title: Graphene oxide induces apoptotic cell death in endothelial cells by activating autophagy via calcium-dependent phosphorylation of c-Jun N-terminal kinases.
abstract: Despite the rapid expansion of the biomedical applications of graphene oxide (GO), safety issues related to GO, particularly with regard to its effects on vascular endothelial cells (ECs), have been poorly evaluated. To explore possible GO-mediated vasculature cytotoxicity and determine lateral GO size relevance, we constructed four types of GO: micrometer-sized GO (MGO; 1089.9±135.3nm), submicrometer-sized GO (SGO; 390.2±51.4nm), nanometer-sized GO (NGO; 65.5±16.3nm), and graphene quantum dots (GQDs). All types but GQD showed a significant decrease in cellular viability in a dose-dependent manner. Notably, SGO or NGO, but not MGO, potently induced apoptosis while causing no detectable necrosis. Subsequently, SGO or NGO markedly induced autophagy through a process dependent on the c-Jun N-terminal kinase (JNK)-mediated phosphorylation of B-cell lymphoma 2 (Bcl-2), leading to the dissociation of Beclin-1 from the Beclin-1-Bcl-2 complex. Autophagy suppression attenuated the SGO- or NGO-induced apoptotic cell death of ECs, suggesting that SGO- or NGO-induced cytotoxicity is associated with autophagy. Moreover, SGO or NGO significantly induced increased intracellular calcium ion (Ca2+) levels. Intracellular Ca2+chelation with BAPTA-AM significantly attenuated microtubule-associated protein 1A/1B-light chain 3-II accumulation and JNK phosphorylation, resulting in reduced autophagy. Furthermore, we found that SGO or NGO induced Ca2+release from the endoplasmic reticulum through the PLC β3/IP3/IP3R signaling axis. These results elucidate the mechanism underlying the size-dependent cytotoxicity of GOs in the vasculature and may facilitate the development of a safer biomedical application of GOs.
  
  
  
  Graphene oxide (GO) have received considerable attention with respect to their utilization in biomedical applications. However, GO-related safety issues concerning human vasculature are very limited. In this manuscript, we report for the first time the differential size-related biological effects of GOs on endothelial cells (ECs). Notably, Subnanometer- and nanometersized GOs induce apoptotic death in ECs via autophagy activation. We propose a molecular mechanism for the GO-induced autophagic cell death through the PLCβ3/IP3/Ca2+/JNK signaling axis. Our findings could be provide a better understanding of the GO sizedependent cytotoxicity in vasculature and facilitate the future development of safer biomedical applications of GOs.
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Mi-Hee
authors.initials: MH
authors.lastname: Lim
authors.affiliation: Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: In Cheul
authors.initials: IC
authors.lastname: Jeung
authors.affiliation: Harzards Monitoring BNT Research Center, Major in NanoBioEngineering and Bioinformatics, University of Science and Technology, Korea Research Institute of Bioscience and Biotechnology, Daejon, Republic of Korea.
authors.forename: Jinyoung
authors.initials: J
authors.lastname: Jeong
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Sung-Jin
authors.initials: SJ
authors.lastname: Yoon
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Sang-Hyun
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Biomolecular Science, University of Science & Technology, Daejeon, Republic of Korea.
authors.forename: Jongjin
authors.initials: J
authors.lastname: Park
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Functional Genomics, University of Science & Technology, Daejeon, Republic of Korea.
authors.forename: Yu-Seon
authors.initials: YS
authors.lastname: Kang
authors.affiliation: Harzards Monitoring BNT Research Center, Major in NanoBioEngineering and Bioinformatics, University of Science and Technology, Korea Research Institute of Bioscience and Biotechnology, Daejon, Republic of Korea.
authors.forename: Hansu
authors.initials: H
authors.lastname: Lee
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Young-Jun
authors.initials: YJ
authors.lastname: Park
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Hee Gu
authors.initials: HG
authors.lastname: Lee
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Seon-Jin
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Baek Soo
authors.initials: BS
authors.lastname: Han
authors.affiliation: Center for Nanosafety Metrology, Korea Research Institute of Standards and Science, Daejeon, Republic of Korea.
authors.forename: Nam Woong
authors.initials: NW
authors.lastname: Song
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Sang Chul
authors.initials: SC
authors.lastname: Lee
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Functional Genomics, University of Science & Technology, Daejeon, Republic of Korea. Electronic address: jekmin@kribb.re.kr.
authors.forename: Jang-Seong
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Functional Genomics, University of Science & Technology, Daejeon, Republic of Korea. Electronic address: khbae@kribb.re.kr.
authors.forename: Kwang-Hee
authors.initials: KH
authors.lastname: Bae
authors.affiliation: Biotherapeutics Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Biomolecular Science, University of Science & Technology, Daejeon, Republic of Korea. Electronic address: jangskim@kribb.re.kr.
authors.forename: Jeong-Ki
authors.initials: JK
authors.lastname: Min
mesh_terms: D000263:Adenylate Kinase / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D000071186:Beclin-1 / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006108:Graphite / Q000494:pharmacology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D053758:Nanoparticles; D010316:Particle Size; D010766:Phosphorylation / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: C491997:BECN1 protein, human; D000071186:Beclin-1; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D006108:Graphite; D048031:JNK Mitogen-Activated Protein Kinases; D000263:Adenylate Kinase; D002118:Calcium
keywords: Autophagy; Calcium ion; Cytotoxicity; Endothelial cell; Graphene oxide