doi: 10.1158/0008-5472.CAN-08-1401.
RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy
Affiliations
- PMID: 18829555
- PMCID: PMC3070411
- DOI: 10.1158/0008-5472.CAN-08-1401
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RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy
Cancer Res.
.
Abstract
Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small cell lung cancer. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth, and results in increased sensitivity to chemotherapeutic drug-induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.
Figures
(A) Generation of cell lines stably expressing Nrf2 shRNA. (a–c) Real time RT-PCR analysis of Nrf2 expression in A549 and H460 cells stably expressing Nrf2 shRNA. Total RNA from stable clones harboring Nrf2 shRNA or non-targeting luciferase shRNA were analyzed for expression of Nrf2. GAPDH was used as normalization control. (c) Immunoblot detection of Nrf2 in A549 and H460 cells stably transfected with shRNAs targeting Nrf2. (B–C) Comparison of GSR, GPX, GST, G6PDH enzyme activities and total GSH levels between cells expressing Nrf2 shRNA and control cells expressing luciferase shRNA. Data represent mean ± SE (n = 3). *, p < 0.05 relative to the cells expressing luciferase shRNA (by t-test). (D) Western blot analysis of TXN1 and TXNRD1 levels in A549 cells stably transfected with the Nrf2 shRNA and control cells expressing luciferase shRNA.
Inhibition of Nrf2 activity leads to ROS accumulation in A549-Nrf2shRNA and H460-Nrf2shRNA cells. (A–B) Comparison of ROS levels in A549 and H460 cells stably expressing Nrf2 shRNA. Cells expressing non-targeting Luc shRNA were used as control. Pretreatment with 20mM NAC decreased the ROS levels. ROS levels in cells expressing luciferase shRNA were same as the control untransfected cells. (C) ROS levels did not change significantly between the BEAS2B cells transfected with Nrf2 siRNA and the control non-targeting NS siRNA. *, p < 0.01 relative to the cells expressing luciferase shRNA; **, p < 0.01 relative to the cells pretreated with NAC.
Overexpression of Nrf2 confers drug resistance. (A–B) Effect of Nrf2 inhibition on drug accumulation in lung cancer cells. Tritium (3H) labeled etoposide and 14C labeled carboplatin accumulation in A549-Nrf2shRNA and H460-Nrf2shRNA cells was measured at regular time intervals (15–120 mins) after incubation with the drug. A non-targeting luciferase shRNA was used as control. Data are mean of 3 independent replicates, combined to generate the mean ± SE for each concentration. Drug accumulation was significantly higher in cells expressing Nrf2 shRNA. ‘*’, P<0.05 relative to Luc shRNA. (C–D) Enhanced sensitivity of A549-Nrf2shRNA and H460–Nrf2shRNA cells to carboplatin and etoposide. Cells were exposed to drugs for 72h– 96h and viable cells were determined by MTS/ phenazine methosulfate assay. Data is represented as percentage of viable cells relative to the vehicle treated control. Data are mean of 8 independent replicates, combined to generate the mean ± SD for each concentration. Representative experiments are shown.
Nr2 ablation leads to reduced tumorigenic properties in vitro and in vivo. (A) Nrf2 promotes lung cancer cell proliferation. A549-Nrf2shRNA (1500 cells) and H460-Nrf2shRNA (1000 cells) cells were plated in 96 well plates and cellular proliferation was analyzed using the colorimetric MTS assay over the indicated time course. Cancer cells expressing Luc-shRNA were used as control. (B) A549-Nrf2shRNA and H460-Nrf2shRNA expressing cells were also analyzed for anchorage-independent growth. (C–D) A549-Nrf2shRNA and H460-Nrf2shRNA cells were injected in the flank of male athymic nude mice (n = 7 for H460, n=6 for A549). A549 and H460 cells expressing Luc-shRNA were used as control. Weekly measurements were taken from the tumors, and the mean tumor volume was determined after 4–6 weeks. Weight of the tumor was recorded at the termination of the experiment. Mean difference in tumor weight between the Luc-shRNA and Nrf2 shRNA expressing H460 cells was 1.24 gms (95% CI=0.773 to 1.71; “*” P=0.0001). Data was analyzed using two-sample Wilcoxon rank-sum (Mann-Whitney) test. A549-Nrf2 shRNA cells did not form any tumor in nude mice.
Therapeutic efficacy of Nrf2 siRNA in combination with carboplatin. (A) Nude mice were injected subcutaneously with A549 cells and randomly allocated to one of the following groups with therapy beginning 15 days after tumor cell injection: GFP siRNA, GFP siRNA+ carboplatin, Nrf2 siRNA and Nrf2 siRNA+ carboplatin. Mice were treated for 4 weeks and then sacrificed. A dot plot shows the tumor weights upon termination by treatment group. Weights of the GFP siRNA treated tumors were significantly higher compared to Nrf2 siRNA treated tumors (ratio of weights = 2.09, 95% CI: [1.41, 3.10], p = 0.0002), and siRNA treated compared to siRNA+ carboplatin treated tumors (2.13, 95% CI: [1.44, 3.16], p = 0.001). (B) Delivery of naked Nrf2 siRNA duplex into tumor inhibited the expression of Nrf2 and its downstream target genes (HO-1 and GCLm). ‘*’,P<0.05 (Wilcoxon rank-sum test). (C) The proliferative index based on Ki-67 immunoreactivity in A549 tumors. Part “a”, shows large fraction of Ki-67 positive cells in GFP siRNA treated A549 tumors. Part ’b’ shows large number of Ki-67 stained cells in GFP siRNA+ carboplatin treated tumors. Part ‘c’ shows very few Ki-67 positive cells in Nrf2 siRNA treated tumors. Part ‘d’ shows ki-67 stained cells in Nrf2 siRNA+ carboplatin treated A549 tumors. Note that ‘d’ has area of extensive cell death (approximately the right half of the panel), and this massive cellular death was not seen in the other samples.
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