Oridonin-induced ferroptosis and apoptosis: a dual approach to suppress the growth of osteosarcoma cells

Abstract

Background: Osteosarcoma (OS) is one of the most common aggressive bone malignancy tumors in adolescents. With the application of new chemotherapy regimens, finding new and effective anti-OS drugs to coordinate program implementation is urgent for the patients of OS. Oridonin had been proved to mediate anti-tumor effect on OS cells, but its mechanism has not been fully elucidated.

Methods: The effects of oridonin on the viability, clonal formation and migration of 143B and U2OS cells were detected by CCK-8, colony formation assays and wound-healing test. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore the mechanism of oridonin on OS. Western blot (WB), real-time quantitative PCR (qRT-PCR) were used to detect the expression levels of apoptosis and ferroptosis-relative proteins and genes. Annexin V-FITC apoptosis detection kit and flow cytometry examination were used to detect the level of apoptosis. Iron assay kit was used to evaluate the relative Fe²⁺ content. The levels of mitochondrial membrane potential and lipid peroxidation production was determined by mitochondrial membrane potential detection kit and ROS assay kit.

Results: Oridonin could effectively inhibit the survival, clonal formation and metastasis of OS cells. The KEGG results indicated that oridonin is associated with the malignant phenotypic signaling pathways of proliferation, migration, and drug resistance in OS. Oridonin was capable of inhibiting expressions of BAX, cl-caspase3, SLC7A11, GPX4 and FTH1 proteins and mRNA, while promoting the expressions of Bcl-2 and ACSL4 in 143B and U2OS cells. Additionally, we found that oridonin could promote the accumulation of reactive oxygen species (ROS) and Fe²⁺ in OS cells, as well as reduce mitochondrial membrane potential, and these effects could be significantly reversed by the ferroptosis inhibitor ferrostatin-1 (Fer-1).

Conclusion: Oridonin can trigger apoptosis and ferroptosis collaboratively in OS cells, making it a promising and effective agent for OS therapy.

Keywords: Anti-tumor; Apoptosis; Ferroptosis; Oridonin; Osteosarcoma; ROS.