Ungeremine suppresses colon cancer cell proliferation through inhibition of the PI3K/AKT/mTOR signaling pathway.

The present study investigated the anticancer potential of ungeremine against colon cancer HCT116 cells and its mechanism of action, with a focus on the inhibition of the PI3K/AKT/mTOR signalling pathway. HCT116 cells were treated with varying concentrations of Ungeremine (25, 50, 75, 100 µM), and all experiments included untreated control groups for comparison. The cytotoxic effect was initially assessed using the MTT assay, revealing a dose dependent reduction in cell viability. Apoptotic morphological changes were confirmed by Acridine Orange/Ethidium Bromide (AO/EtBr) dual staining. Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) disruption were evaluated using DCF-DA and Rhodamine 123 staining, respectively, demonstrating significant oxidative stress and mitochondrial dysfunction in treated groups. Flow cytometric analysis revealed cell cycle arrest at the G0/G1 phase, particularly at 75 µM. RT-PCR analysis revealed a downregulation of PI3K, AKT, and mTOR gene expression, which was further validated at the protein level by Western blotting. These findings indicate the induction of apoptosis in Ungeremine-treated cells. Molecular docking analysis using AutoDockTools v1.5.6 revealed strong binding affinities of Ungeremine with target proteins AKT (-7.19 kcal/mol), mTOR (-7.27 kcal/mol), and PI3K (-7.33 kcal/mol). Interaction visualization was performed using Discovery Studio 2021. AKT formed two hydrogen bonds with GLU279 and ASN280; in contrast, mTOR and PI3K interacted only through van der Waals forces without hydrogen bond formation. Collectively, these results suggest that Ungeremine exerts its anticancer effect by inducing oxidative stress, mitochondrial dysfunction, cell cycle arrest, and apoptosis, primarily through suppression of the PI3K/AKT/mTOR signaling pathway.