Degraded sulfated galactan derived from Gracilaria fisheri induces immunogenic cell death in MDA‑MB‑231 cells

Sulfated galactan (SG) isolated from Gracilaria fisheri has demonstrated promise for cancer therapy and prevention through inhibition of cancer cell proliferation and migration. However, its structure‑activity relationship remains to be elucidated. The present study evaluated the microstructural characteristics and anticancer activity, particularly immunogenic cell death (ICD)‑inducing potential, of SG and its degraded derivative (DSG) in triple‑negative breast cancer cells. SG and DSG were prepared and structurally characterized using gel permeation chromatography, nuclear magnetic resonance, Fourier‑transform infrared and scanning electron microscopy coupled with energy‑dispersive X‑ray spectroscopy. ICD induction was assessed in human MDA‑MB‑231 breast cancer cells using an MTT assay, phase‑contrast microscopy, Hoechst/propidium iodide dual staining, intracellular reactive oxygen species (ROS) generation assay, and transmission electron microscopy (TEM). In addition, western blot analysis, immunofluorescence staining, and reverse transcription‑quantitative PCR were performed. Structural analyses revealed that SG and DSG share similar backbone structures but differ markedly in sulfate content and molecular weight. Both compounds were non‑toxic to normal breast epithelial MCF‑10A cells and exhibited mild cytotoxicity toward MDA‑MB‑231 cells. DSG treatment induced notable morphological changes in cancer cells, with reduced cell numbers, increased membrane permeability and elevated intracellular ROS levels. TEM revealed DSG‑induced ultrastructural changes consistent with cellular stress and cytotoxicity. DSG also markedly upregulated ICD‑associated proteins [calreticulin (CRT) and Fas receptor (Fas‑R)] and endoplasmic reticulum stress‑related genes (protein kinase RNA‑like endoplasmic reticulum kinase, inositol‑requiring enzyme 1, activating transcription factor (ATF)6, ATF4, eukaryotic initiation factor 2 α subunit, CRT and Fas‑R), with effects similar to the positive control doxorubicin. Therefore, these findings indicated that DSG enhances ICD in triple‑negative breast cancer cells and may potentially serve as a promising ICD‑inducing adjuvant for cancer immunotherapy.