The Mechanism of Ouabain Altering Glycolysis and Affecting Colon Cancer Cells Proliferation by G6PD Signaling Pathway

To elucidate the mechanism of ouabain in the proliferation of colon cancer cells HCT116, the CCK-8 assay, cell colony formation assay, EdU assay, and flow cytometry are employed to detect proliferation capacity, cell colony formation, DNA replication activity, and apoptosis rate, respectively, to investigate the effect of ouabain on the viability of colon cancer cells HCT116 in vitro. To further analyze the influence of ouabain on glycolysis in colon cancer cells HCT116, the glucose oxidase method and lactate detection kit are utilized to measure the changes in glucose consumption level and lactate production level of the cells. Additionally, the mechanism by which ouabain regulates glycolysis in colon cancer cells HCT116 is explored by analyzing the mRNA expression of related metabolic pathway factors via real-time PCR and detecting the expression changes of the glucose metabolism-related protein G6PD through Western Blot. The experimental results reveal that, compared to the control group, ouabain significantly inhibits the proliferation, colony formation ability, and DNA replication activity of colon cancer cells HCT116 (P<0.01), with an IC₅₀ value of 50 nmol/L, and exhibits a concentration-dependent manner. Meanwhile, ouabain also significantly induces apoptosis in colon cancer cells HCT116 (P<0.01). Moreover, ouabain significantly suppresses glycolysis in colon cancer cells HCT116 and downregulates the mRNA levels of glycolytic metabolic pathway-related factors (P<0.01). The results obtained from Western Blot, real-time PCR, and cellular enzyme activity assays indicate that ouabain significantly reduces the mRNA and protein expression levels of G6PD, as well as decreases G6PD enzyme activity in colon cancer cells HCT116 compared to the control group (P<0.01). In conclusion, ouabain significantly inhibits the proliferation of colon cancer cells HCT116, promotes apoptosis, and suppresses glycolysis in these cells. The underlying mechanism appears to involve the inhibition of the G6PD signaling pathway. These findings suggest that ouabain holds potential as a candidate drug for colon cancer treatment, offering new avenues for therapeutic intervention in this disease.