CpG oligodeoxynucleotides enhance chemosensitivity of 5-fluorouracil in HepG2 human hepatoma cells via downregulation of the antiapoptotic factors survivin and livin

Abstract

Background: Recent studies indicated that a synthetic oligonucleotide containing un-methylated CpG oligodeoxynucleotides (CpG-ODN) has a potential function for cancer therapy. In this study, we evaluated the chemosensitizing effects of CpG-ODN in 5-fluorouracil (5-FU)-treated HepG2 human hepatoma cells.

Methods: Cell viability assay were utilized to evaluate the direct cytotoxicity of CpG-ODN in the presence or absence of 5-FU in HepG2 cells, and apoptosis as well as cell-cycle was examined by flow cytometry analysis. The mRNA expression of Bcl-2, Livin and Survivin within HepG2 cells treated with CpG-ODN and/or 5-FU were analyzed by Real Time PCR assay in vitro.

Results: CpG-ODN in combination with 5-FU could decrease cell viability, increase apoptosis and further induce HepG2 cells cycle arrest at S phase when compared with CpG-ODN or 5-FU. CpG-ODN or 5-FU could down-regulate the mRNA expression of Bcl-2 within HepG2 cells. The mRNA expression of Livin and Survivin decreased in cells treated with CpG-ODN alone but increased in cells treated with 5-FU alone. However, CpG-ODN in combination with 5-FU could down-regulate the mRNA expression of Livin and Survivin within HepG2 cells.

Conclusions: Our finding demonstrated that CpG-ODN enhanced the chemosentivity of 5-FU in HepG2 human hepatoma cells at least in part by down-regulating the expression of Livin and Survivin, leading to apoptosis and further inducing cell cycle arrest at S phase. Therefore, CpG-ODN may be a potential candidate as chemosensitizer for human hepatocellular carcinoma.

Figure 1

Effect of CpG-ODN and/or 5-FU treatment on the cell viability in vitro. A: The cell viability of HepG2 cells, BEL-7402 cells and A549 cells treated with indicated concentration of CpG-ODN for 48 h in MTS assay, cell viability was expressed as the percentage of control cells (medium). B: The cell viability of HepG2 cells treated with indicated concentration of CpG-ODN for indicated time in MTS assay, cell viability was then quantified as described above. C: The cell viability of HepG2 cells treated with indicated concentration of non-CpG-ODN and ODN M362 for 48 h in MTS assay, cell viability was then quantified as described above. D: HepG2 cells were treated with indicated concentration of 5-FU for indicated time in MTS assay, results were presented as the inhibitory ratio of 5-FU treated HepG2 cells. E: HepG2 cells treated with indicated concentration of CpG-ODN, 5-FU or CpG-ODN in combination with 5-FU for indicated time, inhibition was then determined by the MTS assay. F: The inhibitory ratio of HepG2 cells treated with a rang of 5-FU doses in the presence and absence of 4μM CpG-ODN or non-CpG-ODN for 24h. In this figure, 5-FU, CpG(2) ,CpG(4) and non-CpG(4) represent that the concentration of 5-FU,CpG-ODN , CpG-ODN and non-CpG-ODN is 7.5 μg/ml , 2 μM ,4 μM, 4 μM, respectively. All results shown were means ±SD from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 versus medium.

Figure 2

Effect of CpG-ODN and/or 5-FU treatment on the cell morphology of HepG2 cells. A: Morphologic changes of HepG2 cells treated with the indicated concentration of CpG-ODN and 5-FU alone or together for 48 h, compared with untreated cells (medium) (100X). B: HepG2 cells treated with the indicated concentration of CpG-ODN and 5-FU alone or together for 48 h, then stained with Hoechst 33258(400 X). Condensed and fragmented nuclei in cells were marked by arrow heads. In this figure, 5-FU, CpG(2) and CpG(4) represent that the concentration of 5-FU, CpG-ODN and CpG-ODN is 7.5 μg/ml, 2 μM, 4 μM, respectively.

Figure 3

Effect of CpG-ODN and/or 5-FU treatment on apoptosis of HepG2 cells. A: The apoptosis was examined using annexin V-FITC/PI staining and flow cytometry analysis. One representative flow cytrometry analysis of apoptosis in HepG2 cells. Fluorescence intensity for annexin V/FITC is plotted on the x-axis, and PI is plotted on the y-axis. FITC^-/PI^-, FITC⁺/PI^-, FITC⁺/PI⁺, FITC^-/PI⁺ was regarded as living, early apoptotic, late apoptotic and necrotic cells, respectively. B: The percentage of apoptotic cells examined by annexin V-FITC/PI staining and flow cytometry analysis. Results are presented as mean ±SD of three separate experiments. ***P<0.001 versus medium. ^###P<0.001 versus 5-FU. In this figure, 5-FU, CpG(2) and CpG(4) represent that the concentration of 5-FU, CpG-ODN and CpG-ODN is 7.5 μg/ml, 2 μM, 4 μM, respectively.

Figure 4

Effect of CpG-ODN and/or 5-FU treatment on HepG2 cells cycle distribution. A: Cells were incubated with CpG-ODN and 5-FU alone or together at indicated concentration for 48 h, and the cell cycle distribution was evaluated using PI staining and flow cytometry analysis. One representative flow cytometry analysis of cell cycle distribution. B: The percentage of cells in G0/G1, S and G2/M phase is expressed as mean ±SD of three independent experiments. *P<0.05,***P<0.001 versus medium. ^###P<0.001 versus 5-FU. In this figure, 5-FU, CpG(2) and CpG(4) represent that the concentration of 5-FU, CpG-ODN and CpG-ODN is 7.5 μg/ml , 2 μM , 4 μM , respectively.

Figure 5

Effect of CpG-ODN and/or 5-FU treatment on anti-apoptotic factors within HepG2 cells. A and B: The cells seeded in six-well plates were treated with the indicated concentration of CpG-ODN (A) or 5-FU (B). After 48 h, total RNA was extracted for Bcl-2 mRNA expression using Real-time quantitative PCR. C and D: The cells seeded in six-well plates were treated with the indicated concentration of CpG-ODN and 5-FU alone or together, the Livin(C) and Survivin(D) mRNA expression were analyzed using Real-time quantitative PCR analysis as described above. Results are presented as mean ±SD of three separate experiments. *P<0.05, ***P<0.001 versus medium. ^#P<0.05, ^##P<0.01, ^###P<0.001 versus 5-FU. In this figure, 5-FU, CpG(2) and CpG(4) represent that the concentration of 5-FU, CpG-ODN and CpG-ODN is 7.5 μg/ml, 2 μM, 4 μM, respectively.