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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]

• Overexpression of CIT is found in tumor tissues in various cancer patients. • We found higher CIT level in bladder cancer tissues than normal bladder tissues. • CIT knockdown represses cell growth and regulates cell cycle in vitro and in vivo. • In summary, CIT may promote bladder cancer through regulation of cell cycle pathway. Our study is to examine the citron rho-interacting, serine/threonine kinase 21 (CIT) in bladder cancer. We examined CIT level in human bladder cancer tissues by immunohistochemical staining. To explore the impact of CIT on cell proliferation and apoptosis, we down-regulated its expression in two human bladder cancer cell lines, 5367 and T24. We examined cell growth in 5367 and T24. We also performed in vivo analysis using T24 cells. We further used microarray expression profiling to investigate genes differentially expressed in T24 cells with CIT down-regulated. In 100 human samples, CIT was expressed by only 2 of 30 (6.7 %) controls in bladder tissues, whereas by 64 of 70 (91.4 %) cancer patients in tumor tissues (p < 0.001). in vitro analysis demonstrated that CIT knockdown represses cell proliferation by 50 % in both cells and colony formation (77 ± 5 vs. 13 ± 2, p = 0.001 for T24, 58 ± 3 vs. 1 ± 1, p < 0.001 for 5637). We also found CIT knockdown could induce cell cycle arrest, and promote apoptosis in both cells. Tumor-volume monitoring and live in vivo bladder cancer imaging in human xenograft model confirmed that CIT knockdown reduces tumor volume (668.4 ± 333.0 vs. 305.7 ± 170.4 mm3, p = 0.02) and weight (0.27 ± 0.15 vs. 0.57 ± 0.32 g, p = 0.02). Microarray analysis revealed that CIT may regulate cell cycle signalling pathway through various cell cycle regulators. In summary, we provided clinical and experimental evidence that CIT may promote bladder cancer through regulation of cell cycle pathway. [ABSTRACT FROM AUTHOR]