Protodioscin enhances Methotrexate-induced senescence and senolytic activity in HepG2 liver cancer spheroids by modulating cell cycle regulators and the TGF-β/p-Smad2-4 signaling pathway.

Protodioscin (PD) is a natural saponin with anti-inflammatory, anticancer, and metabolic regulatory properties. Senolytic agents, which selectively eliminate senescent cells, are gaining attention for enhancing cancer therapy. This study evaluated the senolytic potential of PD in 3D HepG2 spheroids subjected to Methotrexate (MTX)-induced senescence. Spheroids were generated using the hanging drop method with a 1:1 mixture of HepG2 medium and HFF1-conditioned medium. Senescence was induced with 6 µM MTX, followed by 24-h PD treatment at 1.6, 4, and 8.14 µM (10%, 25%, and 50% of IC₅₀). Cell viability was assessed via MTT; ROS, NO, and TGF-β levels via ELISA; apoptosis via Annexin V/PI; and cell death via Calcein-AM. Expression of p16, p21, p27, p53, and p-Smad2/4 was measured by Western blot, and β-Galactosidase activity was used as a senescence marker. PD dose-dependently reduced spheroid viability and, combined with MTX, significantly enhanced apoptosis, cell death, ROS, NO, β-Gal activity, and expression of p16, p27, and p53, while downregulating p-Smad2/4 and TGF-β compared to MTX treatment alone. These findings suggest that PD potentiates MTX-induced senescence through modulation of cell cycle regulators and inhibition of the TGF-β/p-Smad2/4 pathway, highlighting its potential as a senolytic adjuvant in liver cancer therapy.