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.

ABSTRACT: 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

SUBMITTER: Jafaei Souq A 

PROVIDER: S-EPMC12722370 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature