A Study Of The Effect And Molecular Mechanism Of IASPP-mediated ROS Inhibition In Tumor Cells

Tumor is a serious disease that threatens human life and health.The incidence of new cases of malignant tumors in China is 3.804 million cases,that is,about 10,000 people are diagnosed with tumors every day and about 7 people are diagnosed every minute.Tumor has exceeded the cardiovascular disease and become the disease with the highest mortality in China.Chemotherapy tolerance is one of the important reasons for poor prognosis of tumor patients.Therefore,understanding the molecular mechanism of chemotherapy tolerance is an important issue in the field of biological research.Reactive Oxygen Species?ROS?is a partial reduction product of oxygen?O₂?,including superoxide anion?O₂^-?,hydrogen peroxide?H₂O₂?,and hydroxyl radical?HO·?.The appropriate amount of ROS is an important signaling molecule that regulates the normal physiological activities of cells.High levels of ROS can destroy the normal functions of important biological macromolecules such as nucleic acids,proteins and lipids,and promote the occurrence and development of tumors.If ROS levels continue to increase,once exceeded the death threshold,it will cause irreversible oxidative damage,leading to cell death.Studies have shown that the level of ROS is generally higher in tumor tissue than those in normal tissue.Chemotherapy drugs can achieve the purpose of killing tumor cells by further increasing ROS.The tumor cells have precisely regulated anti-oxidant systems and can be utilized to prevent excessive accumulation of intracellular ROS.Thus tumor cells could gain of adaptive survival under high oxidative stress,and tolerance to chemotherapeutic drug-induced killing effects.Therefore,revealing the molecular mechanism of ROS homeostasis regulation in tumor cells is expected to provide new clues for improving the chemotherapy sensitivity of tumor cells.Nrf2?Transcription factor nuclear erythroid factor 2-like 2,NFE2L2,Nrf2?is a core transcription factor that regulates ROS,and can limit the excessive accumulation of ROS.Studies have shown that Nrf2 is abnormally activated in tumor cells,inhibiting the increase of intracellular ROS levels,and promoting the survival and drug resistance of tumor cells.The role of Nrf2 in tumors has received more and more attention,and the molecular mechanism regulating its activity has yet to be perfected,which greatly limits the treatment of tumors targeting Nrf2.iASPP?Inhibitor of Apoptosis Stimulating Protein of P53?acts by inhibiting p53-dependent apoptosis,and its function depends on its precise location in the nucleus.In this study,iASPP was found to be mainly localized in cytoplasm in tumor cells.This result suggests that iASPP has oncogene functions independent of p53.The results showed that the inhibition of iASPP expression,intracellular ROS levels increased;overexpression of iASPP is accompanied by a decrease in ROS,suggesting that iASPP has an important activity of inhibiting ROS.More importantly,this function is universal and occurs in a variety of tissue origination as well as cells in different p53 status.Further studies confirmed that the anti-ROS effect of iASPP depends on the expression and activation of Nrf2 rather than p53.On this basis,this study further explores the role of iASPP in the regulation of Nrf2 and its mechanism.The results showed that iASPP enhanced Nrf2 protein stability by inhibiting Keap1-mediated ubiquitin degradation of Nrf2,promoting nuclear translocation and transcription activity of Nrf2.Further molecular mechanism studies showed that in the N-terminal of iASPP,there is a highly conserved DLT241 motif which is similar to the DLG motif of Nrf2.iASPP competes with the DLG motif of Nrf2 for binding to the DGR domain of Keap1 through this motif.Under multiple chemotherapeutic drugs that promote ROS accumulation,the binding capacity of iASPP to Keap1 is enhanced,together with a decrease of the binding ability of Nrf2 to Keap1 and an increase of the Nrf2 protein amount.This study found that both iASPP and Nrf2 are highly expressed in the tissues of renal cell carcinoma patients,and there is a positive correlation between the two proteins,suggesting that high expression of iASPP in tumor tissue may be an important reason leading to increased Nrf2 levels.Subcutaneous tumor studies in nude mice confirmed that knocking down iASPP can inhibit tumor growth and increase the inhibitory effect of 5-FU on tumor growth,and that p53 does not affect the inhibitory effect of iASPP knockdown on tumor growth.After analyzing the expression levels of Nrf2,Keap1,apoptosis-related proteins and anti-oxidation target genes in mice tumor tissues,it was found that the Nrf2 protein level and its anti-oxidation target genes were significantly inhibited by knockdown of iASPP,and the level of pro-apoptotic protein were increased.Suggesting that iASPP is involved in the regulation of the anti-oxidative activity of Nrf2 in these tumors,the knockdown of iASPP inhibits tumor growth and increases the therapeutic effect of 5-FU is related to apoptosis.Further molecular mechanism studies revealed that iASPP activated Nrf2 anti-ROS activity,inhibited ROS levels,decreased DNA damage,inhibited apoptosis,promoted tumor cell growth and enhanced tumor cell tolerance to 5-FU.In summary,this study demonstrates that cytoplasmic iASPP promotes Nrf2 anti-ROS activity in a p53-independent manner,which promotes tumor growth and chemotherapy tolerance.Increases the understanding of Nrf2 regulation mechanism and provide a new perspective for the clinical treatment of highly resistant tumors represented by renal cell carcinoma.