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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

Author: ShenJunJun
Tutor: YaoDengFu
School: Nantong University
Course: Oncology
Keywords: Hepatocellular carcinoma Nuclear factor-κB Tumor necrosis factor -α Monoclonal antibodies Apoptosis Cell cycle RT-PCR Real-Time PCR Western blot Sequencing
CLC: R735.7
Type: Master's thesis
Year: 2009
Downloads: 53
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Abstract

Purpose: The incidence of hepatocellular carcinoma (HCC) development is more than one cause of a multi-stage process, is closely related to the abnormal expression of a variety of cytokines and signal transduction. Nuclear transcription factor-κB (NF-κB) the occurrence of HCC, play an important role in the development process, especially tumor necrosis factor-α (TNF-α)-mediated NF-κB signaling pathway and hepatocellular carcinoma is closely related to hepatoma cells also produce resistance to many anticancer drugs reasons. NF-κB activation intervention may be one of the ways of targeted therapy in HCC, for this study, the anti-human TNF-α monoclonal antibody (TNF-α monoclonal antibody) on NF-κB activation pathway intervention observed hepatoma cells proliferation. Methods: HepG2 cells cultured in vitro, and to give a series of concentrations of TNF-α monoclonal antibody, two-color flow cytometry and AnnexinV-FITC/PI notation detect cell cycle phase and apoptosis changes; nested RT NF-κB-PCR amplification of gene fragment sequencing confirmed Quantitative Real-Time PCR to detect changes in the NF-κB gene; enzyme-linked immunosorbent assay for quantitative analysis of TNF-α in the cell culture medium and and nucleoprotein of NF-κB, the level of semi-quantitative immunohistochemical method and Western blot detection of the nucleoprotein of NF-κB expression. Results: mAb role in liver cancer cells, apoptosis was significantly increased (P lt; 0.01), a significant increase in the proportion of G1 phase cells (P lt; 0.01), while the proportion of S and G2 phase cells did not change significantly. Total RNA from HepG2 cells and NF-κB genes were significantly higher than normal liver cells (P lt; 0.01). The mAb after, HepG2 cells, NF-κB gene copy number was significantly lower than the control group (P lt; 0.01). The NF-κB gene sequencing analysis of the amplified fragment and the source of sequence homology is completely consistent. The expression levels of nuclear NF-κB and expression intensity decreased significantly after treatment (P lt; 0.01), and training was significantly lower TNF-α was a significant positive correlation (r = 0.89, P = 0.000). The role of monoclonal antibodies effects were dose-and time-dependent manner, the effect is most obvious in the first 24 h and at high concentrations (P lt; 0.01). Conclusion: TNF-α monoclonal antibody can significantly inhibit the activation of NF-κB signaling pathway, reducing the gene expression of NF-κB, and induce apoptosis of liver cancer cells, resulting in cell cycle arrest in G1 phase. TNF-α monoclonal antibody on NF-κB activation intervention in vitro can inhibit the growth of liver cancer cells, induction of apoptosis, is expected to become the HCC targeted therapy.

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

Abstract

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