Inhibition Of GRP78 Abrogates Radioresistance In Oropharyngeal Carcinoma Cells After EGFR Inhibition By Cetuximab

Aims: The aim of the present study is to study the mechanism of resistance of oropharyngeal carcinoma cells to the combination of radiotherapy and EGFR-targeted therapy and explore ways to improve efficacy of the therapy for patients who are resistant to EGFR-targeted therapy combined with radiotherapy.Method:1.Western blot test to assess the level of protein expression of ERS signaling pathway,DNA double-strand breaks and apoptosis,such as GRP78,IRE1α,PERK,ATF6,LC3 B,Atg16L1,and cleaved-PARP etc.2.RT-PCR to assess radiation-induced expression of ERS chaperonin GRP78 m RNA transcription in oropharyngeal carcinoma cells.3.Colony formation assay to test survival of oropharyngeal carcinoma cells.4.CCK-8 test to assess the cells proliferation activity.Results:1.Cetuximab increased the radiosensitivity of Fadu cells,with a radiation sensitization ratio of 1.14,but showed no radiosensitization effects in Detroit562 cells.Even if the concentration of cetuximab was increased to 100μg / ml,there was no significant effect.Further,.2.Cetuximab inhibited the radiation-induced expression of GRP78,IRE1α,and ATF6 protein in Fadu cells but did not show this effect in Detroit562 cells.In contrast,cetuximab inhibited radiation-induced PERK protein expression in both cell lines.3.Silencing IRE1α and ATF6 inhibited GRP78 protein expression,while silencing PERK had no significant effect on GRP78 protein expression.4.Cetuximab inhibited radiation-induced expression of GRP78 m RNA in Fadu cells(P < 0.05)but did not have significant effects in Detroit562 cells.5.Radiation induced increased expression of GRP78 in oropharyngeal carcinoma cells in a time-dependent manner.6.Silencing GRP78 increased the radiosensitivity of Fadu and Detroit562 cells,and the radiosensitization ratios(SER)of Fadu and Detroit562 cells were 1.19 and 1.21.7.Silencing GRP78 inhibited the radiation-induced expression of the DNA double-strand break repair protein DNA-PK and autophagy marker LC3 B and the autophagy related protein Atg16L1.8.Ly294002 and 3-MA could increase the radiosensitivity of oropharyngeal carcinoma cells.The results showed that Ly294002 and 3-MA inhibited the radiation-induced expression of anti-apoptotic protein Bcl-2,while they increased the radiation-induced expression of the apoptosis marker protein cleaved PARP.The results of the MTT analysis also showed that Ly294002 and 3-MA further reduced the proliferation of cells inhibited by radiation alone.9.Silencing GRP78 combined with cetuximab treatment significantly inhibited the proliferation of Detroit562 cells,and the effect was more pronounced than radiation alone or radiation combined with cetuximab(P < 0.05 compared with IR;P < 0.01 compared with IR + cetuximab).Conclusion: Cetuximab inhibited radiation-induced oropharyngeal squamous cell DNA double strand break repair and autophagy,induced apoptosis and thus increased the radiosensitivity by inhibiting radiation-induced activation of the ERS signalling pathway proteins IRE1α/ATF6-GRP78 in oropharyngeal squamous cell.