4-Methoxydalbergione triggers ferroptosis in bladder cancer by suppressing the AKT/mTOR/GPX4 axis

Due to its high recurrence and pronounced metastatic potential, bladder cancer (BC) remains a significant and persistent clinical challenge. Here, we investigate the anti-tumour effects of 4-Methoxydalbergione (4MOD), a naturally occurring flavonoid obtained from Dalbergia sissoo Roxb. In T24 human and MBT2 mouse BC cell lines, 4MOD dose-dependently suppressed proliferation, migration, and invasion; colony formation was additionally suppressed in T24 cells. These effects were associated with a reversal of the epithelial- mesenchymal transition (EMT) process, as evidenced by elevated E‑cadherin and reduced N‑cadherin and vimentin expression. Moreover, 4MOD induced both apoptotic cell death and ferroptosis. The induction of ferroptosis was evidenced by mitochondrial shrinkage, accumulation of reactive oxygen species (ROS) and the decreased levels of glutathione peroxidase 4 (GPX4). Caspase and ferroptosis inhibition experiments demonstrated that ferroptosis is the predominant driver of 4MOD‑induced cell death. Mechanistically, 4MOD inhibited AKT and mTOR signaling, as shown by reduced p‑AKT and p‑mTOR levels. Ferroptosis‑specific rescue with Ferrostatin‑1 (Fer‑1) and functional rescue with the AKT activator SC79 indicated that AKT/mTOR inhibition is causally linked to GPX4 downregulation and ferroptosis induction. In a BALB/c‑nu mouse xenograft model, 4MOD markedly suppressed tumour growth without causing significant body weight loss or histopathological abnormalities in liver and kidney tissues, correlating with reduced GPX4, elevated E‑cadherin, decreased vimentin, increased malondialdehyde (MDA) levels, and reduced Ki‑67 positivity in tumour tissues. Notably, 4MOD synergized with the ferroptosis inducer erastin to enhance ferroptosis, apoptosis, and anti‑proliferative activity. Collectively, these findings demonstrate that 4MOD induces ferroptosis via the AKT/mTOR‑GPX4 axis, highlighting its therapeutic potential for bladder cancer.