Celastrol induces mitochondrial apoptosis in hepatocellular carcinoma by suppressing LRP6-associated Wnt/β-catenin signaling.

Celastrol, a quinone methide triterpenoid derived from the root bark of Tripterygium wilfordii Hook. f., has antitumor activity, but its molecular mechanisms in hepatocellular carcinoma (HCC) remain incompletely defined. This study investigated whether celastrol suppresses HCC through LRP6-associated modulation of Wnt/β-catenin signaling and mitochondrial apoptosis. Public datasets and immunohistochemical data were used to assess LRP6 expression. Huh7 and PLC/PRF/5 cells were used to assess cell viability, apoptosis, mitochondrial membrane potential, Mitochondrial Permeability Transition Pore (mPTP) opening, ATP production, Reactive Oxygen Species (ROS) accumulation, and pathway protein expression. Molecular docking, 100 ns molecular dynamics simulation, and cellular thermal shift assay were used to evaluate the possibility that LRP6 as a potential functional binding protein for celastrol, whereas co-immunoprecipitation was used to assess the effect of celastrol on the LRP6-GRP94 interaction. Wnt/β-catenin involvement was tested using CHIR99021, IWR-1, and LRP6 siRNA and antitumor activity was evaluated in an orthotopic Huh7 xenograft model. LRP6 was upregulated in HCC and associated with poorer overall survival and Wnt pathway enrichment. Celastrol inhibited HCC cell proliferation, disrupted mitochondrial homeostasis, and promoted mitochondrial apoptosis. Computational analyses generated a plausible celastrol-LRP6 association model that remained conformationally stable during the 100 ns simulation, whereas CETSA showed celastrol-associated thermal stabilization of cellular LRP6. Celastrol decreased LRP6/p-LRP6, β-catenin, c-Myc, and cyclin D1 levels; Wnt activation partially reversed apoptosis, whereas Wnt inhibition or LRP6 knockdown enhanced it. Celastrol also suppressed orthotopic tumor growth. These findings suggest that celastrol induces mitochondrial apoptosis in HCC, at least partly, by suppressing LRP6-associated Wnt/β-catenin signaling.