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Activation of NRF2 Signaling in Breast Cancer Stem Cell-Enriched Population
whether the adaptive gene responses that accompany Alkbh8 deficiency may be of prognostic significance. Analysis for the Alkbh8 adaptive genes of illumineseq gene expression data from 37 cancer types in The Cancer Genome Atlas revealed a significant decrease in survival percentage of only patients with the kidney cancer, clear cell renal cell carcinoma and increase of the adapt gene signature with tumor grade. Our observation that global selenoprotein suppression can accelerate the senescence phenotype and bioenergetics function, suggests that SECdependent regulation of these processes may be critical for limiting insults that drives tumor development or age related tissue degeneration.
doi: 10.1016/j.freeradbiomed.2016.10.324 284
Expression of FXYD3 in MCF-7 breast cancer cells was ~8-fold and ~2-fold higher than in non-cancer MCF-10A cells and MDA-MB468 cancer cells, respectively. A ~50% reduction in FXYD3 expression increased glutathionylation of the β1 Na+/K+-ATPase subunit and reduced Na+/K+-ATPase activity by ~50%, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the β1 subunit of Na+/K+-ATPase and glutathionylation-induced inhibition of Na+/K+-ATPase. Treatment of MCF-7 and MDA-MB468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments up-regulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na+/K+-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.
doi: 10.1016/j.freeradbiomed.2016.10.326
Activation of NRF2 Signaling in Breast Cancer Stem Cell-Enriched Population Mi-Kyoung Kwak1, In-geun Ryoo1, and Sang-hwan Lee1 1 The Catholic University of Korea, Seoul, Republic of Korea Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and drug efflux transporters. In this study, we investigated the potential involvement of NF-E2-related factor 2 (NRF2) in CSC survival and stress response by using MCF7 breast cancer cells. First, the levels of NRF2 and its target gene expressions were increased in breast CSC-enriched mammosphere culture system. As an underlying mechanism, we demonstrated that enzymatic function and expression of the proteasome are decreased in mammospheres. Additionally, mammospheres retained a higher level of p62 and the silencing of p62 attenuated NRF2 activation in sphere cultured MCF7. Sphere growth and anticancer drugs resistance in mammospheres were diminished in NRF2-knockdown mammospheres. To further confirm NRF2 activation in breast CSC, we isolated CSC surface marker CD44-positive MCF7 cells by flow cytometry. The CD44positive cells showed increased NRF2 protein and target genes, which were accompanied by CSC markers expression. Furthermore, we observed NRF2 activation in CD44-enriched doxorubicin-resistant MCF7 cells. Collectively, these results indicated that NRF2 plays a role in breast CSCs survival and anticancer drug resistance.
doi: 10.1016/j.freeradbiomed.2016.10.325 285 Silencing FXYD3 Protein in Human Breast Cancer Cells Enhances Oxidative Stress of Doxorubicin and Gamma Irradiation Chia-chi Liu1,2 Sydney Medical School, University of Sydney, Australia, 2Kolling Institute of Medical Research, Australia 1
FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na+/K+-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments.
286 Novel Fluorescent Probes for Imaging and Quantitative Analysis of Intracellular Nitric Oxide Zhen Luo1, Jixiang Liu1, Qin Zhao1, Yunting Xi1, Ruogu Peng1, Jennifer Liao1, and Jack Diwu1 1 AAT Bioquest, Sunnyvale, USA Nitric oxide (NO) is an important biological regulator involved in number of physiological and pathological processes. Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO can be rapidly oxidized, thus there is relatively low concentration of NO existing in vivo. Although a variety of fluorescent sensors have been developed to detect NO, their poor photostability, short emission wavelength and low selectivity have limited the applications of these existing reactive oxygen species (ROS) and reactive nitrogen species (RNS) probes in live cells. To address this unmet need, three novel cell-permeable NO probes Nitrixyte Orange, Nitrixyte Red and Nitrixyte NIR were developed for detecting free NO in live cells. Compared to the commonly used diaminofluorescein (DAF-2), Nitrixyte probes have 1) higher selectivity toward NO than other ROS and RNS species; 2) improved sensitivity in detecting endogenous levels of NO; 3) enhanced cell permeability and 4) better photostability. Importantly, long emission wavelength of Nitrixyte Red and Nitrixyte NIR minimize autofluorescence of cells and facilitate multicolor imaging. Taking advantages of Nitrixyte probes, fluorescence imaging was successfully applied to monitor the exogenous NO in various adherent cell lines such as HeLa and CHO-K1, and to visualize the endogenously produced NO in Raw 264.7 macrophage cell line. Fluorescence microplate reader was also successfully used to quantitatively measure NO level in above cell lines. Significantly, all these novel probes were suited for monitoring intracellular NO in suspension cell lines such as T lymphocyte Jurkat cells using flow cytometer, to provide quantitative real-time measurements of NO in individual live cells. These novel fluorescent NO probes provide valuable tools for life science research and medical diagnostic applications.
doi: 10.1016/j.freeradbiomed.2016.10.327
SfRBM / SFRRI 2016
S125
doi: 10.1016/j.freeradbiomed.2016.10.324 284
Expression of FXYD3 in MCF-7 breast cancer cells was ~8-fold and ~2-fold higher than in non-cancer MCF-10A cells and MDA-MB468 cancer cells, respectively. A ~50% reduction in FXYD3 expression increased glutathionylation of the β1 Na+/K+-ATPase subunit and reduced Na+/K+-ATPase activity by ~50%, consistent with the role of FXYD3 to facilitate reversal of glutathionylation of the β1 subunit of Na+/K+-ATPase and glutathionylation-induced inhibition of Na+/K+-ATPase. Treatment of MCF-7 and MDA-MB468 cells with doxorubicin or γ-radiation decreased cell viability and induced apoptosis. The treatments up-regulated FXYD3 expression in MCF-7 but not in MDA-MB-468 cells and suppression of FXYD3 in MCF-7 but not in MDA-MB-468 cells amplified effects of treatments on Na+/K+-ATPase activity and treatment-induced cell death and apoptosis. Overexpression of FXYD3 may be a marker of resistance to cancer treatments and a potentially important therapeutic target.
doi: 10.1016/j.freeradbiomed.2016.10.326
Activation of NRF2 Signaling in Breast Cancer Stem Cell-Enriched Population Mi-Kyoung Kwak1, In-geun Ryoo1, and Sang-hwan Lee1 1 The Catholic University of Korea, Seoul, Republic of Korea Cancer stem cells (CSCs) are known to express high levels of antioxidant proteins and drug efflux transporters. In this study, we investigated the potential involvement of NF-E2-related factor 2 (NRF2) in CSC survival and stress response by using MCF7 breast cancer cells. First, the levels of NRF2 and its target gene expressions were increased in breast CSC-enriched mammosphere culture system. As an underlying mechanism, we demonstrated that enzymatic function and expression of the proteasome are decreased in mammospheres. Additionally, mammospheres retained a higher level of p62 and the silencing of p62 attenuated NRF2 activation in sphere cultured MCF7. Sphere growth and anticancer drugs resistance in mammospheres were diminished in NRF2-knockdown mammospheres. To further confirm NRF2 activation in breast CSC, we isolated CSC surface marker CD44-positive MCF7 cells by flow cytometry. The CD44positive cells showed increased NRF2 protein and target genes, which were accompanied by CSC markers expression. Furthermore, we observed NRF2 activation in CD44-enriched doxorubicin-resistant MCF7 cells. Collectively, these results indicated that NRF2 plays a role in breast CSCs survival and anticancer drug resistance.
doi: 10.1016/j.freeradbiomed.2016.10.325 285 Silencing FXYD3 Protein in Human Breast Cancer Cells Enhances Oxidative Stress of Doxorubicin and Gamma Irradiation Chia-chi Liu1,2 Sydney Medical School, University of Sydney, Australia, 2Kolling Institute of Medical Research, Australia 1
FXYD3, also known as mammary tumor protein 8, is overexpressed in several common cancers, including in many breast cancers. We examined if such overexpression might protect Na+/K+-ATPase and cancer cells against the high levels of oxidative stress characteristic of many tumors and often induced by cancer treatments.
286 Novel Fluorescent Probes for Imaging and Quantitative Analysis of Intracellular Nitric Oxide Zhen Luo1, Jixiang Liu1, Qin Zhao1, Yunting Xi1, Ruogu Peng1, Jennifer Liao1, and Jack Diwu1 1 AAT Bioquest, Sunnyvale, USA Nitric oxide (NO) is an important biological regulator involved in number of physiological and pathological processes. Altered NO production is implicated in various immunological, cardiovascular, neurodegenerative and inflammatory diseases. As a free radical, NO can be rapidly oxidized, thus there is relatively low concentration of NO existing in vivo. Although a variety of fluorescent sensors have been developed to detect NO, their poor photostability, short emission wavelength and low selectivity have limited the applications of these existing reactive oxygen species (ROS) and reactive nitrogen species (RNS) probes in live cells. To address this unmet need, three novel cell-permeable NO probes Nitrixyte Orange, Nitrixyte Red and Nitrixyte NIR were developed for detecting free NO in live cells. Compared to the commonly used diaminofluorescein (DAF-2), Nitrixyte probes have 1) higher selectivity toward NO than other ROS and RNS species; 2) improved sensitivity in detecting endogenous levels of NO; 3) enhanced cell permeability and 4) better photostability. Importantly, long emission wavelength of Nitrixyte Red and Nitrixyte NIR minimize autofluorescence of cells and facilitate multicolor imaging. Taking advantages of Nitrixyte probes, fluorescence imaging was successfully applied to monitor the exogenous NO in various adherent cell lines such as HeLa and CHO-K1, and to visualize the endogenously produced NO in Raw 264.7 macrophage cell line. Fluorescence microplate reader was also successfully used to quantitatively measure NO level in above cell lines. Significantly, all these novel probes were suited for monitoring intracellular NO in suspension cell lines such as T lymphocyte Jurkat cells using flow cytometer, to provide quantitative real-time measurements of NO in individual live cells. These novel fluorescent NO probes provide valuable tools for life science research and medical diagnostic applications.
doi: 10.1016/j.freeradbiomed.2016.10.327
SfRBM / SFRRI 2016
S125