Abstract: Objective To investigate the effect of metformin （MET） in bladder cancer cell metastasis and its regulation on adenosine monophosphate activated protein kinase (AMPK)/kinesin family member 1B (KIF1B) signaling pathway. Methods The effects of MET on the proliferation and migration of bladder cancer cell lines SW780, RT4, and UMUC3 were evaluated using the CCK⁃8 assay and real time cellular analysis (RTCA). The protein expression levels of AMPK/P⁃AMPK, mTOR, AKT/P⁃AKT, and KIF1B in MET⁃treated bladder cancer cells were analyzed via Western blot. Additionally, a lung metastasis model was established in C57BL/6J mice using MB49 cells to assess the intervention effects of MET on the metastasis process in vivo, along with its regulation of AMPK and KIF1B expression. Results MET was found to reduce the proliferation of various bladder cancer cell lines in a concentration⁃dependent manner, with median inhibition concentration （IC50） values of (26.0±1.4) mmol/L, (32.9±5.3) mmol/L, and (20.0±3.4) mmol/L for SW780, RT4, and UMUC3 cell lines, respectively. Treatment with MET at concentrations of 5 mmol/L for 72 hours significantly inhibited the migration of SW780 and UMUC3 cells (all P<0.05). When MET administration upregulated P⁃AMPK protein expression and downregulated KIF1B, AKT, and mTOR protein expression in RT4 and UMUC3 cells. Animal experiments further corroborated MET significantly delayed the formation time of tumor [(34.5±8.3) d vs (24.8±3.7) d， P<0.05）] and the median survival time of mice （40 d vs 28 d， P=0.016）. In addition, the protein expression of P⁃AMPK increased and KIF1B decreased in the lung tissue of lung metastasis mouse models (all P<0.05). Conclusions MET effectively inhibits the metastasis of bladder cancer cells both in vitro and in vivo. This mechanism may involve the activation of the AMPK pathway, which subsequently downregulates the expression of KIF1B, suggesting that the AMPK/KIF1B signaling pathway plays an important role in the MET⁃induced inhibition of bladder cancer metastasis.

Key words: Bladder cancer, Metformin, Metastasis, Adenosine monophosphate activated protein kinase, Kinesin family member 1B