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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Pancreas. Author manuscript; available in PMC Nov 26, 2012.
Published in final edited form as:
PMCID: PMC3506252
NIHMSID: NIHMS419626
Nrf2 regulates drug resistance in pancreatic cancer cells
Young Bin Hong, PhD,* Hyo Jin Kang, MS,* Sun Young Kwon, MD, PhD, Hee Jeong Kim, MS, Kun Young Kwon, MD, PhD, Chi Heum Cho, MD, PhD, Jong-Min Lee, MD, PhD, Bhaskar V.S. Kallakury, MD, and Insoo Bae, PhD§
Young Bin Hong, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA.
§Corresponding author: Insoo Bae, Ph.D, Tel, 1-202-687-5267; Fax, 1-202-687-2847 ; ib42/at/georgetown.edu
*These authors contributed equally to this work
Objective
To investigate the molecular basis of drug resistance in pancreatic cancer.
Methods
The expression of Nrf2 levels in pancreatic cancer tissues and cell lines was analyzed. Clinical relevance between Nrf2 activation and drug resistance was demonstrated by measuring cell viability after Nrf2 and ABCG2 regulation by over-expression or knockdown of these genes. Activity of ABCG2 was measured by Hoechst 33342 staining.
Results
Abnormally elevated Nrf2 protein levels were observed in pancreatic cancer tissues and cell lines relative to normal pancreatic tissues. Increasing Nrf2 protein levels either by over-expression of exogenous Nrf2 or by activating endogenous Nrf2 resulted in increased drug resistance. Conversely, a reduction in endogenous Nrf2 protein levels or inactivation of endogenous Nrf2 resulted in decreased drug resistance. These changes in drug resistance or sensitivity were also positively correlated to the expression levels of Nrf2 downstream genes. Similarly, the expression of ABCG2 was correlated with drug resistance.
Conclusions
Since the intrinsic drug resistance of pancreatic cancers is, in part, due to abnormally elevated Nrf2 protein levels, further research on regulating Nrf2 activity may result in the development of novel pancreatic cancer therapies.
Keywords: Nrf2, drug resistance, pancreatic cancer
Pancreatic cancer is an extremely life-threatening disease. In 2005, estimated deaths from pancreatic cancers (31,800) were approximately equal to its incidence (32,180) in the United States (1). Among patients diagnosed with pancreatic cancer, only 15–20% can be treated with surgical resection, the only chance of a cure. The remaining 80–85% of patients has locally advanced, unresectable or metastatic disease (2). Chemotherapy and radiation therapy or both are used as treatment options for unresectable pancreatic cancers (1). However, pancreatic cancer has been reported to be highly resistant to both radiation and chemotherapy (1, 3). Since current therapeutic approaches are minimally effective, better understanding of the molecular basis of drug resistance in pancreatic cancers may lead to better treatments.
The innate drug resistance of many pancreatic cancers may have multiple causes. One known mechanism of drug resistance involves the ATP binding cassette (ABC) transporter family of proteins (reviewed in 4). The function of ABC transporters is to translocate solutes across cellular membranes (5). Forty-eight ABC transporter genes, classified into seven subfamilies, are encoded in the human genome (5). The ABCB subfamily endows tumors with resistance against a wider variety of drug types than other ABC subfamilies (4). For example, elevated ABCB1 expression is associated with doxorubicin and docetaxel resistance in mammary gland cancer in the conditional double (BRCA1 plus TP53) knock-out mouse model (6). In addition, transfection of ABCB4 and ABCB11 into hepatocyte and insect cells caused increased chemoresistance (7, 8).
Several ABC transporter genes are transcriptionally regulated by nuclear factor erythroid 2-like 2 protein (NFE2L2, Nrf2) (9, 10). Nrf2, which associates with Kelch-like ECH associated protein 1 (Keap1) in the cytoplasm, is known to be a principal transcription factor of intracellular antioxidants and phase II detoxification enzymes, thus playing a key role in cellular responses to oxidative stress (1113). Under oxidative stress, Nrf2 is activated through hyper-phosphorylation, then it translocates into nucleus, where it binds the antioxidant response element (ARE) creating the Keap1-Nrf2-ARE pathway (14). A number of antioxidant genes (e.g., NQO1, HO-1, SOD1, and GCLC) and drug resistance genes (e.g., some of ABCC family and ABCG2) are reported to be controlled by Nrf2 (11).
In lung cancer, recent studies have demonstrated that the activation of Nrf2 results in enhanced resistance against chemotherapeutic agents and that the down regulation of Nrf2 renders these cells more susceptible to therapy (15, 16). Also, protein levels of HO-1, a Nrf2 target gene, was suppressed by Nrf2-specific siRNA, resulting in enhanced chemosensitivity in lung cancer (17). Furthermore, elevated levels of ABCC1, 2 and 3 (multidrug resistance protein; MRP1, 2 and 3), which are downstream targets of Nrf2, were correlated with increased drug resistance in these studies (18). Here, we analyzed Nrf2 expression levels in pancreatic cancer tissue and cell lines and identified the role of Nrf2 in drug resistance of these tissues.
Cell culture
PANC-1 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin. AsPC-1, Capan-1 and Colo-357 were cultured in Roswell Park Memorial Institute (RPMI) 1640 media supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and fetal bovine serum (20% for AsPC-1; 15% for Capan-1; and 10% for Colo-357) with 1% sodium pyruvate. All cell culture reagents were purchased from BioWhittaker, Inc. (Walkersville, MD). All pancreatic cancer cell lines were obtained from the tissue culture shared resources (TCSR) of Georgetown University Medical Center (GUMC). Human pancreatic duct epithelial (HPDE) cells, HPDE6-C7 and HPDE6-C11 from Dr. Tsao (19), were cultured in keratinocyte serum-free (KSF) medium supplemented by epidermal growth factor and bovine pituitary extract (LifeTechnologies, Inc., Grand Island, NY).
Immunohistochemical staining of Nrf2 protein
Immunohistochemistry was performed to detect the expression of Nrf2 using rabbit polyclonal anti-Nrf2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunohistochemical staining for 5 normal and 20 pancreatic ductal adenocarcinoma tissues was carried out on 4 µm sectioned, formalin-fixed, and paraffin embedded human pancreas tissues. Ten of twenty pancreatic carcinomas were offered from the histopathology & tissue shared resoures (HTSR) of GUMC (Washington, DC) and the others from Keimyung University Hospital (Daegu, Korea). The sections were deparaffinized routinely in xylene and rehydrated through a series of graded alcohols to distilled water. The slides were covered with 10mM sodium citrate buffer, pH 6.0 and then heated at 95°C, 10 min for antigen retrieval. Endogenous peroxidase activity was quenched by applying 0.3% hydrogen peroxide for 10 min, followed by washing with buffer. The sections were then incubated for 10 min in 10% goat serum to reduce non-specific antigen-antibody reaction. Previously 1:100 diluted Nrf2 antibody was applied and incubated on the slides for 2 hr at room temperature. After washing with appropriate buffer, the reaction was visualized using a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) at a dilution 1:200 and then with the Vectastain ABC reagent (Vector Laboratories). The slides were counterstained with Meyer’s hematoxylin, dehydrated through graded alcohols, placed in xylene, and coverslipped.
Scoring Nrf2 expression
The immunoreactivity of Nrf2 was evaluated by nuclear and cytoplasmic expression with semi-quantitative method: i) the percentage of distinct nuclear staining and ii) the intensity of cytoplasmic staining. The nuclear staining was scored as 1 (<5%), 2 (5–50%), and 3 (>50%) at the percentage of positive tumor cells on 10 high powered fields at ×400 magnification. No staining or less than 5% of nuclei staining was considered negative. The cytoplasmic staining of tumor cells was divided into four categories by degree of staining intensity; mild or faint, moderate, and strong intensity. Also, it was regarded that less than faint or uncertain cytoplasmic intensity was negative.
Western blotting
Western blots were prepared and analyzed essentially as described previously (20). Briefly, cells were cultured for 24 hrs, lysed and centrifuged. Proteins in the supernatants were separated on SDS-PAGE gels which were then used to Western blots which were analyzed using various primary antibodies: anti-Nrf2 rabbit polyclonal; anti-GCLC mouse monoclonal; anti-Lamin B mouse monoclonal; anti-β-tubulin mouse monoclonal; anti-actin mouse monoclonal (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and anti-ABCG2 mouse monoclonal (Abcam, Inc., Cambridge, MA). Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Sigma, St. Louis, MO). Membrane bound secondary antibodies were visualized by enhanced chemiluminescence (ECL) detection kits (Santa Cruz Biotechnology).
DNA binding assay
For measuring DNA binding activity, TransAM™ Nrf2 assay kit (Active Motif, Carlsbad, CA) was used. Nuclear extracts (10 µg) from AsPC-1, PANC-1, Colo-357, HPDE6-C7 and –C11 cells were incubated for 1 hr at room temperature in 96-well plates, on which DNA oligonucleotides containing ARE consensus binding sequences (5’-GTCACAGTGACTCAGCA GAATCTG-3’) were immobilized. After binding and multiple washing steps, anti-Nrf2 antibody was added for 1 hr at room temperature. Horseradish peroxidase antibody was used as a secondary antibody for colorimetric detection of ARE bound Nrf2 at 450 nm for 5 min with a reference wavelength at 655 nm.
Transfection of DNA and siRNA
For transient expression experiments, cells were transfected with expression vectors for Flag-Nrf2, a dominant negative Nrf2, ABCG2 and pCDNA3 (Invitrogen, Carlsbad, CA) as a control. For Nrf2 knock-down, exponentially proliferating cells were transfected with chemically synthesized control siRNA (5’-gacgagcggcacgugcacauu-3’) or Nrf2-specific siRNA (5’-gaguaugagcuggaaaaacuu-3’), both purchased from Dharmacon, Inc (Lafayette, CO). DNA and siRNA transfection using Lipofectamine 2000 or Lipofectamine Plus (Invitrogen), respectively, were performed as described in Bae at al. (20).
Cell viability assays
Cell viability was measured by MTT assays as previously described (20). Briefly, cells were seeded in 96-well plates at densities of 3 × 104 cells per well, grown overnight, then treated with different concentrations of Camptothecin, Cisplatin and PEITC (Sigma) for 24 hrs and then assayed for MTT dye reduction. Cell viability values were calculated relative to the control cells (100%) and expressed as means ± SE of three independent experiments.
Quantitative real-time RT PCR
Pancreatic cell line knock-down was done with Nrf2-specific siRNA or with a control, non-specific siRNA, for 3 days. Then total mRNAs was extracted with RNAzol B (Tel-Test, Friendswood, TX) and used as the template for RT using Superscript II reverse transcriptase (LifeTechnologies, Inc. Rockville, MD) according to the manufacturer’s instructions. The RT primer sequences were : 5′-GCTATCCCTGTACGCCTCTG-3′ (forward) and 5′-ACATCTGCT GGAAGGTGGAC-3′ (reverse) for β-actin; 5′-AAACCACCCTGAAACGACAG-3′ (forward), and 5′-AGCGGCTTGAATGTTTGTCT-3′ (reverse) for Nrf2; 5′-CTGGGGAGTGA TTTCTGC AT- 3′ (forward) and 5′-AGGAGGGGGCTTAAATCTCA-3′ (reverse) for GCLC; 5′-TTATCCGTGGTGTGTCTGGA-3′ (forward) and 5′-CCTGCTTGGAAG GCTCTATG-3′ (reverse) for ABCG2; 5′-ACCAAGACGTATCAGGTGGCC-3′ (forward), and 5′-CTGTCTGGGCATCCAGGAT-3′ (reverse) for MRP1; 5′-TGAA AGGCTACAAGCGTCCT-3′ (forward) and 5′-GAGCCGCAGTGAATAAGAGG-3′ (reverse) for MRP2; 5′-GAGGGATGAATTTGGCTTCA-3′ (forward) and 5′-CAGGG CTGCTGAGACACATA-3′ (reverse) for MRP4; and 5′-ACCCGTTGTTGCCATCTTAG-3′ (forward), and 5′-TCTGTCAACAGCCACTGAGG-3′ (reverse) for MRP5. The real-time PCR reactions were performed in 20 µl volumes that included 1 µl RT products as template, 10 µl of SYBR Green PCR master mix (Applied Biosystems, Warrington, UK) and 20 pmol of each primer described above. The PCR amplifications (40 cycles of 95°C for 30s and 60°C for 60s) were performed using the ABI 7900 (Applied Biosystems) in the genomics & epigenomics shared resources (GESR) of GUMC. For each target gene the average Ct values calculated from triplicate PCR reactions were normalized to the average Ct values for β-actin. These normalized values were then used to calculate a value expressing the extent of knock-down relative to the non-specific control siRNA according to the formula 2−(meanΔΔCt).
Quantification of Hoechst positive cells
The Hoechst staining method was used as described in Kim et al. (21). AsPC-1 cells transfected with DNA or siRNA were incubated in the presence of 10 µM of Hoechst 33342 (Sigma) for 90 min. After washing with phosphate-buffered saline, fluorescence-containing cells were counted under a microscope (Carl Zeiss, Germany). At least 2000 Hoechst positive (light blue) or negative (dark blue) cells were counted for each samples of three independent experiments.
Nrf2 is over-expressed in human pancreatic cancer tissue
Nrf2 expression in normal pancreas samples revealed two types of immunoreactivity (Fig.1A). The acini of normal pancreas tissue showed diffused cytoplasmic granular staining with mild or moderate intensity without nuclear expression, and normal pancreatic ductal epithelial cells demonstrated focal and weak cytoplasmic staining without nuclear expression in all of 5 cases of normal pancreas tissue (Fig. 1A). However, pancreatic ductal carcinoma showed either nuclear or cytoplasmic positive expression of Nrf2 (Fig. 1B-I). The scoring of Nrf2 immunoreactivity in nucleus (Fig. 1B-E) and cytoplasm (Fig. 1F-I) of pancreatic ductal carcinomas was determined by four degree of staining intensity. Seventy percent (14/20) of the tumor showed positive staining in nucleus, and 80% (16/20) revealed moderate or strong cytoplasmic intensity for Nrf2 (Table 1).
Figure 1
Figure 1
Differential expression of Nrf2 in human pancreatic tissues and cells
Table 1
Table 1
Pancreatic ductal carcinomas show nuclear and/or cytoplasmic immunoreactivity of Nrf2.
Nrf2 is over-expressed in pancreatic cancer cell line
We used Western blots to examine Nrf2 levels in various pancreatic cancer and normal cell lines. At least two types of Nrf2 (a slower migrating form and a faster migrating form) were identified. In two normal pancreatic cell lines, HPDE6-C7 and HPDE6-C11, there is no detectable slower migrating form of Nrf2, while all the pancreatic cancer cell lines, AsPC-1, Capan-1 (data not shown), Colo-357, and PANC-1, have this slower migrating form of Nrf2, as well as ABCG2 and HO-1 (Fig 2A). To characterize the slower migrating band we fractioned cells into nuclear and cytosolic fractions. The slower migrating Nrf2 protein was detected only in the nuclear fractions of all the pancreatic cancer cell lines, but not in the two normal cell lines (Fig 2B). In our unpublished data, we observed increased accumulation of the slower migrating Nrf2 protein after treatments with oxidizing compounds, tert-Butylhydroquinone (t-BHQ), which promotes nuclear localization and activation of Nrf2 (22). Both HPDE6-C11 and AsPC-1 cells showed increased amount of the slower migrating form of Nrf2 in accordance with t-BHQ induction and this caused increased accumulation of Nrf2 target gene, GCLC (Fig 2C). To determine whether the slower migrating nuclear Nrf2 is phosphorylated, we treated nuclear fractions of t-BHQ treated cells with λ-phosphatase. The relative amount of the slower migrating form of Nrf2 in both AsPC-1 and Colo-357 cells were decreased by the 30 min incubation with λ-phosphatase, although this did not result in a corresponding increase in the faster migrating Nrf2 species (Fig 2D). The DNA binding assay showed that nuclear Nrf2 of pancreatic cancer cell lines have higher ARE-binding activity than that of normal cell lines (Fig 2E). Therefore, Nrf2 is over-expressed and transcriptionally active, which might cause over-expression of downstream genes in pancreatic cancer cell lines.
Figure 2
Figure 2
Human pancreatic cancer cell lines accumulate Nrf2. (A) Western blot (WB) analysis of total lysates reveal that pancreatic tumor cell lines (AsPC-1, Colo-357 and PANC-1) have elevated level of Nrf2 and its target proteins when compared with normal pancreatic (more ...)
Increased amount of Nrf2 confers drug-resistance to cancer cell lines
To investigate the possible role of Nrf2 in drug resistance, we determined the changes of drug resistance levels after over-expression of Nrf2 in AsPC- 1 and Colo-357 cells. Transfection of Flag-Nrf2 significantly increased the percentage of AsPC-1 and Colo-357 cell that remained viable after Cisplatin and Camptothecin treatments (Fig 3A–D). The viability of Flag-Nrf2-transfected AsPC-1 cells increased after both 50 µM (from 50% to 64%) and 100 µM (from 39% to 55%) of Cisplatin treatments (Fig 3A). The viability of Flag-Nrf2 transfected AsPC-1 cells increased in 10 µM (from 52% to 76%) and in 25 µM (from 43% to 60%) Camptothecin (Fig 3B). Similarly, over-expression of Nrf2 in Colo-357 also significantly increased viability against Cisplatin and Camptothecin exposure (Fig 3C and D). Transfection efficiencies, which were measured using GFP-Nrf2 vector, were 34.4% in AsPC-1 and 16.5% in Colo-357 cells (data not shown).
Figure 3
Figure 3
Nrf2 over-expression increased drug resistance in pancreatic cancer cells. (A–D) AsPC-1 and Colo-357 transiently transfected with an Nrf2 expression vector (Flag-Nrf2) for 24 hrs showed increased resistance to Cisplatin (A and C) and Camptothecin (more ...)
As an independent way to test whether activation of Nrf2 is related to drug resistance, we pretreated AsPC-1 cells with 100 µM t-BHQ, which increased the slower migrating Nrf2 protein (Fig. 2C). Cells pretreated with t-BHQ for 16 hr had higher drug resistance than DMSO treated cells (Fig. 3E–H). Eighty seven percent of AsPC-1 cell pretreated with t-BHQ survived after 24 hr treatment of 100 µM Cisplatin, while only 53% of the control cells survived (Fig. 3E). Cells resistant to Camptothecin are also increased when pretreated with t-BHQ. Over 80% of AsPC cells survived after 24 hr incubation of 10 µM Camptothecin while less than 50% cell survived in DMSO pretreated cells (Fig. 3F). Pre-incubation with 100 µM t-BHQ showed similar effects on Colo-357 cells. Resistance of Colo-357 cells to Cisplatin is also significantly elevated when pretreated with t-BHQ (Fig. 3G). Likewise, 73% of Colo-357 cells pretreated with t-BHQ survived after 24hr incubation with 10 µM Camptothecin, while only 61% survived after DMSO pretreatment (Fig 3H).
Reducing Nrf2 level renders pancreatic cell lines more sensitive to drugs
Since elevated Nrf2 levels increased drug resistance in pancreatic cancer cell lines, we further investigated whether either reducing Nrf2 amounts or inhibiting Nrf2 activity would sensitize cells to chemotherapeutic drugs. First, we inhibited Nrf2’s transcription regulation activity by transfection of dominant negative mutants of Nrf2 (DN-Nrf2). Transfection of DN-Nrf2 reduced expression level of Nrf2 target proteins (HO-1 and GCLC) in AsPC-1 and Colo-357 (Supplementary 1). In AsPC-1 cells, Nrf2 inactivation renders cells more vulnerable to Cisplatin and phenethyl isothiocyanate (PEITC) (Fig. 4A and B). The viability of AsPC-1 cells treated with 50 µM Cisplatin was significantly reduced from 51% to 30% in cells transfected with DN-Nrf2 compared with control vector (Fig 4A). The viability of AsPC-1 cells against 10 µM PEITC was also reduced from 70% to 44% (Fig. 4B). Transfection of the DN-Nrf2 reduced viability of Colo-357 against Cisplatin and PEITC (Fig. 4C–D). To confirm whether reduced viability measured by MTT assay resulted from cell growth arrest or cell death, we directly counted cell numbers during Cisplatin incubation for up to 72 hrs (Supplementary 2). Cell numbers of AsPC-1 and Colo-357 transfected with DN-Nrf2 reduced to 23% (AsPC-1) and 20% (Colo-357) after incubation with 100 µM Cisplatin for 72 hrs, while control DMSO treated cells increased to 195% (AsPC-1) and 170% (Colo-357). There was less than 5% difference between control vector and DN-Nrf2 transfected cells when incubated with DMSO. However, incubation of 100 µM Cisplatin increased the gap of cell numbers up to 2-fold in both cells.
Figure 4
Figure 4
Inhibition of Nrf2 activity with dominant negative Nrf2 (DN-Nrf2) or knock-down with Nrf2 siRNA decreased drug resistance. (A–D) Transfection with DN-Nrf2 mutants increased sensitivity of AsPC-1 (A and B) and Colo-357 (C and D) to Cisplatin (A (more ...)
Reducing Nrf2 protein levels showed the same results. Knock-down of Nrf2 with Nrf2-specific siRNA reduced Cisplatin and Camptothecin resistance in AsPC-1 and Colo-357 (Fig. 4E–H). In AsPC-1 cells, transfection with Nrf2-specific siRNA dramatically reduced the viability from 71% to 40% in treatments of 50 µM Cisplatin (Fig. 4E). Similarly, the viability of Colo-357 against Cisplatin and Camptothecin was significantly decreased when Nrf2 levels were reduced (Fig. 4G and H). Therefore, we confirmed that reducing the total amount or the transcriptionally active form of Nrf2 rendered pancreatic cancer cells more vulnerable to chemotherapeutic drugs.
Nrf2 knock-down reduces mRNA levels of multidrug resistance proteins
As reduced or inactivated Nrf2 increased drug sensitivity, we analyzed the effect of Nrf2 knock-down on the expression level of drug resistance genes. After knock-down of Nrf2 using Nrf2-specific siRNA, we measured mRNA levels of GCLC, ABCG2 and several ABCC family mRNA, as well as Nrf2 in AsPC-1, Colo-357 and PANC-1 cells using real-time PCR (Fig 5A). The ratios of change (Nrf2 siRNA versus control siRNA) in these genes showed that all the mRNA expression levels of multidrug resistance genes, as well as Nrf2 were significantly reduced after knock-down of Nrf2 in three pancreatic cell lines. Reduced mRNA level of Nrf2 and GCLC were also correlated with reduced protein levels (Fig 5B).
Figure 5
Figure 5
Nrf2 knock-down reduces expression of several multidrug resistance genes. (A) Y-axis shows the percentage of mRNA expression levels of the Nrf2 and its target genes in the AsPC-1, Colo-357 and PANC-1 cells treated with Nrf2-siRNA, compared with those (more ...)
ABCG2 expression correlates with Camptothecin resistance
As ABCG2 is reported to have function in Camptothecin resistance (23), and we found that it is regulated by Nrf2 in transcription level (Fig. 5A), we analyzed the effect of Nrf2 and ABCG2 protein levels on Hoechst dye (a known substrate of ABCG2) efflux pumping using fluorescence microscopy (Fig. 6A–C). Transfection with expression vectors for either Nrf2 (35%) or ABCG2 (28%) gene significantly reduced the percentage of Hoechst positive AsPC-1 cells compared with transfection of control DNA (60%) (Fig. 6A). In contrast, Nrf2 knock-down increased the percentage of Hoechst positive cell to 92–99% (Fig. 6B). The increased Hoechst positive cells by knock-down of Nrf2 in turn, decreased after over-expression of ABCG2 (Fig 6C). As ABCG2 protein levels showed a positive correlation with Hoechst efflux activity, we analyzed the cell viability against Camptothecin with changing expression levels of ABCG2. We reduced ABCG2 level through knock-down with Nrf2-specific siRNA then restored ABCG2 with transfection of ABCG2 DNA. In 50 µM of Camptothecin treatment, AsPC-1 cells pretreated with control siRNA and ABCG2 DNA showed highest viability (67%) and cells pretreated with Nrf2 siRNA and control DNA showed lowest viability (43%) (Fig. 6D). Changes of protein expression levels after knock-down of Nrf2 and over-expression of ABCG2 were confirmed by Western blotting (Fig. 6E).
Figure 6
Figure 6
Nrf2 Knock-down affects Hoechst 33342 efflux. (A) The cell numbers of Hoechst 33342 positive decreased after over-expression of Nrf2 or ABCG2. (B) The cell numbers of Hoechst 33342 positive increased after knock-down of Nrf2 or ABCG2. (C) Restoring ABCG2 (more ...)
In this report, we showed for the first time that Nrf2 protein levels are elevated in human pancreatic cancer tissue and cell lines and that it accumulates in pancreatic cell nuclei and/or cytoplasm of some patient samples. We also observed nuclear localized Nrf2, which is regarded to be the active form and directly induces downstream genes in pancreatic cancer cell lines. Although we did not show the mechanism of elevated Nrf2 expression in pancreatic cancer, we can postulate the relevance of oxidative stress. During oxidative stress, Nrf2 activity is increased and expressions of antioxidant genes are elevated as well (14). If some pancreatic cancers are derived from chronic pancreatitis, the tumor cells may have obtained their defense systems during survival of oxidative stress (24).
Pancreatic cancer is classified as a lifestyle disease (25). Pancreatic cancer risk is significantly associated with high energy and alcohol intake and exposure to cigarette smoke (26). Chronic pancreatitis is also associated with an increased risk of developing pancreatic cancer (25, 27). When protective mechanisms against tumorigenesis such as DNA repair, apoptosis and immunemediated destruction fail, chronic pancreatitis can develop into pancreatic cancer (28). Furthermore, the pathogenesis of chronic pancreatitis shares characteristics with pancreatic cancer as both have elevated level of cytokines (29). In acute and chronic pancreatitis, oxidative stress is considered to play a key role in pathogenesis of inflammation. Oxidative stress triggers the activation of MAPK, NF-κB and STAT3, resulting in the activation of oxidant-sensitive response signaling pathways in the acute pancreatitis model (30). Likewise, reactive oxygen species (ROS) formation in chronic pancreatitis is directly linked to inflammatory pathogenesis (31). In pancreatic cancer cells, oxidative stress also generates various ROS, including superoxide radicals, hydrogen peroxide and hydroxyl radical (32). These ROS as along with cytokines and mediators of the inflammatory pathway have been linked to tumorigenesis by increasing cell cycling caused by loss of tumor suppressor function and by stimulating oncogene expression (33).
We showed that drug resistance in pancreatic cancer results, in part, from elevated levels of Nrf2 and its target proteins. On the other hand, we also report that repression of Nrf2 activity sensitize pancreatic cancer cells to chemotherapeutic drugs. In accordance with our data, Nrf2-knockout mouse embryonic fibroblasts were more sensitive to Cisplatin treatment (34). Therefore, reducing Nrf2 activity should arise as an important potential treatment for pancreatic cancer. Keap1 has been well characterized as a negative regulator of Nrf2. Keap1, as a component of E3 ubiquitin ligase, ubiquitinates Nrf2 and degrades it via a proteasome-mediated protein degradation pathway (35). In lung cancer, the Nrf2–Keap1 regulation system has been reported to be impaired in many tissues and carcinoma cell lines (36, 37). Several protein kinases such as protein kinase C (PKC) (38), extracellular signal-regulated kinases (ERK) (39), p38 mitogen-activated protein kinase (MAPK) (40), and phosphatidylinositol 3-kinase (PI3K) (41) were reported to be involved in signal pathways during the activation of Nrf2-related antioxidant system. As a preliminary study, we observed that blocking several protein kinases using chemical inhibitors reduce Nrf2 related drug resistance (data not shown).
Regarding drug resistance in breast cancer, ABCG2 (BCRP) has been well characterized as a mitozantrone resistant gene (MXR) (42). In this study, we targeted ABCG2 transporter as a direct regulator of Camptothecin resistance. Reducing ABCG2 expression level sensitized pancreatic cells to Camptothecin and transient over-expression of ABCG2 conferred drug resistance. Therefore, regulation of ABCG2 should also be considered in treating pancreatic cancer. Recently, the importance of ABCG2 has been reported along with the identification of “side population” cells in many cancers (43). The side population is distinguished by their low Hoechst 33342 dye staining and their potency as stem cells (44). As expression level of ABCG2 is elevated in side population cells, expression level of Nrf2 can also be elevated in some cancer stem cells. As Nrf2 by itself is involved in cell proliferation (45), over-expression of Nrf2 may serve to benefit to cancer stem cell. Therefore, further research on regulating Nrf2 activity may provide a novel therapeutic strategy to overcome drug resistance in pancreatic cancer.
2
Supplementary 1. Inhibition of Nrf2 activity by DN-Nrf2 vector was confirmed by measuring Nrf2 target gene products (e.g., HO-1). After 24 hr of transfection with control or DN-Nrf2 vector, cells were harvested, and total cell lysates were used for immunoblotting assay analysis as described in Material and Methods.
Supplementary 2. Inhibition of Nrf2 activity with dominant negative Nrf2 (DN-Nrf2) reduced cell numbers in AsPC-1 (A) and Colo-357 (B). AsPC-1 and Colo-357 cells (1 × 104) were transfected with control or DN-Nrf2 vector for 24 hr. Then, the transfected cells were incubated with DMSO or 100 µM Cisplatin. Alive cells were counted under a light-microscope at indicated times. *, p < 0.05; **, p < 0.01.
Acknowledgements
Dr. Bae has been supported by Susan G. Komen for the Cure (BCTR119906 and FAS0703858) and by World Class University (WCU) grant (R31-2008-000-100069-0). We appreciate Dr. Thomas L. Mattson and BioMedText, Inc./Dr. Rashmi Nemade for helpful discussions and editing. We also appreciate Dr. Manabu Furukawa (Nebraska University Medical Center) and Dr. Jawed Alam (Ochsner Medical Center) who kindly provided us Nrf2 and DN-Nrf2 DNA plasmid constructs, respectively.
Abbreviations
Nrf2nuclear factor (erythroid-derived 2)-like 2
ABCG2ATP-binding cassette, sub-family G member 2
MRPmultidrug resistance protein

Contributor Information
Young Bin Hong, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA.
Hyo Jin Kang, 1Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA.
Sun Young Kwon, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA. Department of Pathology, Keimyung University, School of Medicine, Daegu, Korea.
Hee Jeong Kim, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA.
Kun Young Kwon, Department of Pathology, Keimyung University, School of Medicine, Daegu, Korea.
Chi Heum Cho, Department of Obstetrics and Gynecology, Keimyung University, School of Medicine, Daegu, Korea.
Jong-Min Lee, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA. Department of Obstetrics and Gynecology, East-West Neo Medical Center, Kyung Hee University, Seoul, Korea.
Bhaskar V.S. Kallakury, Department of Pathology, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA.
Insoo Bae, Department of Oncology and Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington DC, 20057-1469, USA. Department of Nanobiomedical Science, Dankook University, Chunan, Korea.
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