Cis-3-hexen-1-ol suppresses bladder cancer progression by inducing mitophagy via the PCK2/AMPK/mTOR signaling pathway

Cis-3-hexen-1-ol is a naturally occurring green leaf volatile widely distributed in aromatic and medicinal plants. However, its antitumor activity and underlying mechanisms in bladder cancer (BLCA) remain largely unexplored.

The antitumor effects of cis-3-hexen-1-ol were evaluated using human BLCA cell lines and mouse xenograft and metastasis models. Cell proliferation, migration, invasion, mitochondrial function, and mitophagy were assessed using CCK-8, EdU incorporation, transwell assays, mitochondrial membrane potential and ROS assays, transmission electron microscopy, and immunofluorescence. Transcriptomic profiling, molecular docking, co-immunoprecipitation, immunoblotting, and in vitro kinase assays were performed to elucidate the underlying molecular mechanisms.

Cis-3-hexen-1-ol significantly inhibited BLCA cell proliferation, migration, and invasion in vitro and suppressed tumor growth and lung metastasis in vivo without overt toxicity. Treatment induced mitochondrial dysfunction and robust mitophagy, as evidenced by mitochondrial fragmentation, loss of membrane potential, increased mitochondrial ROS, and enhanced recruitment of autophagy markers. Mechanistically, cis-3-hexen-1-ol activated mitophagy through the PCK2/AMPK/mTOR signaling axis. Genetic silencing of PCK2 or pharmacological inhibition of autophagic flux using 3-methyladenine markedly attenuated the antitumor and pro-mitophagic effects. Notably, biochemical analyses demonstrated that PCK2 undergoes autophosphorylation and directly phosphorylates AMPK in vitro, supporting a kinase-dependent regulatory mechanism.

These findings identify cis-3-hexen-1-ol as a novel mitophagy-inducing phytochemical that suppresses BLCA progression via activation of the PCK2/AMPK/mTOR pathway, highlighting its potential as a natural-product lead for targeted bladder cancer therapy.