Celastrol overcomes 5-fluorouracil resistance in osteosarcoma cells through p53-mediated apoptotic pathway modulation and P-glycoprotein inhibition: A comprehensive mechanistic study.

Introduction: Osteosarcoma, a prevalent malignant bone tumor in children and adolescents, is hindered by chemoresistance, particularly to 5-fluorouracil (5-FU), driven by mechanisms such as P-glycoprotein (P-gp) overexpression and altered p53 signaling. Celastrol, a triterpenoid, exhibits anti-tumor properties, but its potential to overcome 5-FU resistance in osteosarcoma remains underexplored.

Methods: We investigated the synergistic effects of celastrol and 5-FU in human osteosarcoma cell lines with varying p53 statuses (U-2OS, wild-type; SaOS-2, p53-null; HOS, mutant p53) using MTT assays for cytotoxicity, Chou-Talalay method for synergy, and Cell Death Detection ELISA for apoptosis. Gene expression (p53, Bax, Bcl-2, caspase-9) was quantified via qRT-PCR, P-gp levels by Western blot, and P-gp efflux activity by Rhodamine 123 assays. Non-malignant hFOB 1.19 cells served as controls.

Results: Celastrol and 5-FU exhibited potent cytotoxicity, with combination therapy reducing IC₅₀ values 3.7- to 11.9-fold across cell lines, showing strong synergy. The combination significantly enhanced apoptosis and modulated p53-dependent (U-2OS) and -independent (SaOS-2, HOS) pathways, upregulating Bax and caspase-9 while downregulating Bcl-2. Celastrol reduced P-gp expression and increased intracellular drug accumulation, comparable to verapamil.

Conclusion: Celastrol synergizes with 5-FU to overcome chemoresistance in osteosarcoma by enhancing p53-mediated and -independent apoptosis and inhibiting P-gp-mediated drug efflux. These findings suggest a promising low-toxicity therapeutic strategy, warranting further in vivo and clinical investigations.