Objective: To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFa)/nuclear factor-kappa B (NF-kappaB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism.
Methods: A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats. Human subjects with various liver diseases were enrolled in the study, and serum and peripheral blood nuclear cells were collected for analysis. HepG2 cells were cultured in vitro and treated with anti-TNFa (monoclonal antibody, mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-kappaB to inhibit its activation. The liver cell line L02 was used as a control. Changes in protein and gene expression levels of NF-kappaB and TNFa were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR, respectively. Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay, respectively.
Results: TNFa and NF-kappaB expression showed increasing trends during the malignant transformation of rat hepatocytes, and the differential expression patterns showed association with histopathological alterations in the hepatocytes. Following treatment with the TNFa mAb, the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45% +/- 4.07% vs. 5.63% +/- 0.93%, q =10.07, P less than 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23% +/- 1.29% vs. untreated control cells: 59.00% +/- 1.02%, q =10.98, P less than 0.01). The decreased expression of TNFa and NF-kappaB in cell culture supernatants was positively correlated with the dose of treatment (r =0.89, P less than 0.01), with the most robust decreases being achieved with the highest concentrations ( P less than 0.01). NF-kappaB expression was significantly higher in the HepG2 cells than in the L02 cells, and transfection of p65-siRNA reduced the mRNA (93%) and protein (62%) levels and increased the cell apoptosis index (to 85%).
Conclusion: Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFa/NF-kappaB signaling pathway, which promotes cell apoptosis and blocks cell cycling.