c-MET inhibition enhances the response of the colorectal cancer cells to irradiation in vitro and in vivo

Abstract

The aim of the present study was to investigate the effect of hepatocyte growth factor receptor (c-MET) inhibition on the viability of colon cancer cells and xenografts exposed to irradiation using short hairpin (sh)RNA or the c-MET inhibitor PHA665752. The underlying mechanisms were also investigated. Human colorectal adenocarcinoma HT-29 cells were infected with a lentivirus expressing shRNAs against c-MET and were irradiated at 0, 2, 4, 6 and 8 Gy. The viability of the cells was assessed by alamarBlue® assays. Mice bearing human colon carcinoma SW620 xenografts were randomly selected to receive 2.5% dimethyl sulfoxide (DMSO), 25 mg/kg PHA665752 intraperitoneally once every 2 days for 3 weeks, irradiation at 10 Gy, or 25 mg/kg PHA665752 intraperitoneally once every 2 days for 3 weeks followed 24 h later by irradiation at 10 Gy. The mean tumor volume (MTV) was measured. The apoptotic rate of cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays, and double stranded break marker antibody γ-H2AX and hypoxia inducible factor (HIF)-1α expression was examined by immunohistochemistry. alamarBlue assays revealed that c-MET downregulation by shRNA markedly accentuated the irradiation-induced reduction in the viability of HT-29 cells compared with HT-29 cells irradiated at the same doses (P<0.05). A combination of irradiation and PHA665752 caused an additional reduction in the MTV (382.8±42.4 mm3; P<0.01 vs. irradiation and PHA665752, 998.0±180.6 and 844.8±190.0 mm3, respectively). TUNEL assays revealed that irradiation and PHA665752 alone caused significant apoptosis of the SW620 cells in the tumor xenografts (P<0.01 vs. DMSO). The apoptotic index in the tumor xenografts of mice treated with a combination of irradiation and PHA665752 was significantly increased compared with mice treated with either agent alone (P<0.01). The combination of irradiation and PHA665752 was also associated with a marked increase in γ-H2AX levels and a significant decrease in HIF-1α expression in the xenografts (P<0.01). In conclusion, c-MET inhibition sensitizes colorectal cancer cells to irradiation by enhancing the formation of DNA double strand breaks and possibly alleviating tumor hypoxia.

Keywords: c-MET inhibitor; colorectal cancer; radiation; sensitivity; small interfering RNA.

Figure 1.

Human colorectal adenocarcinoma HT-29 cells were transfected with the lentiviral vectors pSD400-c-MET-shRNA or pSD400-scr-shRNA inducibly expressing shRNA against c-MET or scr, respectively. Transfected cells were treated with DOX at the indicated doses for induction of shRNA expression. (A) Immunoblotting assays were performed to examine c-MET expression. The results are representative of at least 3 independent experiments. (B) Transfected cells were irradiated at the indicated doses and cell viability was examined by Alamar Blue assays. The data are expressed as the mean ± standard deviation of ≥3 independent experiments. c-MET, hepatocyte growth factor receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DOX, doxycycline; shRNA, short hairpin RNA; scr, scrambled shRNA.

Figure 2.

(A) Mice bearing human colon carcinoma SW620 xenografts were treated with 2.5% DMSO, PHA665752, irradiation or the combination of irradiation and PHA665752. Tumor growth was monitored by measuring the mean tumor volume. P<0.01, PHA665752, irradiation or a combination of irradiation and PHA665752 vs. DMSO; P<0.01, the combination of irradiation and PHA665752 vs. irradiation or PHA665752. (B-E) Mice were treated as in (A) and the apoptotic rate of the xenograft tissue was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay: (B) The DMSO group; (C) the irradiation group; (D) the PHA665752 group; and (E) the combination of irradiation and PHA665752 group. (F) The percentage of apoptotic cells in the tumor xenografts. *P<0.01, the combination of irradiation and PHA665752 vs. DMSO, irradiation or PHA665752. DMSO; dimethyl sulfoxide; PHA665752, hepatocyte growth factor receptor inhibitor; PHA, PHA665752; Rad, irradiation.

Figure 3.

Mice bearing human colon carcinoma SW620 xenografts were treated with 2.5% DMSO, PHA665752, irradiation or the combination of irradiation and PHA665752. γ-H2AX expression in the tumor xenograft was examined using immunohistochemistry in the (A) DMSO, (B) irradiation, (C) PHA665752, and (D) combination of irradiation and PHA665752 groups. (E) Quantification of γ-H2AX expression. *P<0.01, combination of irradiation and PHA665752 vs. DMSO, irradiation or PHA665752. γ-H2AX, a double stranded break marker; DMSO; dimethyl sulfoxide; PHA665752, hepatocyte growth factor receptor inhibitor; Rad, irradiation; PHA, PHA665752.

Figure 4.

Mice bearing human colon carcinoma SW620 xenografts were treated with 2.5% DMSO, PHA665752, irradiation or the combination of irradiation and PHA665752. HIF-1α expression in the tumor xenograft was examined using immunohistochemistry: (A) The DMSO group; (B) the irradiation group; (C) the PHA665752 group; and (D) the combination of irradiation and PHA665752 group. (E) Quantification of HIF-1α expression. *P<0.01, combination of irradiation and PHA665752 vs. DMSO, irradiation or PHA665752. HIF-1α, hypoxia inducible factor; DMSO; dimethyl sulfoxide; PHA665752, hepatocyte growth factor receptor inhibitor; Rad, irradiation; PHA, PHA665752.