To investigate the antitumor effects and underlying molecular mechanisms of the p53 reactivator APR-246 in bladder cancer cells.

Human and murine bladder cancer cell lines with different p53 statuses were treated with APR-246 to assess drug sensitivity. p53-knockdown mutant cell models were established to evaluate the dependency of APR-246 on p53. For knockdown experiments, cells transduced with lentiviral shScramble were named as Scramble group, whereas cells transduced with shp53 were named as shp53 group. For overexpression experiments, cells transduced with empty pLKO-NC lentivirus were served as the control (NC) , and cells transduced with lentivirus overexpressing p53R248S were named as the OE-p53R248S group. Cell viability assay, ROS staining, and Western blot were performed to analyze cell death patterns and related protein expression. Cells were treated with DMSO (control) , 20 μmol/L APR-246, or 20 μmol/L APR-246 combined with 5 mmol/L NAC. Flow cytometry and a subcutaneous xenograft model in C3H mice were used to validate the biological effects in vitro and in vivo. Differences between two groups were assessed using the independent-samples t test. Comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) , followed by Dunnett's t test for multiple comparisons between each treatment group and the control group.

APR-246 significantly induced cell death in bladder cancer cell lines (EJ: IC₅₀ = 14.09 μmol/L; T24: IC₅₀ = 13.56 μmol/L; UMUC3: IC₅₀ = 15.14 μmol/L; SW780: IC₅₀ = 29.71 μmol/L; MB49: IC₅₀ = 7.02 μmol/L; MBT2: IC₅₀ = 1.12 μmol/L; all P < 0.05) , whereas mutant p53 cells exhibited greater sensitivity to the compound. Knockdown of p53 attenuated cellular responses to APR-246 (UMUC3: 17.69 ± 0.21 vs 24.16 ± 0.24; EJ: 12.24 ± 0.17 vs 19.61 ± 0.29; T24:11.39 ± 0.26 vs 16.24 ± 0.35; all P < 0.05) .Overexpression of mutant p53 reduced drug responsiveness [UMUC3: (18.93 ± 0.57) vs (13.42 ± 0.45) μmol/L; EJ: (13.05 ± 0.32) vs (8.21 ± 0.27) μmol/L; T24: (13.23 ± 0.46) vs (9.21 ± 0.42) μmol/L; all P < 0.05]. Cell death induced by APR-246 was primarily mediated through ROS-dependent ferroptosis and apoptosis. Treatment with APR-246 could downregulate the expression of SLC7A11 and GPX4 (P < 0.05) and increased the expression level of cleaved PARP (P < 0.05) . Furthermore, in bladder cancer cells harboring p53 missense mutations, APR-246 upregulated the expression of p21 and induced cell cycle arrest at the G0/G1 phase [ (37.44 ± 0.65) % vs (53.32 ± 0.83) %; P < 0.05]. In vivo studies further confirmed that APR-246 markedly inhibited tumor growth (P < 0.05) .

APR-246 exerts potent antitumor activity in bladder cancer by reactivating mutant p53 and inducing ROS-dependent ferroptosis and cell cycle arrest, providing a promising molecular target and experimental basis for precision therapy of bladder cancer.