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. 2026 May 25;43(1):21. doi: 10.1007/s10719-026-10219-z

3’-sialyllactose inhibits the migration and invasion of LPS-induced B16F10 mouse melanoma cells through inactivation of the NF-KB signaling pathway

Hee-Do Kim 1, Hyunju-Choi 1, Yu-Chan Chang 2, Cheorl-Ho Kim 1,✉
PMCID: PMC13201296  PMID: 42183904

Abstract

Malignant melanoma has a high mortality rate and is aggressive. The development, metastasis, and angiogenesis of melanoma have all been connected to toll-like receptor 4 (TLR4). However, signal transduction mediated by TLR4 for accelerating melanoma progression is fully unclear. Because of this, the current research has been carried out to explore drug candidate possibility using 3’-Sialyllactose (3’-SL). We investigated the inhibitory effect of 3’-SL on migration and invasion, which are associated with treatment difficulty and mortality. The suppression of matrix metalloproteinases 9 (MMP-9) expression and activity by 3’-SL in B16F10 murine melanoma cells and concurrently downregulated the MAPK signaling pathway involved in this regulation. Therefore, our results demonstrate that 3’-SL may be a good candidate for the development of therapeutic agents to suppress malignancy associated with metastasis and invasion.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10719-026-10219-z.

Keywords: 3’-sialyllactose (3’-SL), Lipopolysaccharide (LPS), MMP-9, NF-KB Signaling Pathway

Introduction

Melanoma is projected to remain the fifth most frequently diagnosed cancer type in 2025, with new subtypes continuing to be reported. Among these, metastatic melanoma remains particularly lethal, exhibiting a five-year survival rate of approximately 35% [1, 2]. Melanoma represents the most aggressive and malignant type of skin cancer, distinguished by extensive cellular heterogeneity [3]. This heterogeneity contributes to therapeutic resistance and leads to unpredictable disease progression. Ultraviolet (UV) radiation exposure constitutes a major environmental risk factor for melanoma, as cumulative UV-induced DNA damage results in mutations within genomic regions that regulate cellular proliferation and repair. Such mutations can initiate the malignant transformation of melanocytes [4–6]. The ongoing effects of global warming, including elevated ambient temperatures and greater UV exposure, have been linked to the rising incidence of skin cancer worldwide [7]. Malignant melanoma displays rapid metastatic potential, frequently spreading to atypical organs such as the brain and heart [8, 9].

Tumor invasion and metastasis, major determinants of cancer-related mortality, involve complex, multistep processes mediated by numerous molecular factors, most notably matrix metalloproteinases (MMPs) [10]. Degradation of the basement member and extracellular matrix (ECM) facilitates tumor cell migration and proliferation within metastatic niches, and MMPs are further implicated in several physiological and pathological events, including inflammation, fibrosis, angiogenesis, invasion, and metastasis. Among the MMP family, MMP-2 and MMP-9 have been extensively associated with tumor invasiveness, with MMP-9 serving as a critical determinant of metastatic aggressiveness [11, 12].

Human breast milk contains numerous bioactive constituents that support infant growth, immune function, and gastrointestinal development [13]. Among these, sialyllactose (SL) is sialylated human milk oligosaccharides (HMOs) composed of lactose and sialic acid. Structurally, 3’-sialyllactose (3’-SL; α-NeuNAc-(2→3)-β-D-Gal-(1→4)-D-Glc) and 6’-sialyllactose (6’-SL; 6’-SL; α-NeuNAc-(2→6)-β-D-Gal-(1→4)-D-Glc) differ in their glycosidic bonding at the α-2,3 or α-2,6 position, respectively [14]. Recent studies have identified diverse biological functions of 3’-SL, including modulation of immune homeostasis, attenuation of inflammation, and prevention of atopic dermatitis through downregulation of pro-inflammatory cytokines mediated by regulatory T cells [15, 16].

Current therapeutic strategies for melanoma encompass surgical excision, chemotherapy, radiotherapy, immune checkpoint blockade, and targeted molecular therapies [17–21]. However, due to the highly heterogeneous nature of melanoma, the search for novel therapeutic agents and mechanisms remains imperative. Despite the expanding knowledge of 3’-SL biological effects, its potential role in melanoma pathogenesis has not yet been explained yet [22]. Therefore, the present study aimed to investigate the consequences of 3’-SL on metastasis-related processes in lipopolysaccharide (LPS)-stimulated B16F10 melanoma cells. The results revealed that 3’-SL significantly reduced MMP-9 expression and suppressed the mRNA and protein levels of inducible nitric oxide synthesis (iNOS) and cyclooxygenase-2 (COX-2), while inhibiting the activation of associated signaling pathways. Collectively, these findings propose that 3’-SL may function as a prospective therapeutic alternative for the treatment of metastatic melanoma by targeting inflammation-mediated signaling and metastasis.

Materials and methods

Reagents

3’-Sialyllactose was purchased from GeneChem Inc. (Daejeon, South Korea). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), and penicillin-streptomycin were acquired from WelGENE (Daegu, South Korea). 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide (MTT), Coomassie blue R-250, DAPI staining solution, Griess reagent, lipopolysaccharide (LPS) (Escherichia coli 0111:B4), and gelatin compound were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for MyD88, COX-2, iNOS p38, NF-KB, Lamin B, β-Actin, and MMP-9 were purchased from Santa Cruz Biotechnology (Paso Robles, CA, USA). Antibodies specific to TLR4, ERK, phosphorylated ERK (p-ERK), phosphorylated IKB (p-IKB), and IKB were acquired from Cell Signaling Technology (Beverly, MA, USA). Protein molecular weight markers were purchased from SMOBIO (Hsinchu City, Taiwan).

Cell culture

B16F10 murine melanoma cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37 ℃ in a humidified incubator containing 5% CO2. The culture medium was refreshed every two days.

MTT assay

The cytotoxic effect of 3’-SL was evaluated using the MTT assay. A total of 1Inline graphic104 B16F10 cells/well were seeded into 96-well plate and cultured overnight to allow cell attachment. Following incubation, the medium was replaced with fresh DMEM containing 5 µg/mL LPS, a combination of 3’-SL and LPS or 3’-SL alone. After 24 h of treatment, MTT reagent was added to each well, and the cells were further incubation for 4 h at 37 ℃. Subsequently, the medium containing residual MTT was carefully removed, and the resulting formazan crystals were dissolved in DMSO. The absorbance of each sample was recorded at 570 nm using a VersaMax microplate reader (Molecular Devices, San Jose, CA, USA) following a 10 min solubilization period at room temperature.

Gelatin zymography

B16F10 melanoma cells were seeded into 6-well cell culture plate and allowed to adhere overnight. After incubation, the cells were exposed for 24 h to either 3’-SL alone or a combination of 3’-SL and LPS in serum-free DMEM. Following treatment, the conditioned media were collected and subjected to electrophoresis on 10% SDS-polyacrylamide gels containing gelatin as a substrate. The gels were subsequently washed with 2.5% Triton X-100 buffer to remove residual SDS and then incubated overnight at 37 ℃ in an activation buffer composed of 50 mM Tris-HCl (pH 7.6), 10 mM CaCl2, and 0.01% NaN3. After incubation, the gels were stained with Coomassie Blue R-250 solution containing 45% methanol, 45% distilled water, and 10% acetic acid, followed by destaining in a solution of 60% distilled water, 30% methanol, and 10% acetic acid. The appearance of clear lytic bands against the blue background indicated gelatinolytic (collagenase) activity.

Reverse transcription polymerase chain reaction (RT-PCR) analysis

Total RNA was isolated using TRIzol reagent from Invitrogen (Carlsbad, CA, United States). Subsequently, 1 µg of total RNA was reverse transcribed into complementary (cDNA) employing the RT Premix kit from Bioneer (Daejon, CA, South Korea). The resulting cDNA was used as a template for the amplification of target genes by polymerase chain reaction (PCR) with gene-specific oligonucleotide primers. Amplified PCR products were separated on 1.5% agarose gels and visualized under ultraviolet (UV) illumination to confirm gene expression. The primer sequences were designed as follow: GAPDH (murine) : forward (5′-TCCACCACCCTGTTGCTGTA-3′) and reverse (5′-AATGTGTCCGTCGTGGATCT-3), MMP-9 (murine): forward (5′-CCTGTGTGTTCCCGTTCATCT-3′) and reverse (5′-CGCTGGAATGATCTAAGCCCA-3′), iNOS (murine): forward (5′-ATGTCCGAAGCAAACATCAC-3′) and reverse (5′-TAATGTCCAGGAAGTAGGTG-3′), and COX-2 (murine): forward (5′-GGAGAGACTATCAAGATAGT-3′) and reverse (5′-TGATCTTCATTTTTTACGCGTGAATT-3′). The intensity of the visualized DNA bands was assessed using an imaging system obtained from Davinch-K Co., and densitometric analysis was performed using the Image J software (Bethesda, MD, United States).

Protein extracts and western blot analysis

Whole-cell lysates of B16F10 melanoma cells were prepared using in 1% NP-40 lysis buffer supplemented with 100 mM sodium orthovanadate, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 M HEPES (pH 7.45), 1.5 M NaCl, and a protease inhibitor cocktail tablet. Cytosolic fractions were obtained by using cells in 10% NP-40 buffer containing 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 10 mM KCl, 0.5 mM PMSF, and 10 mM HEPES (pH 7.9). The lysates were centrifuged at 13,000 rpm for 10 min at 4 ℃ to separate cytoplasmic proteins. Nuclear fractions were extracted using a nuclear lysis buffer composed of 20 mM HEPES (pH 7.9), 0.5 mM PMSF, 0.4 M NaCl, 1 mM DTT, 1 mM EDTA, and 1 mM EGTA. The nuclear pellets were resuspended in the same buffer, vortexed for 15 min, and subsequently centrifuged at 13,000 rpm to obtain the nuclear protein fractions in the supernatants. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories Hercules, CA, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked at room temperature in 5% skim milk prepared in TBST buffer (2 M Tris-HCl, 4 M NaCl, pH 7.5 and 0.01% Tween 20) and incubated overnight at 4℃ with the appropriate primary antibodies. After washing with TBST, membranes were incubated with horseradish (HRP)-conjugated secondary antibodies at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system.

Wound healing assay

The wound healing assay was performed to evaluate the migratory capacity of B16F10 melanoma cells. Cells were seeded into 6-well plates and grown to approximately 90% confluence. A linear scratch was created across the cell monolayer using a sterile pipette tip and detached or damaged cells were gently removed by rinsing twice with phosphate buffered saline (PBS). The culture medium was then replaced with fresh medium, and cells were exposed to the indicated treatments for 24 h. After incubation, wound closure was monitored and imaged using a DS-Fi3 camera (Nikon, Japan).

Transwell migration assay

The transwell migration assay was employed to assess the migratory potential of B16F10 melanoma cells. Cells were harvested, resuspended in serum-free medium, and seeded into the upper chamber of transwell inserts (Corning, Costar). The lower chamber was supplemented with complete medium containing 10% FBS and either 3’-SL alone or a combination of 3’-SL and LPS. After incubation for 24 h at 37 ℃, the inserts were removed, and the cells that had migrated to the underside of the membrane were fixed with 4% paraformaldehyde, permeabilized in 100% methanol, and stained with 5% crystal violet solution. Excess dye was rinsed off with PBS, and the stained membranes were subsequently immersed in 100% methanol to solubilize the retained dye. The absorbance of the extracted solution was then measured at 570 nm to quantitatively determine the degree of cell migration.

Nitric Oxide Assay

The production of nitric oxide (NO) was quantified using Griess reaction. B16F10 murine melanoma cells were seeded into culture plates and allowed to adhere overnight. The cells were subsequently treated with either 3’-SL alone or a combination of 3’-SL and LPS for 24 h. After treatment, equal volumes of the culture supernatant and Griess reagent were mixed, and the absorbance was measured at 540 nm using a microplate reader. Following a 10 min incubation at room temperature, the concentration of NO was calculated based on a standard curve.

Measurement of pro-inflammatory cytokines

B16F10 melanoma cells were seeded into culture plates and allowed to adhere overnight. The cells were subsequently treated with either 3’-SL alone or in combination with LPS for 24 h. After incubation, the culture supernatants were collected for analysis. The concentrations of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6), were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (Affymetrix, eBioscience (San Diego, CA, United States) according to the manufacturer’s protocol.

Immunofluorescence

B16F10 melanoma cells were cultured on sterile coverslips placed in 12-well culture plates and allowed to adhere overnight. The cells were then treated with either 3’-SL alone or in combination with LPS for 24 h. After treatment, the cells were fixed with 4% paraformaldehyde at 37℃ for 10 min, followed by permeabilization with 0.2% Triton X-100 for 10 min at room temperature. Non-specific binding sites were blocked with bovine serum albumin (BSA) for 30 min at room temperature. Subsequently, the cells were incubated overnight at 4 ℃ with the specific primary antibody, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 1 h at room temperature in the dark. The nuclei were counterstained with DAPI solution for 10 min at room temperature. Fluorescence images were acquired using a Zeiss LSM 700 confocal laser microscope (Carl Zeiss, Oberkochen, Germany).

Statistical analysis

All experiments were performed independently at least three times, and representative data are presented. Statistical analyses were performed using GraphPad Prism. Differences among groups were evaluated by one-way analysis of variance (ANOVA), and statistical significance was determined based on ANOVA results.

Results

Effect of 3’-SL on cell viability in LPS-induced B16F10 murine melanoma cells

To assess potential cytotoxic effects, B16F10 melanoma cells were treated with a concentration of 3’-SL (0, 20, 40, 60, 80, and 100 µM), either independently or together with LPS. As depicted in Fig. 1, cell viability remained unchanged across all tested concentrations in both treatment groups. These observations imply that 3’-SL does not elicit cytotoxic responses on B16F10 cells within the examined range. Accordingly, these concentrations were deemed appropriate for subsequent experimental analyses.

Fig. 1.

Fig. 1

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Evaluation of the cytotoxic effect of 3’-SL in the presence or absence of LPS in B16F10 melanoma cells. (a) Schematic diagram of 3’-SL. (b) Cytotoxicity of 3’-SL with or without LPS stimulation in B16F10 melanoma cells. B16F10 cells were treated with various concentrations of 3’-SL either with or without 5 µg/mL LPS, and cellular viability was determined through MTT assay. Data was presented from three independent measurements

Regulatory Role of 3’-SL in MMP-9 Expression, Enzyme Activity, and Cell Migration in LPS-induced B16F10 Melanoma Cells

Exposure to LPS promotes the induction of host MMPs. In particular, MMP–9 contributes to the degradation of extracellular structures, participates in immune response, and governs cell motility [23, 24]. In order to additional clarification of the regulatory effect of 3’-SL on migration of cells caused by LPS, B16F10 melanoma cells were exposed to LPS in combination with increasing concentrations of 3’-SL. The activity and protein expression of MMP-9 were determined by using gelatin zymography and western blot assay, respectively. As presented in Fig. 2a, LPS stimulation markedly enhanced MMP-9 enzyme activity, whereas the addition of 3’-SL led to a gradual decline in activity in a way that depends on concentration. Consistent with this observation, the mRNA and protein levels of MMP-9 exhibited similar patterns of suppression, as depicted in Fig. 2b and c. Consistent with previous results, the wound-healing and transwell assays in Fig. 2d and e, respectively, showed that 3’-SL effectively suppressed the migratory capacity of B16F10 cells. These collective findings imply that 3’-SL mitigates the LPS-driven induction of MMP-9 expression and activity in B16F10 melanoma cells [25].

Fig. 2.

Fig. 2

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Modulatory effect of 3’-SL on LPS-induced migration of B16F10 melanoma cells through downregulating MMP–9 expression and enzyme activity. B16F10 cells were treated with 3’-SL at various consistencies of 0, 20, 40, and 80 µM, in the presence or absence of 5 µg/mL LPS. (a) Zymographic analysis of cell culture supernatants conducted experiments to assess MMP-9 activity. (b) RT-PCR analysis of MMP-9 mRNA levels. (c) Western blot assay of MMP–9 protein levels using MMP-2 and β-actin as internal controls. Quantification was performed utilizing ImageJ software. (d) Wound Healing Assay. A linear scratch was generated across the confluent B16F10 cell monolayer using a sterile 10-㎕ pipette tip. The wound closure was monitored and imaged under a phase-contrast microscope camera after 24 h of treatment with 80 µM of 3’-SL. (e) Transwell Migration Assay. The migrated cells were fixed, stained with crystal violet, and subsequently quantified by measuring the absorbance at 570 nm using a microplate reader. The results shown mean ± SEM and represent three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared with LPS-treated cells. All data were derived from reflections of at least three distinct replicates

3’-SL suppresses LPS-induced NO production, as well as levels of COX-2 and iNOS protein expressions, in B16F10 melanoma cells

NO is synthesized by the enzyme nitric oxide synthases (NOSs), which exist in three isoforms: neuronal NOS (nNOS), inducible NOS (iNOS), endothelial NOS (eNOS). Among them, iNOS is upregulated in response to specific stimuli such as LPS, leading to elevated production of COX-2, NO, and pro-inflammatory cytokines [26–30]. The activation of MMP-9 is facilitated by NO generation brough on by LPS stimulation [31–33]. To determine whether LPS enhances NO generation and to elucidate the regulatory effect of 3’-SL on this process, the drugs were applied to B16F10 cells under the indicated conditions. Following LPS exposure, both the mRNA and protein expression levels of COX-2 and iNOS were markedly elevated, as presented in Fig. 3, but were subsequently reduced in response to 3’-SL treatment. A similar inhibitory trend was observed in NO production. Collectively, these findings suggest that 3’-SL can downregulate NO synthesis as well as iNOS and COX-2 expressions, thereby attenuating the signaling cascade associated with MMP-9 activation in LPS-stimulated B16F10 cells.

Fig. 3.

Fig. 3

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3’-SL inhibits NO production and COX − 2 and iNOS expression on LPS-induced B16F10 cells. B16F10 cells were treated with 3’-SL at diverse concentrations of 0, 20, 40, and 80 µM, in the presence or absence of 5 µg/mL LPS. (a) The amount of Nitric oxide (NO) levels was determined by Griess assay. (b) COX − 2 and iNOS mRNA levels were detected by RT − PCR analysis. (c) COX − 2 and iNOS protein levels were detected by Western blot, respectively. GAPDH and β–actin were used for internal controls. Quantification was performed utilizing ImageJ software. The results shown mean ± SEM and represent three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared with LPS-treated cells. All data were derived from reflections of at least three distinct replicates

Secretion of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) that promote inflammation is potentially suppressed by 3’-SL

To explore whether the impact of 3’-SL on TNF-α, IL-6, and IL-1β levels are regulated through TLR4, we further observed the effect of 3’-SL in various concentrations, both with and without LPS on B16F1F10 cells. By using ELISA, pro-inflammatory cytokines were detected. When LPS is recognized by surfaced glycoproteins like Toll-like receptors (TLRs), macrophages are known to produce and release pro-inflammatory cytokines like IL-1β, TNF-α, and IL-6 during LPS-mediated inflammation [34, 35]. Following treatment with 3’-SL and LPS, supernatants were collected to measure the production of pro-inflammatory cytokines. Production of cytokines (TNF-α, IL-1β, and IL-6) in B16F10 murine melanoma cells was reduced in a dose-dependent way, as illustrated in Fig. 4a and b.

Fig. 4.

Fig. 4

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3’-SL inhibits cytokine (TNF-α, IL-6, and IL-1β) levels induced by 5 µg/mL LPS. B16F10 cells were treated with 3’-SL at various consistencies of 0, 20, 40, and 80 µM, in the presence or absence of 5 µg/mL LPS. (a) Cytokine secretion was quantified by ELISA. (b) mRNA levels of TNF-α, IL-6, and IL-1β were detected by RT − PCR analysis. GAPDH was used for internal control. Quantification was performed utilizing ImageJ software. The results shown mean ± SEM and represent three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared with LPS-treated cells. All data were derived from reflections of at least three distinct replicates

3’-SL inhibits LPS-induced mitogen-activated protein kinase (MAPK) signaling pathways (JNK, p38, and ERK) in B16F10 melanoma cells

LPS is recognized as a prototypical ligand of TLR4. Upon binding of LPS to TLR4 on the cell surface, ERK, JNK, and p38, plays a central part in regulating cell proliferation, differentiation, and the expression of pro-inflammatory cytokines [36–39]. The downstream targets of TLR4 signal pathway were examined by using the Western blotting in order to investigate the molecular system behind the inhibitory effect of 3’-SL. In B16F10 melanoma cells, LPS treatment stimulated the expression of TLR4 and MyD88 protein. Notably, treatment with 3’-SL attenuated this upregulation in a concentration-dependent manner, as depicted in Fig. 5a. Further study into the molecular mechanisms was conducted by examining the alterations in downstream signaling pathways. In Fig. 5b, phosphorylation levels of ERK, JNK, and p38 were significantly increased in LPS-stimulated B16F10 melanoma cells, indicating LPS-induced activation of the MAPK signaling pathway. Conversely, 3′-SL treatment resulted in a concentration-dependent decrease in phosphorylation of the three MAPK components.

Fig. 5.

Fig. 5

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3’-SL inhibits LPS-induced TLR4 expression and MAPK signaling pathways such as JNK, p38, and ERK on B16F10 melanoma cells. B16F10 cells were treated with 3’-SL at diverse consistencies of 0, 20, 40, and 80 µM, in the presence or absence of 5 µg/mL LPS. Western blot analysis was conducted using antibodies specific for TLR4, MyD88, p-JNK, JNK, p-p38, p38, p-ERK, and ERK with β–actin as loading control. Quantification was performed utilizing ImageJ software. The results shown mean ± SEM and represent three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared with LPS-treated cells. All data were derived from reflections of at least three distinct replicates

These results suggest that 3′-SL inhibits LPS-induced MAPK activation. Since the MAPK pathway plays a key role in regulating inflammatory cytokine expression and cell motility, the inhibition of MAPK phosphorylation by 3′-SL may be mechanistically linked to the observed reduction in inflammatory mediators and inhibition of melanoma cell migration and invasion. Therefore, the results in Fig. 5b support the role of 3′-SL as a modulator that effectively attenuates MAPK signaling under inflammatory conditions.

3’-SL reduced LPS-induced NF-KB translocation in B16F10 melanoma cells

NF-KB serves as a pivotal transcriptional factor that modulates governs the expression of several genes associated with cytokine synthesis and pro-inflammatory enzyme production [40]. It is also implicated in the modulation of MMP-9, a key mediator involved in ECM remodeling and cell migration in both physiological and pathological contexts [41]. To further elucidate this regulatory pathway, NF-KB expression was evaluated through Western blot analysis following subcellular fractionation into nuclear and cytoplasmic components. The analysis revealed a pronounced reduction in the nuclear accumulation of the NF-KB p65 subunit, accompanied by elevated cytoplasmic levels of its inhibitory protein, IKB. These observations indicate that 3’-SL impedes the nuclear translocation of the p65 subunit. Consistent with these biochemical results, immunofluorescence microscopy in Fig. 6b demonstrated diminished nuclear localization of NF-KB upon treatment of 3’-SL. Collectively, these findings imply that 3’-SL effectively suppresses NF-KB signaling by preventing p65 nuclear translocation, irrespective of the presence of LPS stimulation.

Fig. 6.

Fig. 6

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3’-SL inhibits LPS-induced NF-KB translocation in B16F10 cells. (a) Western blotting of cytoplasmic and nuclear extracts using antibodies against NF-KB, IKB, and p-IKB with Lamin B and β-actin as internal controls. Quantification was performed utilizing ImageJ software. (b) Immunofluorescence staining of NF-KB using FITC-conjugated secondary antibody and DAPI nuclear counterstain, visualized by confocal microscopy. The results shown mean ± SEM and represent three independent experiments. * p < 0.05 and ** p < 0.01 indicate significant differences compared with LPS-treated cells. All data were derived from reflections of at least three distinct replicates

Discussion

3’-SL is a type of human milk oligosaccharides (HMOs) and prebiotic that contains the monosaccharide N-acetylneuraminic acid (Neu5Ac), which is linked to the 3’ position of the galactose residue in lactose (Fig. 1a). Previous studies have reported that 3’-SL exerts multiple physiological functions, including the prevention of arthritis and the promotion of intestinal microbial growth and activity. In particular, its anti-inflammatory properties have been shown to protect human chondrocytes and mitigate the progression of osteoarthritis [42, 43]. In the present study, the cytotoxic effects of 3’-SL on melanoma cells were examined. Results from cell viability assays demonstrated that 3’-SL exhibited no significant cytotoxicity toward B16F10 melanoma cells.

Melanoma is a malignant skin cancer that develops because of accumulated genetic mutations and alterations in the microenvironment of tumors. NF-KB plays a crucial part in the progression of melanoma by enhancing anti-apoptotic signaling [44]. One of the contributing factors to elevated NF-KB activity in melanoma is the overexpression of Toll-like receptor 4 (TLR4), which recognizes a wide variety of molecular patterns derived from pathogens. TLR4 is frequently overexpressed in several types of cancer and plays multifaceted roles in tumor initiation, progression, and immune modulation. Lipopolysaccharide (LPS), a prototypical ligand of TLR4, has been shown to activate this receptor, resulting in enhanced migration and invasion of cancer cells, including those of the breast, esophagus, and colon cancer [35, 45].

Building on these findings, this study investigated the effect of 3’-SL on TLR4-mediated MAPK and NF-KB signaling pathways, inflammatory cytokine production, and metastasis in an LPS-induced B16F10 melanoma cell model. The expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at both the mRNA and protein levels was markedly downregulated following 3’-SL treatment, accompanied by a significant reduction in nitric oxide (NO) production. Furthermore, 3’-SL suppressed the production of cytokines that promote inflammation and attenuated the activation of MAPK and NF-KB pathways [46]. These effects were associated with a pronounced inhibition of MMP-9 activity, as evidenced by zymography and transcriptional analysis.

B16F10 melanoma cells, which are widely used as a model for studying tumor invasion and metastasis, have been reported to express pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. The production of these cytokines is primarily regulated by the MAPK and NF-KB signaling cascades [35]. The signaling molecule NO, which is generated by iNOS, controls physiological functions such as secretion of hormone and angiogenesis. However, undue NO production can lead to aberrant cellular changes that promote tumorigenesis [31]. Upregulation of iNOS, COX-2, and MMP-9 is known to contribute to enhanced invasiveness and decreased cell survival, ultimately facilitating metastasis.

Although the diverse physiological functions of 3’-SL have been well documented, its potential role in melanoma invasion and metastasis remains largely unexplored. The present findings reveal that 3’-SL significantly inhibited NO production and cytokine secretion in LPS-stimulated B16F10 murine melanoma cells. Moreover, treatment with 3’-SL reduced the MAPK pathway protein phosphorylation like ERK, JNK, and p38, and blocked the nuclear translocation of NF-KB, which controls the expression of multiple pro-inflammatory and metastatic genes. Synthetically, these findings imply that 3’-SL exerts an anti-metastatic effect on melanoma cells by suppressing LPS-triggered activation of the TLR4, MAPK, and NF-KB signaling pathways. In conclusion, although the present study demonstrates that 3′-SL effectively suppresses LPS-induced MAPK and NF-KB signaling in melanoma cells, the precise molecular mechanisms remain to be clarified. Future studies will be required to determine whether 3′-SL interacts with specific receptors, such as siglecs, and to assess whether similar effects are observed with structurally related carbohydrates, including lactose or free sialic acid. Elucidating the receptor and structural specificity of 3′-SL will be essential for further understanding its mode of action and therapeutic potential.

In Fig. 7, the putative inhibitory mechanism of 3’-SL has been schematically illustrated to depict LPS-stimulated B16F10 murine melanoma cells. 3’-SL regulates LPS induced cell migration and invasion via inhibiting of MAPK and NF-KB signaling pathways.

Fig. 7.

Fig. 7

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Schematic illustration depicting the putative inhibitory mechanism of 3’SL in LPS-stimulated B16F10 murine melanoma cells. 3’SL regulates cell migration and invasion via inhibiting of MAPK and NF-KB signaling pathways

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (PDF 1.41 MB) (1.4MB, pdf)

Author contributions

Conceptualization,C.-H K, H.-D.K; writing—original draft preparation, H.-D.K; writing—review and editing, Y.-C C, C.-H.K.; visualization, H.-D.K.; supervision, C.-H.K.; project administration, C.-H.K., H.-J.C; funding acquisition, C.-H.K. All authors have read and agreed to the published version of the manuscript.

Funding

Open Access funding enabled and organized by SungKyunKwan University. This study has in part been supported by the National Research Foundation of Korea (NRF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

Not applicable.

Declarations

Informed Consent

Not applicable.

Competing interests

The authors declare no competing interests.

Institutional Review Board

Not applicable.

Footnotes

Hee-Do Kim and Hyunju-Choi equally contributed as co-first authors.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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