Curcumin Induces Pyroptosis-Associated Molecular Changes in Osteosarcoma Cells Correlating with the ROS/NLRP3/CASPASE-1/GSDMD Axis with Concomitant PI3K/AKT Suppression and Apoptosis Activation

Curcumin, a natural polyphenolic compound derived from turmeric, exhibits broad-spectrum anticancer activities, but its ability to induce pyroptosis in osteosarcoma remains unknown. Osteosarcoma is the most common primary malignant bone tumor in children and adolescents, and novel therapeutic strategies are urgently needed to overcome osteosarcoma chemoresistance. Aim: This study aimed to investigate whether curcumin induces pyroptosis-associated molecular changes in human osteosarcoma cells and to explore the underlying molecular mechanisms, focusing on the ROS/NLRP3/CASPASE-1/GSDMD axis and the PI3K/AKT signaling pathway. Methods: Human osteosarcoma U2OS and MG63 cells were treated with curcumin (20–40 μmol·L⁻¹ for 24 h). Cell viability was assessed by CCK-8 assay. Pyroptotic morphology was observed by scanning electron microscopy. Lactate dehydrogenase (LDH) release was measured colorimetrically, and IL-1β/IL-18 secretion was quantified by ELISA. Mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygen species (ROS) levels were analyzed by flow cytometry. Protein expression levels of NLRP3, cleaved CASPASE-1, GSDMD-N, PI3K, AKT, p-AKT, Bax, Bcl-2 and cleaved CASPASE-3 were detected by Western blotting. Pharmacological validation was performed using the pan-caspase inhibitor Z-VAD-FMK. Results: Curcumin significantly inhibited the proliferation of U2OS and MG63 cells in a dose- and time-dependent manner. Scanning electron microscopy revealed characteristic pyroptotic features including cell swelling, membrane pore formation, and rupture. Curcumin treatment markedly increased LDH release and elevated IL-1β/IL-18 secretion. Mechanistically, curcumin induced mitochondrial membrane depolarization and ROS accumulation, upregulated NLRP3, cleaved CASPASE-1, and GSDMD-N expression, and concomitantly reduced PI3K/AKT pathway activity. Additionally, curcumin upregulated pro-apoptotic Bax, downregulated anti-apoptotic Bcl-2, and activated cleaved CASPASE-3. The pan-caspase inhibitor Z-VAD-FMK partially reversed curcumin-induced cytotoxicity, confirming that caspase-dependent apoptosis contributes to the overall anticancer effect. Conclusions: This study provides evidence that curcumin induces both apoptosis and pyroptosis-associated molecular changes in human osteosarcoma cells. The pyroptotic effect involves the ROS/NLRP3/CASPASE-1/GSDMD axis, accompanied by PI3K/AKT suppression, while caspase-dependent apoptosis also plays an important role. These findings uncover a previously unreported mechanism of curcumin’s anti-osteosarcoma activity and suggest that targeting multiple cell death pathways may represent a promising strategy to overcome apoptosis resistance in osteosarcoma.