Polyphenol extract of Phyllanthus emblica (PEEP) induces inhibition of cell proliferation and triggers apoptosis in cervical cancer cells

Abstract

Background: The aim of this study is to investigate the effects of polyphenol extract from Phyllanthus emblica (PEEP) on cervical cancer cells and to explore the underlying mechanism.

Methods: MTT assay was used to measure inhibition of proliferation of cervical cancer (HeLa) cells after treatment with PEEP at concentrations of 0, 50, 100, 150, and 200 mg/ml for 48 hours. HeLa cells were treated with PEEP (150 mg/ml) for 48 hours in the following analysis. Karyomorphism was assessed by immunofluorescence using DAPI staining, and cell apoptosis and cell cycle were assessed using flow cytometry. Three apoptotic marker proteins, namely, Fas, FasL, and cleaved caspase-8, were assessed by western blotting.

Results: PEEP inhibited the growth of HeLa cells, and the optimum concentration of PEEP was 150 mg/ml. In addition, the karyomorphism of HeLa cells after treatment with PEEP was abnormal. Furthermore, PEEP induced arrest of the HeLa cell cycle at G2/M phase, and triggered apoptosis. PEEP also induced significant Fas and FasL activation, and cleavage of caspase-8.

Conclusions: Our study indicates that PEEP is effective in inhibiting HeLa cell proliferation by inducing cell cycle arrest at G2/M phase and inducing apoptosis.

Figure 1

Polyphenol extract of Phyllanthus emblica (PEEP) inhibits HeLa cell proliferation. HeLa cells were treated with different concentrations of PEEP (50, 100, 150, 200 mg/ml) for 48 hours. Quantification of the number of viable cells was determined by MTT assay. The data are expressed as the means ± SD obtained from three independent experiments.

Figure 2

Polyphenol extract of Phyllanthus emblica (PEEP) induces cell cycle arrest of HeLa cells at G2/M phase. HeLa cells were treated with 0 mg/ml PEEP (A), 150 mg/ml PEEP (B) for 48 hours, and the cell cycle stage was determined by flow cytometry. The panels of M1, M2, and M3inside the image are the marks of stage borders of G₀/G₁, S, and G₂/M, respectively.

Figure 3

Polyphenol extract of Phyllanthus emblica (PEEP) changes the karyomorphism of HeLa cells. HeLa cells were seeded onto coverslips, and treated with 0 mg/ml PEEP (A), 150 mg/ml PEEP (B) for 48 hours before harvesting. DAPI staining was used to highlight the nucleus. The panels inside the image represent the size of 10 μm.

Figure 4

Polyphenol extract of Phyllanthus emblica (PEEP) induces HeLa cell apoptosis. HeLa cells were treated with 0 mg/ml PEEP (A), 150 mg/ml PEEP (B) for 48 hours. The cells were harvested and resuspended in Guava Nexin. Cell cycle stage was determined by flow cytometry. The panels inside the image are marks of the quadrants, cells in the left lower quadrant are living cells, in the right upper quadrant are dead cells, and in the right lower quadrant are apoptotic cells.

Figure 5

Polyphenol extract of Phyllanthus emblica (PEEP) induces activation of Fas, FasL, and cleaved caspase-8. Western blotting analysis of Fas, FasL, and cleaved caspase-8 (Δ-caspase-8), in HeLa cells exposed to PEEP for 48 hours.