Molecular mechanisms of ampelopsin from Ampelopsis megalophylla induces apoptosis in HeLa cells

Abstract

Ampelopsin (AMP) is an active ingredient of flavonoid compounds that is extracted from Ampelopsis megalophylla Diels et Gilg. The present study aimed at investigating the antitumor activities of AMP and the possible underlying molecular mechanisms in HeLa cells. A total of three types of tumor cell were selected to screen antitumor activities for AMP using the MTT assay. Flow cytometry was used to analyze the cell apoptotic proportion and the cell cycle. Rhodamine 123 staining was used to determine changes in mitochondrial transmembrane potential. Western blot analysis was used to determine the expression of apoptosis-associated proteins. The results of the present study demonstrated that AMP may inhibit the viability of HeLa cells in a dose- and time-dependent manner. Changes in morphology were observed using fluorescence microscopy. In addition, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) double staining revealed that AMP induced apoptosis in a concentration-dependent manner and PI staining indicated that HeLa cells were arrested in S phase. Furthermore, western blot analysis demonstrated that AMP treatment induced apoptosis through activation of caspases 9 and 3, which was validated by the increasing ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein to Bcl-2. Additionally, the loss of mitochondrial transmembrane potential and the release of cytochrome c suggested that AMP-induced apoptosis was associated with the mitochondrial pathway. Taken together, these results indicate that AMP may induce apoptosis via the mitochondrial signaling pathway in HeLa cells.

Keywords: Ampelopsis megalophylla Diels et Gilg; HeLa cells; ampelopsin; apoptosis; mitochondrial signaling pathway.

Figure 1.

Effect of AMP on HeLa cells as determined using the MTT assay. HeLa cells were treated with various concentrations of AMP (10, 20, 40, 80, 160 and 320 µM) for 24, 48 and 72 h (*P<0.05). AMP, ampelopsin.

Figure 2.

Morphological alterations of the cell nucleus determined using fluorescence microscopy and Hoechst 33258 staining. Representative micrographs of HeLa cells undergoing apoptosis induced by treatment with various ampelopsin concentrations (between 30 and 70 µM) for 24 h (40X objective).

Figure 3.

Effect of AMP on the cell cycle of HeLa cells determined using flow cytometry with PI staining. The cells were treated with a variety of concentrations of AMP (between 30 and 70 µM) for 12 h. AMP, ampelopsin; PI, propidium iodide.

Figure 4.

Analysis of AMP-induced apoptosis in HeLa cells using the Annexin V-fluorescein isothiocyanate/propidium iodide staining assay. Cells were treated with a variety of concentrations of AMP (between 30 and 70 µM) for 8 h. Each image has four quadrants: Q1, necrotic cells; Q2 and Q3, apoptotic cells; Q4, viable cells. AMP, ampelopsin; FL1-H, cells stained by Annexin V-fluorescein isothiocyanate; FH2-H, cells stained by propidium iodide.

Figure 5.

AMP induces a decrease in mitochondrial membrane potential. Mitochondrial membrane potential (indicated by the fluorescence intensity of rhodamine-123) of HeLa cells was determined following incubation with AMP (concentrations between 30 and 70 µM) for 24 h. Data are presented as the mean ± standard deviation (n=3; *P<0.05, **P<0.01 vs. control). AMP, ampelopsin.

Figure 6.

(A) AMP induced Bax, Bcl-2, caspase 9 and caspase 3 protein expression levels in HeLa cells, analyzed using western blotting. HeLa cells were treated with AMP (between 30 and 70 µM) for 12 h. (B) Protein expression of Bax and Bcl-2 in Hela cells. (C) Protein expression of caspase 9 in Hela cells. (D) Protein expression of caspase 3 in Hela cells. Data are presented as the mean ± standard deviation (n=3; *P<0.05 vs, control). GAPDH was used as a loading control. AMP, ampelopsin. Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated protein.

Figure 7.

Release of Cyt-c in HeLa cells determined using western blot analysis. HeLa cells were treated with a variety of concentrations of AMP (between 30 and 70 µM) for 12 h. Data are presented as the mean ± standard deviation (n=3; *P<0.05 vs. control). GAPDH was used as a loading control. Cyt-c, cytochrome c; AMP, ampelopsin.