Objective To investigate the effect of the antibody neutralization of astacin-like metalloendopeptidase (ASTL) on the radiosensitivity of human cervical carcinoma ME-180 cells.

Methods ME-180 cells and HeLa cells were grouped as follows in accordance with different experimental treatments: (1) ME-180 cells were divided into the control group, irradiation (4 Gy) group, ASTL antibody group, and ASTL antibody + irradiation (4 Gy) group. The number and proliferative capacity of viable cells were detected via 5-ethynyl-2'deoxyuridine nucleoside (EdU) staining and 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Immunofluorescence and Western blot analyses were used to detect the localization of ASTL in the cells at 0, 24, 48, and 72 h after irradiation. (2) ME-180 cells were divided into two groups: the irradiation group and the ASTL antibody + irradiation group irradiated with 0, 2, 4, and 8 Gy γ-rays. The proliferative capacity of the cells was detected through the clone formation assay. (3) HeLa cells were divided into two groups: the irradiation group and the ASTL antibody + irradiation group irradiated with 0, 2, 4, and 8 Gy γ-rays. MTT assay was used to detect the proliferative capacity of the cells. (4) ME-180 cells were divided into two groups: the sh-control group and the sh-ASTL transfection group. Protein kinase B expression levels were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses; at the same time, ME-180 cells were treated with 0, 5, 10 nmoL/L antibodies respectively, and the effect of antibody neutralization on target gene expression was detected by western blot test. (5) ME-180 cells were divided into two groups: the control group and the irradiation (4 Gy) group. Western blot analysis was used to detect ASTL and AKT expression.For the relative experiments, the cells were treated with ¹³⁷Cs γ-ray irradiation at the dose rate of 1 Gy/min. Independent samples t-test was used for comparison between two groups.

Results (1) The results of the EdU staining experiments showed that neutralizing ASTL significantly inhibited the proliferation of ME-180 cells after irradiation (t=9.25, P<0.05). The results of the MTT assay validated that the proliferative capacity of the ASTL antibody + irradiation group (4 Gy) was significantly (t=5.17, 10.32, 14.27; all P<0.05) reduced compared with that of the irradiation group after 24, 48, and 72 h of irradiation. The results of immunofluorescence and Western blot analyses revealed that the cytoplasmic localization of ASTL in ME-180 cells was significantly enhanced after 24 h of 4 Gy irradiation, and ASTL was relocalized on the cell membrane after 48–72 h of irradiation. (2) The results of the clone formation assay demonstrated that ASTL antibodies could significantly reduce the survival rate of ME-180 cells after 8 Gy irradiation (t=7.63, P<0.05). (3) The results of the MTT assay showed that ASTL antibodies could significantly reduce the survival rates of HeLa cells after 2, 4, and 8 Gy irradiation (t=4.27, 9.66, 15.71; all P<0.05). (4) qRT-PCR analysis revealed that the expression level of AKT was significantly (t=13.94, P<0.05) down-regulated after ASTL interference, whereas the results of Western blot analysis showed that ASTL interference or ASTL antibody addition could reduce the protein expression levels of AKT and its target genes. (5) The results of Western blot analysis demonstrated that in ME-180 cells, the levels of the ASTL protein and AKT and its target genes significantly increased after 4 Gy irradiation.

Conclusion The radiosensitivity of human cervical cancer ME-180 cells was correlated with the level of ASTL, and ASTL antibody or sh-ASTL could enhance the radiosensitivity of ME-180 cells after irradiation.