Identification of an inhibitory mechanism of luteolin on the insulin-like growth factor-1 ligand-receptor interaction

Abstract

Using single-molecule force measurement and fluorescence imaging, we have demonstrated that luteolin has an inhibitory effect on IGF-1 ligand-receptor binding, the initial step in IGF-1 signaling. This inhibition mechanism, which was confirmed by flow cytometry and molecular docking, could play a role in cancer therapy.

Figure 1

Measurements of IGF-1R monomers and dimers. (A) A typical single-molecule image of IGF-1R-GFP on the MCF7 cell membrane. The spots enclosed with green circles represented the signals from individual IGF-1R-GFP molecules. Scale bar: 4 μm. (B and C) Distributions of the fluorescence intensity of individual IGF-1R-GFP spots from the stimulated cells without (B) and with (C) 100 μM luteolin treatment. For IGF-1 stimulation experiment, the transfected cells were incubated with 50 ng/mL IGF-1 (R&D) in the serum-free DMEM for 15 min at 37°C. The solid curves show the fitting of the Gaussian function with the arrowheads indicating the peak positions. The two peaks represented monomers and dimers, respectively, and the numbers in the parentheses are the fractions. (D) Frequency of one- and two-step bleaching events under various conditions. Data is expressed as mean ±SD. *P<0.05.

Figure 2

Measurements of the binding between IGF-1 and IGF-1R in living cells. (A) Representative force curves obtained with IGF-1-modified AFM tips on MCF7 cells in the absence (1) or presence (2) of luteolin or after blocking the binding with extracellular domain of IGF-1R (IGF-1RECD) (3). (B and C) Histograms of binding forces of IGF-1/ IGF-1R with untreated cells (B) and the cells treated with 100 μM luteolin (C) (grey bars, experimental data; solid line, theoretical Gaussian distribution curves). (D) The binding probability of IGF-1 and IGF-1R when the cells are treated without or with 100 μM luteolin or the blocking reagent. Data is expressed as mean ±SD. *P<0.05. (E) Fluorescence intensity by flow cytometry using biotinylated IGF-1 and avidin-FITC. The cells were treated without or with luteolin (100 μM) or negative control reagent.

Figure 3

Predicted binding model of the extracellular domain of IGF-1R/IGF-1 complexed with luteolin (sticks) and four binding sites (PHE1, THR516, GLY520 and ASP533).