Abietic acid induces ferroptosis via the activation of the HO-1 pathway in bladder cancer cells

Background: Bladder cancer (BC) is a common urological malignancy that still lacks effective treatments. Abietic acid (AA) is an abietane diterpene that possesses various biological activities, including antitumor activity. This study aimed at evaluating the effects of AA on BC cells.Materials and methods: The 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to assess the effects of AA on the viability of BC cells. Annexin-V and FITC staining was used to assess cellular death. The type of cell death was determined by the administration of various specific cell death inhibitors. Commercial kits were used to measure the levels of reactive oxygen species (ROS), intracellular iron, malon-dialdehyde (MDA), and glutathione (GSH). Real-time polymerase chain reaction (RT-PCR) and western blot analysis were used to assay mRNA and protein levels, respectively. The role of glutathione peroxidase 4 (GPX4) in the antitumor effects of AA was evaluated using the forced expression of GPX4 in BC cells. The impact of HO-1 on the antitumor effects of AA was examined by gene silencing and pharmacological inhibition of the protein. Finally, the antitumor effects of AA were evaluated in xenograft models.Results: AA selectively inhibited the viability of BC cells but not normal cells. AA-induced ferroptosis in BC cells was evidenced by the upregulation of ROS, intracellular iron, and MDA. AA treatment led to the downregulation of GPX4 and the upregulation of HO-1 in BC cells. Forced expression of GPX4 or inhibition of HO-1 resulted in decreased ferroptosis triggered by AA in BC cells. AA also showed synergistic effects with various chemother-apeutic agents against BC and inhibited the growth of BC cells in vivo.Conclusion: This study revealed AA-induced ferroptosis in BC cells both in vitro and in vivo. AA might be applied as a promising agent for the treatment of BC.