title: 3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions.
abstract: Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.
authors.affiliation: Department of Urology, Kangwon National University Hospital, Kangwon National University School of Medicine, Chuncheon, Gangwon-do, Republic of Korea.
authors.forename: Hongzoo
authors.initials: H
authors.lastname: Park
authors.affiliation: Marine Biodiversity Institute of Korea, Seocheon, Chungcheongnam-do, Republic of Korea.
authors.forename: Dae-Sung
authors.initials: DS
authors.lastname: Lee
authors.affiliation: Marine Biodiversity Institute of Korea, Seocheon, Chungcheongnam-do, Republic of Korea.
authors.forename: Mi-Jin
authors.initials: MJ
authors.lastname: Yim
authors.affiliation: Department of Biochemistry, College of Oriental Medicine, Dongeui University, Busan, Republic of Korea.
authors.forename: Yung Hyun
authors.initials: YH
authors.lastname: Choi
authors.affiliation: Department of Microbiology, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Saegwang
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Microbiology, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Su-Kil
authors.initials: SK
authors.lastname: Seo
authors.affiliation: Department of Internal Medicine, Busan Paik Hospital, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Jung Sik
authors.initials: JS
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Won Hee
authors.initials: WH
authors.lastname: Jang
authors.affiliation: Department of Biochemistry, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Sung Su
authors.initials: SS
authors.lastname: Yea
authors.affiliation: Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Gangwon-do, Republic of Korea.
authors.forename: Won Sun
authors.initials: WS
authors.lastname: Park
authors.affiliation: Department of Molecular Microbiology and Immunology, Warren Alpert School of Medicine, Providence, RI, USA.
authors.forename: Chang-Min
authors.initials: CM
authors.lastname: Lee
authors.affiliation: Department of Biomedical Engineering and Center for Marine-Integrated Biomedical Technology (BK21 Plus), Pukyong National University, Busan, Republic of Korea.
authors.forename: Won-Kyo
authors.initials: WK
authors.lastname: Jung
authors.affiliation: Department of Microbiology, College of Medicine, Inje University, Busan, Republic of Korea.
authors.forename: Il-Whan
authors.initials: IW
authors.lastname: Choi
mesh_terms: D016588:Anticarcinogenic Agents / Q000494:pharmacology*; D015687:Cell Hypoxia / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival; D003035:Cobalt / Q000494:pharmacology; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology*; D008268:Macular Degeneration / Q000503:physiopathology; D008928:Mitochondria / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D009593:Nitroimidazoles / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D016588:Anticarcinogenic Agents; C497442:HIF1A protein, human; D006835:Hydrazones; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007211:Indoles; D016328:NF-kappa B; D009593:Nitroimidazoles; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C555826:YCG 063; D003035:Cobalt; D048051:p38 Mitogen-Activated Protein Kinases; C018021:cobaltous chloride; C016392:3,3'-diindolylmethane