Effects of cordycepin on HepG2 and EA.hy926 cells: Potential antiproliferative, antimetastatic and anti-angiogenic effects on hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a hypervascular tumor and accumulating evidence suggests that angiogenesis plays an important role in HCC development. Cordycepin, also known as 3'-deoxyadenosine, is a derivative of adenosine, and numerous cellular enzymes cannot differentiate the two. The aim of the present study was to determine whether cordycepin regulates proliferation, migration and angiogenesis in a human umbilical vein endothelial cell line (EA.hy926) and in a hepatocellular carcinoma cell line (HepG2). MTT was used to assess cell proliferation. Apoptosis was analyzed by flow cytometry (propidium iodide staining). Transwell and wound healing assays were used to analyze the migration and invasion of HepG2 and EA.hy926 cells. Angiogenesis in EA.hy926 cells was assessed using a tube formation assay. Cordycepin strongly suppressed HepG2 and EA.hy926 cell proliferation in a dose- and time-dependent manner. Cordycepin induced EA.hy926 cell apoptosis in a dose-dependent manner (2,000 μg/ml: 50.20±1.55% vs. 0 μg/ml: 2.62±0.19%; P<0.01). Cordycepin inhibited EA.hy926 cell migration (percentage of wound healing area, 2,000 μg/ml: 3.45±0.29% vs. 0 μg/ml: 85.48±0.84%; P<0.05), as well as tube formation (total length of tubular structure, 1,000 μg/ml: 107±39 μm vs. 0 μg/ml: 936±56 μm; P<0.05). Cordycepin also efficiently inhibited HepG2 cell invasion and migration. High-performance liquid chromatography analysis of the cytosol from EA.hy926 cells showed that cordycepin was stable for 3 h. In conclusion, cordycepin not only inhibited human HepG2 cell proliferation and invasion, but also induced apoptosis and inhibited migration and angiogenesis in vascular endothelial cells, suggesting that cordycepin may be used as a novel anti-angiogenic therapy in HCC.

Keywords: angiogenesis; apoptosis; cordycepin; hepatocellular carcinoma; invasion; vascular endothelial cells.

Figure 1

Effects of cordycepin on HepG2 and EA.hy926 cell viability. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay was performed to measure cell viability (the relative growth rate) in (A) EA.hy926 cells and (B) HepG2 cells following treatment with cordycepin at concentrations from 125 to 2,000 μg/ml for 1, 2, 3, 4 and 5 days. EA.hy926 and HepG2 cells treated without cordycepin (0 μg/ml) were used as the negative control. The data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^**P<0.01, vs. the negative control.

Figure 2

Cordycepin induced apoptosis in EA.hy926 cells. Apoptosis was determined by flow cytometric assay [propidium iodide (PI) staining]. (A) Representative flow cytometric plots. EA.hy926 cells were treated with various concentrations of cordycepin (0, 250, 500, 1,000 and 2,000 μg/ml) for 24 h. (B) The number of apoptotic cells divided by the total number of cells (counted manually), expressed as the percentage of total cells. The data are presented as the mean ± standard deviation of three independent experiments. ^*P<0.05 and ^**P<0.01, vs. the negative control (0 μg/ml).

Figure 3

Effects of cordycepin on migration and invasion of HepG2 cells. The migrating and invading abilities of HepG2 cells were examined by Transwell chamber assay. HepG2 cells were treated with various concentrations of cordycepin (0, 125, 250, 500 and 1,000 μg/ml) for 12 h (migration assay) or 24 h (invasion assay). (A and C) Migrating or invading cells were photographed under an inverted fluorescence microscope (A, ×100; C, ×200). Blue represents DAPI staining. (B and D) Quantification of the numbers of migrating or invading cells are presented as the mean ± standard deviation of three independent experiments performed in triplicate. ^*P<0.05 and ^**P<0.01, vs. the negative control (0 μg/ml).

Figure 4

Effect of cordycepin on the migration of EA.hy926 cells by wound healing assay. EA.hy926 cells were treated with various concentrations of cordycepin (0, 125, 500 and 2,000 μg/ml) for 6 h. (A) Migration of EA.hy926 cells were photographed under a light microscope (scale bar, 100 μm) and the data were digitally recorded. (B) The percentages of wound healing area are presented as the means ± standard deviation of three independent experiments. ^***P<0.001, vs. the negative control (0 μg/ml).

Figure 5

Cordycepin inhibited tube formation of EA.hy926 cells. EA.hy926 cells were treated with different concentrations of cordycepin (0, 125, 250, 500 and 1,000 g/ml) and added to 24-well plates precoated with Matrigel for 24 h. (A) Tube formation of EA.hy926 cells were photographed under a light microscope (magnification, ×100). (B) Quantification of the total length of tubular structure under different cordycepin concentrations. ^*P<0.05, vs. the negative control (0 μg/ml); ^#P<0.05, vs. cells treated with 125 μg/ml cordycepin; ^&P<0.05, vs. cells treated with 250 μg/ml cordycepin.

Figure 6

Intracellular cordycepin concentrations in EA.hy926 endothelial cells quantified by high-performance liquid chromatography. Under the chromatographic conditions used, cordycepin had a retention time of 8.96 min. (A) Cordycepin standard curve (100 μg/ml). (B) Intracellular concentration of cordycepin after 0.5 h. (C) Intracellular concentration of cordycepin after 1 h. (D) Intracellular concentration of cordycepin after 3 h. (E) Cytosol of EA.hy926 cells without cordycepin. Arrows show the locations of cordycepin in chromatographic graphs.