Mechanism Of CpG-ODN Inhibited Proliferation And Enhanced Chemosensitivity In HepG2Human Hepatoma Cells

BackgroundRecent studies indicated that a synthetic oligonucleotide containing un-methylatedCpG-oligodeoxynucleotide(CpG-ODN) has a potential function for cancer therapy. In thisstudy, we investigated the anti-proliferative effects of CpG-ODN in human hepatoma cellsHepG2in vitro. Meanwhile, we evaluated the chemosensitizing effects of CpG-ODN inchemotherapeutics-treated HepG2human hepatoma cells.MethodsThe inhibition of CpG-ODN on human hepatoma cells HepG2and Bel-7402、humanlung cancer cells A549were evaluated by MTS assay. Meanwhile, the dose and time-effectsrelationship of HepG2cells treated with CpG-ODN were discussed. The mRNA expressionof TLR9within HepG2cells、Bel-7402cells and A549cells treated with CpG-ODN wereanalyzed by Real Time PCR assay in vitro. Meanwhile, we evaluated the expression ofanti-apoptotic factor Bcl-2within HepG2cells induced by CpG-ODN.Cell viability assay were utilized to evaluate the direct cytotoxicity of CpG-ODN inthe presence or absence of5-FU in HepG2cells, the morphologic change of nuclear stainedwith Hoechst33258were detected by fluorescence microscopy, and apoptosis as well ascell-cycle was examined by flow cytometry analysis. The mRNA expression of Livin andSurvivin within HepG2cells treated with CpG-ODN and/or5-FU were analyzed by RealTime PCR assay in vitro.ResultsThe results showed that CpG-ODN significantly decreased the viability of HepG2cells in a dose-dependent manner. However, CpG-ODN had no effect on the viability ofBel-7402cells and A549cells. The expression of TLR9didn’t be influenced on HepG2 cells、Bel-7402cells and A549cells treated with CpG-ODN. The Bcl-2protein, belongs tothe antiapoptotic factors of Bcl-2family, were related to the tumorigenesis. Thedownregulation of Bcl-2expression may promote apoptotic response to anticancer drugs.Real-time quantitative PCR experiments showed that CpG-ODN alone could down-regulatethe expression of Bcl-2within HepG2cells.CpG-ODN in combination with5-FU could decrease cell viability, increase apoptosisand further induce HepG2cells cycle arrest at S phase when compared with CpG-ODN or5-FU alone. The Livin and Survivin protein, belong to the inhibitors of apoptotic proteins(IAPs), were highly expressed in tumor tissue but lowly in normal tissue, and the inductionof apoptosis was generally associated with down-regulation of Survivin and Livin withintumor cells. The overexpression of Survivin or Livin was closely related to chemoresistance,and inhibition of Survivin or Livin improved the sensitivity of tumor to chemotherapy. ThemRNA expression of Livin and Survivin decreased in cells treated with CpG-ODN alonebut increased in cells treated with5-FU alone. However, CpG-ODN in combination with5-FU could down-regulate the mRNA expression of Livin and Survivin within HepG2cells.ConclusionCpG-ODN can significantly inhibit the growth of HepG2cells via down-regulation ofantiapoptotic factor Bcl-2. Meanwhlie, Our finding demonstrated that CpG-ODN enhancedthe chemosentivity of5-FU in HepG2human hepatoma cells at least in part bydown-regulating the expression of Livin and Survivin, leading to apoptosis and furtherinducing cell cycle arrest at S phase. Therefore, CpG-ODN may be a potential candidate aschemosensitizer for human hepatocellular carcinoma.