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Reactive oxygen species production has a critical role in hypoxia-induced Stat3 activation and angiogenesis in human glioblastoma

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Abstract

Glioblastoma is the most aggressive primary brain tumor with hypoxia-associated morphologic features including pseudopalisading necrosis and endothelial hyperplasia. It has been known that hypoxia can activate signal transducer and activator of transcription 3 (Stat3) and subsequently induce angiogenesis. However, the molecular mechanism underlying hypoxia-induced Stat3 activation has not been defined. In this study, we explored the possible implication of reactive oxygen species (ROS) in hypoxia-driven Stat3 activation in human glioblastoma. We found that hypoxic stress increased ROS production as well as Stat3 activation and that ROS inhibitors (diphenyleneiodonium, rotenone and myxothiazol) and an antioxidant (N-acetyl-l-cysteine) blocked Stat3 activation under hypoxic conditions. To determine a major route of ROS production, we tested whether nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) is involved in hypoxia-induced ROS production. Nox4 expression was found to be increased at both mRNA and protein levels in hypoxic glioblastoma cells. In addition, siRNA-mediated knockdown of Nox4 expression abolished hypoxia induced Stat3 activation and vascular endothelial growth factor expression, which is associated with tumor cells’ ability to trigger tube formation of endothelial cells in vitro. Our findings indicate that elevated ROS production plays a crucial role for Stat3 activation and angiogenesis in hypoxic glioblastoma cells.

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A2001528).

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Authors and Affiliations

  1. Department of Neurosurgery, College of Medicine, Korea University, #126, 5-ga, Anam-Dong, Seongbuk-Gu, Seoul, 136-705, Korea

    Mi Ok Yu, Kyung-Jae Park, Dong-Hyuk Park, Yong-Gu Chung & Shin-Hyuk Kang

  2. School of Life Sciences and Biotechnology, Korea University, 5-1 Anam-Dong, Seongbuk-Gu, Seoul, 136-701, Korea

    Mi Ok Yu & Sung-Gil Chi

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Fig. S1 An increase of various intracellular ROS production by hypoxic stress. a U87 cells were incubated under hypoxic condition for 6 and 24 h and stained with 5 μM DCFDA, 30 μM Mitosox and 10 μM DHE for 30 min under normoxic condition. ROS levels were measured by flow cytometry

Fig. S2 Knockdown of siNox4 in glioblastoma and its effect on Stat3 activation and related ROS production. a U87 cells were transfected with vehicle, siControl, siNox4-1 or siNox4-2 and incubated for 96 h. Whole-cell lysates were extracted and subjected to immunoblot. b Vehicle, siControl or siNox4-1-transfected U87 cells were collected at the indicated times after transfection and Nox4 protein was examined by immunoblot. c At 48 h after siControl or siNox4-1 transfection, U87 cells were exposed to hypoxic stress for 6 h. Phospho-Stat3 expression was analyzed by immunofluorescence stain. d siNox4-1 transfected U87 cells were incubated under normoxic or hypoxic condition for 6 h and stained with 10 μM DHE for 30 min. Cytoplasmic superoxide production was measured by flow cytometry

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Yu, M.O., Park, KJ., Park, DH. et al. Reactive oxygen species production has a critical role in hypoxia-induced Stat3 activation and angiogenesis in human glioblastoma. J Neurooncol 125, 55–63 (2015). https://doi.org/10.1007/s11060-015-1889-8

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  • DOI: https://doi.org/10.1007/s11060-015-1889-8

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