Inhibitory Influences On Proliferation Of Hepg2 Cells Through The Intervention Of Nuclear Transcription Factor-κb Activation By Anti-human Tumor Necrosis Factor-α Monoclonal Antibody

Objective: Hepatocellular carcinoma (HCC) is characterized by multicause and multistage process of tumor progression, which is closely related to abnormal-expression and signal transduction of many cellular factors. Nuclear transcription factor-κB (NF-κB) plays an important role in the occurrence and development of HCC, especially the activation of NF-κB signal pathway induced by tumor necrosis factor-α(TNF-α) is closely related to hepatocarcinogenesis, and is also responsible for drug ressitance of HCC cells againt many chemotherapy drugs. Intervention of NF-κB activation maybe is one of the targeted therapies of HCC. The aim of this study was to investigate the influences on proliferation of HepG2 cells through the intervention of NF-κB activation pathway by anti- human TNF-αmonoclonal antibody (TNF-αmab).Methods: The human HepG2 cell line was cultured in vitro and intervented by different concentrations of TNF-αmab. The alterations of cell cycles and apoptosis were quantitively analyzed by flow cytometry and AnnexinV-FITC/PI double dyeing assay, respectively. The gene fragment of NF-κB was amplified by nested RT-PCR, and confirmed by sequencing, and the copy of NF-κB gene was quantitively measured by Real-Time PCR with absolute stardand. The changes of TNF-αin supernatant of cell culture fluid and NF-κB in nucleoprotein were analyzed by enzyme linked immunosorbent assay, and the NF-κB expression was also semi-quantitively analyzed by western blot assay and immunohistochemistry, respectively.Results: After given TNF-αmab, the apoptotic percentage of HepG2 cells in mab group was more than that in control one (P < 0.01), and the cell cycle changed greatly at the proportion of G1 phase (P < 0.01) but not in S and G2 phase. The levels of total RNA and NF-κB gene in HepG2 cells were higher than those in normal hepatocytes (P < 0.01). The copy of NF-κB gene in mab group was significantly lower than that in control (P < 0.01). The amplified fragment of NF-κB gene identified by sequencing, was in accordance with its original sequence. The expression level and density of nucleoprotein NF-κB in mab groups were obviously lower than those in control ones (P < 0.01), which were positively correlated with the decreased TNF-αin supernatant of cell culture fluid (r = 0.89, P = 0.000). The effect of mab was dose- and time-dependent, which was most effective in the first 24 hours and at the highest concentration (P < 0.01).Conclusions: TNF-αmab can effectively inhibit the activation of NF-κB signal pathway, reduce the expression of NF-κB gene, induce the HCC cells apoptosis, and lead to G1 phase arrest. Intervention of NF-κB activation pathway by TNF-αmab can suppress the growth of HCC cells and induce apoptosis in vitro, which is expected to become a new method of HCC targeted therapy.