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. 2011 Apr 13:10:37.
doi: 10.1186/1476-4598-10-37.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

Adam Lister  1 , Taoufik Nedjadi, Neil R Kitteringham, Fiona Campbell, Eithne Costello, Bryony Lloyd, Ian M Copple, Samantha Williams, Andrew Owen, John P Neoptolemos, Chris E Goldring, B Kevin Park
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Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

VSports在线直播 - Adam Lister et al. Mol Cancer. .

Abstract

Background: Nrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer. VSports手机版.

Results: Keap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2) V体育安卓版. The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0. 001). .

Conclusions: Expression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics. V体育ios版.

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Figures

Figure 1
Figure 1
Basal expression levels of Keap1, Nrf2 and Nrf2-regulated genes GCLC and AKR1c1 and GSH amongst a panel of pancreatic cancer cell lines. A, Immunoblot detection of basal Keap1 protein in MiaPaca-2, Panc-1, FAMPAC, Paca-2 and Suit-2 cells. B, Immunoblot detection of basal Nrf2 in cells untreated or treated with the proteosome inhibitor MG132 (10 μM) for 2 h in order to visualize Nrf2 protein. The band labelled 'non-specific' was not depleted following transfection with 10 nM Nrf2-targeting siRNA Nrf2 (data not shown). Beta actin was used as a reference control for blots A and B. C, Immunoblot detection of basal GCLC and AKR1c1. D, Total basal glutathione levels. * = P < 0.05,** = P < 0.01,*** = P < 0.001.
Figure 2
Figure 2
Response of Nrf2-target genes to siRNA depletion of Nrf2 in FAMPAC, MiaPaca-2 and Suit-2 pancreatic cancer cells. Cells were transfected with 10 nM Nrf2-targeting siRNA, or Stealth RNAi control, for 96 h. A, qRTPCR analysis of relative Nrf2 mRNA expression. GAPDH was used as a reference control. B, Immunoblot detection of GCLC and AKR1c1 in three parallel experiments. Beta-actin is used as reference control. C, Total glutathione levels. Data are the means ± S.D. of four discrete experiments. * = P < 0.05,** = P < 0.01.
Figure 3
Figure 3
Effect of siRNA depletion of Nrf2 on proliferation of FAMPAC, MiaPaca-2 and Suit-2 pancreatic cancer cells. Cells were transfected with 10 nM Nrf2-targeting siRNA, or Stealth RNAi control, for up to 120 h. A, Cell survival was measured at 120 h using the MTS test. Data is shown as cell viability versus Stealth RNAi control. B, Cell numbers were quantified at the indicated time points using Trypan blue staining. Data are the means ± S.D. of four discrete experiments. ** = P < 0.01,*** = P < 0.001.
Figure 4
Figure 4
Effect of siRNA depletion of Nrf2 on cell cycle progression and apoptosis status of Suit-2 and FAMPAC pancreatic cancer cells. Cells were transfected with 10 nM Nrf2-targeting siRNA, or Stealth RNAi control, for 72 h. A, Flow cytometric analysis of cell cycle status in propidium iodide stained cells. Data are the means ± S.D of four discrete experiments. * = P < 0.05. B, Micrographs depicting cells analysed in A. C, Flow cytometric analysis of apoptotic status in Annexin 5/propidium iodide stained cells.
Figure 5
Figure 5
Effect of siRNA depletion of Nrf2 on sensitivity of Suit-2 and FAMPAC pancreatic cancer cells to chemo- and radiotherapies. Cells were transfected with 10 nM Nrf2-targeting siRNA, or Stealth RNAi control, for 48 h followed by exposure to gemcitabine (A), 5 flurouracil (5-FU) (B), cisplatin (C) or gamma radiation (D) for 72 h, at the indicated concentrations. Cell viability was measured using the MTS assay. Graphs represent data as a surviving fraction versus non-drug treated Stealth RNAi control transfected cells. IC50 values are expressed as μM. Data are the means ± S.D. of four discrete experiments. * = P < 0.05,** = P < 0.01,*** = P < 0.001.
Figure 6
Figure 6
Immunohistochemical analysis of Nrf2 and Keap1 in pancreatic tissues. A and B, Pancreatic cancer tissue showing strong and weak Nrf2 levels, respectively. E and F, Pancreatic cancer tissue showing Keap1 expression. C and G, Pancreatic cancer tissue showing absence of detectable Nrf2 and Keap1, respectively. D and H, Benign pancreatic tissue showing Nrf2 expression in ductal cells and acinar cells (D) and absence of Keap1 in ductal cells, with positive islet cells (H). Scale bars = 50 μm.
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