MDPI Acta Medica Okayama 2073-4409 9 3 2020 HMGB1 Translocation in Neurons after Ischemic Insult: Subcellular Localization in Mitochondria and Peroxisomes 643 EN Dengli Wang Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Keyue Liu Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Yusuke Fukuyasu Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Kiyoshi Teshigawara Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Li Fu Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Hidenori Wake Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Aiji Ohtsuka Department of Human Morphology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University Masahiro Nishibori Department of Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University High mobility group box-1 (HMGB1), a nonhistone chromatin DNA-binding protein, is released from neurons into the extracellular space under ischemic, hemorrhagic, and traumatic insults. However, the details of the time-dependent translocation of HMGB1 and the subcellular localization of HMGB1 through the release process in neurons remain unclear. In the present study, we examined the subcellular localization of HMGB1 during translocation of HMGB1 in the cytosolic compartment using a middle cerebral artery occlusion and reperfusion model in rats. Double immunofluorescence microscopy revealed that HMGB1 immunoreactivities were colocalized with MTCO1(mitochondrially encoded cytochrome c oxidase I), a marker of mitochondria, and catalase, a marker of peroxisomes, but not with Rab5/Rab7 (RAS-related GTP-binding protein), LC3A/B (microtubule-associated protein 1 light chain 3), KDEL (KDEL amino acid sequence), and LAMP1 (Lysosomal Associated Membrane Protein 1), which are endosome, phagosome, endoplasmic reticulum, and lysosome markers, respectively. Immunoelectron microscopy confirmed that immune-gold particles for HMGB1 were present inside the mitochondria and peroxisomes. Moreover, HMGB1 was found to be colocalized with Drp1 (Dynamin-related protein 1), which is involved in mitochondrial fission. These results revealed the specific subcellular localization of HMGB1 during its release process under ischemic conditions. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 2073-4409 9 3 2020 Cell Stress Induced Stressome Release Including Damaged Membrane Vesicles and Extracellular HSP90 by Prostate Cancer Cells 755 EN Takanori Eguchi Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Chiharu Sogawa Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Kisho Ono Department of Oral and Maxillofacial Surgery, Okayama University Hospital Masaki Matsumoto Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University Tran Manh Tien Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Yuka Okusha Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Benjamin J. Lang Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Kuniaki Okamoto Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University Stuart K. Calderwood Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School Tumor cells exhibit therapeutic stress resistance-associated secretory phenotype involving extracellular vesicles (EVs) such as oncosomes and heat shock proteins (HSPs). Such a secretory phenotype occurs in response to cell stress and cancer therapeutics. HSPs are stress-responsive molecular chaperones promoting proper protein folding, while also being released from cells with EVs as well as a soluble form known as alarmins. We have here investigated the secretory phenotype of castration-resistant prostate cancer (CRPC) cells using proteome analysis. We have also examined the roles of the key co-chaperone CDC37 in the release of EV proteins including CD9 and epithelial-to-mesenchymal transition (EMT), a key event in tumor progression. EVs derived from CRPC cells promoted EMT in normal prostate epithelial cells. Some HSP family members and their potential receptor CD91/LRP1 were enriched at high levels in CRPC cell-derived EVs among over 700 other protein types found by mass spectrometry. The small EVs (30-200 nm in size) were released even in a non-heated condition from the prostate cancer cells, whereas the EMT-coupled release of EVs (200-500 nm) and damaged membrane vesicles with associated HSP90 alpha was increased after heat shock stress (HSS). GAPDH and lactate dehydrogenase, a marker of membrane leakage/damage, were also found in conditioned media upon HSS. During this stress response, the intracellular chaperone CDC37 was transcriptionally induced by heat shock factor 1 (HSF1), which activated the CDC37 core promoter, containing an interspecies conserved heat shock element. In contrast, knockdown of CDC37 decreased EMT-coupled release of CD9-containing vesicles. Triple siRNA targeting CDC37, HSP90 alpha, and HSP90 beta was required for efficient reduction of this chaperone trio and to reduce tumorigenicity of the CRPC cells in vivo. Taken together, we define "stressome" as cellular stress-induced all secretion products, including EVs (200-500 nm), membrane-damaged vesicles and remnants, and extracellular HSP90 and GAPDH. Our data also indicated that CDC37 is crucial for the release of vesicular proteins and tumor progression in prostate cancer. No potential conflict of interest relevant to this article was reported.