Ganoderma Microsporum immunomodulatory protein suppresses osteosarcoma growth through redox imbalance and ER...

Osteosarcoma (OS) is an aggressive bone malignancy with limited therapeutic advances and persistent chemoresistance, highlighting the need for safer and more effective treatments. Ganoderma microsporum immunomodulatory protein (GMI) is a fungal-derived bioactive molecule with reported antitumor activity, yet its effects in OS remain unknown. Here, we investigated the anticancer potential and mechanisms of GMI in osteosarcoma. GMI selectively reduced the viability of HOS and U2OS cells while exhibiting low toxicity toward normal osteoblasts. GMI induced morphological alterations, chromatin condensation, reduced colony formation, apoptosis, and G2/M arrest. RNA sequencing identified 292 upregulated and 158 downregulated genes, indicating suppression of DNA repair, proliferation, cell-cycle progression, and mitochondrial pathways, alongside activation of autophagy-related signatures. Mechanistically, GMI elevated intracellular reactive oxygen species (ROS), and N-acetylcysteine (NAC) attenuated GMI-induced apoptosis and restored viability. GMI-induced ER stress was associated with JNK activation, and sustained JNK activation disrupts the balance of BCL-2 family proteins by repressing anti-apoptotic BCL-2 and augmenting pro-apoptotic members, thereby driving mitochondria-dependent apoptosis in osteosarcoma cells. In addition, GMI treatment increases the formation of acidic vesicular organelles and autophagosome-like structures, accompanied by elevated LC3B-II and reduced p62 levels. Pharmacological blockade of autophagy using Baf A1, CQ, or 3-MA further augments GMI-induced apoptosis and exacerbates the reduction in cell viability. Finally, GMI significantly inhibited tumor growth in a xenograft model without affecting body weight. These findings identify GMI as a promising natural therapeutic candidate for osteosarcoma and warrants further preclinical and translational evaluation.