Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression

ORIGINAL ARTICLE

Inhibition of the Nrf2 transcription factor by the alkaloidtrigonelline renders pancreatic cancer cells more susceptibleto apoptosis through decreased proteasomal gene expressionand proteasome activityA Arlt1,4, S Sebens2,4, S Krebs1, C Geismann1, M Grossmann1, M-L Kruse1, S Schreiber1,3 and H Schafer1

Evidence accumulates that the transcription factor nuclear factor E2-related factor 2 (Nrf2) has an essential role in cancerdevelopment and chemoresistance, thus pointing to its potential as an anticancer target and undermining its suitability inchemoprevention. Through the induction of cytoprotective and proteasomal genes, Nrf2 confers apoptosis protection in tumorcells, and inhibiting Nrf2 would therefore be an efficient strategy in anticancer therapy. In the present study, pancreatic carcinomacell lines (Panc1, Colo357 and MiaPaca2) and H6c7 pancreatic duct cells were analyzed for the Nrf2-inhibitory effect of the coffeealkaloid trigonelline (trig), as well as for its impact on Nrf2-dependent proteasome activity and resistance to tumor necrosisfactor-related apoptosis-inducing ligand (TRAIL) and anticancer drug-induced apoptosis. Chemoresistant Panc1 and Colo357cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strongtert-butylhydroquinone (tBHQ)-inducible Nrf2 activity and drug resistance. Trig efficiently decreased basal and tBHQ-induced Nrf2activity in all cell lines, an effect relying on a reduced nuclear accumulation of the Nrf2 protein. Along with Nrf2 inhibition, trigblocked the Nrf2-dependent expression of proteasomal genes (for example, s5a/psmd4 and a5/psma5) and reduced proteasomeactivity in all cell lines tested. These blocking effects were absent after treatment with Nrf2 siRNA, a condition in which proteasomalgene expression and proteasome activity were already decreased, whereas siRNA against the related transcription factor Nrf1 didnot affect proteasome activity and the inhibitory effect of trig. Depending on both Nrf2 and proteasomal gene expression, thesensitivity of all cell lines to anticancer drugs and TRAIL-induced apoptosis was enhanced by trig. Moreover, greater antitumorresponses toward anticancer drug treatment were observed in tumor-bearing mice when receiving trig. In conclusion, representingan efficient Nrf2 inhibitor capable of blocking Nrf2-dependent proteasome activity and thereby apoptosis protection in pancreaticcancer cells, trig might be beneficial in improving anticancer therapy.

Oncogene (2013) 32, 4825–4835; doi:10.1038/onc.2012.493; published online 29 October 2012

Keywords: chemoresistance; oxidative stress; tumorigenesis; pancreas

INTRODUCTIONA great number of malignant tumors,1–11 for example, colonic,thyroid, endomethrial, lung, ovarian, breast and pancreatic cancer,exhibit an increased activity of the antioxidative transcriptionfactor nuclear Factor E2-related factor 2 (Nrf2). This enhanced Nrf2activation has been shown to originate from rare gain-of-functionmutations of Nrf2 itself12,13 and from loss-of-function mutations,promoter hypermethylation or micro RNA targeting of theNrf2 inhibitory protein Keap1/INRF.14–16 Besides these geneticand epigenetic alterations, an exaggerated Nrf2 activity may alsoresult from cellular adaptation to metabolic stress, for example,fumarate accumulation leading to Keap1 succination17,18 or tooxidative stress,19–21 for example, emerging along with persistentinflammation during chronic colitis or pancreatitis. As a conse-quence of the enhanced Nrf2 activity, tumor cells acquire protec-tion from apoptosis1,20–22 and are more capable of proliferation,

both conditions favoring tumorigenesis on one hand and makingtumor cells more refractory to chemo- and radiotherapy on theother hand.2,5,23 By inducing a battery of phase II enzymes anddetoxification genes that protect cells from anticancer drugtoxicity, Nrf2 directly confers chemoresistance, as quite recentlyreported for several types of tumors,24,25 including pancreaticadenocarcinoma (PDAC).3,4,9 A recent study also identified a roleof NRF2 in promoting tumor angiogenesis through the HIF-1a/VEGF pathways, not only underscoring the potential of Nrf2 tosustain tumor growth and survival,26 but also providing aninteresting insight into how hypoxia-related signals and oxidativestress adaptation may be linked to each other.

Another mode of action by which Nrf2 activation favorstumorigenesis relates to the induction of proteasomal geneshaving an impact on the ubiquitine–proteasome signaling path-way. It is well known that alterations in proteasome activity, which

1Department of Internal Medicine I, Laboratory of Molecular Gastroenterology & Hepatology, UKSH, Kiel, Germany; 2Institute of Experimental Medicine, InflammatoryCarcinogenesis Research Group, Kiel, Germany and 3Institute of Clinical Molecular Biology, UKSH, Kiel, Germany. Correspondence: Professor H Schafer, Department of InternalMedicine I, Laboratory of Molecular Gastroenterology & Hepatology, Bldg. 6, UKSH-Campus Kiel, Schittenhelmstr. 12, Kiel, Schleswig-Holstein, D-24105, Germany.E-mail: [email protected] authors contributed equally to this work.This work is part of a doctoral thesis (SK).Received 13 December 2011; revised 30 August 2012; accepted 13 September 2012; published online 29 October 2012

Oncogene (2013) 32, 4825–4835& 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13

www.nature.com/onc

is crucial for cellular homeostasis and regular cell growth, areinvolved in tumor development. Upregulation of proteasomalgene expression in tumors has been previously reported,1,27–30

and in this context Nrf2 has an important role owing to itsinducing effect on proteasomal gene expression and thereby on26S/20S proteasome activity.1,31–34 Recent studies identified anumber of proteasomal genes (for example, a5/PSMA5, PSMB5,b1/PSMB6 and s5a/PSMD4) transcriptionally regulated by Nrf2through one or multiple antioxidant response elements (ARE)within their gene promoters.35,36 In colon cancer, greater Nrf2activity and increased proteasomal gene expression correlate withan elevated proteasome activity,1 and human colonocytes andcolon cancer cells acquire an increased apoptosis protectionfrom elevated proteasomal gene expression and proteasomeactivation.1,28,30

Thus, evidence has accumulated that Nrf2 exhibits profoundprotumorigenic activity37,38 and its targeting may therefore havegreat potential in antitumor therapy,23,39 particularly in over-coming chemoresistance, for example, in PDAC. However, Nrf2 hasalso gained attention in terms of its use as a chemopreventivetarget, because the activation of Nrf2 by certain antioxidantssuch as sulforaphane and oltipraz leads to protection from toxicDNA damage and thereby from tumorigenesis.40,41 Obviously,in normal cells exhibiting tightly controlled Nrf2 activation, theimpact of Nrf2 is rather preventive against cancer, but inpermanently stressed or even transformed cells exhibitingderegulated Nrf2 activity its impact is rather protumorigenic.37,42

Besides the induction of phase II enzyme expression through Nrf2,accounting for direct detoxification of anticancer drugs, theimpact of Nrf2 on the ubiquitine–proteasome signaling pathwayconfers general apoptosis resistance that manifests in protectionfrom various apoptotic stimuli, for example, death ligands, and inenhanced proliferation. One goal therefore would be to establishnovel compounds that selectively block Nrf2 in tumor cells.Recently, certain signaling pathways—for example, throughretinoic acid receptor alpha or estrogen-related receptor beta—have been described that interfere with Nrf2 activation,43,44 but inPDAC cells these nuclear receptors are not expressed. Thus, othernatural compounds that have been identified to inhibit Nrf2, forexample, the alkaloid trigonelline (trig),45 would be attractive toolsfor the sensitization of tumor cells to apoptosis.

In the present study, we elucidated the effects of trig on Nrf2-dependent proteasome activity and antiapoptotic protection inPDAC cell lines (MiaPaca2, Panc1 and Colo357), as well as in thehuman pancreatic duct cell line H6c7. It could be shown that, in allcell lines investigated, trig efficiently suppressed Nrf2 activityalong with a decreased proteasome activity, leading to anelevated sensitivity to apoptosis induction in vitro and in vivo,indicating its suitability as an Nrf2 inhibitor adding to anticancertherapy.

RESULTSBasal and inducible Nrf2 activity in PDAC and H6c7 cellsMiaPaca2, Panc1 and Colo357 PDAC cells, as well as the humanpancreatic duct cell line H6c7, were analyzed for basal andinducible Nrf2 activity. Western blot analysis revealed greaternuclear protein level of Nrf2 (visible as 100 kDa band) in Panc1 andColo357 cells as compared with MiaPaca2 and H6c7 cells(Figure 1a). Treatment with 50mM tBHQ for 8 h increased theamount of Nrf2 in nuclear extracts from all cell lines (Figure 1a),exerting the greatest effect in MiaPaca2 and H6c7 cells. Similarly,ARE-luciferase assays demonstrated (Figure 1b) that untreatedPanc1 and Colo357 cells possess greater Nrf2 activity thanMiaPaca2 and H6c7 cells. Upon treatment with tBHQ for 8 h, allfour cell lines exhibited an increase of ARE-driven luciferaseactivity that was strongest in MiaPaca2 and H6c7 cells.

Effect of trigonelline on Nrf2 activity in PDAC and H6c7 cellsARE-luciferase assays conducted with Panc1 and Colo357 cellssubject to trig treatment at various concentrations (0.01–10 mM) for16 h revealed a dose-dependent inhibition of ARE-driven lucifer-ase expression by trig. The greatest inhibition was seen atconcentrations between 0.1 and 1 mM (Figure 1c). Similarly,ARE-driven luciferase expression could be dose-dependentlyinhibited by trig in all cell lines treated with 50 mM tBHQ for 8 hand preincubated (1 and 16 h) with trig before tBHQ administra-tion. The 16-h preincubation with trig was less efficient than the1-h preincubation. At the latter time point and at a dose of 0.1 mM,most significant inhibitory effects were observed in MiaPaca2 cellsexhibiting a 54% decrease of ARE-driven luciferase expression incomparison with vehicle-treated cells. In the other cell lines, theinhibitory effect of trig at 0.1 mM was somewhat lower, rangingbetween 35 and 50% inhibition. At higher doses (1 and 10 mM), theinhibition of ARE-driven luciferase by trig was not stronger or evenless pronounced.45

Trig affects nuclear localization of Nrf2 in PDAC and H6c7 cellsTo elucidate the mechanisms by which trig interferes with Nrf2activation, its effect on the subcellular distribution of Nrf2 wasinvestigated. The inhibitory impact of trig on Nrf2 could beverified by a decline in the nuclear level of Nrf2 protein that wasseen in Panc1 and Colo357 cells treated with the compound for8 h (Figure 2a). Similarly, H6c7 and MiaPaca2 cells stimulated withtBHQ exhibited a decreased accumulation of Nrf2 protein in thenucleus when subjected to pretreatment with trig (Figure 2b). Incontrast, no differences in the amount of Nrf2 protein weredetected in total cellular extracts (Figures 2a and b) Thus, thehigher basal and induced level of nuclear Nrf2 protein in Panc1 orColo357 cells and H6c7 or MiaPaca2 cells, respectively, weremarkedly affected by trig, indicating that the effect of the drugon the decline in Nrf2-driven gene transcription was relatedto a decrease in its nuclear localization. Immunofluorescencemicroscopy verified the reduced nuclear accumulation of Nrf2protein in the PDAC cell lines and H6c7 cells when subjected totrig treatment (Supplementary Figure S1).

When treated with leptomycin-B (LMB), an inhibitor of thecrm1-dependent nuclear export of proteins, tBHQ-stimulatedPanc1, Colo357, H6c7 and MiaPaca2 cells still exhibited lowerlevels of nuclear Nrf2 protein after administration of trig, whereasprotein levels of Keap1 were not affected (Figure 2c, lower panel).Therefore, the effect of trig did not depend on the nuclear exportof Nrf2 and/or Keap1,46 but rather on the import of Nrf2 into thenucleus. This is underlined by the increased level of Nrf2 protein inthe cytoplasm if trig was administered to LMB- and tBHQ-treatedcells (Figure 2c, upper panel). Similarly, ARE-luciferase assaysrevealed a decreased ARE-driven reporter gene expression in allfour cell lines subjected to LMB and tBHQ treatment if trig hadbeen administered (Figure 2d).

Effect of trig on proteasome activity in PDAC and H6c7 cellsNext, we investigated the effect of trig on basal and tBHQ-inducedproteasome activity. For this purpose, PDAC and H6c7 cellssubjected to tBHQ treatment or not and in the absence orpresence of trig were submitted to the Suc-LLVY-AMC proteasomeassay. As shown in Figure 3a, proteasome activity was induced bytBHQ in all the tested cell lines. In the presence of 100 nM trig,basal proteasome was significantly suppressed in Colo357 andPanc1 cells (39% and 57%, respectively), and tBHQ-inducedproteasome activity was markedly reduced by trig in all the celllines. This effect was most prominent in H6c7, MiaPaca2and Colo357 cells (58%, 64% and 72% decrease, respectively)and somewhat lower in Panc1 cells (36% decrease).

To confirm the Nrf2 dependency of the blocking effect of trigon proteasome activity, all cell lines were transfected with control

Nrf2 inhibition and pancreatic cancerA Arlt et al

4826

Oncogene (2013) 4825 – 4835 & 2013 Macmillan Publishers Limited

and Nrf2 siRNA and the proteasome activity was determined. Asshown in Figure 3b, tBHQ-induced proteasome activity wassignificantly lower in Nrf2 siRNA-treated H6c7, MiaPaca2, Colo357and Panc1 cells (28%, 41%, 29% and 32% decrease, respectively)when compared with control siRNA-treated cells. Notably, theaddition of 100 nM trig only marginally affected tBHQ-inducedproteasome activity in all the cell lines subjected to Nrf2 siRNAtreatment. Moreover, transfection of H6c7, MiaPaca2, Colo357 andPanc1 cells with siRNA targeting the related transcription factorNrf147 did not significantly affect tBHQ-induced proteasome activityin Colo357 and Panc1 cells (Figure 3b) or even slightly increasedproteasome activation in H6c7 and MiaPaca2 cells. In contrast toNrf2 knockdown, after silencing of Nrf1 expression, trig treatmentwas still effective in the inhibition of tBHQ-induced proteasomeactivity in H6c7, MiaPaca2, Colo357 and Panc1 cells (44%, 45%, 31%and 27% decrease, respectively).

Altered Nrf2-dependent expression of proteasomal genes in PDACand H6c7 cells subjected to treatment with trigTo elucidate whether the effect of trig on Nrf2-dependentproteasome activation relates to alterations of proteasomal geneexpression, real-time PCR was conducted for the analysis of theexpression of the proteasomal 19S and 20S subunit proteins S5a(psmd4) and a5 (psma5), respectively. As shown in Figure 4a, the

basal expression level of S5a and a5 were significantly lower inColo357 and Panc1 cells subjected to trig treatment as comparedwith untreated cells. Similarly, the increase of s5a and a5 mRNAlevels in response to tBHQ treatment was suppressed by trig inMiaPaca2 and H6c7 cells. A similar alteration in response to trigtreatment was seen when analyzing the expression of glutamate–cysteine ligase catalytic subunit as a member of phase II enzymesamong the Nrf2 target genes.

Western blot analysis confirmed the trig-dependent declineof s5a and a5 expression in Colo357 and Panc1 cells, as well as intBHQ-treated MiaPaca2 and H6c7 cells (Figure 4b).

The impact of trig on TRAIL and etoposide-induced apoptosis inPDAC and H6c7 cellsTo study the impact of the suppressed Nrf2 activation on deathligand or anticancer drug-induced apoptosis, Colo357 and Panc1cells were treated with 100 nM trig for 1 h followed by theadministration of tumor necrosis factor-related apoptosis-inducingligand (TRAIL) or etoposide, and apoptosis was determined byassaying caspase 3/7 activation. Etoposide was used becausethis anticancer drug exerts the greatest sensitization in manychemoresistant PDAC cell lines.48,49 Whereas both cell lines per sewere only moderately sensitive to TRAIL-induced apoptosis(Figure 5a), exhibiting an increase of caspase 3/7 activation by

Figure 1. Nrf2 activity in PDAC and H6c7 cells and its inhibition by trig. (a) Nuclear extracts from the indicated cell lines either left untreated ortreated with 50mM tBHQ for 8 h were submitted to western blotting for the detection of Nrf2 and Hsp90 as loading control. A representativeout of four independent experiments is shown. (b) The indicated cell lines were transfected with either an empty or an ARE-driven fireflyluciferase (ff ) construct together with a constitutively renilla luciferase (rl)-expressing construct. Cells were either left untreated or weretreated with 50mM tBHQ for 8 h. Then, luciferase activity was determined and relative luciferase units (RLU) were calculated from the ff/rl ratios.Data are expressed as n-fold activity of ARE-driven RLU normalized to the empty vector RLU. Mean values±s.d. from four independentexperiments are shown; *Po0.05. (c) The indicated cell lines were transfected with either an empty or an ARE-driven firefly luciferase (ff )construct together with a constitutively renilla luciferase (rl) expressing construct. Then, the cells were treated with trig at the indicated dosesfor 16 h or not (left panel), or cells were treated with 50 mM tBHQ for 8 h either preincubated (� 1 h and � 16 h, respectively) with trig at theindicated doses, or not (center & right panels). Luciferase activity was determined and relative luciferase units (RLU) were calculated from theff/rl ratios. Data are expressed as the percentage of specific ARE-driven RLU normalized to the empty vector RLU. Mean values±s.d. from sixindependent experiments are shown; *Po0.05, **Po0.01.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4827

& 2013 Macmillan Publishers Limited Oncogene (2013) 4825 – 4835

1.8 and 2.7-fold, respectively, pretreatment with trig signifcantlyenhanced apotosis by TRAIL (2.8 and 4.3-fold increase of caspase3/7 activity, respectively). Treatment with etoposide elicited a 1.4and 1.8-fold increase in caspase3/7 activity in Colo357 and Panc1cells (Figure 5a), respectively, and preincubation with trig led to anincreased apoptosis response to etoposide (2.3 and 3.1-fold highercaspase3/7 activity, respectively). Moreover, tBHQ pretreatment ofMiaPaca2 and H6c7 cells significantly reduced apoptoticresponses to TRAIL (from 5.5 to 3.6 and 4.4 to 3.5-fold caspase3/7 activity, respectively) and to etoposide (from 6.7 to 4.5 and 4.8to 3.6-fold caspase 3/7 activity, respectively). In the presence oftrig, tBHQ-induced protection from TRAIL and etoposide-inducedapoptosis were diminished (Figure 5b).

To confirm these apoptosis effects, poly(ADP-ribose)–polymerase 1(PARP1) cleavage was analyzed by western blotting. As shown inFigure 5a (lower panels), PARP1 cleavage after etoposide and TRAILtreatment was enhanced by trig in Colo357 and Panc1 cells.Moreover, the decrease of etoposide- and TRAIL-induced PARP1

cleavage in MiaPaca2 and H6c7 cells after tBHQ treatment wasblocked by pretreatment with trig (Figure 5b, lower panels).

The apoptosis-sensitizing effect of trig depends on Nrf2 andproteasomal gene expressionTo elucidate whether the apoptosis-sensitizing effect of trigdepends on Nrf2 and proteasomal gene expression, Panc1and MiaPaca2 cells were chosen for knockdown experiments.After transfection with Nrf2 siRNA, the sensitivity of Panc1 cellstoward TRAIL or etoposide-induced apoptosis was enhanced, asshown by a greater caspase 3/7 activation (Figure 6a). Theaddition of trig was not capable of substantially increasing TRAIL-or etoposide-induced apoptosis in Nrf2 siRNA-transfected cellsany further, as shown by similar rates of caspase 3/7 activity intrig-treated and untreated cells subjected to Nrf2 knockdown(Figure 6a). In addition, the diminishing effect of tBHQ treatmenton TRAIL- and etoposide-induced apoptosis in MiaPaca2 cells was

Figure 2. Trig treatment decreases nuclear protein level of Nrf2 independent of its nuclear export. (a) Panc1 and Colo357 cells were treatedwith 0.5mM trig (trig) for 24 h, or (b) H6c7 and MiaPaca2 cells were treated with 50 mM tBHQ for 8 h after preincubation with trig (0.5mM) for 1 hor not. Then, total cell lysates or nuclear extracts were prepared and submitted to western blots for the detection of Nrf2 and Hsp90 or lamin-A/C. Representative results of two independent experiments are shown. (c) Panc1, Colo357, MiaPaca2 and H6c7 cells were treated with 50 mMtBHQ for 8 h in the presence of 20 ng/ml of the crm1 inhibitor LMB, either preincubated with trig (0.5 mM) or not. Then, cytosolic (c) or nuclear(n) extracts were prepared and submitted to western blots for the detection of Nrf2, Keap1 and tubulin or lamin-A/C. Representative results oftwo independent experiments are shown. (d) Panc1, Colo357, MiaPaca2 and H6c7 cells either transfected with an empty or an ARE-drivenfirefly luciferase vector were treated with 50 mM tBHQ for 8 h in the presence of 20 ng/ml LMB, either preincubated with trig (0.5 mM) or not.Then, luciferase activity was determined and relative luciferase units (RLU) were calculated from the ff/rl ratios. Data are expressed as thepercentage of specific ARE-driven RLU normalized to the empty vector RLU. Mean values±s.d. from three independent experiments areshown, *Po0.05.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4828

Oncogene (2013) 4825 – 4835 & 2013 Macmillan Publishers Limited

less pronounced after knocking down Nrf2 expression, as shownby greater caspase3/7 activation in response to both apoptosisstimuli (Figure 6b). Moreover, the addition of trig under Nrf2knockdown was not capable of substantially increasing TRAIL- or

Figure 3. Effect of trig on proteasome activity in PDAC and H6c7cells. (a) The indicated cell lines were either left untreated or weretreated with 50mM tBHQ for 24 h, in the absence or presence of0.1mM trig, added 1h before. Then, the cells were submitted to theSuc-LLVY-AMC proteasome assay. Fluoresence units were normal-ized to the protein content determined in parallel. Data areexpressed as n-fold AMC activity/mg protein and represent meanvalues±from six indepedent experiments; *Po0.05. (b) Theindicated cell lines were treated with control, Nrf2 and Nrf1 siRNAs.After 48 h, cells were either left untreated or were treated with 50 mMtBHQ in the absence or presence of 0.1mM trig. The efficacy of thesiRNAs was validated by qPCR analysis (Supplementary Figure S5).Data are expressed as n-fold AMC-activity/mg protein and representmean values±s.d. from four indepedent experiments, *Po0.05.

Figure 4. Altered Nrf2-dependent expression of proteasomal genesin PDAC and H6c7 cells subjected to treatment with trig. (a, b)Colo357 and Panc1 cells were treated with 0.1mM trig alone for16 h, or MiaPaca2 and H6c7 cells were treated with 50 mM tBHQ for16 h, or not, either in the absence or presence of 0.1mM trig.(a) Total RNA was submitted to reverse transcription and real-timePCR for the detection of the proteasomal genes s5a and a5, as wellas the phase II enzyme glutamate–cysteine ligase catalytic subunit(GCLC). For normalization, beta actin was analyzed as control. Dataare expressed as normalized mRNA level and represent the meanvalues±s.d from four independent experiments performed induplicate; *Po0.05. (b) Total cell lysates were submitted to westernblotting for the detection of s5a and a5 proteins, as well as Hsp90 asloading control. A representative result out of four is shown.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4829

& 2013 Macmillan Publishers Limited Oncogene (2013) 4825 – 4835

etoposide-induced apoptosis in tBHQ-treated MiaPaca2cells. Similar findings were obtained when analyzing Panc1and MiaPaca2 cells by PARP1 western blotting (SupplementaryFigure S2).

When transfecting Panc1 cells with s5a or a5 siRNA, theapoptosis in response to TRAIL and etoposide was significantlyincreased as compared with control siRNA-transfected cells,and the addition of trig only weakly enhanced TRAIL- oretoposide-dependent caspase 3/7 activation (Figure 7a). Similarly,MiaPaca2 cells subjected to tBHQ treatment were more sensitiveto TRAIL- and etoposide-induced apoptosis if s5a and a5

expression was knocked down. Moreover, tBHQ-treated MiaPaca2cells were almost refractory to trig (Figure 7b) when treatedwith s5a or a5 siRNA. Similar findings were obtained whenanalyzing Panc1 and MiaPaca2 cells by PARP1 western blotting(Supplementary Figure S3).

Greater antitumor response through combined trig/etoposidetreatment in Colo357 and Panc1 tumor bearing-SCID–beige miceTo verify the chemotherapy sensitizing activity of trig, asubcutaneous xenograft tumor model in female severe combined

Figure 5. Impact of trig on TRAIL- and etoposide-induced apoptosis in PDAC and H6c7 cells. (a) Colo357 and Panc1 cells were treated with0.1mM trig alone for 16 h, or (b) MiaPaca2 and H6c7 cells were treated with 50 mM tBHQ for 16 h, or not, either in the absence or presence of0.1mM trig. Then, the cells were either left untreated or were treated with 20 mg/ml etoposide or 10 ng/ml TRAIL for 24 h and 6 h, respectively.Apoptosis was determined by analyzing caspase 3/7 activity (upper panel) or by western blot analysis of PARP1 cleavage (lower panel) usingtubulin as loading control. Data are expressed as n-fold caspase 3,7 activity normalized to the cellular protein content and represent the meanvalue±s.d. from six independent experiments performed in duplicate; *Po0.05. The western blots are representative of three independentexperiments.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4830

Oncogene (2013) 4825 – 4835 & 2013 Macmillan Publishers Limited

immune deficiency (SCID)-beige mice was employed usingColo357 and Panc1 cells. Three weeks after tumor cell inoculation,when the tumors had reached a size of 5� 5 mm2 or more, tumor-bearing animals were treated (i.p) either with 0.9% NaCl only (ascontrol), etoposide alone (10 mg/kg body weight) or with acombination of etoposide and trig (1 mg/kg body weight). Duringand after treatment, the etoposide/trig combination groups(III) exhibited a reduced gain of tumor sizes (Figure 8a)when compared with the NaCl (I)- and etoposide-treated (II)groups, which exhibited continuing tumor growth. After scari-fication of mice, tumor volume (Colo357: 391±155 mm3; Panc1:

113±45 mm3) and weight (Colo357: 336±136 mg; Panc1:84±38 mg) were found to be significantly lower (Figure 8b) inthe combination group as compared with the NaCl group(Colo357: 671±318 mm3 and 549±293 mg, respectively; Panc1:328±203 mm3 and 220±118 mg, respectively) and the etoposidegroup (Colo357: 661±461 mm3 and 536±264 mg, respectively;Panc1: 216±126 mm3 and 124±74 mg, respectively).

In line with these findings and the in vitro data, immunohis-tochemical analysis detected a lower nuclear level of activatedNrf2 protein in tumors from the combination group whencompared with tumors from the other two groups (Figure 8c).

DISCUSSIONPancreatic ductal adenocarcinoma (PDAC) is still one of the mostaggressive and deadly tumors characterized by rapid growth andinfiltration of surrounding tissues. At the time of diagnosis, mostpatients suffer from advanced disease and are already in anincurable state. Besides a surgical intervention accessible for only10–20% of PDAC patients, no other therapeutical measure iscurrently available offering cure from the disease.50 On one hand,this limitation of therapeutic options is due to the rapid andextensive tumor spread of PDAC giving rise to metastases. On theother hand, this is due to the profound resistance of PDAC cellstoward anticancer therapy. This therapy resistance relies on an

Figure 6. The apoptosis-sensitizing effect of trig depends on Nrf2.The indicated cell lines were treated with control or Nrf2 siRNA for48 h. Then, (a) Panc1 cells were treated with 0.1mM trig for 16 hfollowed by adminstration of 20 mg/ml etoposide and 10ng/mlTRAIL for 24 h and 6 h, respectively, or (b) MiaPaca2 cells weretreated with 50mM tBHQ or not for 16 h either in the presence orabsence of 0.1mM trig (added 1 h before), followed by etopside andTRAIL treatment. Apoptosis was determined by analyzing caspase 3/7 activity. Data are expressed as n-fold caspase 3/7 activitynormalized to the cellular protein content and represent the meanvalue±s.d. from four independent experiments performed induplicate; *Po0.05.

Figure 7. The apoptosis-sensitizing effect of trig depends onproteasomal gene expression. The indicated cell lines were treatedwith control, s5a or a5 siRNA siRNA for 48 h. Then, (a) Panc1 cellswere treated with 0.1 mM trig for 16 h followed by the administrationof 20 mg/ml etoposide and 10 ng/ml TRAIL for 24 h and 6h,respectively, or (b) MiaPaca2 cells were treated with 50mM tBHQ ornot for 16 h either in the presence or absence of 0.1mM trig (added1h before), followed by etopside and TRAIL treatment. Apoptosiswas determined by analyzing caspase 3/7 activity. Data areexpressed as n-fold caspase 3/7 activity normalized to the cellularprotein content and represent the mean value±s.d. from fourindependent experiments performed in duplicate; *Po0.05.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4831

& 2013 Macmillan Publishers Limited Oncogene (2013) 4825 – 4835

efficient apoptosis protection, reversible by, for example,proteasome inhibitors,48 as well as on the capability of PDACcells to get rid of the anticancer drugs. Among the mechanisms bywhich tumor cells—including PDAC cells—gain apoptotic protec-tion and exclusion to anticancer drugs, the activation of theKeap1/Nrf2 signaling pathway has an important role that is not yetfully defined. Nrf2, either being induced by the cellular alterationsdescribed above or even by anticancer drugs themselves,24 canlead to protection from anticancer drugs by inducing a battery ofdetoxifying enzymes and proteins, as well as by increasing theproteasome activity and thereby apoptosis protection.1,48

As shown in the present study, a line of PDAC cells, as well asthe human pancreatic duct cell line H6c7, is efficiently protectedfrom apoptosis by basal and/or tBHQ-induced Nrf2 activity. Inaccordance with recent data,4 Colo357 and Panc1 cells arecharacterized by higher basal Nrf2 activity, rendering these cellsless sensitive to chemotherapy-induced apoptosis. This Nrf2-dependent protection includes an increase in proteasomal geneexpression, as well as greater proteasome activity, providing thecells with an accelerated turnover of regulatory proteins involvedin growth regulation and survival.1,22,51 Given this significantimpact of Nrf2 on the resistance of PDAC cells against apoptosis,

inhibition of Nrf2 might be an attractive tool to overcome thisresistance and to sensitize PDAC cells to anticancer therapy.

Thus, we tested the natural compound trig45 for its capacity toinhibit Nrf2 and to sensitize PDAC cells to apoptosis and PDACtumors to chemotherapy. Among all four cell lines tested, trigexerted the greatest inhibitory effect on Nrf2 activity atsubmaximal doses (0.1–1 mM); higher doses were less efficient oreven ineffective. This Nrf2 inhibitory potential of trig following abiphasic dose dependency with the highest efficiency atsubmicromolar doses has been reported recently.45 Ourinvestigations further revealed that trig decreases the nuclearlevel of Nrf2 protein (Figure 2), whereas the overall amount of Nrf2expression was not altered. As the effect of trig on nuclear Nrf2protein level, as well as on ARE-dependent luciferase expression,was still present in all the investigated cell lines if stimulated withtBHQ and treated with the nuclear export inhibitor LMB, one canconclude that trig impairs the nuclear import of Nrf2. Underconditions of Nrf2 activation through its release from Keap1inhibition—here by tBHQ treatment—the inhibitory effect of trigon nuclear accumulation of Nrf2 was still observed even if itscrm1-dependent nuclear export was blocked by LMB.46 Moreover,the addition of the proteasome inhibitor Mg132 (Supplementary

Figure 8. Greater antitumor response through combined trig/etoposide treatment in Colo357 and Panc1 tumor-bearing SCID–beige mice.SCID mice bearing subcutaneously inoculated Colo357 or Panc1 tumors were treated daily (i.p. injection) for 4 days with 0.9% NaCl (group I) orwith 10mg/kg body weight etoposide either alone (group II) or in combination with 1mg/kg body weight trig (group III). One day before,animals already received 0.9% NaCl (group I & II) or trig (group III). After 3 days, the same treatment protocol was applied. (a) Tumor sizes(mm2) were analyzed weekly until the scarification of mice 3 weeks after the start of treatment (the treatment periods are indicated by thearrows). Data are expressed as mean±s.d. (n¼ 6); *Po0.05 compared with group I. At the end of experiment, (b) the volume and weight offreshly excised tumors were determined. Data are expressed as mean±s.d. (n¼ 6); *Po0.05 compared with group I. (c) Tumor cryosectionswere stained with a Phoshpo-Nrf2 (Ser40) antibody detecting activated and mainly nuclear Nrf2. Representative images are shown in thelower panel. Expression scores were calculated and the mean±s.d. are shown (upper panel); *Po0.05 compared with group I.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4832

Oncogene (2013) 4825 – 4835 & 2013 Macmillan Publishers Limited

Figure S4), which prevents Keap1-dependent intranuclear degra-dation of Nrf252,53, did not affect this function of trig. This indicatesthat trig affects the nuclear import of Nrf2, but not its export orintranuclear stability. Along with the strong inhibitory effect of trigon Nrf2 activation, a suppression of proteasome activity was alsonoted with this compound. Notably, siRNA-mediated knockdownof Nrf2 but not of Nrf1 expression abrogated the inhibitory effectof trig on proteasome activity, indicating an exclusive Nrf2dependency under these conditions. This allows to speculatethat the reported role of Nrf1 in proteasome activity47,54 isrestricted to a feedback effect upon proteasome inhibition. In fact,the Nrf1 protein is much more susceptible to proteasomaldegradation than Nrf2.20,47 The latter, however, is particularlyinvolved in the upregulation of the proteasome upon antioxidantstimulation and during cellular stress response.20,55,56

Moreover, proteasomal gene expression was decreased in theinvestigated cell lines subjected to trig treatment. As proteasomeactivation contributes to antiapoptotic protection by Nrf2 intumor cells,1,22,51 not only the cellular response to anticancerdrugs depending on phase II enzyme and detoxification geneexpression, but also the responsiveness to death ligands would beaffected by Nrf2 inhibition. Accordingly, Nrf2-dependent protectionto anticancer drugs and TRAIL-induced apoptosis was significantlyabrogated in all cell lines when pretreated with trig. Besides thesiRNA-mediated knockdown of Nrf2 expression, the knockdown ofproteasomal genes such as a5 and s5a similarly increasedapoptosis and abrogated the sensitizing effect of trig. Thus,the antiapoptotic action of Nrf2 manifests not only through thereported detoxification of anticancer drugs42 but also throughproteasome activation, thereby broadly protecting cells fromapoptosis induction. In this manner, the blockade of Nrf2 byphytochemicals—such as trig—would greatly enhance the tumorkilling effects by TRAIL and anticancer drugs. By using asubcutaneous xenograft tumor model in SCID-beige mice, it couldbe further demonstrated that the chemosensitizing effect of trigalso operates in vivo. Accordingly, Colo357 and Panc1 tumor-bearing mice that had received the combination therapy withetoposide and trig exhibited a significantly greater reduction intumor sizes (Figure 8) than tumor-bearing mice subjected totreatment with the chemotherapeutic drug etoposide alone. Thisfinding underscores the suitability of trig to efficiently enhancethe antitumor response of anticancer drugs.

Hence, trig has the potential to be used in combination therapyof highly resistant tumors such as pancreatic cancer. As no adverseeffects of this natural coffee constituent have been reported so far,and as its suitability for application in humans has been alreadyshown in diabetes and metabolic syndrome patients,57,58 furthertesting of trig in clinical trials seems to be promising.

MATERIALS AND METHODSMaterialsTrig and tert-butylhydroquinone (tBHQ) were purchased from Sigma(Deisenhofen, Germany). LMB and Killer-TRAIL were from Enzo Life-Science/Alexis (Lorrach, Germany). N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyr-osyl-7-amido-4-methylcumarin (Suc-LLVY-AMC) and Mg132 were fromBiomol (Taunusstein, Germany). Etoposide (Vepesid) was from Bristol-Myers/Sqibb (Munich, Germany).

Cell cultureThe human pancreatic ductal epithelial cell line H6c7 (kindly provided byMS Tsao, Ontario Cancer Center, Toronto, Canada) was cultured asdescribed.59 The human PDAC cell lines MiaPaca2, Panc1 (both from ATCC/LSC) and Colo357 (donated by H Kalthoff, Experimental Cancer ResearchInstitute, UKSH-Campus Kiel) were cultured in RPMI 1640 containing 10%FCS, 1% L-glutamine and 1% sodium pyruvate (all from PAA-Laboratories,Colbe, Germany). Cells were cultured at 37 1C, 5% CO2 and 85% humidity.

Fluorometric proteasome assayFor the determination of proteasomal activity in living cells, a fluorometricassay using the proteasome substrate Suc-LLVY-AMC in the absence orpresence of the proteasome inhibitor Mg132 was performed as describedrecently.1 All measurements were recorded in duplicates.

Western blottingNuclear and cytosolic extracts or total cell lysates were prepared asdescribed before.60 After electrophoresis and semidry electroblotting ontoPVDF membranes, the following primary antibodies were used forimmunodetection at 1:1000-fold dilution in 5% (w/v) nonfat milk powderand 0.05% Tween20 in TBS (Tris-buffered saline: 50 mM Tris/HCl, pH 7.6, and150 mM NaCl): Nrf2, Keap1, lamin-A/C and Hsp90 (Santa CruzBiotechnology, Heidelberg, Germany), PARP1 (Cell Signaling Technology,Frankfurt, Germany) and tubulin (Sigma). After incubation overnight at4 1C, the blots were exposed to the appropriate horse-radish peroxidase-conjugated secondary antibody (Santa Cruz) diluted (1:1000) in blockingbuffer and developed using the Dura detection kit (Perbio Sciences, Bonn,Germany). Data acquisition was done with the Chemidoc-XRS geldocumentation system (BioRad, Munich, Germany) using the QuantityOne software (Bio-Rad). Hsp90, lamin-A/C and tubulin served as loading control.

RNA preparation and real-time PCRIsolation of total RNA and reverse transcription into single-stranded cDNAwas carried out as described.20 cDNA was subjected to real-time PCR(iCycler; BioRad) using the SYBR-Green assay20 with gene-specific primersat a final concentration of 0.2mM. The primer sequences and PCRconditions can be appreciated from Supplementary Table S1.

siRNA treatmentFor siRNA (Qiagen, Hilden, Germany) treatment, cells grown in 12-wellplates were submitted to lipofection using 6 ml of the HiPerfect reagent(Qiagen) and 150 ng/well of either negative control siRNA, Nrf2(no. SI03246614), Nrf1 (no. SI00657909), S5a (no. SI03019331) or a-5 siRNA(no. SI100043316).

Dual Luciferase assayARE-driven reporter gene expression in cells was determined using acommercial pathway detection kit (SABioscience/Qiagen) and as describedrecently.20

Caspase-3/-7 assayApoptosis induced by Killer-TRAIL or etoposide was determined by themeasurement of caspase3/7 activity (Promega, Mannheim, Germany)according to the manufacturer’s instructions and as described.20 All assayswere performed in duplicates. Caspase3/7 activity was normalized to theprotein content of the analyzed cell lysates.

Subcutaneous xenograft tumor model in SCID miceColo357 and Panc1 cells (2� 106/200ml saline) were inoculated into theshaved flank of 8-week-old female SCID–beige mice (body weight around20 g; Charles River, Sulzfeld, Germany). When the tumors had reached asize of 5� 5 mm2 or more, the animals were randomized into three groups(n¼ 6) receiving one of the following treatments, which were given byintraperitoneal injections (200ml): group I, 0.9% NaCl (day 1–5 &9–13); group II, 10 mg/kg body weight etoposide in 0.9% NaCl (day 2–5& 10–13); and group III, 0.02 mg/kg body weight trig in 0.9% NaCl (day 1–5& 9–13) and 0.2 mg/kg body weight etoposide in 0.9% NaCl (day 2–5 &9–13). Before, during and after treatment, tumor sizes were measuredweekly until the scarification of mice (day 21 after start of treatment). Next,the tumors were excised, weighed and snap-frozen in liquid nitrogen. Theexperiments were approved by the local animal research authority.

ImmunohistochemistrySix-micrometer cryostat sections were mounted on uncovered glass slides,air-dried overnight at room temperature, fixed in chilled acetone(Merck, Darmstadt, Germany) for 10 min and air-dried again for 10 min.Next, the slides were washed in PBS. To avoid nonspecific binding, sectionswere treated with 4% bovine serum albumin (BSA) (Serva, Heidelberg,Germany) for 20 min followed by incubation with the monoclonal rabbit

Nrf2 inhibition and pancreatic cancerA Arlt et al

4833

& 2013 Macmillan Publishers Limited Oncogene (2013) 4825 – 4835

Ser40-Phospho-Nrf2 antibody (Epitomics, Berlin, Germany) at 1:200 dilutionin 1% BSA/PBS. After primary antibody incubation (overnight, 4 1C), thesections were washed three times in PBS and then treated with EnVisionperoxidase conjugates (DakoCytomation, Hamburg, Germany) for 30 min.Thereafter, the sections were washed three times in PBS. Next, peroxidasesubstrate reaction was performed with the AEC peroxidase substrate kit(DakoCytomation) according to the manufacturer’s instructions. Subsequently,sections were washed in water, counterstained in 50% haemalaun (Merck)and mounted with glycerol-gelatin. The same protocol was performed fornegative controls, either omitting the first antibody or using an isotype-matched control antibody. Evaluation of Nrf2 expression was doneby scoring the percental distribution (0¼ 0%, 1¼ 1–10%; 2¼ 10–50%;3¼ 50–90% and 4¼490%) and expression intensity (0¼ none, 1¼ low;2¼ medium and 3¼ high) in each section. The expression score wascalculated by multiplication of the intensity and distribution scores.

StatisticsThe data represent the mean±standard deviation and wereanalyzed by Student’s t-test; P-values o0.05 were considered statisticallysignificant.

ABBREVIATIONSARE, antioxidant response element; LMB, leptomycin-B; Nrf2 & -1,nuclear factor E2-related factor 2 & -1; PARP1, poly(ADP-ribose)–polymerase 1; PDAC, pancreatic ductal adenocarcinoma; SCID,severe combined immunodeficiency; Suc-LLVY-AMC, N-succinyl-L-leucyl-L-leucyl-L-valyl-L-tyrosyl-7-amido-4-methylcumarin; tBHQ,tertbutylhydroxyquinone; TRAIL, tumor necrosis factor-relatedapoptosis-inducing ligand; trig, trigonelline

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSTechnical assistance by Frauke Grohmann, Dagmar Leisner and Iris Bauer isacknowledged. The project was funded by the German Research Society DFG (SCHA677/9-1), the Sander Foundation (2010.076.1) and the German Cluster of Excellence‘Inflammation-at-Interfaces’.

REFERENCES1 Arlt A, Bauer I, Schafmayer C, Tepel J, Muerkoster SS, Brosch M et al. Increased

proteasome subunit protein expression and proteasome activity in colon cancerrelate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2).Oncogene 2009; 28: 3983–3996.

2 Akhdar H, Loyer P, Rauch C, Corlu A, Guillouzo A, Morel F. Involvement of Nrf2activation in resistance to 5-fluorouracil in human colon cancer HT-29 cells.Eur J Cancer 2009; 45: 2219–2227.

3 DeNicola GM, Karreth FA, Humpton TJ, Gopinathan A, Wei C, Frese K et al.Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumor-igenesis. Nature 2011; 475: 106–109.

4 Hong YB, Kang HJ, Kwon SY, Kim HJ, Kwon KY, Cho CH et al. Nuclear factor(erythroid-derived 2)-like 2 regulates drug resistance in pancreatic cancer cells.Pancreas 2010; 39: 463–472.

5 Jiang T, Chen N, Zhao F, Wang XJ, Kong B, Zheng W et al. High levels of Nrf2determine chemoresistance in type II endometrial cancer. Cancer Res 2010; 70:5486–5496.

6 Kim TH, Hur EG, Kang SJ, Kim JA, Thapa D, Lee YM et al. NRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1alpha.Cancer Res 2011; 71: 2260–2275.

7 Kim SK, Yang JW, Kim MR, Roh SH, Kim HG, Lee KY et al. Increased expression ofNrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancercells. Free Radic Biol Med 2008; 45: 537–546.

8 Konstantinopoulos PA, Spentzos D, Fountzilas E, Francoeur N, Sanisetty S,Grammatikos AP et al. Keap1 mutations and Nrf2 pathway activation in epithelialovarian cancer. Cancer Res 2011; 71: 5081–5089.

9 Lister A, Nedjadi T, Kitteringham NR, Campbell F, Costello E, Lloyd B et al. Nrf2 isoverexpressed in pancreatic cancer: implications for cell proliferation and therapy.Mol Cancer 2011; 10: 37.

10 Singh A, Misra V, Thimmulappa RK, Lee H, Ames S, Hoque MO et al.Dysfunctional KEAP1-NRF2 interaction in non-small-cell lung cancer. PLoS Med2006; 3: e420.

11 Solis LM, Behrens C, Dong W, Suraokar M, Ozburn NC, Moran CA et al. Nrf2 andKeap1 abnormalities in non-small cell lung carcinoma and association withclinicopathologic features. Clin Cancer Res 2010; 16: 3743–3753.

12 Hu Y, Ju Y, Lin D, Wang Z, Huang Y, Zhang S et al. Mutation of the Nrf2 gene innon-small cell lung cancer. Mol Biol Rep 2012; 39: 4743–4747.

13 Kim YR, Oh JE, Kim MS, Kang MR, Park SW, Han JY et al. Oncogenic NRF2mutations in squamous cell carcinomas of oesophagus and skin. J Pathol 2010;220: 446–451.

14 Wang R, An J, Ji F, Jiao H, Sun H, Zhou D. Hypermethylation of the Keap1 gene inhuman lung cancer cell lines and lung cancer tissues. Biochem Biophys ResCommun 2008; 373: 151–154.

15 Eades G, Yang M, Yao Y, Zhang Y, Zhou Q. miR-200a regulates Nrf2 activationby targeting Keap1 mRNA in breast cancer cells. J Biol Chem 2011; 286:40725–40733.

16 Nioi P, Nguyen T. A mutation of Keap1 found in breast cancer impairs its ability torepress Nrf2 activity. Biochem Biophys Res Commun 2007; 362: 816–821.

17 Kinch L, Grishin NV, Brugarolas J. Succination of Keap1 and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinomatype 2. Cancer Cell 2011; 20: 418–420.

18 Adam J, Hatipoglu E, O’Flaherty L, Ternette N, Sahgal N, Lockstone H et al. Renalcyst formation in Fh1-deficient mice is independent of the Hif/Phd pathway:roles for fumarate in KEAP1 succination and Nrf2 signaling. Cancer Cell 2011; 20:524–537.

19 Kim J, Cha YN, Surh YJ. A protective role of nuclear factor-erythroid 2-relatedfactor-2 (Nrf2) in inflammatory disorders. Mutat Res 2010; 690: 12–23.

20 Sebens S, Bauer I, Geismann C, Grage-Griebenow E, Ehlers S, Kruse ML et al.Inflammatory macrophages induce NRF2 dependent proteasome activity incolonic NCM460 cells and thereby confer anti-apoptotic protection. J Biol Chem2011; 286: 40911–40921.

21 Singh S, Vrishni S, Singh BK, Rahman I, Kakkar P. Nrf2-ARE stress responsemechanism: a control point in oxidative stress-mediated dysfunctions and chronicinflammatory diseases. Free Radic Res 2010; 44: 1267–1288.

22 Du ZX, Yan Y, Zhang HY, Liu BQ, Gao YY, Niu XF et al. Proteasome inhibitioninduces a p38 MAPK pathway-dependent antiapoptotic program via Nrf2 inthyroid cancer cells. J Clin Endocrinol Metab 2011; 96: E763–E771.

23 Singh A, Bodas M, Wakabayashi N, Bunz F, Biswal S. Gain of Nrf2 function in non-small-cell lung cancer cells confers radioresistance. Antioxid Redox Signal 2010; 13:1627–1637.

24 Shim GS, Manandhar S, Shin DH, Kim TH, Kwak MK. Acquisition of doxorubicinresistance in ovarian carcinoma cells accompanies activation of the NRF2 path-way. Free Radic Biol Med 2009; 47: 1619–1631.

25 Zhang DD. The Nrf2-Keap1-ARE signaling pathway: the regulation and dualfunction of Nrf2 in cancer. Antioxid Redox Signal 2010; 13: 1623–1626.

26 Kim TH, Hur EG, Kang SJ, Kim JA, Thapa D, Lee YM et al. NRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced activation of HIF-1a.Cancer Res 2011; 71: 2260–2275.

27 Chen L, Madura K. Increased proteasome activity, ubiquitin-conjugating enzymes,and eEF1A translation factor detected in breast cancer tissue. Cancer Res 2005; 65:5599–5606.

28 Hu XT, Chen W, Zhang FB, Shi QL, Hu JB, Geng SM et al. Depletion of theproteasome subunit PSMA7 inhibits colorectal cancer cell tumorigenicity andmigration. Oncol Rep 2009; 22: 1247–1252.

29 Ren S, Smith MJ, Louro ID, McKie-Bell P, Bani MR, Wagner M et al. The p44S10locus, encoding a subunit of the proteasome regulatory particle, is amplifiedduring progression of cutaneous malignant melanoma. Oncogene 2000; 19:1419–1427.

30 Rho JH, Qin S, Wang JY, Roehrl MH. Proteomic expression analysis of surgicalhuman colorectal cancer tissues: up-regulation of PSB7, PRDX1, and SRP9 andhypoxic adaptation in cancer. J Proteome Res 2008; 7: 2959–2972.

31 Kwak MK, Wakabayashi N, Itoh K, Motohashi H, Yamamoto M, Kensler TW.Modulation of gene expression by cancer chemopreventive dithiolethionesthrough the Keap1-Nrf2 pathway. Identification of novel gene clusters for cellsurvival. J Biol Chem 2003; 278: 8135–8145.

32 Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler TW. Antioxidantsenhance mammalian proteasome expression through the Keap1-Nrf2 signalingpathway. Mol Cell Biol 2003; 23: 8786–8794.

33 Kapeta S, Chondrogianni N, Gonos ES. Nuclear erythroid factor 2-mediated pro-teasome activation delays senescence in human fibroblasts. J Biol Chem 2010;285: 8171–8184.

34 Schaedler S, Krause J, Himmelsbach K, Carvajal-Yepes M, Lieder F, Klingel K et al.Hepatitis B virus induces expression of antioxidant response element-regulatedgenes by activation of Nrf2. J Biol Chem 2010; 285: 41074–41086.

Nrf2 inhibition and pancreatic cancerA Arlt et al

4834

Oncogene (2013) 4825 – 4835 & 2013 Macmillan Publishers Limited

35 Kwak MK, Kensler TW. Induction of 26S proteasome subunit PSMB5 by thebifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not theAhR/Arnt-XRE, pathway. Biochem Biophys Res Commun 2006; 345: 1350–1357.

36 Kwak MK, Itoh K, Yamamoto M, Kensler TW. Enhanced expression of thetranscription factor Nrf2 by cancer chemopreventive agents: role of antioxidantresponse element-like sequences in the nrf2 promoter. Mol Cell Biol 2002; 22:2883–2892.

37 Lau A, Villeneuve NF, Sun Z, Wong PK, Zhang DD. Dual roles of Nrf2 in cancer.Pharmacol Res 2008; 58: 262–270.

38 Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation,and cancer: how are they linked? Free Radic Biol Med 2010; 49: 1603–1616.

39 Taguchi A, Politi K, Pitteri SJ, Lockwood WW, Faca VM, Kelly-Spratt K et al. Lungcancer signatures in plasma based on proteome profiling of mouse tumormodels. Cancer Cell 2011; 20: 289–299.

40 Hayes JD, McMahon M. NRF2 and KEAP1 mutations: permanent activation of anadaptive response in cancer. Trends Biochem Sci 2009; 34: 176–188.

41 Osburn WO, Kensler TW. Nrf2 signaling: an adaptive response pathway forprotection against environmental toxic insults. Mutat Res 2008; 659: 31–39.

42 Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y et al. Nrf2 enhancesresistance of cancer cells to chemotherapeutic drugs, the dark side of Nrf2.Carcinogenesis 2008; 29: 1235–1243.

43 Wang XJ, Hayes JD, Henderson CJ, Wolf CR. Identification of retinoic acid as aninhibitor of transcription factor Nrf2 through activation of retinoic acid receptoralpha. Proc Natl Acad Sci USA 2007; 104: 19589–19594.

44 Zhou W, Lo SC, Liu JH, Hannink M, Lubahn DB. ERRbeta: a potent inhibitor of Nrf2transcriptional activity. Mol Cell Endocrinol 2007; 278: 52–62.

45 Boettler U, Sommerfeld K, Volz N, Pahlke G, Teller N, Somoza V et al. Coffeeconstituents as modulators of Nrf2 nuclear translocation and ARE (EpRE)-dependent gene expression. J Nutr Biochem 2011; 22: 426–440.

46 Kannan S, Jaiswal AK. Low and high dose UVB regulation of transcription factorNF-E2-related factor 2. Cancer Res 2006; 66: 8421–8429.

47 Steffen J, Seeger M, Koch A, Kruger E. Proteasomal degradation is transcriptionallycontrolled by TCF11 via an ERAD-dependent feedback loop. Mol Cell 2010; 40:147–158.

48 Arlt A, Vorndamm J, Breitenbroich M, Folsch UR, Kalthoff H, Schmidt WE et al.Inhibition of NF-kappaB sensitizes human pancreatic carcinoma cells to apoptosisinduced by etoposide (VP16) or doxorubicin. Oncogene 2001; 20: 859–868.

49 Fritsche P, Seidler B, Schuler S, Schnieke A, Gottlicher M, Schmid RM et al. HDAC2mediates therapeutic resistance of pancreatic cancer cells via the BH3-only pro-tein NOXA. Gut 2009; 58: 1399–1409.

50 Stathis A, Moore MJ. Advanced pancreatic carcinoma: current treatment andfuture challenges. Nat Rev 2010; 7: 163–172.

51 Elangovan M, Oh C, Sukumaran L, Wojcik C, Yoo YJ. Functional differencesbetween two major ubiquitin receptors in the proteasome; S5a and hRpn13.Biochem Biophys Res Commun 2010; 396: 425–428.

52 Nguyen T, Sherratt PJ, Nioi P, Yang CS, Pickett CB. Nrf2 controls constitutiveand inducible expression of ARE-driven genes through a dynamic pathwayinvolving nucleocytoplasmic shuttling by Keap1. J Biol Chem 2005; 280:32485–32492.

53 Niture SK, Jaiswal AK. Prothymosin-a mediates nuclear import of the INrf2/Cul3_Rbx1 complex to degrade nuclear Nrf2. J Biol Chem 2009; 284:13856–13868.

54 Radhakrishnan SK, Lee CS, Young P, Beskow A, Chan JY, Deshaies RJ. Transcriptionfactor Nrf1 mediates the proteasome recovery pathway after proteasome inhi-bition in mammalian cells. Mol Cell 2010; 38: 17–28.

55 Xie Y. Feedback regulation of proteasome gene expression and its implications incancer therapy. Cancer Metastasis Rev 2010; 29: 687–693.

56 Nguyen T, Nioi P, Pickett CB. The Nrf2-antioxidant response elementsignaling pathway and its activation by oxidative stress. J Biol Chem 2009; 284:13291–13295.

57 van Dijk AE, Olthof MR, Meeuse JC, Seebus E, Heine RJ, van Dam RM. Acute effectsof decaffeinated coffee and the major coffee components chlorogenic acid andtrigonelline on glucose tolerance. Diabetes Care 2009; 32: 1023–1025.

58 Olthof MR, van Dijk AE, Deacon CF, Heine RJ, van Dam RM. Acute effects ofdecaffeinated coffee and the major coffee components chlorogenic acid andtrigonelline on incretin hormones. Nutr Metab (Lond) 2011; 8: 10.

59 Geismann C, Arlt A, Bauer I, Pfeifer M, Schirmer U, Altevogt P et al. Binding of thetranscription factor slug to the L1CAM promoter is essential for transforminggrowth factor-beta1 (TGF-beta)-induced L1CAM expression in human pancreaticductal adenocarcinoma cells. Int J Oncol 2011; 38: 257–266.

60 Muerkoster S, Arlt A, Sipos B, Witt M, Gro�mann M, Kloppel G et al. Increasedexpression of the E3-ubiquitin ligase receptor subunit bTRCP1 relates to con-stitutive NF-kB activation and chemoresistance in pancreatic carcinoma cells.Cancer Res 2005; 65: 1316–1324.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Nrf2 inhibition and pancreatic cancerA Arlt et al

4835

& 2013 Macmillan Publishers Limited Oncogene (2013) 4825 – 4835