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Patients with bladder cancer (BLCA) still show high recurrence after surgery and chemotherapy. Hesperetin (HE), as a natural compound, has attracted researchers' attention due to its low toxicity and easy access. However, the inhibitory effect of HE on BLCA remains unknown. The hub genes and enrichment pathways regulated by HE in the treatment of BLCA were predicted by network pharmacology. The molecular docking of HE and hub proteins was visualized. Colony and CCK8 assays were used to test cell proliferation, and BLCA migration was confirmed by transwell and wound healing assays. In addition, the occurrence of apoptosis and ferroptosis was demonstrated by Hoechst staining, transmission electron microscopy (TEM) and ROS (reactive oxygen species) assay. Western Blotting was performed to validate the hub proteins, target functions and pathways. SRC, PIK3R1 and MAPK1 were identified as hub targets for HE in BLCA, involving the PI3k/AKT pathway. Furthermore, HE inhibited the proliferation and migration of BLCA cells. The MMP2/MMP9 proteins were significantly inhibited by HE. The increased expression of Bax and cleaved caspase-3 indicated that HE could promote BLCA cell apoptosis. In addition, Hoechst staining revealed concentrated and illuminated apoptotic nuclei. The activation of ROS and the decline of GPX4 expression suggested that HE might induce ferroptosis as an anti-BLCA process. Shrunk mitochondria and apoptotic bodies were observed in BLCA cells treated with HE, with reduced or absent mitochondrial cristae. We propose for the first time that HE could inhibit the proliferation and migration of BLCA cells and promote apoptosis and ferroptosis. HE may act by targeting proteins such as SRC, PIK3R1 and MAPK1 and the PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Background: We evaluated antitumoral effect of combined chemotherapy and interleukin-12 (IL-12) gene therapy in in vitro and in vivo experimental urothelial bladder cancer (UBC) model. Materials and methods: EJ UBC cells were transfected with recombinant IL-12 genes using a liposomal transfection agent. Pirarubicin (THP) was added to the experimental samples at a final concentration of 20 mg/l. Four groups were assigned in vitro: untreated cells, transfected cells, untransfected cells plus THP and transfected cells plus THP. Death rates (DR) and cellular micromorphologic changes were evaluated. Bladder tumor model was established by subcutaneous injection of EJ cells to the nude mice. Four groups were assigned in vivo: control group; THP group; IL-12 gene group and IL-12 gene plus THP group. After injection of combined THP and IL-12 gene therapy, tumor size and IL-12 levels were evaluated. Results: In vitro study: DR in the THP + IL-12 gene therapy group (58.2 ± 15.8%) was significantly higher than transfected group (12.2 ± 5.6%; P = 0.01) and untransfected cells plus THP group (33.4 ± 7.8; P = 0.046). A higher amount of apoptotic changes and necrosis on transmission electron microscope analysis were observed in transfected cells plus THP group. In vivo study: A significant tumor attenuation was found in IL-12 gene in combination with THP group when compared with any other groups that were treated without Il-12 or THP ( P < 0.05). IL-12 levels in serum were significant high in IL-12 gene groups ( P < 0.01). Conclusion: The combination of THP chemotherapy and IL-12 gene therapy showed an additive antitumoral effect on bladder cancer cells in vitro and in vivo. Further investigation should be focused on high-level transgene protocols in vivo . [ABSTRACT FROM AUTHOR]