[Mechanism of periplogenin in promoting Nrf2 degradation and inducing ferroptosis to inhibit bladder cancer].

This study aimed to investigate the inhibitory effect of periplogenin(PPG) on bladder cancer cells and elucidate its anti-tumor mechanism via the induction of ferroptosis. The effects of PPG on the proliferation of T24 and MBT-2 bladder cancer cells were assessed using cell counting kit-8(CCK-8) and 5-ethynyl-2'-deoxyuridine(EdU) assays. Ferroptosis was evaluated by measuring the glutathione/oxidized glutathione(GSH/GSSG) ratio, Fe~(2+), and malondialdehyde(MDA) levels, as well as mitochondrial superoxide production using the MitoSOX~(TM) Red fluorescent probe. Western blot and immunofluorescence were performed to detect the expression of ferroptosis-related proteins, including heme oxygenase 1(HMOX1), solute carrier family 7 member 11(SLC7A11), glutathione peroxidase 4(GPX4), and nuclear factor erythroid 2-related factor 2(Nrf2). The regulatory mechanism of PPG on Nrf2 was further investigated using cycloheximide(CHX) to inhibit de novo protein synthesis, the proteasome inhibitor MG132 to block proteasomal degradation, and co-immunoprecipitation to assess Nrf2 ubiquitination. A mouse xenograft model was established by subcutaneously injecting MBT-2 cells into BALB/C mice. The animals were randomly divided into a control group and two PPG treatment groups(low-dose: 10 mg·kg~(-1); high-dose: 20 mg·kg~(-1)). Starting from day 5 when tumors became palpable, PPG was administered once daily for 30 consecutive days. Tumor growth was monitored, and the expression of Ki67, GPX4, and Nrf2 in tumor tissues was analyzed by immunohistochemistry. Compared with the control group, PPG significantly inhibited proliferation and reduced the viability of T24 and MBT-2 cells. PPG treatment led to a significant reduction in the GSH/GSSG ratio, while Fe~(2+) and MDA levels were markedly increased, along with enhanced mitochondrial superoxide generation, suggesting the induction of ferroptosis. Western blot and immunofluorescence results showed that PPG significantly downregulated the expression of HMOX1, SLC7A11, GPX4, and Nrf2. CHX and MG132 treatments, along with ubiquitination assays, demonstrated that PPG promoted Nrf2 ubiquitination and proteasomal degradation. In vivo, PPG significantly suppressed tumor growth and reduced the expression of Ki67, GPX4, and Nrf2 in xenograft tumors compared with the control group. PPG induces ferroptosis in bladder cancer cells by promoting Nrf2 ubiquitination and proteasomal degradation, thereby inhibiting tumor cell growth.