Downregulation of cytochrome c oxidase 1 induced radioresistance in esophageal squamous cell carcinoma

Abstract

Comprehensive gene screening with transposons is a novel procedure for the systematic identification of resistant genes. The present study aimed to use this technique to identify candidate radioresistant genes in esophageal squamous cell carcinoma. A transposon is a base sequence that can translocate to another location in the genome at random. By inserting the cytomegalovirus promotor as a transcriptional activator in the transposon, the following gene in the new location becomes overexpressed and the gene located at the transposon insertion site is downregulated. Consequently, various transposon-tagged cells, which have differentially overexpressed or downregulated genes using the transposon method can be obtained. Following the irradiation of transposon-tagged cells, candidate radioresistant genes can be selected in order to detect the location of the transposon in the cells that have survived. A total of 11 genes were detected as candidate radioresistant genes. Cytochrome c oxidase 1 (MT-CO1), an enzyme involved in apoptosis through the activation of the caspase cascade, was one of the candidate genes identified. The relative expression level of MT-CO1 was 0.12 in MT-CO1-downregulated cells which was significantly lower compared with the expression level in parent TE4 cells (P<0.001). The survival rate was 28.7% in MT-CO1-downregulated cells and 10.5% in parent TE4 cells 9 days following 5-Gy irradiation. The activity of cytochrome c and caspase-3 following irradiation was significantly lower in the MT-CO1-downregulated radioresistant cells compared with in TE4 cells. In conclusion, the novel gene screening technique demonstrated to be useful for detecting candidate radioresistant genes in esophageal squamous cell carcinoma. The results of the present study revealed that the downregulation of MT-CO1 induced radioresistance occurs by inhibiting the activation of the caspase cascade in radioresistant esophageal cancer cells.

Keywords: cytochrome c oxidase 1; esophageal cancer; gene screening; radioresistance; transposon.

Figure 1.

Downregulation of MT-CO1. (A) The detected base sequence was matched with a part of the MT-CO1 gene sequence. If a transposon is inserted into the MT-CO1 gene, MT-CO1 may be downregulated and the following gene (MT-CO2 or MT-CO3) may be overexpressed. (B) The expression level of MT-CO1 was 3.4 in the RRCs, in which the transposon had transported into the MT-CO1 gene, and 28.0 in the parent TE4 cells. There was a significant difference between the two groups (P<0.001). ND2, MT-CO2, and MT-CO3 were not overexpressed in the RRCs. (Light bars, parent cells; dark bars, RRCs). Error bars represent the standard deviation of the mean. MT-CO1, cytochrome c oxidase 1; RRCs, radioresistant cells.

Figure 2.

Radioresistance in the cells with downregulation of MT-CO1. Cell survival rates were measured at 1, 3, 5, 7, and 9 days after 7-Gy irradiation. The survival rates of MT-CO1-downregulated cells were significantly higher in days 5, 7 and 9 (P<0.001). Error bars represent the standard deviation of the mean. MT-CO1, cytochrome c oxidase 1; RRCs, radioresistant cells.

Figure 3.

Cytochrome c oxidase assay. The activity of cytochrome c oxidase was measured as the reduction in absorbance at 550 nm, a characteristic wavelength for ferrocytochrome c. Cytochrome c oxidase activity was significantly reduced because of MT-CO1 downregulation in the RRCs (*P=0.023). Although cytochrome c oxidase activity was increased in both the parent and RRCs after 5-Gy irradiation, it was significantly lower in the RRCs (^#P=0.005). Error bars represent the standard deviation of the mean. MT-CO1, cytochrome c oxidase 1; RRCs, radioresistant cells.

Figure 4.

Caspase-3 activity after irradiation. After 5-Gy irradiation, the parent (TE4) cells and RRCs (MT-CO1-downregulated cells) were incubated with colorimetric substrate DEVD-pNA for 24 h. Caspase-3 activity was measured as the absorbance at 405 nm and was found to be higher in parent cells after irradiation (*P<0.001). Error bars represent the standard deviation of the mean. MT-CO1, cytochrome c oxidase 1; RRCs, radioresistant cells.