Establishment Of Nrf2-ARE Activity Based Screening And Confirmation Platform And Their Experimental Applications In Environmental Oxidative Stressors Assessment And Disease Prevention

Objective:The nuclear factor erythroid 2-related factor 2(Nrf2)is a key transcription factor regulating a wide array of genes for antioxidant and detoxification enzymes in response to oxidative and xenobiotic stress.This cytoprotective role of Nrf2 has been implicated in many diseases associated with oxidative stress.Nrf2-knockout mice are more prone to developing diseases related to oxidative stress and cancers.Thus,a large number of Nrf2-antioxidat response element(ARE)activators have been identified and used to induce Nrf2expression or activity to enhance the cellular oxidative defense capacity against diseases.To date,four Nrf2 activators have entered clinical trials and the only one has been approved by FDA is DMF which is applied on the treatment of multiple sclerosis.However,constitutive activation of Nrf2 was found in a variety of cancers in recent years.Aberrant activation of Nrf2 is correlated with cancer progression,chemoresistance,and radioresistance.Therefore,suppressing Nrf2 activity may be an effective way to sensitize cancer cells to chemotherapy and radiotherapy.In light of this,numerous studies are trying to find effective Nrf2 inhibitors for use in cancer chemotherapy.But most of them could not move forward to clinical application due to their unclear inhibitory effects on Nrf2 and concerns on specificity and safety.Human beings are exposed to many kinds of environmental chemicals in their daily life,which could lead to adverse health effects.Most of these health problems are caused by chemical-induced oxidative damage.As a core regulator of cellular redox homeostatic,Nrf2-ARE signaling pathway always replies to environmental oxidative stressors by diverse response mechanisms.Some chemicals could adaptively activate Nrf2 pathway by stimulating radical oxygen species(ROS)production.While others could directly act on Nrf2 activity and disturb the cellular redox balance.Thus,the examination of cellular ROS levels and ARE activity are crucial to the assessment of environmental oxidative stressors.Based on that,we could develop possible preventive strategies or provide basic experimental evidence for chemical-induced adverse effect.Methods:1.Establishment of Nrf2-ARE regulators and environmental oxidative stressor screening and confirmation platform:HepG2 and HaCat cell lines are transducted by lentivirus wrapping a recombinant plasmid with repeated AREs driven luciferase reporter gene.Then we develop a series of efficient and feasible screening and confirmation strategies based on our experience of Nrf2-related studies.The HepG2-ARE cells were seeded in 384-well plate and treated with 7500 chemicals from commercial chemical libraries for 6 h.And then measurement of ARE luciferase activity was carried out by plate reader.The assessment of cellular ROS levels was achieved by DCFH-DA probe.The HepG2 cells were seeded in 96-well plate and treated by chemicals for 1h and 6 h.The results were indicated by fluorescence intensity of probe by plate reader.2.The confirmation of potential Nrf2-ARE regulators in second screening experiment:We chose 13 potential Nrf2-ARE inhibitors for second screening by detecting ARE luciferase activity.HepG2 cells were treated with 1.25,2.5 and 5μM chemicals for 6 h.Then we performed 24 h cell viability assay to obtain the non-toxic concentration of each chemical.The cancer cell line HepG2,A549 and non-cancer cell line HaCat cell line were treated with potential Nrf2-ARE inhibitors on non-toxic concentration.The protein levels of Nrf2 were detected after 4 h and the mRNA levels of Nrf2 and its downstream genes were detected after 6 h.Regarding to the confirmation experiments of Nrf2-ARE activators,we have identified several effective activators.The data is not shown here due to the process of patent application.3.The specificity and inhibitory mechanism of potential Nrf2-ARE inhibitors on Nrf2pathway:We treated A549 cells with the potential Nrf2-ARE inhibitors on the same concentration of their Nrf2 inhibitory effect and detect other short-life protein like p53 and other mRNA like NFE2L1 regulated PSM family.In the experiements regarding to Nrf2inhibiting mechanism,A549 cells were treated with protein synthesis inhibitor CHX or RNA synthesis inhibitor ActD with/without potential Nrf2-ARE inhibitors.We collected the cells in different time points and measured the mRNA and protein levels of Nrf2.4.The role of Nrf2 in lung cancer progression and chemoresistance:We collected clinical fresh lung cancer and adjacent tissue samples from 10 patients subjected to surgery.The Nrf2 and its downstream gene expression was detected by extraction of RNA and protein.And then we examined the basal levels of Nrf2 and the cell viability treated by antitumor drugs in three different human lung cancer cell lines.We also detected the change of cell viability treated by antitumor drugs in Nrf2 silencing cells.Then,we employed the mouse xenograft tumor model.Sixteen C57BL/6 mice were randomly divided into two groups.Xenograft tumors were initiated by subcutaneous inoculation of1×10~6 3LL(SCR or Nrf2-KD)cells per mouse in the right dorsal thoraric(upper back)region.The body weight and tumor volume was measured every other day after seven days.At the end of the experiments(20 days),mice were sacrificed and tumors were dissected and weighted.5.The experimental application of Nrf2-ARE inhibitors in lung cancer cells:We treated A549 and 3LL cells with different concentration of antitumor drugs with/without Nrf2-ARE inhibitors for 24 h and detected the cell viability.In mouse xenograft tumor model performed by normal 3LL cells,mice were given either PBS(control),antitumor drugs,TPL,or both(combination;COM)by intraperitoneal(ip.)injection every other day for 10 days(5 injections total).The methods are same with 4.The serum was collected for detection of ALT and AST.Results:1.Establishment of cell lines stable expressing ARE luciferase reporter and the effect of chemical library on ARE luciferase activity:We treated the HepG2-ARE cells which stable expressed ARE luciferase reporter with confirmed Nrf2 activators curcumin and tBHQ and the ARE activity significantly increased after 6 h(p<0.05).Then,we obtained a huge amount of results by treating cells with chemical library.We defined the chemicals which increased ARE activity to 150%as potential ARE activators and decreased ARE activity to 50%as potential ARE inhibitors.2.Establishment of environmental oxidative stressor assessment platform and ROS detection:We employed three typical chemicals related to Nrf2 pathway,Arsenate,Ethionamide and Camptothecin to elucidate our screening and assessment strategies.As a positive control,the well-known oxidative stressor arsenate increased the cellular ROS levels(p<0.05)which proved a successful method to detect ROS by 96-well system.Then we treated cells with the other two chemcals and measured the ROS levels.Based on the ARE activity and ROS levels,we classified them into different catalogs.3.Potential Nrf2-ARE inhibitors TPL and ANM decreased Nrf2-ARE activity:In the second screening experiment,we found that TPL and ANM showed an inhibitory effect on ARE-luciferase activity at extreme low concerntration(0.05μM).Then we found non-toxic TPL dose-dependently decreased the mRNA levels of Nrf2 and its downstream genes and the protein levels of Nrf2 in HepG2 and A549 cells(p<0.05),while there is no effect on Nrf2 in HaCat cells.Non-toxic ANM inhibited the expression of Nrf2 protein in all of those cells but not Nrf2 mRNA levels.4.The specificity and inhibitory mechanism of potential Nrf2-ARE inhibitors on Nrf2pathway:ANM inhibited other short-life protein for example p53 and Survivin in A549with the same concentration of Nrf2 inhibitory effect;while TPL had no effect on other short-life protein and also for the mRNA levels of NFE2L1 and its downstream genes.Nrf2-ARE inhibitors TPL had no effect on stability of Nrf2 mRNA and protein.Compared to the control cells collected at the same time point after ActD or CHX treatment,TPL treatment did not affect the mRNA and protein levels of Nrf2.5.Constitutive activation of Nrf2 accelerate lung cancer progression and decrease its sensitivity to antitumor drugs:In clinical lung cancer samples,we found the mRNA and protein levels Nrf2 and its downstream gene GCLC increased in cancer tissue compared to adjacent tissue in some patients,but there is no significant difference(p>0.05).We need to collect samples from more patients and to statically analyze the data by cancer type and stage in the future.Within the three lung cancer cell lines,A549 got the highest Nrf2 basal levels and lowest sensitivity to antitumor drug induced cytotoxicity,while opposite to H1299 and H1975 was in the middle(p<0.05).In the Lewis lung cancer cell line 3LL,Nrf2-KD cell got lower cell viability when treated with same concentration of antitumor drugs compared to SCR(p<0.05).In mouse xenograft model,the rate of growth of xenograft tumors after inoculation of Nrf2-KD cells was significantly slower and showed less tumor weight at the end of experiment than SCR(p<0.05).6.TPL sensitized lung cancer cells to antitumor drug-induced cytotoxicity by inhibiting Nrf2 pathway:Non-toxic concentration of TPL increased the sensitivity of A549and 3LL cells to cytotoxicity induced by the antitumor drugs(p<0.05).In the xenograft model,antitumor alone or TPL alone did not significantly alter tumor growth compared to vehicle group.However,the combination group markedly reduced tumor volume and tumor weight(p<0.05).There is no obvious adverse health effect based on body weight and serum ALT and AST levels.Conclusion:1.The screening and confirmation platform based on the ARE luciferase activity and cellular ROS determination was successfully developed.2.A novel Nrf2-ARE inhibitor TPL,with low toxicity and high specificity,was identified by using Nrf2-ARE regulator screening and confirmation platform.TPL sensitized cancer cell to antitumor drugs induced cytotoxicity by inhibiting Nrf2 pathway in both in vitro and in vivo models,which provide a new potential molecular target for clinical cancer treatment.