ABSTRACT
Ferroptosis is a new type of programmed cell death, which has been involved in the progression of tumours. However, the regulatory network of ferroptosis in pancreatic cancer is still largely unknown. Here, using datasets from GEO and TCGA, we screened HSPB1, related to the P450 monooxygenase signalling, a fuel of ferroptosis, to be a candidate gene for regulating pancreatic cancer cell ferroptosis. We found that HSPB1 was enriched in the exosomes derived from human pancreatic cancer cell lines SW1990 and Panc-1. Then, hypoxic SW1990 cells were incubated with exosomes alone or together with HSPB1 siRNA (si-HSPB1), and we observed that exosomes promoted cell proliferation and invasion and suppressed ferroptosis, which was reversed by si-HSPB1. Moreover, we found a potential binding affinity between HSPB1 and FUS, verified their protein interaction by using dual-colour fluorescence colocalization and co-IP assays, and demonstrated the promoting effect of FUS on oxidative stress and ferroptosis in hypoxic SW1990 cells. Subsequently, FUS was demonstrated to bind with and stabilize the mRNA of Nrf2, a famous anti-ferroptosis gene that negatively regulates the level of P450. Furthermore, overexpressing FUS and activating the Nrf2/HO-1 pathway (using NK-252) both reversed the inhibitory effect of si-HSPB1 on exosome functions. Finally, our in vivo studies showed that exosome administration promote tumour growth in nude mice of xenotransplantation, which was able to be eliminated by knockdown of HSPB1. In conclusion, exosomal HSPB1 interacts with the RNA binding protein FUS and decreases FUS-mediated stability of Nrf2 mRNA, thus suppressing hypoxia-induced ferroptosis in pancreatic cancer.
Subject(s)
Ferroptosis , HSP27 Heat-Shock Proteins , NF-E2-Related Factor 2 , Pancreatic Neoplasms , RNA-Binding Protein FUS , Animals , Humans , Mice , Cell Line, Tumor , Cell Proliferation , Exosomes/metabolism , Ferroptosis/genetics , Gene Expression Regulation, Neoplastic , Heat-Shock Proteins , HSP27 Heat-Shock Proteins/metabolism , HSP27 Heat-Shock Proteins/genetics , Mice, Nude , Molecular Chaperones/metabolism , Molecular Chaperones/genetics , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Protein Binding , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Protein FUS/metabolism , RNA-Binding Protein FUS/geneticsABSTRACT
Ferroptosis is expected to be a therapeutic target for cancers including pancreatic cancer. We aimed to screen genes that regulate ferroptosis and doxycycline resistance in pancreatic cancer and to explore the underlying mechanisms. Bioinformatics analysis was performed to identify genes that respond to ferroptosis in two human pancreatic cancer cells with GOT1 knocked down or not. 325 and 842 genes were upregulated in MiaPaCa and Tu8902 cells in response to GOT1 knockdown, with 43 genes shared. Among the 43 genes, 14 genes were identified to interact with ferroptosis key genes. MB and HMOX1 were the genes most sensitive to Erastin and doxycycline. Moreover, MB and HMOX1 expression was higher in human normal pancreatic duct epithelial cells than in pancreatic cancer cells. MB and HMOX1 proteins physically bound and promoted each other's expression. By interacting with HMOX1, MB suppressed pancreatic cancer cell proliferation, colony formation and invasion, and promoted cell ferroptosis and sensitivity to erastin and doxycycline. Silencing HMOX1 reversed the promoting effect of MB on cell ferroptosis and sensitivity to doxycycline. A pancreatic cancer xenograft model was established by subcutaneous injection of Panc-1 cells transfected with or without Ad-MB, and doxycycline was administered intraperitoneally. Overexpression of MB enhanced the inhibitory effect of doxycycline on xenograft growth. In conclusion, MB facilitated doxycycline sensitivity in pancreatic cancer cells through promoting HMOX1-mediated ferroptosis.
Subject(s)
Ferroptosis , Pancreatic Neoplasms , Humans , Heme Oxygenase-1/genetics , Myoglobin , Doxycycline/pharmacology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/geneticsABSTRACT
OBJECTIVE: To investigate HMGB-1 expression and its extracellular release of cultured primary hepatic parenchymal cells (HC) and Kupffer cells (KC) that were induced by lipopolysaccharides (LPS). METHODS: Primary hepatic parenchymal cells and Kupffer cells were cultured in flasks, and some cells were treated with 500 microg/L LPS for 24 hours (induced group) and some were not treated with LPS and served as controls. All of the cells were repeatedly frozen-thawed, and the expression levels of HMGB1-mRNA and HMGB1 proteins were detected by semi-quantitative RT-PCR and Western blot respectively. Then HC and KC were subcultured in 24-well culture plates for 6 h, 12 h, 24 h and 48 h, and the HMGB1 protein in culture fluids was detected by Western blot at each time point. RESULTS: Compared with the cells in the control group, the expression levels of HMGB1-mRNA in the induced group were significantly increased in both HC and KC at 24 h (t=31.32 and 45.90, P<0.05) and the protein levels of HMGB1 showed the same results (t=46.19 and 38.44, P<0.05). There was a small quantity of HMGB1 protein in the culture fluids of two control groups and the induced group of HC. However the HMGB1 protein in the induced group of KC were obviously increased with prolonged culture time (F=42.74, P<0.05). Compared with the control group, the level of HMGB1 protein in the induced group of KC was not increased at 6 h (t=9.57, P>0.05) but was significantly increased at 12 h, 24 h and 48 h (t=21.95, 32.39, 44.16, respectively P<0.05). CONCLUSION: LPS could increase HMGB1 expression of HC and KC and HMGB1 release from KC, but not from HC. The results suggest that KC play an important role in triggering inflammation and liver injury.