Osteosarcoma (OS) is an aggressive primary bone malignancy with limited therapeutic progress and frequent chemoresistance. The search for safer agents with novel mechanisms remains a priority. Ganoderma microsporum immunomodulatory protein (GMI) is a fungal-derived bioactive molecule previously reported to have antitumor activity in other cancer types; its effects in osteosarcoma were not defined prior to this study. The authors investigated GMI's anticancer potential and underlying mechanisms in OS cell lines and an in vivo xenograft model.
The study evaluated GMI in human osteosarcoma cell lines HOS and U2OS and compared effects to normal osteoblasts. GMI selectively reduced viability in HOS and U2OS while exhibiting low toxicity toward normal osteoblasts, indicating some tumor selectivity in the tested models.
GMI-treated osteosarcoma cells showed morphological alterations consistent with cytotoxic stress, including chromatin condensation. Functional assays demonstrated reduced colony formation capacity. Cell-cycle analysis revealed GMI induced G2/M arrest, and assays of cell death confirmed induction of apoptosis as a principal mode of cell killing.
RNA sequencing of treated cells identified 292 upregulated and 158 downregulated genes. The gene expression pattern indicated suppression of pathways involved in DNA repair, cell proliferation, cell-cycle progression, and mitochondrial function, together with activation of signatures related to autophagy. These transcriptomic findings support the observed phenotypes of growth inhibition, cell-cycle blockade, mitochondrial dysfunction, and autophagic activity.
Mechanistic experiments showed that GMI treatment elevated intracellular reactive oxygen species (ROS). Co-treatment with the antioxidant N-acetylcysteine (NAC) attenuated GMI-induced apoptosis and restored cell viability, indicating that ROS generation is a key mediator of GMI cytotoxicity in osteosarcoma cells.
GMI induced markers of endoplasmic reticulum (ER) stress, which was associated with activation of the stress-responsive kinase JNK. The study reports sustained JNK activation disrupted the balance of BCL-2 family proteins by repressing anti-apoptotic BCL-2 and augmenting pro-apoptotic members. This shift favored mitochondria-dependent apoptosis, linking ER stress and JNK signaling to the intrinsic apoptotic pathway in GMI-treated OS cells.
Treatment with GMI increased the formation of acidic vesicular organelles and autophagosome-like structures. Biochemical markers of autophagy were altered: LC3B-II levels rose while p62 decreased, consistent with autophagic flux or autophagosome formation. The transcriptomic signature also showed autophagy-related activation. Together, these data indicate that GMI triggers autophagic processes in osteosarcoma cells.
To test the functional role of autophagy, the investigators used pharmacologic autophagy inhibitors—Bafilomycin A1 (Baf A1), chloroquine (CQ), and 3-methyladenine (3-MA). Blockade of autophagy using these agents further augmented GMI-induced apoptosis and exacerbated reductions in cell viability. These findings imply that autophagy acts, at least in part, as a cytoprotective response to GMI; inhibiting autophagy enhances the pro-apoptotic effects of GMI in osteosarcoma cells.
In a xenograft tumor model, systemic or local GMI treatment significantly inhibited tumor growth relative to control. Importantly, GMI administration did not affect body weight in the treated animals, suggesting tolerability in the experimental conditions reported. The xenograft results extend the in vitro findings and support further preclinical evaluation.
The study identifies GMI as a natural product-derived candidate with selective antitumor activity against osteosarcoma in vitro and antitumor efficacy in vivo. Mechanistically, GMI increases intracellular ROS, induces ER stress with sustained JNK activation, shifts BCL-2 family protein balance to favor mitochondrial apoptosis, and triggers autophagy that appears cytoprotective. Pharmacologic autophagy inhibition potentiated apoptosis, suggesting potential combination strategies. The authors conclude that GMI warrants further preclinical and translational evaluation as a potential therapeutic approach for osteosarcoma.
Notes on reported details and limitations
All factual statements above reflect findings reported in the source abstract. Specific experimental parameters (doses, treatment schedules, detailed quantitative results, and statistical values) were not reported in the abstract and therefore are not included here. The full text should be consulted for methodological details and complete data supporting these conclusions.