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, 2014, 539607

Apoptosis Induction by Polygonum Minus Is Related to Antioxidant Capacity, Alterations in Expression of Apoptotic-Related Genes, and S-phase Cell Cycle Arrest in HepG2 Cell Line

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Apoptosis Induction by Polygonum Minus Is Related to Antioxidant Capacity, Alterations in Expression of Apoptotic-Related Genes, and S-phase Cell Cycle Arrest in HepG2 Cell Line

Mohd Alfazari Mohd Ghazali et al. Biomed Res Int.

Abstract

Polygonum minus (Polygonaceae) is a medicinal herb distributed throughout eastern Asia. The present study investigated antiproliferative effect of P. minus and its possible mechanisms. Four extracts (petroleum ether, methanol, ethyl acetate, and water) were prepared by cold maceration. Extracts were subjected to phytochemical screening, antioxidant, and antiproliferative assays; the most bioactive was fractionated using vacuum liquid chromatography into seven fractions (F1-F7). Antioxidant activity was measured via total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) assays. Antiproliferative activity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Most active fraction was tested for apoptosis induction and cell cycle arrest in HepG2 cells using flow cytometry and confocal microscopy. Apoptotic-related gene expression was studied by RT-PCR. Ethyl acetate extract was bioactive in initial assays. Its fraction, F7, exhibited highest antioxidant capacity (TPC; 113.16 ± 6.2 mg GAE/g extract, DPPH; EC50: 30.5 ± 3.2 μg/mL, FRAP; 1169 ± 20.3 μmol Fe (II)/mg extract) and selective antiproliferative effect (IC50: 25.75 ± 1.5 μg/mL). F7 induced apoptosis in concentration- and time-dependent manner and caused cell cycle arrest at S-phase. Upregulation of proapoptotic genes (Bax, p53, and caspase-3) and downregulation of antiapoptotic gene, Bcl-2, were observed. In conclusion, F7 was antiproliferative to HepG2 cells by inducing apoptosis, cell cycle arrest, and via antioxidative effects.

Figures

Figure 1
Figure 1
Flowchart of study.
Figure 2
Figure 2
Early apoptosis in HepG2 cells exposed to EtOAc crude extract of P. minus. HepG2 cells (1 × 106/well) were incubated with EtOAc crude extract of P. minus (18–100 μg/mL) for different periods (24, 48, and 72 h) at 37°C. Apoptosis was detected by flow cytometry using annexin-V and FITC. Results are mean ± S.D. (n = 3). Values with different alphabets are significantly different (P < 0.05, ANOVA and Bonferroni test).
Figure 3
Figure 3
Percentage of HepG2 cells in early apoptosis upon exposure to F7 of P. minus. HepG2 cells (1 × 106/well) were incubated with F7 of P. minus (12.5–37.5 μg/mL) for different periods (24, 48, and 72 h) at 37°C. Apoptosis was detected by flow cytometry using annexin V-FITC. Results are mean ± S.D (n = 3). Values with different alphabets are significantly different (P < 0.05, ANOVA and Bonferroni test).
Figure 4
Figure 4
Histogram of annexin-V and FITC/PI flow cytometry of HepG2 cells exposed to F7. HepG2 cells (1 × 106/well) were incubated with F7 of P. minus (25 μg/mL, equivalent to IC50) or (a) control for (b) 24, (c) 48 and (d) 72 h at 37°C. Lower left quadrant in each panel represents viable cells which excluded PI and were negative for annexin V-FITC binding. Upper right quadrant contains nonviable, necrotic cells or late stage apoptotic cells, positive for annexin V-FITC/PI uptake. Lower right quadrant contains early apoptotic cells, annexin V-FITC positive and PI negative. One experiment is representative of three independent experiments.
Figure 5
Figure 5
Fluorescence confocal imaging of TUNEL positive staining in HepG2 cells exposed to F7 at 48 h. HepG2 cells (0.5 × 106/well) were treated with F7 of P. minus (12.5–37.5 μg/mL) for 48 h at 37°C. Red stain shows viable, healthy cells while intense green stain indicates apoptotic cell with DNA fragmentation. One experiment is representative of three independent experiments.
Figure 6
Figure 6
Effect of F7 on expression levels of apoptotic-related genes in HepG2 cells. HepG2 cells (1 × 106/well) were incubated with F7 of P. minus (12.5–37.5 μg/mL) for 48 h at 37°C. Results are mean ± S.D (n = 3). Values with different alphabets are significantly different (P < 0.05, ANOVA and Bonferroni test).
Figure 7
Figure 7
Effect of F7 of P. minus on cell cycle progression in HepG2 cells. Cells (1 × 106/well) were treated with F7 (12.5–37.5 μg/mL) for (top lane) 24 h or (bottom lane) 48 h at 37°C. Data are representative of three independent experiments. *Significantly different from untreated control cells (P < 0.05, ANOVA and Bonferroni test).

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