Foretinib Enhances the Radiosensitivity in Esophageal Squamous Cell Carcinoma by Inhibiting Phosphorylation of c-Met

Abstract

As a crucial event involved in the metastasis and relapse of esophageal cancer, c-Met overexpression has been considered as one of the culprits responsible for the failure in patients who received radiochemotherapy. Since c-Met has been confirmed to be pivotal for cell survival, proliferation and migration, little is known about its impact on the regulation of radiosensitivity in esophageal cancer. The present study investigated the radiosensitization effects of c-Met inhibitor foretinib in ECA-109 and TE-13 cell lines. Foretinib inhibited c-Met signaling in a dose-dependent manner resulting in decreases in the cell viability of ECA-109 and TE-13. Pretreatment with foretinib synergistically prompted cell apoptosis and G2/M arrest induced by irradiation. Moreover, decreases ability of DNA damage repair was also observed. In vivo studies confirmed that the combinatorial use of foretinib with irradiation significantly diminishes tumor burden compared to either treatment alone. The present findings implied a crucial role of c-Met in the modulation of radiosensitization in esophageal cancer, and foretinib increased the radiosensitivity in ECA-109 and TE-13 cells mainly via c-Met signaling, highlighting a novel profile of foretinib as a potential radiosensitizer for the treatment of esophageal cancer.

Keywords: Esophageal cancer; Foretinib; Radiosensitivity; c-Met.

Fig 1

The effects of c-Met inhibitor foretinib on cell viability, survival, phosphorylation of c-Met and downstream PI3K/Akt pathway in ESCC cells. (A) Foretinib inhibited cell proliferation in a time and dose-dependent manner in ESCC cells. (B) The clonogenic survival of ESCC cells was suppressed significantly by foretinib at 0, 2, 4, 6, 8 Gy. (C) Western blotting results demonstrated that foretinib blocked the phosphorylation of c-Met and Akt obviously.

Fig 2

The effect of c-Met inhibitor foretinib on cell apoptosis in ESCC cells. (A) Flow cytometry was used to detect the apoptosis in ESCC cells treated with foretinib or irradiation. (B) Combination treatment increased the rate of apoptosis versus the use of foretinib or irradiation alone (*, P<0.05; **, P<0.01). (C) Western blotting results showed that foretinib resulted in improvement of Bax and degradation of Bcl-2 and Bcl-xl combined with irradiation.

Fig 3

The effect of c-Met inhibitor foretinib on cell cycle in ESCC cells. (A) The cell cycle distribution of ESCC cells was examined by flow cytometry. (B) Foretinib increased the percentage of G2/M phase with increasing concentrations and enhanced G2/M arrest induced by irradiation. (C) Foretinib increased the expression of phosphorylated Cdc2 and decreased the expression of Cyclin B1. Irradiation improved the expression of phosphorylated Cdc2 and Cyclin B1.

Fig 4

The effect of c-Met inhibitor foretinib on DNA damage repair in ESCC cells. (A) γH2AX foci was evaluated by immunofluorescence. (B) Foretinib treatment group significantly delayed the DNA damage repair compared with control group after irradiation at 2, 8, 24h (*, P<0.05).

Fig 5

Foretinib enhances the radiosensitization of ESCC in vivo. ECA-109 xenograft-bearing female nude mice were divided into four groups: control, IR, foretinib, and a combination treatment group. Seven days following administration of foretinib, the mice were irradiated with a single fraction of 6 Gy X-rays. (A) Representative images of ECA-109 xenograft-bearing nude mice. (B) Measurements of tumor size. Data represent mean and SD (**, P<0.01). (C) Relative tumor proliferation rate T/C (%). T/C (%) was calculated according to the formula: T_RTV/C_RTV×100% (*, P<0.05; **, P<0.01). (D) Measurements of tumor weight. Data represent mean and SD (*, P<0.05; **, P<0.01).

Fig 6

Characterizing the mechanisms of radiosensitization mediated by foretinib. Foretinib surpressed the proliferation and survival of ESCC through inhibiting the phosphorylation of c-Met. Foretinib enhanced the radioresponse via impairing the DNA repair ability of c-Met. Foretinib increased the radiosensitivity through inducing G2/M arrest and apoptosis postirradiation.