A Small Molecule Targeting Glutathione Activates Nrf2 And Inhibits Cancer Cell Growth Through Promoting Keap1 S-glutathionylation And Inducing Apoptosis

BACKGROUND AND OBJECTIVEAs one of the three major killers endangering human health,cancer is seriously endangering the health and well-being of human beings.At present,the world has not yet found a radical and effective cure for multiple cancers,and the incidence of cancer and death rates are increasing year by year with the more common cancers including high mortality lung cancer,high incidence of gastric carcinoma,gynecological malignant cervical cancer and primary glioma Etc.are seriously devastating human life and health.The current scientific research mainly focuses on how to interfere with tumor cell division and inhibit the generation of blood vessels around tumor cells to prevent the growth and spread of tumor cells.On the other hand,many scientists are trying to induce tumor cell death through various biological and chemical methods to achieve the important purpose of treating cancer.Here,inducing tumor cell apoptosis has become the main line of most research.Apoptosis is an important way of cell death.As a programmed death of cell,it is a suicidal behavior of cells.Currently,there are many drugs that can treat tumors,the mechanism of which is basically to initiate a suicide activity of tumor cells by initiating the endogenous or exogenous apoptotic pathway of the tumor cells.Induction of suicide apoptosis in tumor cells can avoid damage to surrounding normal cells while avoiding secondary and metastatic tumor cells.Therefore,how to efficiently and specifically induce target tumors in this suicide apoptosis has become a research hotspot.In addition,inducing tumor cells to undergo other non-apoptotic deaths,such as autophagy and necrosis,is also a method of treating tumors.Glutathione(GSH),a tripeptide containing y-amide bond and thiol,consists of three amino acids:glutamic acid,cysteine and glycine.It exists in every cell of the body.Glutathione includes both forms of reduced glutathione(G-SH)and oxidized glutathione(G-S-S-G).Under normal physiological conditions,one body is mainly reduced glutathione,accounting for about 95%of the total human glutathione content.In the normal body of glutathione,it can help enhance the antioxidant function of cells to maintain the body’s normal immune function and promote the body’s integrated detoxification.The nuclear factor E2-related factor 2(Nrf2)is one of the nuclear transcription factors of the cap-’n’-collar alkaline-leucine zipper(b-ZIP)protein family and consists of seven Nrf2-ECH homologs(Neh)domains,each of which has a different function.Nrf2 contains a highly conserved sequence that interacts with antioxidant response elements(AREs)and regulates gene expression of detoxification enzymes and antioxidant enzymes in a positive direction,thereby protecting cells from oxidative and teratogenic stresses.Relevant reports indicate that the level of glutathione(GSH)is abnormally increased in many cancer cells and that the successful depletion of GSH in tumor cells by a chemical small molecule that specifically targets GSH is a new strategy for the treatment of cancer.Recently,we synthesized and demonstrated the new compound 2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)cyclohexa-2,5-diene-Diketone(PBQC)can target and specifically damage the intracellular GSH content of tumor cells.At the same time,PBQC can induce S-glutathionylation of Nrf2-associated protein Keap1 and promote Nrf2 nuclear translocation as well as pro-apoptotic genes expression,the successful promotion of tumor cell apoptosis,inhibition of tumor cell growth.Our results show that the novel chemical small molecule PBQC has the potential to be developed into a novel anticancer drug.STUDY CONTENTS1.Identify a novel fluorescent chemical small molecule PBQC that promotes Nrf2 activity;2.To study the inhibitory effect of PBQC on the growth of tumor cells(Hela,A549,U87);3.Investigate the effect of compound PBQC on GSH depletion in Hela cells and detect its mechanism of S-glutathionelation of Keapl protein;4.To study the mechanism that compound PBQC promotes Nrf2 entry into the nucleus;5.To explore the effect of compound PBQC on the expression of antioxidant genes in cells and the changes of intracellular ROS;6.To study the mechanism of PBQC promoting tumor cell apoptosis gene expression;7.To detect compound PBQC promotes tumor cell apoptosis.METHODS1.Synthesis of PBQC2.Cell culture3.Luciferase assay4.Cell-morphological observation5.Cell viability assay(SRB)6.Co-immunoprecipitation7.Immunofluorescence microscopy8.Detecting ROS levels9.RT-PCR analysis10.LDH assay11.Hoechst 33258 staining12.TUNEL assay13.Western blot analysis14.Measurement of glutathione(GSH)15.RNA interference16.Statistical analysisRESULTS1.PBQC up-regulated Nrf2 activity.2.PBQC inhibited the growth of tumor cells while it did not affect the normal vascular endothelial cell viability.3.PBQC up-regulated the expression level of Nrf2 in HeLa cells.4.PBQC promoted Keapl s-glutathionylation and Nrf2 nuclear translocation.5.PBQC up-regulated the expressions of anti-oxidant genes and decreased the intracellular ROS level.6.The varying degree of Nrf2 activation had different effects on the downstream Bcl-2 and Bax gene expressions.7.Significant activation of Nrf2 by PBQC promoted NQO1 and p53 expressions.8.PBQC induced cancer cell apoptosis.CONCLUSIONS1.We found a new Nrf2 activator,2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)cyclohexa-2,5-diene-1,4-dione(PBQC);2.Compound PBQC significantly inhibited the growth of tumor cells,the most obvious for human cervical cancer cells;3.Based on the sensing mechanism of PBQC toward GSH,we know the consumption of intracellular glutathione is increased by compound PBQC;4.Compound PBQC can cause Keapl S-glutathionylation and promote Nrf2 nuclear translocation;5.Compound PBQC down-regulated Bcl-2/Bax ratio and promoted cell apoptosis.