentrations of 1 and 2.5 μg/mL compared to the control. (B) In HaCaT cells, no effect on proliferation induced by MrL is observed at the concentrations and times used. To evaluate morphological characteristics and colony formation, SCC-152 and HaCaT cells (4.5 × 10⁵ cells per well) are cultured and stimulated with MrL under the conditions described above. Morphological characteristics are observed under an inverted microscope at 40×, and microphotographs are recorded at 24, 48, and 72 h. (C) MrL in SCC-152 cells causes a favorable effect on the preservation of colony integrity and morphology at all concentrations and times used. Colonies without lectin (control) show morphological alterations with poorly defined edges and an increase in the number of individual and dispersed cells. After 72 h, a total loss of colony integrity is observed, with a greater presence of dispersed cells. (D) MrL in HaCaT cells do not induce favorable changes in cell morphology or monolayer formation at any of the concentrations and incubation times used. MrL at a concentration of 5 μg/mL induces a negative effect on cell morphology after 24 h, while at 48 and 72 h, a loss of cell adhesion is observed compared to the control. The results represent the mean ± S.E.M. of 3 independent experiments. Statistical analysis is performed by ANOVA followed by Dunnett’s post-test. * p < 0.05; ** p < 0.01; n.s., non-significant.