Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Despite advances in surgery and multiagent chemotherapy, outcomes remain poor for patients with metastatic or recurrent disease. Regorafenib, an oral multikinase inhibitor targeting pathways involved in tumor proliferation, angiogenesis, and metastasis, has shown clinical promise in osteosarcoma; however, its mechanisms of action are not fully understood.

We investigated the effects of regorafenib in a human osteosarcoma cell line (MG63) and a patient-derived primary cell line (APR1). Cells were treated at their respective IC_{5 0} concentrations (26 μM for MG63 and 42 μM for APR1). Regorafenib-induced changes in cell viability, apoptosis, migration, invasion, and cell-cycle distribution were assessed. Mitochondrial bioenergetics were evaluated using Seahorse XF technology. Molecular effects were analyzed by assessing gene and protein expression related to cancer-associated pathways and by RT² Profiler PCR arrays targeting key oncogenic genes.

Regorafenib significantly reduced cell viability, migration, and invasion and induced apoptosis in both cell models. MG63 cells exhibited greater sensitivity, reflected by a lower IC_{5 0} value. Cell-cycle analysis revealed G2/M arrest in MG63 cells and G0/G1 accumulation in APR1 cells, indicating distinct adaptive responses. At the molecular level, regorafenib modulated the expression of apoptotic regulators and noncoding RNAs. Suppression of the PI3K/AKT/mTOR pathway occurred predominantly via inhibition of protein phosphorylation rather than transcriptional downregulation. Metabolic analyses showed reduced ATP production through both oxidative phosphorylation and glycolysis in MG63 cells, while APR1 cells preserved glycolytic activity. Transcriptomic profiling revealed differential regulation of genes associated with oncogenesis, hypoxia, DNA damage response, and angiogenesis.

These findings demonstrate the multifaceted antitumor activity of regorafenib in osteosarcoma and highlight cell context-dependent responses. The study provides new insights into molecular and metabolic mechanisms underlying regorafenib activity and supports further investigation of this agent in osteosarcoma therapy.