Sodium Selenite Induces Apoptosis Of Xuanwei Lung Cancer Cells Resistant To Cisplatin Through Keap1/Nrf2/GSH

Objectives:1.To construct human lung adenocarcinoma A549 and Yunnan Xuanwei lung adenocarcinoma XWLC-05 stable cisplatin-resistant cell lines and analyze their biological characteristics.2.To clarify whether sodium selenite induced cisplatin-resistant cell apoptosis of Xuanwei lung cancer depends on the production of ROS.3.To investigate whether sodium selenite induces apoptosis of cisplatin-resistant Xuanwei lung cancer cells by regulating the Keap1/Nrf2/GSH.Methods:1.Cisplatin resistance of human lung adenocarcinoma A549 and Yunnan Xuanwei lung adenocarcinoma XWLC-05 were induced by low-dose maintenance and dose escalation method in vitro,and A549/CDDP and XWLC-05/CDDP cell lines were constructed.The morphological changes were observed by microscope.MTT was used to calculate the IC50 and drug resistance index,and Western blot was used to detect the drug resistance marker protein P-glycoprotein(P-gp)to verify the stable drug resistance of the cells.Cell functional experiments(proliferation,scratching,cloning and invasion)were used to analyze the malignant biological characteristics of drug-resistant cells.2.Single dose and dose gradient combination of sodium selenite and cisplatin were applied to XWLC-05/CDDP resistant cells and XWLC-05 parent cells,and the combined effect of the two drugs was analyzed by CompuSyn software to find the optimal combination concentration;XWLC-05/CDDP resistant cells and XWLC-05 parent cells were treated with sodium selenite alone or in combination with cisplatin.Cell cycle and apoptosis were detected by flow cytology.The mRNA expressions of Bcl-2,Bax and Caspase-3 were detected by RT-qPCR to verify whether sodium selenite induced apoptosis of mitochondrial pathway.DCFH-DA probe was used to detect the ROS level in cells to clarify whether the apoptosis induced by sodium selenite was related to ROS.In the sodium selenite group,ROS scavenging agent(NAC)was added,and cell apoptosis was detected by flow cytometry to further clarify whether sodium selenite induced drug-resistant cell apoptosis was dependent on ROS production.3.Cisplatin resistant cells and parent cells were treated with sodium selenite alone or in combination with cisplatin,and the activity of GSH in cells was detected by DTNB enzymatic hydrolysis method to verify whether the production of sodium selenite mediated ROS was related to intracellular GSH level;The mRNA levels of Keap1,Nrf2 and GCLC were detected by RT-qPCR.After Nrf2 agonist(NK-252)and Nrf2 inhibitor(ML385)were treated with sodium selenite,The protein expression levels of Nrf2,GCLC and Caspase-3 in the cells were detected by Western blot,To investigate whether sodium selenite induces apoptosis of cisplatin-resistant cells through Keap1/Nrf2/GSH.Results:1.XWLC-05/CDDP and A549/CDDP resistant cells were successfully constructed.The cisplatin IC50 of XWLC-05/CDDP and A549/CDDP resistant cells were 5.90μg/ml and 15μg/ml,respectively,and the drug resistance indexes were 1.865 and 2.470,respectively.Compared with the parent cells,the drug-resistant cells were irregular in morphology,significantly increased in cell volume,the cytoplasm and intracellular granules were increased,and multinucleated and lobulated nuclei were common.Cell function experiments showed that XWLC-05/CDDP and A549/CDDP resistant cells had stronger proliferation,migration and invasion abilities than their parents(P<0.05).2.The optimal combined effect concentration of sodium selenite and cisplatin was 0.5μg/ml sodium selenite+4μg/ml cisplatin.The proliferation inhibition of XWLC-05 parent cells by sodium selenite was associated with S phase arrest(P<0.05),and G2/M phase arrest was also significantly increased in XWLC-05/CDDP resistant cells(P<0.05).Sodium selenite significantly increased the ratio of Bax/Bcl-2 mRNA in XWLC-05/CDDP resistant cells and XWLC-05 parent cells(P<0.05),and induced the overexpression of Caspase-3 mRNA(P<0.05),suggesting that sodium selenite mediated cell apoptosis through endogenous mitochondrial pathway.The level of ROS in untreated XWLC-05/CDDP resistant cells was significantly higher than that in XWLC-05 parent cells(P<0.05).Under the treatment of sodium selenite,the production of ROS in XWLC-05/CDDP drug-resistant cells and parent cells increased in a time-dependent manner,and the ROS level in drug-resistant cells was significantly higher than that in parent cells(P<0.05).NAC intervention could significantly reverse the apoptosis induced by sodium selenite(P<0.05),suggesting that the apoptosis induced by sodium selenite in cisplatin resistant lung cancer cells was dependent on ROS production.3.The content of GSH in untreated XWLC-05/CDDP resistant cells was significantly higher than that in XWLC-05 parent cells(P<0.05),indicating that intracellular GSH level was related to cisplatin resistance.Sodium selenite significantly reduced the GSH level in XWLC-05/CDDP and XWLC-05 cells(P<0.05),suggesting that the increase of ROS induced by sodium selenite was related to the down-regulation of GSH activity.The mRNA expression levels of Keapl,Nrf2 and GCLC in XWLC-05/CDDP resistant cells and XWLC-05 parent cells were down-regulated by sodium selenite(P<0.05),suggesting that sodium selenite may mediate ROS production by inhibiting GSH activity.The effect of sodium selenite on the down-regulation of Nrf2 and GCLC proteins and the up-regulation of caspase-3 protein could be reversed by Nrf2 agonist(NK252)(P<0.05),which initially demonstrated that sodium selenite induced apoptosis of cisplatin-resistant cells through the Keap1/Nrf2/GSH.Conclusions:1.Cisplatin-resistant cell lines of Yunnan Xuanwei lung cancer XWLC-05/CDDP were successfully constructed by low-dose maintenance and dose escalation method in vitro.2.Sodium selenite regulates GSH level through the Keap1/Nrf2 and induces ROS-dependent apoptosis in cisplatin resistant lung cancer cells.