Polysaccharides isolated from Bangia fuscopurpurea induce apoptosis and autophagy in human ovarian cancer A2780 cells

Abstract

Although ovarian cancer is common, its prognosis remains poor because of drug resistance and early metastasis. Polysaccharides extracted from Bangia fuscopurpurea (BFP) are potential anti-cancer agents, but the mechanisms underlying their effects in human ovarian cancer remain unclear. Here, we investigated the mechanisms of action of BFP polysaccharides in A2780 ovarian cancer cells using cell migration, invasion, apoptosis, and autophagy assays. Transwell assays indicated that BFP inhibited cell migration and invasion. Flow cytometry analysis showed that BFP treatment induced apoptosis and reactive oxygen species production, while significantly reducing mitochondrial membrane potential. Reverse transcription-polymerase chain reaction and Western blot analyses revealed changes in the expression of apoptosis- and autophagy-related cellular mRNAs and proteins, respectively, following BFP treatment for 24 h. Transmission electron microscopy revealed that BFP induced autophagy in A2780 cells. These findings demonstrate that BFP may be useful for developing functional foods for cancer therapy.

Keywords: Bangia fuscopurpurea; apoptosis; autophagy; ovarian cancer; polysaccharide.

FIGURE 1

Bangia fuscopurpurea (BFP) treatment abrogated migration and invasion capacity of A2780 cells (200×). (a) A2780 cells were incubated with 0.01 µg/mL taxol (positive control, PC), with different concentrations of BFP (1, 3, and 9 µg/mL), or with the Dulbecco's modified Eagle medium (DMEM; negative control, NC) for 24 h. Next, cell migration and invasion were detected in a Transwell assay. Quantification of migrated (b) and invaded (c) cells. *p < .05 and **p < .01 versus. NC group (mean ± SD, n = 3)

FIGURE 2

Effects of BFP treatment on cell cycle progression in A2780 cells determined using flow cytometry. A2780 cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control (NC) group was established. (a) Representative graph of (propidium iodide) PI‐stained cells sorted using flow cytometry. (b) Quantification of the percentage of cells in various phases of the cell cycle using flow cytometry

FIGURE 3

BFP treatment induced apoptosis. A2780 cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC), BFP (1, 3, and 9 µg/mL), NAC (100 µM), NAC (100 µM) + BFP (1, 3, and 9 µg/mL), and a negative control (NC) group was established. (a) Cell apoptosis was evaluated using flow cytometry after staining with Annexin V‐FITC/PI (V‐fluorescein isothiocyanate). Cells in quadrants Q1‐1, Q1‐2, Q1‐3, and Q1‐4 represent necrotic, late apoptotic, viable (live), and early apoptotic populations, respectively. (b) Quantification of apoptotic cells. **p < .01 versus. NC group (mean ± SD, n = 3), ^## p < .01 versus. BFP (1 µg/mL) group (mean ± SD, n = 3), ^&& p < .01 versus. BFP (3 µg/mL) group (mean ± SD, n = 3), ^$$ p < .01 versus. BFP (9 µg/mL) group (mean ± SD, n = 3)

FIGURE 4

Effect of BFP treatment on the mitochondrial membrane potential (MMP) of A2780 cells. Cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control group (NC) was established. (a) MMP was evaluated by flow cytometry after staining the cells with DiO. (b) Quantification of MMP. *p < .05 and **p < .01 versus. NC group (mean ± SD, n = 3)

FIGURE 5

Effect of BFP treatment on reactive oxygen species (ROS) generation in A2780 cells. Cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control group (NC) was established. (a) Reactive oxygen species (ROS) production was measured using flow cytometry after staining the cells with 2′,3′‐dichlorodihydrofluorescein diacetate (DCFH‐DA). (b) Quantification of ROS production. *p < .05 and **p < .01 versus. NC group (mean ± SD, n = 3)

FIGURE 6

Effect of BFP treatment on the expression of cleaved caspase‐3 (C‐caspase‐3), cleaved caspase‐9 (C‐caspase‐9), caspase‐3, caspase‐9, BAX, and BCL2 in A2780 cells. A2780 cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control (NC) group was established. (a–f) Quantitative analysis of C‐caspase‐3, C‐caspase‐9, caspase‐3, caspase‐9, BAX, and BCL2 proteins. (g) Expression of C‐caspase‐3, C‐caspase‐9, caspase‐3, caspase‐9, BAX, and BCL2 proteins was confirmed using Western blotting, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was used as a loading control. *p < .05 and **p < .01 versus. NC group (mean ± SD, n = 3)

FIGURE 7

Effect of BFP treatment on the protein expression of LC3, Beclin‐1, and P62 in A2780 cells. The cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control (NC) group was established. (a–c) Quantitative analysis of LC3, Beclin‐1, and P62 proteins. (d) Expression of LC3, Beclin‐1, and P62 proteins was confirmed using Western blotting, and GAPDH was used as a loading control. *p < .05 and **p < .01 versus. NC group (mean ± SD, n = 3)

FIGURE 8

Transmission electron microscopy (TEM) analysis of A2780 cells. Cells were subjected to treatment with 0.01 µg/mL taxol (positive control, PC) and BFP (1, 3, and 9 µg/mL), and a negative control (NC) group was established. Mitochondrion, red arrow; lysosome, blue arrow; autophagosome, yellow arrow