Identification Of A Novel Nrf2 Inhibitor And Study On The Mechanisms Of Its Growth-inhibitory Effect On Cancer Cells

BACKGROUND AND OBJECTIVESCancer is a major contributor to deaths worldwide,with incidence and mortality increasing every year.Lung cancer and leukemia are at the forefront of cancer death.Chemotherapeutics can interfere with cell division,inhibit tumor angiogenes:is and(or)induce cancer cell death,which is important for cancer treatment.Emerging evidence has demonstrated the induction of apoptotic cell death by many drugs through the extrinsic or intrinsic signaling pathway.It is increasingly apparent that other cell death modes,such as autophagy and necrosis,can be initiated by some anticancer drugs as well.Autophagy,also called type ? programmed cell death,is a conserved biological process in eukaryotes.Autophagy is implicated in many physiological or pathological processes,including cancer,infection and aging.Therefore,it is necessary to understand the mechanisms by which apoptosis or autophagy is regulated,which may provide powerful basis for cancer treatment.Nuclear factor-erythroid 2-related factor 2(NFE2L2,also called Nrf2)is a ubiquitously expressed transcriptional factor that belongs to the eap'n'collar family of basic leucine-zipper(b-ZIP)proteins.The precise mechanism of Nrf2 in tumorigenesis has been actively explored in vitro and in vivo because of the pivotal role of Nrf2 as a defense mechanism under oxidative stress.Highly constitutive activation of Nrf2 is associated with increased risk of various human cancers including lung cancer and acute myeloid leukemia(AML).To date,many Nrf2 inhibitors,such as all-trans retinoic acid,retinoic acid receptor a agonists,luteolin and brusatoL have been reported.In this scenario,discovery or synthesis of more Nrf2 inhibitors will be an attractive therapeutic strategy to improve cancer therapyThe pyrazole structure is a core scaffold in medicinal chemistry,with multiple modification sites in its five-membered heterocyclic ring.Pyrazole and its derivatives have been highlighted for the development of novel anticancer therapeutics.Hydroxamic acid derivatives exert properties of antioxidation,anti-inflalmation,antimicrobe and antitumor.Among the hydroxamic acid derivatives,trichostatin A and a novel acetohydroxanic acid SAHA have been applied in tumor clinical therapy.Nonetheless,the antitumor effect of pyrazolyl hydroxamic acid derivatives has remained unclear.Hence,based on the previous work,we synthesized a series of novel pyrazolyl hydroxamic acid derivatives and identified a novel Nrf2 inhibitor,1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide(4f),Furthermore,we investigated the growth-inhibitory effects on three AML cell lines(THP-1,HL-60 and U937)and non-small cell lung cancer(NSCLC)cell line A549.The results revealed that comound 4f inhibited cancer cell growth via triggering apoptosis or autophagy.Therefore,as a promising lead compound for novel chemotherapeutics,the pyrazolyl hydroxamic acid derivative 4f may be provide the new approach for cancer therapy.STUDY CONTENTSL.To identify the pyrazolyl hydroxamic acid derivatives with inhibiting Nrf2 activity2.To investigate the inhibitory effects and mechanism of compound 4f on the growth of three AML cell lines(THP-1,HL-60 and U937)3.To screen the pyrazolyl hydroxamic acid derivatives with growth-inhibitory effect onA549 cells and investigate the biological activities4.To clarify the possible action mechanism of compound 4f in autophagy regulationMETHODS1.Cell cultureAll cells,including human AML cell lines(THP-1,HL-60 and U937),human NSCLC cell line(A549),human cervical carcinoma cells(HeLa)stably expressing an antioxidant response element(ARE)-luciferase reporter plasmid,human glioblastoma cell line(U87)stably expressing a green fluorescent protein-microtubule associated protein light chain 3(GFP-LC3)construct,human umbilical vein endothelial cells(HUVECs)and SV40 T-antigen imortalized murine islet endothelial cells(MSI),were cultured in normal condition.2.Identification of Nrf2 inhibitors2.1 The Nrf2 activity was examined by use of the luciferase assay system.2.2 The expression of NrfZ downstream target genes,heme oxygenase 1(HO-1)and glutamate cysteine ligase catalytic subunit(GCLC),was examined by RT-PCR.2.3 The protein level of Nrf2 was detected by Western blot in the presence of 4f.3.Determination of the growth-inhibitory effects of the pyrazolyl hydroxamic acid derivatives on cancer cellsin vitro3.1 Cell viability assayCell viability of three AML cell lines was assessed by using Cell Counting Kit-8(CCK-8)kit;other cell viability was assessed by Sulforhodamine B(SRB)or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay.3.2 Detection of apoptosis1)Apoptosis in AML cells was examined by flow cytometry(FCM).2)Cleavage of caspase-3 and cleavage of poly(ADP-ribose)polymerase(PARP)in AML cells were detected by Western blot.3)Apoptotic cells in tumor sections were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay.4)Chromatin condensation and DNA fragmentation in A549 cells was detected by Hoechst 33258 staining.3.3 Detection of cell cycle distributionThe cell cycle distribution was examined by FCM analysis.3.4 Detection of autophagy1)Acidic vesicles accumulated in A549 cells were detected by AO staining.2)LC3-? and p62 protein levels were detected by Western blot.3)U87 human glioblastoma cells stably expressing GFP-LC3 were applied to monitor the formation of autophagosomes by use of irnmunocytochemical method.3.5 Detection of necrosis1)Necrosis in AML cells was examined by FCM.2)The activity of lactate dehydrogenase(LDH)was examined using a LDH kit in A549 cells.3.6 Detection of mitochondrial apoptotic signaling pathway:1)B-cell lymphoma-2(Bce-2)mRNA level was detected by RT-PCR.2)The protein levels of Bcl-2 and Bcl-2-associated X protein(Bax)were exam:ined by Western blot.4.Detection of the inhibitory effect of compound 4f on tumor growth and blood vessel development in vivo4.1 A chicken embryo model was used to evaluate the effect of compound 4f on tumor growth.4.2 Fertile gelatin sponges were placed onto the chick embryo chorioallantoic membrane(CAM)to evaluate the effect of 4f on blood vessel development in vivo.5.Detection of intracellular redox balance influenced by compound 4f5.1 Detection of GSH The reduced GSH level was detected by a probe specific to reduced GSH.5.2 Detection of GSH synthesis The mRNA levels of GCLC and GCLM were examined by RT-PCR.6.Detection of the relationship between Nrf2 and apoptosis induced by compound 4f6.1 Cell viability,cleaved caspase-3 and cleaved PARP protein levels were examined in THP-1 cells after treatment with an Nrf2 activator tert-butylhydroquinone(tBHQ).6.2 Cell viability,cleaved caspase-3 and cleaved PARP protein levels were determined in Nrf2-overexpressed THP-1 cells.7.Detection of the relationship between GSH and autophagy induced by compound 4fWestern blot was used to detect LC3 protein level when cells were treated with N-acetyl-L-cysteine(NAC)in absence or presence of compound 4f in A549 cells.RESULTS1.Pyrazolyl hydroxamic acid derivative 4f is a novel Nrf2 inhibitorCompound 4f(1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide)at 5 and 10 ?M(12 and 24 h)inhibits Nrf2 activity by use of an ARE-lukiferase reporter approach from a series of novel pyrazolyl hydroxamic acid derivatives(4a-41).The mRNA levels of HO-1 and GCLC were decreased by compound4f in Hela cells.2.The pyrazolyl hydroxamic acid derivative 4f induces apoptosis via inhibiting Nrf2 in AML cells2.1 Compound 4f(5 and 10 ? M)had a profound growth-inhibitory effect on three AML cell lines,THP-1,HL-60 and U937.The values of IC50 were 5.33,8.94 and 8.98?M,respectively.2.2 Flow cytometry of AML cells revealed increased apoptosis with 4f(10 ?M)treatment for 48 h.The protein levels of cleaved caspase-3 and cleaved PARP were enhanced,and cell cycle was arrested at S phase in all three AML cell types.2.3 We found a similar anti-growth effect of 4f in a chick embryo model and more apoptotic cells in tumor sections in comparison with control group.2.4 Nrf2 protein level was downregulated by 4f(5 and 10 ?M,12 and 24 h).And upregulation of Nrf2 by tBHQ or Nrf2 overexpression could ameliorate 4f-inducedcell growth inhibition and apoptosis.2.5 Treatment with 4f reduced both Bcl-2 expression and Bcl-2/Bax ratio,which indicated that 4f induces apoptosis,at least in part,via mitochondrial-mediated signaling pathway.2.6 Compound 4f suppressed the growth of HUVECs and MS1 cells in vitro and blood vessel development in vivo.3.The pyrazolyl hydroxamic acid derivatives inhibit A549 cell growth3.1 The series of derivatives 4a-41(5 and 10?M,24 and 48 h)had inhibitory effects on the growth of A549 cells in dose-and time-dependent manners.Comopounds 4b,4f,4h and 4j had more effective anti-growth activity and IC50 values were 1.72,1.82,4.21 and 2.56 ?M,respectively.3.2 The series of compounds 4a-41 at 10 ?M resulted in acidic vesicles accumulation,but no obvious chromosomal condense and DNA fragment in A549 cells.3.3 Treatment with compound 4j at 10 ?M for 48 h caused LDH release,indicating that necrosis may be involved in the process.However,the similar effect was not observed in other groups.3.4 Compound 4b,4f or 4h blocked A549 cell cycle at G1 phase.3.5 Compound 4b,4f or 4h promoted LC3-? conversion to LC3-? and decreased p62 level,demonstrating that autophagy is induced in A549 cells.4.The mechanism of the pyrazolyl hydroxalnic acid derivative 4f in regulating autophagy in A549 cells4.1 Compounds 4f increased GFP-LC3 puncta in U87 cells and LC3-? protein level in time-and dose-dependent manners in A549 cells.4.2 The phosphorylation of p70S6K and 4EBP1,two downstream targets of mTOR,was suppressed following exposure to 4f(5 and 10 ?M,6 and 12 h).Hence,compound 4f triggers autophagy in A549 cells through mTOR signaling pathway.4.3 Compound4f decreased Nrf2 protein level in A549 cells.4.4 Compound 4f led to GSH depletion and downregulated GCLC and GCLM gene expression,suggesting that GSH synthesis is suppressed by 4f.Treatment with NAC could partially inhibit 4f-induced GSH depletion and LC3-? increase.Therefore,GSH is involved in 4f-induced autophagy.CONCLUSIONS1.The pyrazolyl hydroxamic acid derivative 4f,1-(4-(tert-Butyl)benzyl)-3-(4-chlorophenyl)-N-hydroxy-1H pyrazole-5-carboxamide,is identified as a novel Nrf2 inhibitor.2.Compound 4f inhibits Nrf2 and triggers apoptosis via mitochondria signaling pathway in three AML cells(THP-1,HL-60 and U937).3.The pyrazolyl hydroxamic acid derivatives 4b,4f and 4h exert better effects on A549 cell growth inhibition,block cell cycle progression and induce autophagy.4.Compound 4f suppresses Nrf2 and subsequent GSH de novo synthesis,which ultimately leads to intracellular redox unbalance and induces mTOR-dependent autophagy inA549 cells.