SG and DSG were derived from Gracilaria fisheri, which was collected from the Shrimp Genetic Improvement Center (Surat Thani, Thailand) between October and December 2024. The polysaccharides were prepared according to previously established methods (21,22). SG had a molecular weight of 217 kDa, whereas DSG had a molecular weight of 8 kDa, as determined by gel permeation chromatography (Fig. S1). The structures of SG and DSG were confirmed by ¹H- and ¹³C-nuclear magnetic resonance (Bruker Corporation) analyses. Both compounds consist of alternating 3-linked β-D-galactopyranose and 4-linked 3,6-anhydro-α-L-galactopyranose or α-L-galactopyranose-6-sulfate residues, as presented in Fig. S2, consistent with previous studies (21,22).

The surface morphology and elemental composition of SG and DSG were examined using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX). Samples (5–10 µg) were deposited onto SEM stubs and gold-coated for 2 min at a coating current of 20 mA. Surface morphology was visualized using an SEM (JSM-IT200™; JEOL, Ltd.) and elemental composition was analyzed using an EDX detector (Ultim^® Max 40; Oxford Instruments plc). For EDX measurements, the accelerating voltage was set to 10 kV, with a working distance of ~8.8 mm. Spectra were acquired from randomly selected regions of each sample to ensure representative elemental characterization. At least three independent areas were analyzed and the acquisition time for each spectrum was ~30 sec. Elemental mapping and point analyses were performed using the instrument software (AZtec^® version 6.1; Oxford Instruments) to identify and quantify the elemental distribution on the sample surface. The elemental composition was reported as weight (%).

Sulfate groups in SG and DSG were quantified using the barium chloride (BaCl₂)-gelatin method and confirmed by FTIR spectroscopy. Samples (20 mg) were hydrolyzed with 2 N HCl at 100°C for 2 h, centrifuged (3,000 × g, for 10 min at room temperature), and the supernatants were diluted with Milli-Q water and 0.5 N HCl. BaCl₂-gelatin reagent (1 ml) was added to the diluted supernatants, and the mixtures were incubated at room temperature for 30 min. Absorbance was measured at 550 nm to determine sulfate content as described previously (22). For FTIR analysis, SG and DSG (2 mg) were mixed with dried potassium bromide to prepare pellets and spectra were recorded using a Bruker TENSOR 27 spectrometer (attenuated total reflectance mode) over the range of 400–4,000 cm^-1 at a resolution of 1 cm^-1 with 16 scans.

Mammary epithelial cells (MCF-10A; cat. no. CRL-10317™) and breast cancer cells (MDA-MB-231; cat. no. HTB-26™) were obtained from the American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle Medium (Gibco™; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) and 0.5 M sodium bicarbonate in a humidified incubator at 37°C with 5% CO₂. The cytotoxicity of SG, DSG and doxorubicin [DOXO; Fresenius Kabi (Thailand) Ltd.], used as a positive control, was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates (3×10⁴ cells/well; 200 µl per well) and cultured overnight at 37°C. Cells were then treated with various concentrations of SG or DSG (100–1,000 µg/ml) or DOXO (0.02–1.50 µg/ml) for 24 h at 37°C. Cell viability was assessed using the MTT assay, in which dimethyl sulfoxide (DMSO) was used to solubilize formazan crystals, and absorbance was measured at OD 595 nm. Cytotoxicity was expressed as the 50% cytotoxic concentration.

Morphological changes in MCF-10A and MDA-MB-231 cells following treatment with DOXO, SG or DSG were evaluated. Cells (3×10⁶ cells/well) were seeded in 6-well plates and cultured for 24 h at 37°C, then assigned to four groups: i) Normal control (NC); ii) DOXO (0.5 µg/ml); iii) SG (1,000 µg/ml); and iv) DSG (1,000 µg/ml). After 24 h of treatment, cell morphology was examined using phase-contrast microscopy (Leica DFC 7000 T; Leica Microsystems GmbH). Morphological changes were quantified in three randomly selected fields at ×200 magnification using ImageJ software (version 1.54g; National Institutes of Health). To further assess membrane permeability in MDA-MB-231 cells, Hoechst/propidium iodide (PI) dual staining was performed. Cells (6×10⁴ cells/well) were seeded onto round coverslips in 24-well plates and cultured for 24 h at 37°C, followed by an additional 24 h of treatment at 37°C with DOXO, SG or DSG. Cells were then washed with PBS and stained with Hoechst (Merck KGaA; MilliporeSigma) and PI (BioChemica; PanReac AppliChem) at a 1:1 µg/ml ratio for 30 min at room temperature in the dark. After washing, coverslips were mounted with antifade medium (Invitrogen; Thermo Fisher Scientific, Inc.) and stained cells were visualized using a confocal microscope (ZEISS LSM 800; Carl Zeiss AG), as described previously (21). PI fluorescence intensity was quantified in three randomly selected fields at ×200 magnification using ImageJ software (version 1.54g; National Institutes of Health).

Based on cytotoxicity, morphological and membrane permeability results, intracellular ROS generation was further assessed in MDA-MB-231 cells using an ROS detection assay kit (BioVision, Inc.; Abcam). Following treatment with DOXO, SG or DSG, cells were washed with 100 µl of ROS assay buffer and incubated with 100 µl of 1X ROS labeling solution containing the DCFH-DA fluorophore (BioVision, Inc.; Abcam) diluted in assay buffer at 37°C for 45 min in the dark. Cells were then washed again with ROS assay buffer and fluorescence was measured immediately at excitation/emission wavelengths of 495/529 nm. Intracellular ROS levels were expressed as fold changes compared with the control.

MDA-MB-231 cells were further examined for ultrastructural changes following compound treatment using TEM analysis. After treatment with DOXO, SG or DSG, cells were fixed in Karnovsky's fixative (2.5% glutaraldehyde+2.0% paraformaldehyde, in 0.1 M sodium cacodylate buffer, pH 7.4) for 30 min at room temperature, washed with PBS and centrifuged at 1,500 × g for 1 min at 25°C. Cells were post-fixed with 1% O_SO₄ in 0.1 M phosphate buffer for 1 h at room temperature, washed with PBS and centrifuged at 1,500 × g, for 1 min at room temperature. Cell pellets were embedded in 2% agarose for 20 min at room temperature, cut into small pieces and dehydrated using a graded ethanol series. Samples were infiltrated with propylene oxide and propylene oxide/EPON 812 mixtures, followed by pure EPON 812 overnight, then embedded and polymerized at 60°C for 48 h. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined using JEM-1010-TEM (JEOL Ltd.).

After 6, 12 and 24 h of treatment, MDA-MB-231 cells were harvested and proteins were extracted using a protein lysis buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM phenylmethylsulfonyl fluoride; PMSF) supplemented with a 100X protease inhibitor solution. Protein concentration was determined using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.) by measuring absorbance at 280 nm. Equal amounts of protein (50 µg per sample) were separated by 12.5% SDS-PAGE and transferred onto nitrocellulose membranes (MilliporeSigma; Merck KGaA). Membranes were blocked with 2% BSA (cat. no. BSA-1S; Capricorn Scientific) in PBS for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies (1:1,000 dilution) against CRT (cat. no. MA5-15382), Fas-R (cat. no. MA5-32489) and MHC class I (cat. no. MA5-35712; Invitrogen; Thermo Fisher Scientific, Inc.), followed by HRP-conjugated goat anti-rabbit secondary antibody (cat. no. 31460; 1:2,000 dilution; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Immunoreactive bands were visualized using Clarity™ Western ECL substrate (Bio-Rad Laboratories, Inc.). Protein expression levels were normalized to β-actin (cat. no. AF7018; Affinity Biosciences, Inc.) and quantified by densitometric analysis using Scion Image version 4.0.2 software (Scion Corporation, Frederick, MD, USA). All experiments were performed in triplicate.

Immunofluorescence staining was performed to assess the expression levels and localization of CRT, Fas-R and MHC class I in MDA-MB-231 cells. Cells grown on coated round glass coverslips were treated with DOXO (0.5 µg/ml) or DSG (1,000 µg/ml). After 12 h of treatment, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After washing, cells were incubated overnight at 4°C with primary antibodies against CRT, Fas-R and MHC class I. Cells were then incubated with a FITC-conjugated goat anti-rabbit IgG secondary antibody (1:500; cat. no. 701078; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature. Negative controls were prepared by omitting the primary antibody. Cell membranes were counterstained with CellMask™ Deep Red Plasma Membrane (cat. no. C10046; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions and nuclei were stained using ProLong™ Diamond Antifade Mountant with DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min at room temperature. Immunofluorescence images were acquired using a confocal microscope (ZEISS LSM 800; Carl Zeiss AG). FITC fluorescence intensity for CRT, Fas-R and MHC class I was quantified in three randomly selected fields at ×200 magnification using ImageJ software (version 1.54g; National Institutes of Health).

Total RNA was extracted using TRIzol^® reagent (200 µl; MilliporeSigma) and RNA purity and concentration were assessed by measuring the A260/280 ratio using a NanoDrop™ 2000 spectrophotometer (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA (cDNA) was synthesized from 1 µg of RNA using the RevertAid™ First Strand cDNA Synthesis Kit (Invitrogen; Thermo Fisher Scientific, Inc.) by incubation at 42°C for 60 min, followed by 70°C for 5 min. Gene expression was analyzed by RT-qPCR using synthesized cDNA, SYBR Green PCR Master Mix (Invitrogen; Thermo Fisher Scientific, Inc.) and gene-specific forward and reverse primers. The RT-qPCR thermocycling conditions were as follows: 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. Relative mRNA expression levels were calculated using the 2^-ΔΔCq method (23) and analyzed using Bio-Rad CFX Maestro software version 2.3 (Bio-Rad Laboratories, Inc.). Primers targeting protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), activating transcription factor (ATF)6, ATF4, eukaryotic initiation factor 2 α subunit (eIF2α), CRT, Fas-R, MHC class I and GAPDH were obtained from BIONICS Co., Ltd. Primer sequences were designed using National Center for Biotechnology Information Primer-Basic Local Alignment Search Tool and are listed in Table I.

Data are presented as the mean±standard error of the mean from three or more independent experiments. Statistical significance was assessed using one-way analysis of variance followed by Tukey's multiple comparison test in GraphPad Prism software (version 10.3.1.509; Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

SEM-EDX was used to examine the microstructure and elemental composition of SG and DSG (24). As presented in Fig. 1A and B, SG and DSG exhibited distinct surface morphologies. SG appeared as large lamellar, bundle-like fiber structures, whereas DSG consisted of shorter, fragmented sheet-like structures with irregular shapes. EDX analysis confirmed the presence of carbon, oxygen and sulfur in both samples (Fig. 1C and D). In SG, carbon, oxygen and sulfur contents were 53.25±1.17%, 44.98±1.12% and 1.77±0.09%, respectively, whereas in DSG these values were 52.73±0.36%, 45.05±0.58% and 2.23±0.43%. The sulfate contents of SG and DSG were further quantified as 11.41±0.14% and 12.92±0.07%, respectively (Fig. S1). FTIR analysis revealed characteristic absorption bands between 400 and 4,000 cm^-1, consistent with polysaccharide functional groups (Fig. S2). Notably, absorbance bands at 861.60, 770.07 and 708.49 cm^-1, corresponding to sulfate group stretching vibrations (21), were more notable in DSG (Fig. 1E and F). These results confirmed the sulfated nature of both polysaccharides and suggested that degradation to a lower molecular weight is associated with increased sulfate content within the structure.

The cytotoxic effects of SG and DSG on normal breast epithelial MCF-10A cells and MDA-MB-231 breast cancer cells were evaluated after 24 h of exposure and expressed as a percentage of control at concentrations ranging from 100 to 1,000 µg/ml. SG and DSG exhibited no significant cytotoxicity toward MCF-10A cells at concentrations up to 1,000 µg/ml (Fig. 2A). By contrast, both compounds significantly reduced viability in MDA-MB-231 cells at concentrations of 400–1,000 µg/ml, with DSG exhibiting greater cytotoxic potency compared with SG (Fig. 2B). The IC₅₀ values for SG and DSG in MDA-MB-231 cells were 2,736.17 and 1,927.11 µg/ml, respectively, calculated from the slope and equation obtained from Fig. 2B. DOXO, used as a positive control, significantly reduced the viability of both MCF-10A and MDA-MB-231 cells, with IC₅₀ values of 0.56 and 0.52 µg/ml, respectively (Fig. 2C and D). Since SG and DSG did not achieve 50% cytotoxicity at the highest concentration tested, IC₅₀ values could not be determined within the experimental range. Therefore, a concentration of 1,000 µg/ml was selected for subsequent experiments.

To assess the effects of SG and DSG on cellular morphology, morphological features and membrane permeability in MDA-MB-231 cells were examined. Phase-contrast microscopy revealed that MDA-MB-231 cells treated with DOXO, SG and DSG (Fig. 3A) exhibited clear morphological changes compared with untreated controls, including cell shrinkage, rounding and detachment. By contrast, MCF-10A cells treated with SG or DSG indicated no noticeable morphological alterations compared with controls (Fig. S3). As presented in Fig. 3B, MDA-MB-231 cells treated with SG and DSG displayed prominent cytoplasmic PI fluorescence, similar to that observed in DOXO-treated cells, indicating increased membrane permeability consistent with cell death. Quantitative analysis of morphological changes and PI fluorescence intensity in MDA-MB-231 cells demonstrated a significantly higher incidence in the order of DOXO>DSG>SG (Fig. 3C and D).

Furthermore, ROS levels exceeding the antioxidant capacity of cancer cells can cause biomolecular damage and trigger cell death (25). The present study evaluated whether SG and DSG increase intracellular ROS levels. The effects of DOXO, SG and DSG on intracellular ROS generation in MDA-MB-231 cells are presented in Fig. 3E. Cells treated with DOXO and DSG exhibited significantly higher ROS levels (1.62-fold for DOXO and 1.41-fold for DSG compared with the control), whereas SG-treated cells exhibited ROS levels similar to those of the control. These findings suggested that DSG induces changes in cell morphology and membrane permeability in MDA-MB-231 cells through increased intracellular ROS generation.

To further characterize the ultrastructural effects of SG and DSG on MDA-MB-231 cells, TEM analysis was performed. As presented in Fig. 4, distinct ultrastructural differences were observed among the NC, DOXO-, SG- and DSG-treated groups. Cells in the NC group exhibited well-preserved cellular architecture, including intact nuclear membranes, prominent nucleoli, evenly distributed chromatin and a uniform, electron-lucent cytoplasm (Fig. 4A). By contrast, DOXO-treated cells revealed marked ultrastructural damage, characterized by cytoplasmic condensation, extensive vacuolization and accumulation of electron-dense bodies, consistent with severe cellular stress and cytotoxicity (Fig. 4B). SG-treated cells largely maintained ultrastructural features similar to those of the NC group, indicating minimal cytotoxic effects (Fig. 4C). By contrast, DSG-treated cells retained nuclear integrity but exhibited notable cytoplasmic alterations, including extensive vacuolization, electron-dense inclusions and organelle disorganization (Fig. 4D). These findings indicated that DSG induces notable ultrastructural damage in MDA-MB-231 cells, with cytotoxic effects similar to those observed with DOXO.

ICD is a regulated form of cell death that stimulates tumor-specific immunity through the release of DAMPs from dying cancer cells, thereby activating immune responses within the tumor microenvironment. ICD can be induced by several conventional therapies, including chemotherapy, targeted therapy, radiotherapy and photodynamic therapy (15). Therefore, the present study examined the effects of DSG on ICD-related marker expression and the underlying mechanisms in MDA-MB-231 cells. DSG treatment increased the expression levels of CRT and Fas-R, as presented in Figs. 5 and 6. Western blotting analysis demonstrated that DSG significantly upregulated CRT and Fas-R expression levels at 12 and 24 h, whereas expression level of MHC class I did not differ compared with that in control cells (Fig. 5). The DSG-induced increases in CRT and Fas-R expression levels were similar to those observed in DOXO-treated cells, a known ICD inducer (26). Specifically, CRT expression increased to 1.46±0.41-fold at 6 h, 1.39±0.24-fold at 12 h and 2.09±0.26-fold at 24 h following DSG treatment compared with the control. Fas-R expression increased to 1.38±0.09-fold at 12 h and 2.14±0.74-fold at 24 h following DSG treatment.

Furthermore, confocal immunofluorescence staining was performed to confirm the expression levels and subcellular localization of CRT, Fas-R and MHC class I in MDA-MB-231 cells. Immunoreactivity of CRT and Fas-R was markedly increased in cells treated with DOXO and DSG compared with control cells, with both proteins predominantly localized at the cell surface membrane. By contrast, no significant differences in MHC class I immunoreactivity were observed among the experimental groups (Fig. 6). Quantitative analysis of FITC fluorescence intensity further revealed that DSG significantly increased CRT and Fas-R expression compared with the control (Fig. 6D), supporting the ICD-inducing activity of DSG in MDA-MB-231 cells.

Furthermore, the expression levels of key mRNAs involved in ICD-inducing mechanisms in MDA-MB-231 cells, including those associated with ER stress (15), was evaluated after DSG treatment using RT-qPCR. Compared with the control group, DSG-treated cells exhibited significant upregulation of ER stress- and ICD-related genes, including PERK, IRE1, ATF6, ATF4, eIF2α, CRT and Fas-R, consistent with the effects observed in DOXO-treated cells. By contrast, MHC class I mRNA expression remained unchanged following DSG treatment (Fig. 7). Collectively, these results indicated that DSG is associated with activation ER stress-related ICD signaling pathways at the transcriptional level, supporting its ICD-inducing potential in MDA-MB-231 breast cancer cells.