Isorhamnetin inhibits bladder cancer progression by mediating glycolysis through targeting the SRC/PI3K/Akt axis

This study aimed to investigate the anti-tumor effects of the natural flavonoid Isorhamnetin (ISO) on BC and elucidate its underlying molecular mechanisms. We integrated network pharmacology and bioinformatic analysis to predict the core targets and pathways between BC and ISO. In vitro functional assays were performed using the human BC cell line 5637. Cell viability, proliferation, apoptosis, migration, invasion, and adhesion were assessed via CCK-8, colony formation, flow cytometry, Transwell, and adhesion assays. Glycolytic function was evaluated by measuring extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glucose uptake, and lactate production. Molecular docking and cellular thermal shift assays (CETSA) were used to validate ISO-SRC binding. Western blot and RT-qPCR analyses were conducted to examine protein and mRNA expression of key signaling and glycolytic components. For the rescue experiments, SRC was overexpressed along with treatment using the PI3K inhibitor LY294002. ISO significantly inhibited BC cell proliferation, migration, invasion, adhesion, and glycolysis while inducing G0/G1 phase arrest and apoptosis. Network pharmacology and molecular docking identified SRC as a direct target of ISO, with CETSA confirming their binding. ISO treatment downregulated phosphorylation of SRC and its downstream effectors (PI3K, Akt, mTOR), reduced the expression of glycolytic enzymes (HK2, GLUT1, PFKFB3), and decreased ECAR, OCR, glucose uptake, and lactate production. SRC overexpression reversed the anti-tumor and anti-glycolytic effects of ISO. Furthermore, administration of LY294002 on this basis counteracted the effects induced by SRC overexpression, confirming the critical role of the SRC/PI3K/Akt axis. ISO inhibits the malignant progression of BC by directly targeting SRC to suppress glycolysis mediated by the PI3K/Akt signaling pathway. These findings provide a mechanistic basis for considering ISO as a potential metabolic-targeting agent for BC, warranting further preclinical evaluation.