Effects of Flavonoids from <i>Potamogeton crispus</i> L. on Proliferation, Migration, and Invasion of Human Ovarian Cancer Cells

<div><p>In order to explore the efficient utilization of plant resources from constructed wetlands, the potential anti-metastatic effects of flavonoids from <i>Potamogeton crispus </i>L. were investigated in human ovarian cancer cells (ES-2). Two major flavonoids, luteolin-3ʹ-O-β-D-glucopyranoside and flavone-6-C-β-D-glucopyranoside, were isolated from <i>P</i>. <i>crispus</i> and identified. The effects of these flavonoids on cell proliferation, cell morphology, cell cycle, apoptosis, and cell migration and invasion were then investigated. Furthermore, reverse transcriptase polymerase chain reaction assays and western blotting analysis were conducted to examine the expression level of mRNA and protein. Results indicated that Luteolin-3ʹ-O-β-D-glucopyranoside inhibited ES-2 cell migration and invasion and suppressed the expression of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, and Flavone-6-C-β-D-glucopyranoside had no significant inhibitory effects on ES-2 cells. Thus, this study demonstrated the potential anti-metastatic properties of a <i>P</i>. <i>crispus</i> flavonoid, and provided a scientific approach for the screening of promising natural resources from constructed wetlands to identify useful products for use in the pharmaceutical and healthcare industries.</p></div

Effects of <i>P</i>. <i>crispus</i> flavonoids on ES-2 cell migration.

<p>Cells were exposed to the indicated concentrations of luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP) and flavone-6-C-β-d-glucopyranoside (FL6C-GP), and their migration was quantified using a Transwell chamber. Values represent the means of triplicate experiments. *<i>p</i> < 0.01, as compared to the control; #<i>p</i> < 0.05, as compared with the indicated concentration.</p

Effects of <i>P</i>. <i>crispus</i> flavonoids on ES-2 cell invasion.

<p>Cells were treated with luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP) or flavone-6-C-β-d-glucopyranoside (FL6C-GP), and their invasion was quantified in a Transwell chamber membrane with Matrigel. Values represent the means of experiments performed in triplicate. *<i>p</i> < 0.01, as compared to the control; #<i>p</i> < 0.05, as compared with the indicated concentration.</p

Proliferation inhibition of two <i>P</i>. <i>crispus</i> flavonoids on ES-2 cells.

<p>Cells were incubated with five different flavonoid concentrations or dimethyl sulfoxide (DMSO; control) for 48 h. Data represent the means of triplicate experiments ± standard deviation (SD) * <i>p</i> < 0.01.</p

Quantification of apoptotic ES-2 cells after treatment with <i>P</i>. <i>crispus</i> flavonoids.

<p>(a) Negative control; (b) cells treated with 100 μg/mL flavone-6-C-β-d-glucopyranoside (FL6C-GP); (c) cells treated with 30 μg/mL luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP); (d) cells treated with 60 μg/mL LU3′O-GP. Apoptotic cells are shown in Q3.</p

Luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP) reduced protein expression of MMP-2 and MMP-9 of in ES-2 cells.

<p>(a) LU3′O-GP effects on MMP-2(pro-MMP-2 and activity MMP-2); (b) LU3′O-GP effects on MMP-9 expression. Protein levels were investigated by western boltting analysis. Values represent the means of triplicate experiments. *<i>p</i> < 0.05, as compared to the control.</p

Luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP) reduced mRNA expression of MMP-2 and MMP-9 in ES-2 cells.

<p>(a) LU3′O-GP effects on MMP-2 expression; (b) LU3′O-GP effects on MMP-9 expression. mRNA levels were investigated by reverse transcriptase polymerase chain reaction (RT-PCR), using glyceraldehyde 3-phosphate dehydrogenase as the loading control. RT-PCR products were detected by ethidium bromide staining. Values represent the means of triplicate experiments. *<i>p</i> < 0.05, as compared to the control.</p

Chemical structure of two <i>P</i>. <i>crispus</i> flavonoids

<p>Chemical structure of two <i>P</i>. <i>crispus</i> flavonoids</p

<i>P</i>. <i>crispus</i> flavonoid effects on cell cycle progression in ES-2 cells.

<p>(a) Negative control; (b) cells treated with 100 μg/mL flavone-6-C-β-d-glucopyranoside (FL6C-GP); (c) cells treated with 30 μg/mL luteolin-3′-O-β-d-glucopyranoside (LU3′O-GP); and (d) cells treated with 60 μg/mL LU3′O-GP. Cells were fixed with ethanol and stained with propidium iodide prior to cell cycle distribution analysis by flow cytometry. The number of cells in G0/G1 phase is represented by the first peak, and those in G2/M phase are represented by the second peak. Cells in S phase are present in the area between the G0/G1 and G2/M peaks. These data represent the mean of triplicate experiments. * <i>p <</i> 0.01 as compared with control.</p

Number of days of exposure of <i>Alternanthera philoxeroides</i> ramets under different fluctuation treatments (FF1, FF2, FF3 and FF4) with their respective cycles of fluctuations (0, 3, 6 and 12 cycles) over a 96-day experimental growth period, for any particular level of submergence.

<p>Number of days of exposure of <i>Alternanthera philoxeroides</i> ramets under different fluctuation treatments (FF1, FF2, FF3 and FF4) with their respective cycles of fluctuations (0, 3, 6 and 12 cycles) over a 96-day experimental growth period, for any particular level of submergence.</p