Oncogene (2013) 32, 4825–4835 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity A Arlt1,4, S Sebens2,4, S Krebs1, C Geismann1, M Grossmann1, M-L Kruse1, S Schreiber1,3 and H Scha¨fer1 Evidence accumulates that the transcription factor nuclear factor E2-related factor 2 (Nrf2) has an essential role in cancer development and chemoresistance, thus pointing to its potential as an anticancer target and undermining its suitability in chemoprevention. Through the induction of cytoprotective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would therefore be an efficient strategy in anticancer therapy. In the present study, pancreatic carcinoma cell lines (Panc1, Colo357 and MiaPaca2) and H6c7 pancreatic duct cells were analyzed for the Nrf2-inhibitory effect of the coffee alkaloid trigonelline (trig), as well as for its impact on Nrf2-dependent proteasome activity and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anticancer drug-induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tert-butylhydroquinone (tBHQ)-inducible Nrf2 activity and drug resistance. Trig efficiently decreased basal and tBHQ-induced Nrf2 activity in all cell lines, an effect relying on a reduced nuclear accumulation of the Nrf2 protein. Along with Nrf2 inhibition, trig blocked the Nrf2-dependent expression of proteasomal genes (for example, s5a/psmd4 and a5/psma5) and reduced proteasome activity in all cell lines tested. These blocking effects were absent after treatment with Nrf2 siRNA, a condition in which proteasomal gene expression and proteasome activity were already decreased, whereas siRNA against the related transcription factor Nrf1 did not affect proteasome activity and the inhibitory effect of trig. Depending on both Nrf2 and proteasomal gene expression, the sensitivity of all cell lines to anticancer drugs and TRAIL-induced apoptosis was enhanced by trig. Moreover, greater antitumor responses toward anticancer drug treatment were observed in tumor-bearing mice when receiving trig. In conclusion, representing an efficient Nrf2 inhibitor capable of blocking Nrf2-dependent proteasome activity and thereby apoptosis protection in pancreatic cancer 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 INTRODUCTION both conditions favoring tumorigenesis on one hand and making A great number of malignant tumors,1–11 for example, colonic, tumor cells more refractory to chemo- and radiotherapy on the 2,5,23 thyroid, endomethrial, lung, ovarian, breast and pancreatic cancer, other hand. By inducing a battery of phase II enzymes and exhibit an increased activity of the antioxidative transcription detoxification genes that protect cells from anticancer drug factor nuclear Factor E2-related factor 2 (Nrf2). This enhanced Nrf2 toxicity, Nrf2 directly confers chemoresistance, as quite recently 24,25 activation has been shown to originate from rare gain-of-function reported for several types of tumors, including pancreatic 3,4,9 mutations of Nrf2 itself12,13 and from loss-of-function mutations, adenocarcinoma (PDAC). A recent study also identified a role promoter hypermethylation or micro RNA targeting of the of NRF2 in promoting tumor angiogenesis through the HIF-1a/ Nrf2 inhibitory protein Keap1/INRF.14–16 Besides these genetic VEGF pathways, not only underscoring the potential of Nrf2 to 26 and epigenetic alterations, an exaggerated Nrf2 activity may also sustain tumor growth and survival, but also providing an result from cellular adaptation to metabolic stress, for example, interesting insight into how hypoxia-related signals and oxidative fumarate accumulation leading to Keap1 succination17,18 or to stress adaptation may be linked to each other. oxidative stress,19–21 for example, emerging along with persistent Another mode of action by which Nrf2 activation favors inflammation during chronic colitis or pancreatitis. As a conse- tumorigenesis relates to the induction of proteasomal genes quence of the enhanced Nrf2 activity, tumor cells acquire protec- having an impact on the ubiquitine–proteasome signaling path- tion from apoptosis1,20–22 and are more capable of proliferation, 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, Inflammatory Carcinogenesis Research Group, Kiel, Germany and 3Institute of Clinical Molecular Biology, UKSH, Kiel, Germany. Correspondence: Professor H Scha¨fer, Department of Internal Medicine I, Laboratory of Molecular Gastroenterology & Hepatology, Bldg. 6, UKSH-Campus Kiel, Schittenhelmstr. 12, Kiel, Schleswig-Holstein, D-24105, Germany. E-mail: [email protected] 4These 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 Nrf2 inhibition and pancreatic cancer A Arlt et al 4826 is crucial for cellular homeostasis and regular cell growth, are Effect of trigonelline on Nrf2 activity in PDAC and H6c7 cells involved in tumor development. Upregulation of proteasomal ARE-luciferase assays conducted with Panc1 and Colo357 cells 1,27–30 gene expression in tumors has been previously reported, subject to trig treatment at various concentrations (0.01–10 mM) for and in this context Nrf2 has an important role owing to its 16 h revealed a dose-dependent inhibition of ARE-driven lucifer- inducing effect on proteasomal gene expression and thereby on ase expression by trig. The greatest inhibition was seen at 1,31–34 26S/20S proteasome activity. Recent studies identified a concentrations between 0.1 and 1 mM (Figure 1c). Similarly, number of proteasomal genes (for example, a5/PSMA5, PSMB5, ARE-driven luciferase expression could be dose-dependently b1/PSMB6 and s5a/PSMD4) transcriptionally regulated by Nrf2 inhibited by trig in all cell lines treated with 50 mM tBHQ for 8 h through one or multiple antioxidant response elements (ARE) and preincubated (1 and 16 h) with trig before tBHQ administra- 35,36 within their gene promoters. In colon cancer, greater Nrf2 tion. The 16-h preincubation with trig was less efficient than the activity and increased proteasomal gene expression correlate with 1-h preincubation. At the latter time point and at a dose of 0.1 mM, 1 an elevated proteasome activity, and human colonocytes and most significant inhibitory effects were observed in MiaPaca2 cells colon cancer cells acquire an increased apoptosis protection exhibiting a 54% decrease of ARE-driven luciferase expression in from elevated proteasomal gene expression and proteasome comparison with vehicle-treated cells. In the other cell lines, the 1,28,30 activation. inhibitory effect of trig at 0.1 mM was somewhat lower, ranging Thus, evidence has accumulated that Nrf2 exhibits profound between 35 and 50% inhibition. At higher doses (1 and 10 mM), the 37,38 protumorigenic activity and its targeting may therefore have inhibition of ARE-driven luciferase by trig was not stronger or even 23,39 great potential in antitumor therapy, particularly in over- less pronounced.45 coming chemoresistance, for example, in PDAC. However, Nrf2 has also gained attention in terms of its use as a chemopreventive Trig affects nuclear localization of Nrf2 in PDAC and H6c7 cells target, because the activation of Nrf2 by certain antioxidants such as sulforaphane and oltipraz leads to protection from toxic To elucidate the mechanisms by which trig interferes with Nrf2 DNA damage and thereby from tumorigenesis.40,41 Obviously, activation, its effect on the subcellular distribution of Nrf2 was in normal cells exhibiting tightly controlled Nrf2 activation, the investigated. The inhibitory impact of trig on Nrf2 could be impact of Nrf2 is rather preventive against cancer, but in verified by a decline in the nuclear level of Nrf2 protein that was permanently stressed or even transformed cells exhibiting seen in Panc1 and Colo357 cells treated with the compound for deregulated Nrf2 activity its impact is rather protumorigenic.37,42 8 h (Figure 2a). Similarly, H6c7 and MiaPaca2 cells stimulated with Besides the induction of phase II enzyme expression through Nrf2, tBHQ exhibited a decreased accumulation of Nrf2 protein in the accounting for direct detoxification of anticancer drugs, the nucleus when subjected to pretreatment with trig (Figure 2b). In impact of Nrf2 on the ubiquitine–proteasome signaling pathway contrast, no differences in the amount of Nrf2 protein were confers general apoptosis resistance that manifests in protection detected in total cellular extracts (Figures 2a and b) Thus, the from various apoptotic stimuli, for example, death ligands, and in higher basal and induced level of nuclear Nrf2 protein in Panc1 or enhanced proliferation. One goal therefore would be to establish Colo357 cells and H6c7 or MiaPaca2 cells, respectively, were novel compounds