Inhibition of G6PD-Mediated MAPK Signaling Triggers Mitochondrial Apoptosis and Cell Cycle Arrest in Hepatocellular Carcinoma

Background: Glucose-6-phosphate dehydrogenase (G6PD) has been implicated in tumor progression. This study investigated the biological role of G6PD in the malignant progression of hepatocellular carcinoma (HCC) and explored the underlying molecular mechanisms. Methods: RNA-sequencing data and corresponding clinical data for patients with HCC were obtained from The Cancer Genome Atlas (TCGA). Independent validation datasets were obtained from Gene Expression Omnibus (GEO). We performed bioinformatics analyses to assess the expression of G6PD in HCC, as well as its relationship with clinical features and prognostic outcomes. Immunohistochemistry and western blotting were used to validate G6PD expression in human HCC tissues and cell lines. Stable G6PD-overexpressing and knockdown cell lines were established using lentiviral transduction. Functional assays were performed using G6PD-overexpressing and G6PD-knockdown cell lines to evaluate cell proliferation, cell cycle progression, apoptosis, mitochondrial function, and oxidative stress. Western blotting was conducted to assess the protein levels of key regulators of cell cycle progression, apoptosis, and the MAPK pathway. Finally, a BALB/c nude mouse xenograft model was used to evaluate the in vivo effects of G6PD on tumor growth. Results: G6PD was significantly overexpressed in HCC tissues and was significantly associated with histological grade (p < 0.001), T stage (p = 0.039), vascular invasion (p = 0.002), and pathological stage (p = 0.031). Patients with high G6PD expression exhibited significantly shorter overall, disease-specific, and progression-free survival. Multivariate analysis identified high G6PD expression as an independent predictor of mortality in patients with HCC. G6PD knockdown significantly inhibited HCCLM3 cell proliferation and induced G0/G1 cell-cycle arrest, accompanied by the downregulation of cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), and cyclin D1 protein levels. In addition, G6PD silencing triggered mitochondrial apoptosis, including decreased mitochondrial membrane potential, BCL-2-associated X protein (BAX) and cysteine-aspartic acid protease-3 (caspase-3) upregulation, B-cell lymphoma 2 (BCL-2) downregulation, elevated reactive oxygen species (ROS) levels, and suppressed MEK1/2 and ERK1/2 phosphorylation. In vivo, G6PD knockdown significantly reduced xenograft tumor size and weight and decreased Ki67 and proliferating cell nuclear antigen (PCNA) expression. Conclusions: G6PD is an independent prognostic biomarker in HCC and promotes disease progression through pleiotropic mechanisms, including modulation of the MAPK/ERK pathway, promotion of cell proliferation, and suppression of mitochondrial apoptosis. These findings suggest that G6PD and its downstream signaling pathways may be promising therapeutic targets for HCC.