Brusatol Enhances The Radiosensitivity Of A549 Cell Line By Regulating Nrf2

Objective:To compare the difference of radiosensitivity between H1299,A549 and H460 cell lines,and discover the effect of Brusatol for increasing radiosencitivity of A549 cell line.Methods:MTT assay and clone formation assay were used to compare radiosensitivity of H1299,A549 and H460 cell lines;comet assay showed DNA damage level of A549 and H460 cell lines,post 8 Gy,2 hours;Western blot assay discovered different Nrf2 protein level in H1299,A549 and H460 cell lines;qRT-PCR and Western Blot assay tested Nrf2 and NQO1 mRNA and protein level of A549 cell line,after treated with Nrf2 siRNA;the change of A549 cell line 's Nrf2 protein after irradiation was tested by Western blot and Immunofluorescence;Annixin V-FITC/PI flow cytometry was used to test apoptosis,which was caused by irradiation;MTT assay showed cytotoxicity of Brusatol to H1299 and A549 cell lines;Western blot assay showed Brusatol's effect on Nrf2 protein level in H1299 and A549 cell lines,and the change of Nrf2 protein level in A549 cell line,which was treated with Brusatol and irradiation at the same time;comet assay showed DNA damage,DCFH-DA tested ROS level,MTT and clone formation assay discovered radiosensitivity in A549 cell line,which was treated with Brusatol and irradiation or not.Results The survival fraction of MTT assay and clone formation assay indicated that the radioresistance from strong to weak was H1299,A549,H460;comet assay showed that H460 cell line suffered much more DNA damage than A549 cell line,when exposed to irradiation;Western blot assay showed that A549 cell line had the highest Nrf2 protein level compared with H1299 and H460 cell lines,and Nrf2 protein level was increased by irradiation in A549 cell line;Nrf2 gathered into nuclear after irradiation;Annixin V-FITC/PI flow cytometry proved that the apopotosis rate increased when NrF2 was knocked down and exposed to irradiation;Brusatol can inhibit Nrf2 effectively in A549 cell line,and counteract Nrf2 protein level increase,which was caused by irradiation;Brusatol increased DNA damage,ROS level and radiosensitivity of A549 cell line.Conclusions The radioresistance from strong to weak was H1299,A549,H460.The differenct radiosensitivity of NSCLC cell lines might have some ralationship with their Nrf2 protein level.Brusatol increased A549 cell line radiosensitivity by inhibitting Nrf2 protein,leading to DNA damage and accumulating ROS.