Pristimerin attenuates cell proliferation of uveal melanoma cells by inhibiting insulin-like growth factor-1 receptor and its downstream pathways

Abstract

Uveal melanoma (UM) has a high mortality rate due to liver metastasis. The insulin-like growth factor-1 receptor (IGF-1R) is highly expressed in UM and has been shown to be associated with hepatic metastases. Targeting IGF signalling may be considered as a promising approach to inhibit the process of metastatic UM cells. Pristimerin (PRI) has been demonstrated to inhibit the growth of several cancer cells, but its role and underlying mechanisms in the IGF-1-induced UM cell proliferation are largely unknown. The present study examined the anti-proliferative effect of PRI on UM cells and its possible role in IGF-1R signalling transduction. MTT and clonogenic assays were used to determine the role of PRI in the proliferation of UM cells. Flow cytometry was performed to detect the effect of PRI on the cell cycle distribution of UM cells. Western blotting was carried out to assess the effects of PRI and IGF-1 on the IGF-1R phosphorylation and its downstream targets. The results indicated that IGF-1 promoted the UM cell proliferation and improved the level of IGF-1R phosphorylation, whereas PRI attenuated the effect of IGF-1. Interestingly, PRI could not only induce the G1 phase accumulation and reduce the G2 phase induced by IGF-1, but also could stimulate the expression of p21 and inhibit the expression of cyclin D1. Besides, PRI could attenuate the phosphorylations of Akt, mTOR and ERK1/2 induced by IGF-1. Furthermore, the molecular docking study also demonstrated that PRI had potential inhibitory effects on IGF-1R. Taken together, these results indicated that PRI could inhibit the proliferation of UM cells through down-regulation of phosphorylated IGF-1R and its downstream signalling.

Keywords: Akt; ERK1/2; IGF-1; mTOR; pristimerin; uveal melanoma.

Figure 1

Effects of PRI on proliferation and colony formation of UM cells. A, Chemical structure of PRI. B, UM cells were treated with indicated concentrations of PRI (0‐10 μmol/L) for 24 h, and cell viability was assessed by MTT assay. C, UM cells were treated with various concentrations of IGF‐1 (3‐300 ng/mL) for 24 h, and the cell viability was measured by MTT assay. D, Cells were pre‐treated with various concentrations (0‐3 μmol/L) of PRI for 2 h and then incubated with IGF‐1 for a further 24 h. Cell viability was determined by MTT assay. E, UM cells were seeded in 6‐well plates for 7 days after the treatment of PRI and IGF‐1, fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. F, The statistic results of each colony formation assay. All data are represented as mean ± SD from triplicate wells. *P < .05, **P < .01 vs control, ^## P < .01 vs IGF‐1‐treated alone group

Figure 2

PRI affected the cell cycle progression and the downstream target genes in UM cells. A, UM cells were treated with PRI (1 μmol/L) for 40 min and then were treated with or without IGF‐1 in a serum‐free medium for 40 min. Cells were stained with propidium iodide (PI) and analysed using a flow cytometer. The cell cycle distribution (%) was calculated using FlowJo 7.6. B, Pre‐treatment of UM cells with indicated concentration of PRI for 40 min was followed by treatment with or without 100 ng/mL IGF‐1 for 40 min. The expressions of cyclin D1 and p21 were determined by western blotting. C, Quantification of the immunoblot was performed using densitometric analysis. The results represent prototypical examples of experiments replicated at least three times. *P < .05 vs control groups, ^# P < .01 vs IGF‐1‐treated alone group

Figure 3

Time‐ and dose‐dependent IGF‐1 activated IGF‐1R. A, UM cells were treated with 100 ng/mL IGF‐1 for scheduled time or C, cells were treated with scheduled concentrations of IGF‐1 for 40 min and the phosphorylation of IGF‐1R was determined by western blotting. B and D, show the densitometric analysis of the immunoblot was expressed as the fold of control. *P < .05, **P < .01 vs control groups. The results represent prototypical examples of experiments replicated at least three times

Figure 4

PRI attenuated IGF‐1R activation induced by IGF‐1 in UM cells. A, UM cells were treated with various concentrations of PRI and 100 ng/mL IGF‐1. The levels of p‐IGF‐1R were determined by western blotting. C, UM cells were treated with 1 μmol/L PRI and 100 ng/mL IGF‐1 at various time points. The levels of p‐IGF‐1R were determined by western blotting. B and D, show the densitometric analysis and the p‐IGF‐1R/IGF‐1R ratio was determined. *P < .05 vs control group; ^# P < .05 vs IGF‐1‐treated alone group. The results represent prototypical examples of experiments replicated at least three times

Figure 5

Time‐ and dose‐dependent IGF‐1 increased the phosphorylation levels of mTOR, Akt and ERK1/2 in UM cells. UM cells were treated with IGF‐1 at 100 ng/mL for A, various time points (10‐80 min) and C, various concentrations (0‐100 ng/mL) for 40 min. The phosphorylation of mTOR, Akt and ERK1/2 in UM cells was analysed by western blotting. B and D, Densitometric analysis of the immunoblot was expressed as the fold of control. *P < .05 vs control groups. Results represent prototypical examples of experiments replicated at least three times

Figure 6

PRI attenuated the phosphorylation levels of mTOR, Akt and ERK1/2 in UM cells in a time‐ and dose‐dependent manner. A, Cells were pre‐treated with PRI at 1 μmol/L for a scheduled time and then incubated with 100 ng/mL IGF‐1 for 40 min. The phosphorylation of mTOR, Akt and ERK1/2 were determined by western blotting. B, Cells were pre‐treated with scheduled concentrations (0‐3 μmol/L) of PRI for 40 min and then incubated with IGF‐1 for 40 min. The phosphorylation of mTOR, Akt and ERK1/2 were determined by western blotting. C and D, Densitometric analysis of the immunoblot was expressed as the fold of control. *P < .05 vs control groups, ^# P < .05 vs IGF‐1 treated group. Results represent prototypical examples of experiments replicated at least three times

Figure 7

PRI inhibited IGF‐1R kinase. A, The two‐dimensional diagrams displayed the docking model of PRI in active sites of IGF‐1R kinase. D, The three‐dimensional diagram showed the binding conformation of PRI with IGF‐1R kinase. Atom colours: green‐carbon atoms of PRI, grey‐carbon atoms of residues of IGF‐1R kinase, dark blue‐nitrogen atoms and red‐oxygen atoms