The Role And Mechanism Of Nrf2 In Docetaxel-induced Ferroptosis In Prostate Cancer Cells

Part I Docetaxel induces ferroptosis in castration resistant prostate cancer cells Objective To explore whether ferroptosis occurs in castration resistant prostate cancer cells induced by docetaxel.Method CRPC cell lines PC3 and DU145 were co cultured with docetaxel,ferroptosis inhibitor ferrostatin-1 and apoptosis specific inhibitor Z-VAD-FMK,flow cytometry was used to detect the changes of lipid ROS level after docetaxel treatment;The changes of GSH,GPXs and MDA in CRPC cells treated with docetaxel were detected by corresponding kit;The ultrastructure of CRPC treated cells was observed by electron microscope;The protein level of GPX4 in docetaxel treated CRPC cells was detected by Western blot.Result After docetaxel treatment,the level of lipid ROS in CRPC cells increased significantly and could be reversed by ferroptosis inhibitor ferrostatin-1 and apoptosis specific inhibitor Z-VAD-FMK;In addition,the content of intracellular MDA increased,while GSH and GPX decreased,and the level of GPX4 protein decreased.Transmission electron microscopy showed that the mitochondria of docetaxel treated CRPC cells shrank and became smaller,the density of mitochondrial membrane increased,and the cristae decreased or even disappeared.Conclusion Docetaxel can induce characteristic changes in morphology,metabolism and protein expression of CRPC cells,and cell death can be reversed by ferroptosis inhibitors,indicating that docetaxel can induce ferroptosis of CRPC cells.Part II Mechanism of Nrf2 gene regulating docetaxel induced castration resistant prostate cancer cellsObjective The targets of docetaxel induced ferroptosis in CRPC cells were screened by transcriptome sequencing,and the sensitivity of CRPC cells to docetaxel chemotherapy was accurately adjusted by regulating the expression level of key target molecules.Method Transcriptome sequencing was performed in PC3 control group and docetaxel treatment group.Bioinformatics analysis was performed on the sequencing results.Nrf2 gene was selected according to the research and judgment of differentially expressed genes and their key signal pathways.After constructing silenced Nrf2 CRPC cells,the control group and silencing group were incubated with docetaxel respectively.The proliferation ability of cells was detected by MTT,the level of lipid ROS was detected by flow cytometry,the expression of GPX4 was detected by Western blot,the levels of GSH,GPXs and MDA were detected by kit,and the ultrastructure was observed by electron microscope.Result Transcriptome sequencing took Padj < 0.05 and the absolute value of log FC > 1 as the screening conditions.A total of 1288 genes were up-regulated and 780 genes were down regulated.Among them,the oxidative stress regulating genes were further screened,and the Nrf2 gene with the smallest Padj was screened;Gene set enrichment analysis showed that cholesterol homeostasis,p53 signaling pathway,unfolded protein response and m TORC1 signaling pathway were significantly enriched.The heat map of oxidative stress-related genes also suggested that Nrf2,GPX4,prox4 and other genes played a role in docetaxel chemotherapy-induced ferroptosis of CRPC cells;The sensitivity of CRPC cells to docetaxel chemotherapy increased in the silencing Nrf2 group.Electron microscopy showed that the number of CRPC cells with mitochondrial shrinkage and increased membrane density under ferroptosis stress increased significantly in the silencing Nrf2 group;In the Nrf2 silencing group,the levels of MDA and ROS in docetaxel treated CRPC cells increased significantly,and the levels of GSH and GPXs decreased significantly.Conclusion1.Docetaxel induces ferroptosis in CRPC cells through the classical GSH-GPX system.2.Nrf2 gene plays a protective role in docetaxel induced ferroptosis of CRPC cells.Part III Expression of effector factors after docetaxel combined with Tf R-CAR TObjective To construct Tf R-CAR T cells and explore the antitumor effect of Tf R-CAR T cells through co incubation experiment,so as to lay a theoretical basis for docetaxel combined with Tf R car t in the treatment of prostate cancer.Method The expression of Tf R protein in control group,docetaxel group,silenced Nrf2 CRPC cell group and silenced Nrf2 CRPC cell combined with docetaxel group was detected by Western blot;The expressions of PD-L1,CD8 and FOXP3 in HSPC and CRPC patients before and after docetaxel chemotherapy were analyzed by immunohistochemistry;Tf R-CAR T cells were constructed and divided into four groups:NC group,Tf R-CAR T group,docetaxel group and docetaxel combined with Tf R-CAR T group.Tf R-CAR T and CRPC tumor cells were incubated for 24 hours according to the efficiency target ratio of 1:1,5:1 and 10:1.The cell activity was detected by MTT,and the cytokine secretion in the supernatant of co incubated Tf R-CAR T and CRPC cells was detected by flow CBA multi factor detection technique.Result Western blot showed that the expression of Tf R protein increased after docetaxel treatment,and decreased in the silencing Nrf2 group;Immunohistochemical results showed that PD-L1 and FOXP3 had no significant changes and CD8 + T cell infiltration increased significantly in HSPC and CRPC patients after chemotherapy;MTT showed that docetaxel combined with Tf R-CAR T had stronger killing ability on CRPC cells than docetaxel alone;The results of CBA multi factor detection by flow cytometry showed that docetaxel alone could enhance cytokine secretion,and the cytokine secretion level of Tf R-CAR T cells was higher than that of NC cells,while the cytokine secretion level of docetaxel combined with Tf R-CAR T cells was significantly higher than that of docetaxel alone and TfR-CAR T groups.Conclusion Docetaxel enhanced the expression of Tf R protein in CRPC cells;Docetaxel chemotherapy can change the tumor microenvironment of HSPC and CRPC patients;Docetaxel combined with Tf R-CAR T can improve the ability of killer cells and increase the secretion of cytokines.