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Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Zhang Z, Chen S, Mei H, Xuan J, Guo X, Couch L, Dobrovolsky VN, Guo L, Mei N - Sci Rep (2015)

Bottom Line: In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined.In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II.Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.

ABSTRACT
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

No MeSH data available.


Related in: MedlinePlus

Effects of quercetin on the cell cycle of HepG2 cells.(A) Histograms show DNA content analyses for HepG2 cells treated with the indicated concentrations of quercetin for 24 h by flow cytometric analysis. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. *p < 0.05 representing significant difference from the vehicle control. (C) Expression of cell cycle checkpoint-related proteins was determined in HepG2 cells treated with the indicated concentrations of quercetin for 2, 4, 6, and 24 h. Treated cells were lysed and subjected to Western blot analyses with antibodies against phospho-Chk1 and phospho-Chk2.
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f7: Effects of quercetin on the cell cycle of HepG2 cells.(A) Histograms show DNA content analyses for HepG2 cells treated with the indicated concentrations of quercetin for 24 h by flow cytometric analysis. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. *p < 0.05 representing significant difference from the vehicle control. (C) Expression of cell cycle checkpoint-related proteins was determined in HepG2 cells treated with the indicated concentrations of quercetin for 2, 4, 6, and 24 h. Treated cells were lysed and subjected to Western blot analyses with antibodies against phospho-Chk1 and phospho-Chk2.

Mentions: HepG2 cells were treated with various concentrations of quercetin for 24 h (no significant cytotoxicity was observed under these conditions, Supplementary Fig. 3) and cell cycle profiles were analyzed by measuring the DNA content using flow cytometry (Fig. 7A). Cell cycle distribution analysis indicated that there was a concentration-dependent increase in the number of cells in S-phase (Fig. 7B). Concomitantly, there was a decrease in the number of cells in G1-phase and no significant change in G2/M-phase. These results suggest that quercetin disturbs cell cycle progression, leading to the accumulation of cells in S-phase. In addition, we characterized the DNA damaging effect and cell cycle arrest induced by quercetin. Treatment of HepG2 cells with quercetin resulted in an increase in p-Chk1 and p-Chk2, and the induction of both checkpoint-related proteins was observed as early as 2 h (Fig. 7C).


Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

Zhang Z, Chen S, Mei H, Xuan J, Guo X, Couch L, Dobrovolsky VN, Guo L, Mei N - Sci Rep (2015)

Effects of quercetin on the cell cycle of HepG2 cells.(A) Histograms show DNA content analyses for HepG2 cells treated with the indicated concentrations of quercetin for 24 h by flow cytometric analysis. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. *p < 0.05 representing significant difference from the vehicle control. (C) Expression of cell cycle checkpoint-related proteins was determined in HepG2 cells treated with the indicated concentrations of quercetin for 2, 4, 6, and 24 h. Treated cells were lysed and subjected to Western blot analyses with antibodies against phospho-Chk1 and phospho-Chk2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4588569&req=5

f7: Effects of quercetin on the cell cycle of HepG2 cells.(A) Histograms show DNA content analyses for HepG2 cells treated with the indicated concentrations of quercetin for 24 h by flow cytometric analysis. Treated cells were stained with propidium iodide (PI) and processed for cell cycle analysis. (B) The bar graph depicts the mean percentage of each cell cycle phase ± S.D. from four independent experiments. *p < 0.05 representing significant difference from the vehicle control. (C) Expression of cell cycle checkpoint-related proteins was determined in HepG2 cells treated with the indicated concentrations of quercetin for 2, 4, 6, and 24 h. Treated cells were lysed and subjected to Western blot analyses with antibodies against phospho-Chk1 and phospho-Chk2.
Mentions: HepG2 cells were treated with various concentrations of quercetin for 24 h (no significant cytotoxicity was observed under these conditions, Supplementary Fig. 3) and cell cycle profiles were analyzed by measuring the DNA content using flow cytometry (Fig. 7A). Cell cycle distribution analysis indicated that there was a concentration-dependent increase in the number of cells in S-phase (Fig. 7B). Concomitantly, there was a decrease in the number of cells in G1-phase and no significant change in G2/M-phase. These results suggest that quercetin disturbs cell cycle progression, leading to the accumulation of cells in S-phase. In addition, we characterized the DNA damaging effect and cell cycle arrest induced by quercetin. Treatment of HepG2 cells with quercetin resulted in an increase in p-Chk1 and p-Chk2, and the induction of both checkpoint-related proteins was observed as early as 2 h (Fig. 7C).

Bottom Line: In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined.In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II.Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, USA.

ABSTRACT
Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

No MeSH data available.


Related in: MedlinePlus