Pharmacological and In Silico Analysis of Oat Avenanthramides as EGFR Inhibitors: Effects on EGF-Induced Lung Cancer Cell Growth and Migration

Abstract

Avena sativa L. is a wholegrain cereal and an important edible crop. Oats possesses high nutritional and health promoting values and contains high levels of bioactive compounds, including a group of phenolic amides, named avenanthramides (Avns), exerting antioxidant, anti-inflammatory, and anticancer activities. Epidermal growth factor receptor (EGFR) represents one of the most known oncogenes and it is frequently up-regulated or mutated in human cancers. The oncogenic effects of EGFR include enhanced cell growth, angiogenesis, and metastasis, and down-regulation or inhibition of EGFR signaling has therapeutic benefit. Front-line EGFR tyrosine kinase inhibitor therapy is the standard therapy for patients with EGFR-mutated lung cancer. However, the clinical effects of EGFR inhibition may be lost after a few months of treatment due to the onset of resistance. Here, we showed the anticancer activity of Avns, focusing on EGFR activation and signaling pathway. Lung cancer cellular models have been used to evaluate the activity of Avns on tumor growth, migration, EMT, and anoikis induced by EGF. In addition, docking and molecular dynamics simulations showed that the Avns bind with high affinity to a region in the vicinity of αC-helix and the DGF motif of EGFR, jeopardizing the target biological function. Altogether, our results reveal a new pharmacological activity of Avns as EGFR tyrosine kinase inhibitors.

Keywords: EGFR; avenanthramide C; avenanthramides; classical molecular dynamics simulation; docking simulation; lung cancer; steered molecular dynamics simulation.

Figure 1

Effect of natural Avns on lung cancer cell vitality. A549 (A) and H1299 (B) vitality was evaluated by the MTT assay. Cells were exposed to EGF in presence of increasing concentrations of Avns (10, 50, and 100 µM) for three days. Data are expressed as % over basal control and are representative of three independent experiments run in triplicate. Statistical analysis: +++ p < 0.001 and ++ p < 0.01 vs. CTRL; *** p < 0.001; ** p < 0.01 and * p < 0.05 vs. EGF.

Figure 2

Natural Avns reduce clonogenicity of lung cancer cells promoted by EGF. Percentage of colonies of A 549 (A) and H1299 (B) cells in response to EGF in presence or absence of Avns. Data are expressed as % over basal control and are representative of three independent experiments run in triplicate. Statistical analysis: +++ p < 0.001 and + p < 0.05 vs. CTRL; *** p< 0.001; ** p< 0.01 and * p < 0.05 vs. EGF.

Figure 3

Effect of Avns on anoikis after 24 (A) and 48 (B) h. Cell vitality of A549 in suspension treated with Avns (100 µM) in 0.1% of serum. Results are expressed as fold of increase of dead cells and are representative of three independent experiments run in triplicate. Statistical analysis: +++ p < 0.001 and ++ p < 0.01 vs. CTRL; *** p < 0.001 and ** p < 0.05 vs. EGF.

Figure 4

Avns reduce migration of tumor cells promoted by EGF. A549 migration was evaluated by scratch assay. Data are reported as % of opened area over basal control and are representative of three independent experiments run in triplicate. Statistical analysis: + p < 0.05 vs. CTRL and * p < 0.05 vs. EGF.

Figure 5

Avns regulate E-cadherin and vimentin expression. Representative images and quantification of western blot analysis of A549 cells exposed to Avns (10 and 100 µM, 48 h) (A.D.U: arbitrary densitometry units). Data are expressed as fold increase compared with control and are representative of three independent experiments. Statistical analysis: +++ p < 0.001; ++ p < 0.01 and + p < 0.05 vs. CTRL; *** p < 0.001; ** p < 0.01 and * p < 0.05 vs. EGF.

Figure 6

Regulation of inflammation markers by Avns. Representative images and quantification of western blot analysis of COX-2 expression in A549 cells treated with Avns (10 and 100 µM, 48 h). (A.D.U: arbitrary densitometry units). Data are expressed as fold increase compared to control and are representative of three independent experiments. Statistical analysis: +++ p < 0.001 and + p < 0.05 vs. CTRL; *** p < 0.001 and ** p < 0.01 vs. EGF.

Figure 7

Induction of phosphorylated proteins by Avns. Representative images and quantification of western blot analysis of Akt and ERK 1/2 phosphorylation in A549 cells pre-treated with Avns (10 and 100 µM, 24 h) and 15 min with EGF (25 ng/mL). (A.D.U: arbitrary densitometry units). Data are expressed as fold increase compared to control and are representative of four independent experiments. Statistical analysis: +++ p < 0.001 and ++ p < 0.01 vs. CTRL; *** p < 0.001 and ** p < 0.01 vs. EGF.

Figure 8

Regulation of EGFR phosphorylation by Avns. Representative images and quantification of western blot analysis of p-EGFR in A549 cells treated with Avns (10 and 100 µM, 48 h). (A.D.U: arbitrary densitometry units). Data are expressed as fold increase compared to control and are representative of three independent experiments. Statistical analysis: + p < 0.05 vs. CTRL; ** p < 0.01 and * p < 0.05 vs. EGF.

Figure 9

Effects of Avns on A431 cells vitality and EGFR phosphorylation. (A) MTT assay. Cells were exposed to EGF in the presence of increasing concentrations of Avns (10, 50, and 100 µM) for three days. Data are expressed as % over basal control and are representative of three independent experiments run in triplicate. Statistical analysis: ++ p < 0.01 and + p < 0.01 vs. CTRL; ** p < 0.01 and * p < 0.05 vs. EGF. (B) EGFR phosphorylation. Representative images and quantification of western blot analysis in A431 cells treated with EGF in presence or absence of Avns (10 and 100 µM). (A.D.U: arbitrary densitometry units). Data are expressed as fold increase compared to control and are representative of three independent experiments. Statistical analysis: +++ p < 0.01; ++ p < 0.1 and + p < 0.5 vs. CTRL; *** p < 0.001; ** p < 0.01 and * p < 0.05 vs. EGF. (C) Representative images and quantification of western blot analysis in A431 cells treated with Avns. Data are representative of three independent experiments.

Figure 10

Overview of EGFR docked with AnvA and AvnC. The EGFR 3D structure is depicted in green surface/cartoon. The αC helix region and DFG motif are represented in red and blue surface/cartoon, respectively. AvnA and AvnC were reported as cyan and purple sticks/balls, respectively. The enlargement shows the interaction network of (A) AvnA and (B) AvnC in complex with the binding residues of EGFR after the docking simulation. The residues involved in hydrogen bonds (blue continue line), salt bridge (red dotted line), and hydrophobic interactions (grey dotted line) are indicated as green, orange, and violet sticks/balls, respectively. The yellow sphere, represent the charge centre of atoms forming the salt bridge. To clarify, the hydrogens were hidden.

Figure 11

MD interaction network analysis. The binding residues and the molecular dynamics run time (10,000 frames of 100 ns of MD run time) are reported on the x and y axis, respectively. The hydrophobic, π-stacking, cationic, π-cationic interactions, and the hydrogen bonds (HB-Donor and HB-Acceptor) are reported as blue, red, brown, purple, orange, and green lines, respectively for (A) AvnA, (B) AvnC, and (C) gefitinib.

Figure 12

Steered molecular dynamics simulations. Force profiles of compounds pulled out of the EGFR binding pocket along the unbinding pathway, AvnA (cyan line), AvnC (purple line), and gefitinib (grey line).