Bufalin Targets CENPA-modulated Glycolysis via PI3K/AKT in Hepatocellular Carcinoma: A Bioinformatics, In Vitro and Preliminary In Vivo Evidence.
TL;DR
Findings suggest that bufalin may suppress CENPA-associated glycolysis and HCC cell migration through modulation of PI3K/AKT signaling.
Abstract
Background
Hepatocellular carcinoma (HCC) remains a substantial therapeutic challenge because of its high recurrence rate and resistance to conventional therapies, necessitating the exploration of novel approaches. A crucial aspect of cancer metabolism is the Warburg effect, which promotes tumor progression by increasing glycolysis. Bufalin is a key component of a natural product derived from the venom of toads (Bufo gargarizans), which has anticancer properties. Centromeric protein A (CENPA) has been reported to play a significant role in the progression of many cancers, including HCC. However, the effect of bufalin on CENPA expression via the PI3K/AKT signaling pathway remains to be elucidated. The current study aimed to fill this gap, which could lead to a novel therapeutic approach for HCC.
Methods
Bioinformatics tools were used to identify CENPA as a key glycolysis-related gene (GRG) linked with poor prognosis in patients with HCC. In vitro experiments, such as Cell Counting Kit-8 (CCK-8), wound healing, Transwell migration, RT-qPCR, Western blotting (WB), and transfection assays, were used to assess the impact of bufalin on CENPA expression in HCC cell lines. Additionally, molecular docking was employed to calculate the binding energy between bufalin and CENPA. Preliminary xenograft immunohistochemistry extended these findings in vivo.
Results
Bioinformatics analysis identified CENPA as a glycolysis-related prognostic gene in HCC. Bufalin reduced CENPA mRNA and protein expression, attenuated PI3K/AKT phosphorylation, decreased ECAR, and suppressed HCC cell migration. CENPA knockdown reduced migratory behavior, whereas CENPA overexpression enhanced migration and was partially reversed by bufalin treatment. Molecular docking predicted a favorable interaction between bufalin and CENPA. Pilot xenograft immunohistochemistry further showed reduced CENPA staining in bufalin-treated tumor tissues compared with vehicle-treated controls. In addition, CENPA expression was associated with immune checkpoint-related genes and immune microenvironment features in HCC datasets.
Conclusion
These findings suggest that bufalin may suppress CENPA-associated glycolysis and HCC cell migration through modulation of PI3K/AKT signaling. The preliminary xenograft immunohistochemistry supports the in vivo relevance of CENPA downregulation, although comprehensive animal efficacy, toxicity, and biochemical binding studies are still needed. Overall, this study provides mechanistic proof of concept for the bufalin-CENPA-glycolysis axis in HCC.