Timosaponin A-III Induces ROS-mediated Apoptosis and Triggers Protective Autophagy via the AMPK/mTOR Pathway in Prostate Cancer.

Prostate cancer, timosaponin A‑III, apoptosis, autophagy, reactive oxygen species, AMPK/mTOR.

Introduction: Timosaponin A-III (TAIII) is an effective anti-tumor ingredient extracted from the rhizomes of Anemarrhena asphodeloides. However, the effect of TAIII on prostate cancer cells (PCa) and its underlying mechanisms is rarely investigated. The current study aimed to investigate the anti-tumor effect and potential mechanisms of TAIII in PCa cells.

Methods: The effect of TAIII on the cell proliferation of PCa was evaluated by CCK-8 assay, colony formation assay, and EDU assay. Cell apoptosis and reactive oxygen species (ROS) production were evaluated by flow cytometry. The puncta of LC3 were detected by immunofluorescence analysis. The protein levels of apoptosis, autophagy, and AMPK/mTOR pathway were assessed by western blot. Finally, a PC3 xenograft nude mouse model was constructed to determine the effect of TAIII combined with chloroquine (CQ) in vivo.

Results: Our data showed that TAIII inhibited the proliferation of PCa cells and induced ROS-dependent apoptosis. TAIII treatment dramatically promoted the formation of LC3-positive puncta, and increased the expression of LC3B-II and P62 protein. Moreover, the combination of TAIII with CQ significantly enhanced the pro-apoptosis effect of TAIII in PCa cells and the PC3 xenograft model. In addition, the activation of the AMPK/mTOR pathway and the induction of autophagy induced by TAIII were reversed by Compound C. Suppressing AMPK with Compound C enhanced the apoptosis induced by TAIII in PCa cells.

Discussion: This study establishes TAIII as a potent anti-prostate-cancer agent that kills tumor cells via ROSdriven apoptosis while simultaneously triggering cytoprotective autophagy through the AMPK-mTOR axis. However, TAIII's clinical potential awaits pharmacokinetic, bioavailability, and toxicity evaluation.

Conclusion: TAIII induced ROS-mediated cell apoptosis and promoted cytoprotective autophagy via the AMPK/mTOR pathway in PCa. These findings may provide a new strategy for combining TAIII with CQ together for PCa treatment.