In Vitro Chemopreventive Potential of Phlorotannins-Rich Extract from Brown Algae by Inhibition of Benzo[a]pyrene-Induced P2X7 Activation and Toxic Effects

Abstract

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.

Keywords: A549 cells; P2X7 receptor; cancer; cytoskeleton; phlorotannins; pollution.

Figure 1

Effects of the brown algae extract on the P450 1-dependent monooxygenases of A549 cells incubated with different concentrations of B[a]P (ethoxyresorufin-O-deethylase (EROD) assay). Cells were incubated with either phosphate buffered saline PBS (control, black bars) or the brown algae extract at 0.1% (grey bars) for 20 min. The solutions were then removed, and the cells were incubated with B[a]P for 48 h (A) or 72 h (B). Data correspond to the mean ± SEM of four independent experiments. The significance thresholds were ** p < 0.01, **** p < 0.0001 and $ p < 0.05.

Figure 2

Effects of the brown algae extract on P2X7 receptor activation of A549 cells incubated with different concentrations of B[a]P (YO-PRO-1™ assay). Cells were incubated with either PBS (control, black bars) or the brown algae extract at 0.1% (light grey bars) or Brilliant Blue G (BBG) at 25 µM (dark grey bars) for 20 min. The solutions were then removed, and the cells were incubated with B[a]P for 48 h (A) or 72 h (B). Data correspond to the mean ± SEM of four independent experiments. The significance thresholds were * p < 0.05, ** p < 0.01, **** p < 0.0001, and $ p < 0.0001.

Figure 3

Fluorescence microscopic changes in F-actin cytoskeleton organization in A549 cell line. After B[a]P incubation, the cells were stained using ActinGreen 488 ReadyProbes^®. The data shown are representative of three independent experiments. The images were captured under the same acquisition parameters by EVOS FL fluorescence microscope (Thermo Fisher Scientific). Upper panels: cells incubated in the absence of the brown algae extract, lower panels: cells incubated in the presence of the brown algae extract.

Figure 4

Immunofluorescence analysis of E-cadherin expression in A549 cells. Immunostaining was detected using antibody specific for E-cadherin and Alexa Fluor 488-labeled secondary antibody. The data shown are representative of three independent experiments. The images were captured under the same acquisition parameters by EVOS FL fluorescence microscope (Thermo Fisher Scientific). Upper panels: cells incubated in the absence of the brown algae extract, lower panels: cells incubated in the presence of the brown algae extract.

Figure 5

Effects of the brown algae extract on. reactive oxygen species (ROS) production by A549 cells incubated with different concentrations of B[a]P (H2DCF-DA assay). Cells were incubated with either PBS (control, black bars) or the brown algae extract at 0.1% (grey bars) for 20 min. The solutions were then removed, and the cells were incubated with B[a]P for 48 h. Data correspond to the mean ± SEM of four independent experiments. The significance thresholds were ** p < 0.01 and $$$$ p < 0.0001, $$ p < 0.01.