Trifolirhizin induces apoptosis and suppresses proliferation, invasion and immune evasion in bladder cancer via AKT/NF-κB signaling pathway

Bladder carcinoma (BCa) represents a clinically significant oncological challenge requiring novel therapeutic strategies. Trifolirhizin (TFL), a bioactive pterocarpan flavonoid, demonstrates broad-spectrum anticancer properties, though its efficacy and molecular mechanisms in BCa remain unexplored. The viability of T24/UMUC3 cells was established via cell counting kit-8 (CCK-8) assays. Functional impacts of TFL were assessed through colony formation assay, transwell migration, Hoechst 33342 nuclear staining, cytokine quantification, flow cytometry and immunoblotting. Mechanistic investigations focused on protein kinase B (AKT)/nuclear factor kappa-B (NF-κB) pathway modulation, validated through pharmacological activation with SC79. In vivo experiments were conducted on xenograft models by hematoxylin and eosin (HE) staining, immunohistochemistry and immunoblotting. TFL reduced the BCa cell viability (IC50: 67.41 μM for T24 cells; 72.58 μM for UMUC3 cells) with concurrent suppression of proliferation (the reduction in the number of colony formation), and invasiveness (the decrease in the invaded cell numbers and vimentin expression, and the increase in the E-cadherin level). Apoptotic induction, and enhanced CD8 + T cell-mediated cytotoxicity and the interferon-gamma (IFN-γ) level were observed in TFL-treated BCa cells. AKT/NF-κB phosphorylation was attenuated by TFL, reversible via SC79 co-treatment. Also, SC79 management reversed the changes of indicators involved in proliferation, apoptosis, invasion and immune escape in T24 cells. In vivo administration reduced tumor burden and the vimentin level but increased apoptotic indices and the IFN-γ level. TFL consistently inhibited the expression of AKT/NF-κB pathway in mice xenografted with T24 cells. The study highlighted the effect of TFL as a multimodal therapeutic agent suppressing proliferation, invasion, and immune escape, but enhancing BCa apoptosis through the downregulation of the AKT/NF-κB pathway.