The Role Of Nrf2-ARE Signaling Pathway And Autophagy In Cisplatin Resistance Of Ovarian Cancer

Cisplatin resistance is a major obstacle for ovarian cancer treatment, with the underlying mechanism not well understood. Recent studies have revealed factors which are off-targets involved in cisplatin resistance, including autophagy and Nrf2 transcription factor. However, how these factors affect chemoresistance is unclear. To explore the underlying mechanism is of essential significance for identifying potential targets for ovarian cancer treatment.Autophagy is a kind of programmed cell death which is different from apoptosis. When confronted with stress and hypoxia, the cell engulfs the damaged organelles and molecules with lysosomal system and maintains energy cycling. The dysfunction of autophagy is related with kinds of diseases, including aging and neurodegenerative disease. There exists some controversy in the role of autophagy in cancers that autophagy could inhibit the formation of cancers, while other investigations indicate that autophagy contributes the development of cancer. It is same to the investigation of autophagy and chemoresistance of cancer. The different point is that most studies reveal that autophagy activation correlates with chemoresistance, while only a few studies find that chemoresistance leads to autophagy deficiency. It remains unclear what role of autophagy is in cisplatin resistance in ovarian cancer.Nrf2-ARE (NF-E2-related factor 2-antioxidant response element) signaling pathway protects cells from reactive oxygen species. When induced by oxygen stress, Nrf2 transcription factor could be overexpressed and enters nucleus to provoke the expression of the downstream anti-oxidant response elements. Increasing investigations are evidenced that overexpression of Nrf2 contributes to chemoresistance. Our previous work has confirmed that resistance ovarian cancer cells exhibits high level of autophagy, downregulation of which resensitizing cells to cisplatin. The molecular mechanism about Nrf2-ARE signaling pathway participating in the resistance of ovarian cancer is elusive.Overall, we hypothesize that Nrf2-ARE signaling pathway may regulate autophagy in cisplatin resistance of ovarian cancer. The objective of this project is to explore the role of Nrf2-ARE signaling pathway and autophagy in ovarian cancer cells’cisplatin resistance and try to illustrate the possible regulation between the two.Part I The role of autophagy in cisplatin resistance of ovarian cancer cellsObjective:To explore the role of autophagy in cisplatin resistance of ovarian cancer.Methods:The human ovarian carcinoma cell lines A2780 and its cisplatin resistant variant A2780cp are adopted. Cells are treated with or without cisplatin to investigate the level of autophagy by Western blot, immunofluorescence and transmission electron microscopy. For cells with high level of autophagy, the autophagy pharmacological inhibitor 3-MA and autophagy gene beclin 1 siRNA is used to downregulate the autophagy level, then cell viability and apoptotic analysis is performed.Results:The 50 percent inhibition concentration of cisplatin for 24h of A2780cp cells was 6.5 times higher to cisplatin than the parental cell line, which provides an ideal pair of cell model for studies. In Western blot analysis, A2780cp cells express higher level of autophagy related protein LC3 Ⅰ/Ⅱ and beclin 1 than A2780 cells after different concentrations of cisplatin treatment. In immunofluorescence analysis, the LC3 expression is stronger in A2780cp cells in contrast to A2780 cells. At the same time, A2780cp cells exhibit more autophagosomes than A2780 cells in transmission electron analysis. For A2780cp cells, the co-treatment of 3-MA and cisplatin could increase cisplaitin-induced cell death. But such co-treatment does not affect the apoptosis. Meanwhile, after beclin 1 siRNA transfection into A2780cp cells, both cell death and apoptosis that are induced by cisplatin increase. Conclusions:Autophagy may contribute to cisplatin resistance in ovarian cancer cells. Inhibition of autophagy could resensitize ovarian cancer cells to cisplatin.Part Ⅱ The role of Nrf2-ARE signaling pathway in cisplatin resistance of ovarian cancer cells and possible regulation between Nrf2-ARE signaling pathway and autophagyObjective:To explore the role of Nrf2-ARE signaling pathway in cisplatin resistance of ovarian cancer and try to illustrate the possible relationship between Nrf2-ARE signaling pathway and autophagy.Methods:Examine the expression of essential genes in Nrf2-ARE signaling pathway, Nrf2, Keapl, NQ01 and HO-1 in both A2780 cells and A2780cp cells by real-time PCR and Western blot. For cells with high level of Nrf2, Nrf2 siRNA is transfected and cell viability, together with apoptosis is examined. At the same time, autophagy gene expression is also detected as Nrf2 decrease.Results:The mRNA expression of Nrf2, Keap1, NQ01 and HO-1 are all much higher in A2780cp cells than those in A2780 cells (P<0.05 except for NQ01). Western blot analysis is consistent with mRNA results. For A2780cp cells, Nrf2 siRNA is transfected and all of Nrf2, NQ01 and HO-1 expression are inhibited except for Keapl. It enables A2780cp cells greatly sensitive to cisplatin in cell viability assay. Cells exhibit more apoptotic bodies in Tunel assay and higher level of apoptosis in Annexin-V/PI detection analysis as Nrf2 decrease. We have found that A2780cp cells express high level of autophagy than A2780 cells in previous work. Further, all of autophagy related genes, Atg3, Atg5, Atg12 and p62 expression are examined both in mRNA level and protein level, which are much higher in A2780cp cells than those in A2780 cells. Interestingly, autophagy related genes Atg3, Atg5, Atg6, Atg12 and p62 decrease both in mRNA level and protein level with Nrf2 inhibition. Meanwhile, the formation of autophagosomes is also inhibited in Nrf2 siRNA transfection group in contrast to control siRNA transfection group.Conclusions:Nrf2-ARE signaling pathway plays a role in cisplatin resistance of ovarian cancer cells. Inhibition of Nrf2 contributes to the re-sensitization to cisplatin for resistant cells. Besides, Nrf2-ARE signaling pathway may participate in cisplatin resistance of ovarian cancer cells through the activation of autophagy.