A role of metallothionein-3 in radiation-induced autophagy in glioma cells
Identifieur interne : 000202 ( Pmc/Corpus ); précédent : 000201; suivant : 000203A role of metallothionein-3 in radiation-induced autophagy in glioma cells
Auteurs : Young Hyun Cho ; Seung-Hwan Lee ; Sook-Jeong Lee ; Ha Na Kim ; Jae-Young KohSource :
- Scientific Reports [ 2045-2322 ] ; 2020.
Abstract
Although metallothionein-3 (
Url:
DOI: 10.1038/s41598-020-58237-7
PubMed: 32029749
PubMed Central: 7005189
Links to Exploration step
PMC:7005189Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A role of metallothionein-3 in radiation-induced autophagy in glioma cells</title><author><name sortKey="Cho, Young Hyun" sort="Cho, Young Hyun" uniqKey="Cho Y" first="Young Hyun" last="Cho">Young Hyun Cho</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurosurgery, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Lee, Seung Hwan" sort="Lee, Seung Hwan" uniqKey="Lee S" first="Seung-Hwan" last="Lee">Seung-Hwan Lee</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Biomedical Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Lee, Sook Jeong" sort="Lee, Sook Jeong" uniqKey="Lee S" first="Sook-Jeong" last="Lee">Sook-Jeong Lee</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0470 4320</institution-id><institution-id institution-id-type="GRID">grid.411545.0</institution-id><institution>Department of Bioactive Material Science,</institution><institution>Jeonbuk National University,</institution></institution-wrap>Jeonju, Jeollabuk-do Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Ha Na" sort="Kim, Ha Na" uniqKey="Kim H" first="Ha Na" last="Kim">Ha Na Kim</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Koh, Jae Young" sort="Koh, Jae Young" uniqKey="Koh J" first="Jae-Young" last="Koh">Jae-Young Koh</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurology, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">32029749</idno><idno type="pmc">7005189</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005189</idno><idno type="RBID">PMC:7005189</idno><idno type="doi">10.1038/s41598-020-58237-7</idno><date when="2020">2020</date><idno type="wicri:Area/Pmc/Corpus">000202</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000202</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A role of metallothionein-3 in radiation-induced autophagy in glioma cells</title><author><name sortKey="Cho, Young Hyun" sort="Cho, Young Hyun" uniqKey="Cho Y" first="Young Hyun" last="Cho">Young Hyun Cho</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurosurgery, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Lee, Seung Hwan" sort="Lee, Seung Hwan" uniqKey="Lee S" first="Seung-Hwan" last="Lee">Seung-Hwan Lee</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Biomedical Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Lee, Sook Jeong" sort="Lee, Sook Jeong" uniqKey="Lee S" first="Sook-Jeong" last="Lee">Sook-Jeong Lee</name><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0470 4320</institution-id><institution-id institution-id-type="GRID">grid.411545.0</institution-id><institution>Department of Bioactive Material Science,</institution><institution>Jeonbuk National University,</institution></institution-wrap>Jeonju, Jeollabuk-do Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Kim, Ha Na" sort="Kim, Ha Na" uniqKey="Kim H" first="Ha Na" last="Kim">Ha Na Kim</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author><author><name sortKey="Koh, Jae Young" sort="Koh, Jae Young" uniqKey="Koh J" first="Jae-Young" last="Koh">Jae-Young Koh</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation><affiliation><nlm:aff id="Aff5"><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurology, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</nlm:aff></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">Although metallothionein-3 (<italic>MT3</italic>), a brain-enriched form of metallothioneins, has been linked to Alzheimer’s disease, little is known regarding the role of <italic>MT3</italic> in glioma. As <italic>MT3</italic> plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of <italic>MT3</italic> with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by <italic>MT3</italic> knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, <italic>MT3</italic> knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that <italic>MT3</italic> and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Stupp, R" uniqKey="Stupp R">R Stupp</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wu, Wk" uniqKey="Wu W">WK Wu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dalby, Kn" uniqKey="Dalby K">KN Dalby</name></author><author><name sortKey="Tekedereli, I" uniqKey="Tekedereli I">I Tekedereli</name></author><author><name sortKey="Lopez Berestein, G" uniqKey="Lopez Berestein G">G Lopez-Berestein</name></author><author><name sortKey="Ozpolat, B" uniqKey="Ozpolat B">B Ozpolat</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Amaravadi, Rk" uniqKey="Amaravadi R">RK Amaravadi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Janku, F" uniqKey="Janku F">F Janku</name></author><author><name sortKey="Mcconkey, Dj" uniqKey="Mcconkey D">DJ McConkey</name></author><author><name sortKey="Hong, Ds" uniqKey="Hong D">DS Hong</name></author><author><name sortKey="Kurzrock, R" uniqKey="Kurzrock R">R Kurzrock</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Krizkova, S" uniqKey="Krizkova S">S Krizkova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pedersen, Mo" uniqKey="Pedersen M">MO Pedersen</name></author><author><name sortKey="Larsen, A" uniqKey="Larsen A">A Larsen</name></author><author><name sortKey="Stoltenberg, M" uniqKey="Stoltenberg M">M Stoltenberg</name></author><author><name sortKey="Penkowa, M" uniqKey="Penkowa M">M Penkowa</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sens, Ma" uniqKey="Sens M">MA Sens</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Somji, S" uniqKey="Somji S">S Somji</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Werynska, B" uniqKey="Werynska B">B Werynska</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Juang, Hh" uniqKey="Juang H">HH Juang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kmiecik, Am" uniqKey="Kmiecik A">AM Kmiecik</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Deng, D" uniqKey="Deng D">D Deng</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peng, D" uniqKey="Peng D">D Peng</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tao, Yf" uniqKey="Tao Y">YF Tao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Falnoga, I" uniqKey="Falnoga I">I Falnoga</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mehrian Shai, R" uniqKey="Mehrian Shai R">R Mehrian-Shai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Uchida, Y" uniqKey="Uchida Y">Y Uchida</name></author><author><name sortKey="Takio, K" uniqKey="Takio K">K Takio</name></author><author><name sortKey="Titani, K" uniqKey="Titani K">K Titani</name></author><author><name sortKey="Ihara, Y" uniqKey="Ihara Y">Y Ihara</name></author><author><name sortKey="Tomonaga, M" uniqKey="Tomonaga M">M Tomonaga</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Erickson, Jc" uniqKey="Erickson J">JC Erickson</name></author><author><name sortKey="Hollopeter, G" uniqKey="Hollopeter G">G Hollopeter</name></author><author><name sortKey="Thomas, Sa" uniqKey="Thomas S">SA Thomas</name></author><author><name sortKey="Froelick, Gj" uniqKey="Froelick G">GJ Froelick</name></author><author><name sortKey="Palmiter, Rd" uniqKey="Palmiter R">RD Palmiter</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Carrasco, J" uniqKey="Carrasco J">J Carrasco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Puttaparthi, K" uniqKey="Puttaparthi K">K Puttaparthi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Park, Mh" uniqKey="Park M">MH Park</name></author><author><name sortKey="Kim, Hj" uniqKey="Kim H">HJ Kim</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="West, Ak" uniqKey="West A">AK West</name></author><author><name sortKey="Hidalgo, J" uniqKey="Hidalgo J">J Hidalgo</name></author><author><name sortKey="Eddins, D" uniqKey="Eddins D">D Eddins</name></author><author><name sortKey="Levin, Ed" uniqKey="Levin E">ED Levin</name></author><author><name sortKey="Aschner, M" uniqKey="Aschner M">M Aschner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Jy" uniqKey="Lee J">JY Lee</name></author><author><name sortKey="Kim, Jh" uniqKey="Kim J">JH Kim</name></author><author><name sortKey="Palmiter, Rd" uniqKey="Palmiter R">RD Palmiter</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hwang, Jj" uniqKey="Hwang J">JJ Hwang</name></author><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Kim, Ty" uniqKey="Kim T">TY Kim</name></author><author><name sortKey="Cho, Jh" uniqKey="Cho J">JH Cho</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Cho, Ks" uniqKey="Cho K">KS Cho</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Seo, Br" uniqKey="Seo B">BR Seo</name></author><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Cho, Ks" uniqKey="Cho K">KS Cho</name></author><author><name sortKey="Yoon, Yh" uniqKey="Yoon Y">YH Yoon</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Jh" uniqKey="Lee J">JH Lee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Poole, B" uniqKey="Poole B">B Poole</name></author><author><name sortKey="Ohkuma, S" uniqKey="Ohkuma S">S Ohkuma</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rangwala, R" uniqKey="Rangwala R">R Rangwala</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mahalingam, D" uniqKey="Mahalingam D">D Mahalingam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rosenfeld, Mr" uniqKey="Rosenfeld M">MR Rosenfeld</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rangwala, R" uniqKey="Rangwala R">R Rangwala</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vogl, Dt" uniqKey="Vogl D">DT Vogl</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Knipp, M" uniqKey="Knipp M">M Knipp</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cai, L" uniqKey="Cai L">L Cai</name></author><author><name sortKey="Satoh, M" uniqKey="Satoh M">M Satoh</name></author><author><name sortKey="Tohyama, C" uniqKey="Tohyama C">C Tohyama</name></author><author><name sortKey="Cherian, Mg" uniqKey="Cherian M">MG Cherian</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ding, Zc" uniqKey="Ding Z">ZC Ding</name></author><author><name sortKey="Ni, Fy" uniqKey="Ni F">FY Ni</name></author><author><name sortKey="Huang, Zx" uniqKey="Huang Z">ZX Huang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lee, Sj" uniqKey="Lee S">SJ Lee</name></author><author><name sortKey="Seo, Br" uniqKey="Seo B">BR Seo</name></author><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="El Ghazi, I" uniqKey="El Ghazi I">I El Ghazi</name></author><author><name sortKey="Martin, Bl" uniqKey="Martin B">BL Martin</name></author><author><name sortKey="Armitage, Im" uniqKey="Armitage I">IM Armitage</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bell, Sg" uniqKey="Bell S">SG Bell</name></author><author><name sortKey="Vallee, Bl" uniqKey="Vallee B">BL Vallee</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jacobs, Vl" uniqKey="Jacobs V">VL Jacobs</name></author><author><name sortKey="Valdes, Pa" uniqKey="Valdes P">PA Valdes</name></author><author><name sortKey="Hickey, Wf" uniqKey="Hickey W">WF Hickey</name></author><author><name sortKey="De Leo, Ja" uniqKey="De Leo J">JA De Leo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Koh, Jy" uniqKey="Koh J">JY Koh</name></author><author><name sortKey="Choi, Dw" uniqKey="Choi D">DW Choi</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Sci Rep</journal-id><journal-id journal-id-type="iso-abbrev">Sci Rep</journal-id><journal-title-group><journal-title>Scientific Reports</journal-title></journal-title-group><issn pub-type="epub">2045-2322</issn><publisher><publisher-name>Nature Publishing Group UK</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32029749</article-id><article-id pub-id-type="pmc">7005189</article-id><article-id pub-id-type="publisher-id">58237</article-id><article-id pub-id-type="doi">10.1038/s41598-020-58237-7</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A role of metallothionein-3 in radiation-induced autophagy in glioma cells</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Cho</surname><given-names>Young Hyun</given-names></name><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Seung-Hwan</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Sook-Jeong</given-names></name><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Ha Na</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Koh</surname><given-names>Jae-Young</given-names></name><address><email>jkko@amc.seoul.kr</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff5">5</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Neural Injury Research Center, Asan Institute for Life Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurosurgery, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Biomedical Sciences,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</aff><aff id="Aff4"><label>4</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0470 4320</institution-id><institution-id institution-id-type="GRID">grid.411545.0</institution-id><institution>Department of Bioactive Material Science,</institution><institution>Jeonbuk National University,</institution></institution-wrap>Jeonju, Jeollabuk-do Republic of Korea</aff><aff id="Aff5"><label>5</label><institution-wrap><institution-id institution-id-type="ISNI">0000 0004 0533 4667</institution-id><institution-id institution-id-type="GRID">grid.267370.7</institution-id><institution>Department of Neurology, Asan Medical Center,</institution><institution>University of Ulsan College of Medicine,</institution></institution-wrap>Seoul, Republic of Korea</aff></contrib-group><pub-date pub-type="epub"><day>6</day><month>2</month><year>2020</year></pub-date><pub-date pub-type="pmc-release"><day>6</day><month>2</month><year>2020</year></pub-date><pub-date pub-type="collection"><year>2020</year></pub-date><volume>10</volume><elocation-id>2015</elocation-id><history><date date-type="received"><day>12</day><month>4</month><year>2019</year></date><date date-type="accepted"><day>13</day><month>1</month><year>2020</year></date></history><permissions><copyright-statement>© The Author(s) 2020</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold> This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>.</license-p></license></permissions><abstract id="Abs1"><p id="Par1">Although metallothionein-3 (<italic>MT3</italic>), a brain-enriched form of metallothioneins, has been linked to Alzheimer’s disease, little is known regarding the role of <italic>MT3</italic> in glioma. As <italic>MT3</italic> plays a role in autophagy in astrocytes, here, we investigated its role in irradiated glioma cells. Irradiation increased autophagy flux in GL261 glioma cells as evidenced by increased levels of LC3-II but decreased levels of p62 (SQSTM1). Indicating that autophagy plays a cytoprotective role in glioma cell survival following irradiation, measures inhibiting autophagy flux at various steps decreased their clonogenic survival of irradiated GL261 as well as SF295 and U251 glioma cells. Knockdown of <italic>MT3</italic> with siRNA in irradiated glioma cells induced arrested autophagy, and decreased cell survival. At the same time, the accumulation of labile zinc in lysosomes was markedly attenuated by <italic>MT3</italic> knockdown. Indicating that such zinc accumulation was important in autophagy flux, chelation of zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), induced arrested autophagy in and reduced survival of GL261 cells following irradiation. Suggesting a possible mechanism for arrested autophagy, <italic>MT3</italic> knockdown and zinc chelation were found to impair lysosomal acidification. Since autophagy flux plays a cytoprotective role in irradiated glioma cells, present results suggest that <italic>MT3</italic> and zinc may be regarded as possible therapeutic targets to sensitize glioma cells to ionizing radiation therapy.</p></abstract><kwd-group kwd-group-type="npg-subject"><title>Subject terms</title><kwd>CNS cancer</kwd><kwd>Macroautophagy</kwd><kwd>Mechanisms of disease</kwd><kwd>Cancer in the nervous system</kwd><kwd>Gliogenesis</kwd></kwd-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">https://doi.org/10.13039/501100005006</institution-id><institution>Asan Institute for Life Sciences, Asan Medical Center</institution></institution-wrap></funding-source><award-id>2009-466</award-id><principal-award-recipient><name><surname>Cho</surname><given-names>Young Hyun</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">https://doi.org/10.13039/501100003725</institution-id><institution>National Research Foundation of Korea (NRF)</institution></institution-wrap></funding-source><award-id>2016R1D1A1B04934383</award-id><award-id>NRF-2016R1E1A1A01941212</award-id><award-id>NRF-2017M3C7A1028949</award-id><principal-award-recipient><name><surname>Lee</surname><given-names>Sook-Jeong</given-names></name><name><surname>Koh</surname><given-names>Jae-Young</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">https://doi.org/10.13039/501100003710</institution-id><institution>Korea Health Industry Development Institute (KHIDI)</institution></institution-wrap></funding-source><award-id>HI14C1913</award-id><principal-award-recipient><name><surname>Koh</surname><given-names>Jae-Young</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2020</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1" sec-type="introduction"><title>Introduction</title><p id="Par2">Patients with glioblastoma, the most malignant form of glioma, usually succumb within a few years after the diagnosis despite aggressive chemo and radiotherapies following surgery<sup><xref ref-type="bibr" rid="CR1">1</xref></sup>. Such disheartening therapeutic outcomes desperately call for more effective measures. However, untoward biological characteristics of glioma such as aggressive local invasion and resistance to anticancer therapy, present a daunting obstacle in achieving this goal.</p><p id="Par3">Dysregulation of autophagy has been implicated in cancer cell proliferation, invasion, high histological grades, and poor prognosis in a wide spectrum of cancers. These findings highlight the possibility that autophagy-related molecules may prove useful as prognostic markers and/or therapeutic targets. In addition, many of anticancer drugs along with ionizing radiation (IR) are known to activate autophagy in cancer cells. The functional significance of autophagy activation associated with cancer therapeutics is still controversial. A growing body of evidence supports the possibility that autophagy plays a self-defensive cytoprotective role against toxicity induced by anti-cancer therapies<sup><xref ref-type="bibr" rid="CR2">2</xref>–<xref ref-type="bibr" rid="CR4">4</xref></sup>, although opposing data favoring the cytotoxic effect of autophagy are also available<sup><xref ref-type="bibr" rid="CR2">2</xref>,<xref ref-type="bibr" rid="CR3">3</xref></sup>. Despite these controversies, the current consensus is that the modulation of autophagy may have significant effects in anti-cancer therapeutics. Although a number of intrinsic or extrinsic pathways of autophagy have been delineated, the final destination of all these pathways is the lysosome, where degradation of cargo contents occurs. For this reason, diverse attempts have been made to maximize cancer cell death following the treatments through reducing the rate of autophagy flux with drugs acting on lysosomes<sup><xref ref-type="bibr" rid="CR4">4</xref>,<xref ref-type="bibr" rid="CR5">5</xref></sup>. For instance, currently, several clinical trials are investigating the effect of the combination treatment with lysosomal inhibitors such as chloroquine (CQ) or hydroxychloroquine (HCQ) and various anticancer drugs or IR in various cancer types (<ext-link ext-link-type="uri" xlink:href="http://clinicaltrials.gov">http://clinicaltrials.gov</ext-link>).</p><p id="Par4">Recent evidence indicates that aberrancy in zinc homeostasis is linked to a variety of cancers including breast, esophageal, gastric, colon, nasopharyngeal, prostate, bladder, and skin cancers<sup><xref ref-type="bibr" rid="CR6">6</xref></sup>. As regard to the mechanistic roles of zinc dyshomeostasis in cancer cell proliferation, well known effects of zinc on transcriptional regulation of genes involved in cell growth, proliferation, and apoptosis, and on the oxidative stress have been suggested as such. Since zinc is involved in so many cellular processes, there exist a number of zinc binding proteins in cells. Of many zinc-binding proteins, metallothioneins (<italic>MTs</italic>) are regarded as the major regulators of cellular zinc. Of four isoforms (<italic>MT1</italic>–4), the ubiquitously expressed <italic>MT1</italic> and <italic>MT2</italic> have been most extensively investigated and frequently found to be overexpressed in many cancers such as breast, ovarian, renal, prostate, lung and colorectal cancers as well as soft tissue sarcomas, and associated with poor patient prognosis<sup><xref ref-type="bibr" rid="CR6">6</xref>,<xref ref-type="bibr" rid="CR7">7</xref></sup>. On the contrary, relatively little is known regarding the role of <italic>MT3</italic>, a CNS-enriched Mt isoform, in cancers including glioma. Unlike <italic>MT1</italic> and <italic>MT2</italic>, the status of <italic>MT3</italic> expression has been found rather inconsistent among different cancer types, upregulated in some cancers (i.e. breast, prostate, urinary bladder, and lung cancers)<sup><xref ref-type="bibr" rid="CR8">8</xref>–<xref ref-type="bibr" rid="CR12">12</xref></sup>, while downregulated in others (i.e. gastric and esophageal cancers and leukemia)<sup><xref ref-type="bibr" rid="CR13">13</xref>–<xref ref-type="bibr" rid="CR15">15</xref></sup>. Moreover, data regarding the role of <italic>MT3</italic> are still scant and contradictory, some suggesting malignant phenotype-promoting<sup><xref ref-type="bibr" rid="CR8">8</xref>,<xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR11">11</xref>,<xref ref-type="bibr" rid="CR12">12</xref></sup> or others antitumor effect<sup><xref ref-type="bibr" rid="CR15">15</xref></sup> of <italic>MT3</italic>. As for glioma, induced expression of <italic>MT3</italic> as well as <italic>MT1</italic> and <italic>MT2</italic> following arsenic trioxide treatment on U87-MG glioblastoma cells was demonstrated<sup><xref ref-type="bibr" rid="CR16">16</xref></sup>, which may be postulated as a potential mechanism for glioma resistance. Importantly, Mehrian-Shai <italic>et al</italic>.<sup><xref ref-type="bibr" rid="CR17">17</xref></sup> recently reported that, in glioblastoma patients, high expression of including <italic>MT3</italic> was associated with poor patient survival whereas low <italic>MT</italic> levels corresponded to good prognosis, suggesting the prognostic implications of Mts in glioma.</p><p id="Par5">Since <italic>MT3</italic> was first identified as a neuronal growth inhibitory factor (GIF) that was deficient in brain extracts of Alzheimer’s disease<sup><xref ref-type="bibr" rid="CR18">18</xref></sup>, altered <italic>MT3</italic> expression has been also reported in various neurological disorders such as Parkinson’s disease, Amyotrophic lateral sclerosis (ALS), Down syndrome, and Creutzfeld-Jakob disease<sup><xref ref-type="bibr" rid="CR19">19</xref></sup>. The role of MT3 in the CNS pathologies appears to be either neuroprotective or cytotoxic depending on the experimental models. The neuroprotective effect of MT3, which is presumably mediated by its metal chelating and antioxidative abilities, was observed in epileptic brain injury, cortical cryoinjury, and a mutant superoxide dismutase 1 mouse model of ALS<sup><xref ref-type="bibr" rid="CR20">20</xref>–<xref ref-type="bibr" rid="CR22">22</xref></sup>. On the other hand, the cytotoxic effect of MT3 has been also demonstrated; intracellular zinc released from MT3 may trigger neuronal and astrocytic cell death<sup><xref ref-type="bibr" rid="CR23">23</xref>–<xref ref-type="bibr" rid="CR25">25</xref></sup>. We have previously demonstrated that MT3 plays a key role in regulating the function of lysosomes in astrocytes, in a zinc- and actin-dependent manner, which effects are not shared by MT1 and MT2<sup><xref ref-type="bibr" rid="CR23">23</xref></sup>. Of note, free zinc may contribute to autophagic flux in neurons and astrocytes, as evidenced by the result that oxidative stress induces accumulation of zinc ions in autophagosomes and lysosomes, and the inhibition of zinc accumulation by chelators or MT3 silencing blocks the increases in autophagy flux<sup><xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR26">26</xref>,<xref ref-type="bibr" rid="CR27">27</xref></sup>. Although detailed information as to how zinc increases autophagy flux, one possible mechanism may be that lysosomal zinc accumulation is correlated with subsequent lysosomal acidification<sup><xref ref-type="bibr" rid="CR28">28</xref></sup>. Lysosomal acidity has been found to be defective in neurodegenerative conditions such as Alzheimer’s disease, in which autophagy flux is arrested<sup><xref ref-type="bibr" rid="CR29">29</xref></sup>.</p><p id="Par6">Based on these findings, we hypothesized that glioma cells, as a cancerous counterpart of glial cells of developing brains, might utilize this novel cellular mechanism involving MT3 and zinc as a route for circumventing the toxicity of IR. Here, we explored this intriguing possibility.</p></sec><sec id="Sec2" sec-type="results"><title>Results</title><sec id="Sec3"><title>Increases in autophagy flux in irradiated GL261 glioma cells</title><p id="Par7">To assess the autophagy flux in GL261 glioma cells after irradiation, we measured levels of LC3-II, a marker for autophagy activation, and p62 (SQSTM1), a marker for autophagic/lysosomal degradation. Immunoblots showed that levels of LC3-II gradually increased in GL261 glioma cells following irradiation at 2 Gy, reaching its peak level at 4 h and decreasing thereafter (Fig. <xref rid="Fig1" ref-type="fig">1a</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">1(a)</xref> for the original blot). On the other hand, levels of p62 substantially decreased after irradiation (Fig. <xref rid="Fig1" ref-type="fig">1b</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">1(b)</xref> for the original blot). Moreover, blockade of lysosomal degradation with bafilomycin A1 (BA) resulted in a further increase in LC3-II levels (Fig. <xref rid="Fig1" ref-type="fig">1c</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">1(c)</xref> for the original blot), consistent with an increase in autophagy flux. Morphologically, confocal fluorescence microscopy showed that the number and intensity of RFP-LC3-positive vesicular puncta increased in the cytoplasm of RFP-LC3-transfected GL261 cells following irradiation (Fig. <xref rid="Fig1" ref-type="fig">1d</xref>). All these results indicated that irradiation increased autophagy flux in GL261 glioma cells.<fig id="Fig1"><label>Figure 1</label><caption><p>Induction of autophagy in irradiated GL261 cells. (<bold>a</bold>) Western blots for LC3 at 1 to 5 h after irradiation at 2 Gy. Western blot for β-actin is presented as a loading control. Bars denote the density ratio of LC3-II bands to the corresponding β-actin bands, normalized against the ratio in control (0 h) (mean ± SEM; <italic>n</italic> = 3 cultures; **<italic>P</italic> < 0.01 vs. 0 h). (<bold>b</bold>) Western blots for p62 (SQSTM1) 4 h after irradiation (RT) (mean ± SEM; <italic>n</italic> = 3 cultures; *<italic>P</italic> < 0.05 vs. control). (<bold>c</bold>) The increase in LC3-II levels after irradiation was accelerated when cells were treated with 50 nM BA immediately after irradiation (mean ± SEM; <italic>n</italic> = 3 cultures; *<italic>P</italic> < 0.05 vs. control with BA; **<italic>P</italic> < 0.01 vs. vehicle with RT). (<bold>d</bold>) RFP-LC3 fluorescence (red) in cells before (control) and 4 h after irradiation (RT). The number and intensity of RFP-LC3-positive vesicular puncta noticeably increased after irradiation. Confocal microscopic images were taken from a single Z-section. Scale bar: 10 μm.</p></caption><graphic xlink:href="41598_2020_58237_Fig1_HTML" id="d29e593"></graphic></fig></p></sec><sec id="Sec4"><title>Inhibition of autophagy decreases clonogenic survival of irradiated GL261 cells</title><p id="Par8">To determine the functional significance of autophagy flux increased by irradiation, autophagy flux in irradiated cells was inhibited with pharmacologic inhibitors, BA or 3-methyladenine (3MA), or with siRNA against <italic>Beclin1</italic> (beclin). BA or 3MA was given immediately after irradiation, whereas siRNA was given 24 h prior to the irradiation. In each experiment, effective inhibition of autophagy flux was confirmed by an increase in p62 levels on Western blot analysis (Fig. <xref rid="Fig2" ref-type="fig">2a–c</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">2(a–c)</xref> for the original blot). In colony forming assays, as compared with vehicle alone or negative-transfected controls (NC), cells treated with the drugs or <italic>beclin</italic> siRNA exhibited significantly lesser survival after irradiation (Fig. <xref rid="Fig2" ref-type="fig">2d–f</xref>). Since colony forming assay measures total cell number reflecting not only cell death but cell growth, we measured LDH efflux from dead cells as more direct measure for cell death. Also, we examined other human-derived glioma cell lines, SF295 and U251 cells. In all glioma cell lines, irradiation-induced cell death (LDH release) was increased by above measures inhibiting autophagy flux (Suppl. Fig. <xref rid="MOESM1" ref-type="media">6</xref>).<fig id="Fig2"><label>Figure 2</label><caption><p>Inhibition of autophagy decreases clonogenic survival of irradiated GL261 cells. (<bold>a</bold>–<bold>c</bold>) Western blots for p62 4 h after irradiation at 2 Gy. Cells were treated with 50 nM BA (<bold>a</bold>) or 1 mM 3MA (<bold>b</bold>) immediately after irradiation, or transfected with <italic>beclin</italic> siRNA (<italic>beclin</italic>) or control siRNA (NC) for 24 h and then irradiated (<bold>c</bold>). Bars denote relative density of p62 bands normalized to corresponding β-actin bands in respective experiments (mean ± SEM; <italic>n</italic> = 3 cultures, each; **<italic>P</italic> < 0.01 vs. vehicle or NC with RT). (<bold>d</bold>–<bold>f</bold>) Cells were seeded at a density of 200 cells per well and allowed to attach overnight, and irradiated with treatment of BA (<bold>d</bold>), 3MA (<bold>e</bold>), or transfection with <italic>beclin</italic> siRNA (<italic>beclin</italic>) or control siRNA (NC) (<bold>f</bold>). The relative cell survival rates were calculated against the plating efficiency in control with vehicle (mean ± SEM; <italic>n</italic> = 3 or 4 cultures; *<italic>P</italic> < 0.05 or **<italic>P</italic> < 0.01 vs. vehicle or NC with RT).</p></caption><graphic xlink:href="41598_2020_58237_Fig2_HTML" id="d29e686"></graphic></fig></p></sec><sec id="Sec5"><title>siRNA knockdown of <italic>MT3</italic> blocks lysosomal degradation of autophagic vacuoles in irradiated GL261 cells and decreases cell survival</title><p id="Par9">We have previously demonstrated that <italic>MT3</italic> plays a regulatory role in autophagy flux in primary cultured astrocytes<sup><xref ref-type="bibr" rid="CR23">23</xref></sup>. To examine the role of <italic>MT3</italic> in GL261 glioma cell line, we downregulated <italic>MT3</italic> using siRNA (Fig. <xref rid="Fig3" ref-type="fig">3a</xref>). Compared with GL261 cells that were transfected with NC siRNA, GL261 cells that were transfected with <italic>MT3</italic> siRNA, showed increases in the levels of both LC3-II and p62 after irradiation (Fig. <xref rid="Fig3" ref-type="fig">3b,c</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">3(b,c)</xref> for the original blot), suggesting that autophagy flux was inhibited distal to the autophagosome formation in the <italic>MT3</italic>-downregulated cells. To further confirm this, we observed RFP-LC3-transfected GL261 cells stained with Lysotracker Green under fluorescence microscope. In <italic>MT3</italic> siRNA-transfected cells, both RFP-LC3 fluorescence and Lysotracker fluorescence were increased in lysosomes (Fig. <xref rid="Fig3" ref-type="fig">3d</xref>), consistent with the possibility that lysosomal degradation of autophagic vacuole-associated RFP-LC3 was impaired. Subsequent survival analysis demonstrated that <italic>MT3</italic> knockdown decreased the survival of GL261 glioma cells after irradiation (Fig. <xref rid="Fig3" ref-type="fig">3e</xref>).<fig id="Fig3"><label>Figure 3</label><caption><p>Knockdown of <italic>MT3</italic> blocks lysosomal degradation of AVs in irradiated GL261 cells and decreases cell survival. (<bold>a</bold>) Quantitative analysis of mRNA levels by RT-PCR for <italic>MT3</italic> after 24 h transfection with siRNA against <italic>MT3</italic>. Bars denote the ratio of MT3 mRNA values to the corresponding Gapdh mRNA values, normalized against the ratio in control siRNA (NC) (mean ± SEM; <italic>n</italic> = 3 cultures; <italic>**P</italic> < 0.01 vs. NC). (<bold>b</bold>,<bold>c</bold>) Western blots for LC3 (<bold>b</bold>) and p62 (<bold>c</bold>) 4 h after irradiation at 2 Gy in cells transfected with <italic>MT3</italic> siRNA or control siRNA (NC) (mean ± SEM; <italic>n</italic> = 4 cultures, each; **<italic>P</italic> < 0.01 vs. NC with RT). (<bold>D</bold>) Confocal fluorescence microscopic images of RFP-LC3-transfected cells stained with Lysotracker Green. Cells were transfected with <italic>MT3</italic> siRNA or control siRNA (NC), and irradiated. The fluorescences of RFP-LC3 and Lysotracker in <italic>MT3</italic> siRNA-transfected cells strikingly accumulated and overlapped 4 h after irradiation, indicating that lysosomal degradation of AVs was severely impaired. Images were taken from a single Z-section. Scale bar: 10 μm. (<bold>e</bold>) Clonogenic survival of irradiated cells transfected with <italic>MT3</italic> siRNA or control siRNA (NC). Bars denote the relative survival rates as described in Fig. <xref rid="Fig2" ref-type="fig">2</xref> (mean ± SEM; <italic>n</italic> = 3 cultures; <italic>*P</italic> < 0.05 vs. NC with RT).</p></caption><graphic xlink:href="41598_2020_58237_Fig3_HTML" id="d29e811"></graphic></fig></p><p id="Par10">To confirm the finding, we prepared another siRNA to <italic>MT3</italic>. Both siRNAs reduced Mt3 mRNA levels (Suppl. Fig. <xref rid="MOESM1" ref-type="media">7a</xref>). The second siRNA also augmented radiation-induced cell death of GL261, SF295, and U251 glioma cells (Suppl. Fig. <xref rid="MOESM1" ref-type="media">7b</xref>).</p></sec><sec id="Sec6"><title>Accumulation of labile zinc in lysosomes following irradiation, which is attenuated by <italic>MT3</italic> knockdown</title><p id="Par11">As <italic>MT3</italic> releases zinc upon various stimuli such as oxidative stress, the fact that <italic>MT3</italic> knockdown reduced autophagy flux in irradiated GL261 cells raised a possibility that <italic>MT3</italic>-dependent zinc dynamics may be involved in this phenomenon. In line with our previous observation of zinc accumulation in lysosomes of neurons and astrocytes under oxidative stress<sup><xref ref-type="bibr" rid="CR26">26</xref>,<xref ref-type="bibr" rid="CR27">27</xref></sup>, irradiation of GL261 cells induced an elevation of labile zinc levels inside cells within 2 h as visualized with FluoZin-3 fluorescence microscopy; double staining of these cells with Lysotracker Red revealed that, most of zinc fluorescence was localized inside lysosomes (Fig. <xref rid="Fig4" ref-type="fig">4a</xref>). As expected from our previous study in cultured cortical astrocytes<sup><xref ref-type="bibr" rid="CR23">23</xref></sup>, knockdown of <italic>MT3</italic> markedly attenuated the increase in labile zinc following irradiation (Fig. <xref rid="Fig4" ref-type="fig">4b</xref>). In addition, treatment with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a cell-permeant zinc chelator, almost completely blocked the increase in free zinc levels in lysosomes following irradiation (Fig. <xref rid="Fig4" ref-type="fig">4b</xref>).<fig id="Fig4"><label>Figure 4</label><caption><p>Accumulation of labile zinc in lysosomes following irradiation, which is attenuated by <italic>MT3</italic> knockdown. (<bold>a</bold>) Confocal fluorescence microscopic images of GL261 cells double-stained with FluoZin-3 and Lysotracker Red 2 h after irradiation at 2 Gy. The levels of labile zinc (green) increased in the cytosol after irradiation with its localization to lysosomes or autolysosomes (red) as its signal significantly overlapped (merge) with Lysotracker. Images were taken from a single Z-section. Scale bar: 10 μm. (<bold>b</bold>) Confocal fluorescence microscopic images of GL261 cells stained with FluoZin-3 (green) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; blue). Cells were transfected with <italic>MT3</italic> siRNA or control siRNA (NC), or treated with 1 μM TPEN, and irradiated. Scale bar: 10 μm.</p></caption><graphic xlink:href="41598_2020_58237_Fig4_HTML" id="d29e884"></graphic></fig></p></sec><sec id="Sec7"><title>Chelation of intracellular zinc blocks lysosomal degradation of AVs in irradiated GL261 cells and decreases cell survival</title><p id="Par12">The two findings that labile zinc accumulates in lysosomes or autolysosomes following irradiation and that knockdown of <italic>MT3</italic>, a probable source of zinc, blocks degradation of AVs imply that zinc may have a direct effect on the degradative function of lysosomes, and consequently on cell survival. To test this hypothesis, we examined the effects of depleting or adding labile zinc on these events. In TPEN-treated (zinc-depleted) cells, changes similar to those seen in <italic>MT3</italic>-downregulated cells were observed. First, the levels of both LC3-II and p62 markedly increased after irradiation (Fig. <xref rid="Fig5" ref-type="fig">5a,b</xref>, see Supplementary Fig. <xref rid="MOESM1" ref-type="media">4(a,b)</xref> original blot). Second, striking accumulation of dilated RFP-LC3 (+) AVs in lysosomes was observed (Fig. <xref rid="Fig5" ref-type="fig">5c</xref>). These findings indicate that lysosomal degradation of AVs was reduced, and hence autophagy flux was arrested as in the case of <italic>MT3</italic>-knockdown cells. The clonogenic survival of TPEN-treated cells were also reduced after irradiation (Fig. <xref rid="Fig5" ref-type="fig">5d</xref>). In contrast to TPEN treatment, addition of zinc chloride (Zn) in the media, increased autophagy flux and lysosomal degradation as indicated by decreased levels of both LC3-II and p62 together with attenuated accumulation of AVs after irradiation (Fig. <xref rid="Fig5" ref-type="fig">5a–c</xref>). As expected, raising intracellular zinc with zinc treatment that increased autophagy flux, resulted in the increased cell survival following the irradiation (Fig. <xref rid="Fig5" ref-type="fig">5d</xref>).<fig id="Fig5"><label>Figure 5</label><caption><p>Chelation of intracellular zinc blocks lysosomal degradation of AVs in irradiated GL261 cells and decreases cell survival. (<bold>a</bold>,<bold>b</bold>) Western blots for LC3 (<bold>a</bold>) and p62 (<bold>b</bold>) 4 h after irradiation at 2 Gy in cells treated with 1 μM TPEN or 60 μM Zn (mean ± SEM; <italic>n</italic> = 3 cultures; *<italic>P</italic> < 0.05 or **<italic>P</italic> < 0.01 vs. vehicle with RT). (<bold>c</bold>) Confocal fluorescence microscopic images of RFP-LC3-transfected cells stained with Lysotracker Green. Cells were treated with TPEN or Zn, and irradiated. In TPEN-treated cells, a striking accumulation of dilated AVs mostly fused with lysosomes was noted 4 h after irradiation, which was concordant with the effect of <italic>MT3</italic> knockdown as seen in Fig. <xref rid="Fig3" ref-type="fig">3</xref>. In contrast, addition of Zn attenuated the accumulation of AVs after irradiation. Images were taken from a single Z-section. Scale bar: 10 μm. (<bold>d</bold>) Clonogenic survival of irradiated cells treated with 1 μM TPEN or 60 μM Zn. Bars denote the relative survival rates as described in Fig. <xref rid="Fig2" ref-type="fig">2</xref> (mean ± SEM; <italic>n</italic> = 5 cultures; **<italic>P</italic> < 0.01 vs. vehicle with RT).</p></caption><graphic xlink:href="41598_2020_58237_Fig5_HTML" id="d29e970"></graphic></fig></p></sec><sec id="Sec8"><title>Impaired lysosomal acidification by <italic>MT3</italic> knockdown and zinc chelation in irradiated GL261 cells</title><p id="Par13">In diverse cases of arrested autophagy, lysosomal acidification has been found to be abnormal. To examine this possibility, changes in lysosomal pH were assessed using a fluorescent lysosomal pH indicator dye Lysosensor Yellow/Blue dextran. Consistent with the result showing that <italic>MT3</italic> knockdown induced arrested autophagy in GL261 glioma cells, following irradiation <italic>MT3</italic> deficient glioma cells exhibited lysosomal pH shifting toward more alkaline direction as compared with that in Negative control transfected cells (Fig. <xref rid="Fig6" ref-type="fig">6a</xref>). Similar changes (“alkalinization”) in lysosomal pH following irradiation were observed in zinc-depleted (TPEN-treated) or NH<sub>4</sub>Cl-treated glioma cells (Fig. <xref rid="Fig6" ref-type="fig">6b</xref>). These results support the possibility that zinc and <italic>MT3</italic> play a critical role in the maintenance of acidity of lysosomal lumens in these cells.<fig id="Fig6"><label>Figure 6</label><caption><p>Impaired lysosomal acidification by <italic>MT3</italic> knockdown and zinc chelation in irradiated GL261 cells. (<bold>a</bold>,<bold>b</bold>) Confocal fluorescence microscopic images of irradiated cells stained with a lysosomal pH indicator dye Lysosensor Yellow/Blue dextran (Lysosensor). Cells were transfected with <italic>MT3</italic> siRNA or control siRNA (NC) (<bold>a</bold>) or treated with 1 μM TPEN, 60 μM Zn, or 60 μM NH<sub>4</sub>Cl (<bold>b</bold>), and irradiated at 2 Gy. The emission 510 and 450 nm were assigned the colors blue and yellow, respectively. The less acidic the vacuoles are, the lower the Blue/Yellow ratio is. Note the accumulation of yellow-green fluorescences in irradiated cells transfected with <italic>MT3</italic> siRNA or treated with TPEN or NH<sub>4</sub>Cl. Images were taken from a single Z-section. Scale bar: 10 μm.</p></caption><graphic xlink:href="41598_2020_58237_Fig6_HTML" id="d29e1034"></graphic></fig></p></sec></sec><sec id="Sec9" sec-type="discussion"><title>Discussion</title><p id="Par14">While autophagy has been implicated as an important factor in glioma cell biology, its precise role in radio-sensitivity of glioma has been still controversial as to the end result being beneficial or harmful. The current study presents novel insights into this issue. First, present results demonstrated that <italic>MT3</italic>, possibly by releasing zinc that enters lysosomes, is a key regulator of autophagy flux that changes in response to irradiation. Second, thusly enhanced autophagy flux may be cytoprotective and increase the survival of glioma cells after irradiation. Therefore, downregulation of <italic>MT3</italic> or zinc chelation at the time of irradiation treatment may provide a beneficial effect to glioma patients by blocking the autophagy flux and increasing glioma cell death. Mechanistically, it appears that irradiation releases zinc from <italic>MT3</italic>, which in turn causes lysosomal acidification and increases autophagy flux and lysosomal degradation. These results are in line with the glioma clinical trials with the lysosomal acidification inhibitors, CQ and HCQ<sup><xref ref-type="bibr" rid="CR30">30</xref></sup>, which have shown a modest efficacy<sup><xref ref-type="bibr" rid="CR31">31</xref>–<xref ref-type="bibr" rid="CR35">35</xref></sup>. Present results further support this idea, and suggest that the strategy aiming at the blockade of lysosomal acidification, especially targeting <italic>MT3</italic> and/or zinc, may prove useful to find a supportive measure to current glioma therapy.</p><p id="Par15">In the present study, to examine the effect of autophagy on irradiation-induced death of GL261 cells, we modulated the autophagy flux by several different methods. First, we used standard autophagy inhibitors such as 3MA, BA, and siRNA against <italic>beclin</italic>. All these measures inhibited autophagy flux, and resulted in the increased death of GL261 glioma cells following irradiation. Two other human glioma cell lines, SF295 and U251 cells, showed the same effects. Next, we examined the effects of <italic>MT3</italic> knockdown with siRNA as well as altering intracellular zinc levels with TPEN or zinc. As <italic>MT3</italic> knockdown or zinc depletion produced similar results as the above autophagy inhibitors, we concluded that <italic>MT3</italic> and zinc play a key role in positively modulating the autophagy flux.</p><p id="Par16"><italic>MTs</italic> are a family of small (6–7 kd), intracellular, cysteine-rich proteins with a number of thiol (-SH) groups enabling them to bind metals such as zinc and copper as well as toxic heavy metals. In oncogenesis, <italic>MTs</italic> are generally regarded to function as tumor suppressors in normal cells via chelating carcinogenic metal ions and maintaining redox homeostasis. On the other hand, <italic>MTs</italic> are upregulated in diverse cancer cells, and their levels are positively correlated with the degree of malignancy and the resistance to anticancer therapy<sup><xref ref-type="bibr" rid="CR36">36</xref>,<xref ref-type="bibr" rid="CR37">37</xref></sup>. For example, high levels of <italic>MTs</italic> confer resistance to platinum chemotherapeutics, and hence are regarded as a marker for poor prognosis<sup><xref ref-type="bibr" rid="CR6">6</xref>,<xref ref-type="bibr" rid="CR7">7</xref></sup>. The upregulation of <italic>MTs</italic> may be a response of cancer cells to metabolic stresses associated with aberrant cancer cell biology, and may function as a defense against anti-cancer therapeutics.</p><p id="Par17">There are 4 isoforms of <italic>MTs</italic>. Of these, <italic>MT3</italic> is expressed mainly in the CNS. With approximately 70% sequence homology to other <italic>MTs</italic>, <italic>MT3</italic> contains a unique conserved sequence TCPCP motif at positions 5–9 in the N-terminus. A characteristic conformational organization of the β domain by virtue of the TCPCP motif of <italic>MT3</italic> in its zinc-bound form is suggested to provide a potential interface for protein-protein interactions, which may be responsible for the distinct biological functions of <italic>MT3</italic> such as neuronal growth inhibitory activity<sup><xref ref-type="bibr" rid="CR38">38</xref>,<xref ref-type="bibr" rid="CR39">39</xref></sup>. Other biological functions of <italic>MT3</italic> include roles in glycolytic metabolism, protein chaperone and scaffolding functions, metal transport/buffering, and redox signaling<sup><xref ref-type="bibr" rid="CR25">25</xref>,<xref ref-type="bibr" rid="CR40">40</xref>,<xref ref-type="bibr" rid="CR41">41</xref></sup>. We have previously demonstrated that <italic>MT3</italic> plays a role in the maintenance of autophagy flux in cultured cortical astrocytes, likely by regulating lysosomal functions<sup><xref ref-type="bibr" rid="CR23">23</xref></sup>. Similar to the findings obtained in primary astrocytes, in the present study, siRNA knockdown of <italic>MT3</italic> in GL261 glioma cells induced arrested autophagy following irradiation, likely due to impaired lysosomal degradation. Our results indicated that release of free zinc from <italic>MT3</italic> may be a key event herein, since zinc chelation produced similar effects. Both <italic>MT3</italic> knockdown and zinc chelation induced significant alkalinization of lysosomes, which should inhibit acidic hydrolase activities in lysosomes. Conversely, supplementation of zinc induced lysosomal reacidification. Although it is unclear how intracellular zinc release and its accumulation in lysosomes help acidify lysosomes, one plausible speculation is that a certain Zn<sup>2+</sup>/H<sup>+</sup> antiporter may be functioning in lysosomes. Hence, zinc inside lysosomes may provide energy for proton to enter the lysosomal lumen. Alternatively, zinc may indirectly upregulate the expression of lysosomal proton pumps such as the vacuolar-type ATPase. These intriguing possibilities remain to be tested.</p><p id="Par18">The cytoprotective effect of <italic>MT3</italic> in glioma cells has been reported by others. For instance, elevated <italic>MT3</italic> expression in U87-MG glioblastoma cells than in normal astrocytes and other glioma cell line was correlated with an inactive conformational change of p53, resulting in attenuated apoptosis<sup><xref ref-type="bibr" rid="CR17">17</xref></sup>. Hence, <italic>MT3</italic> may contribute to glioma survival through diverse ways including increases in autophagy flux through lysosomal acidification. Further studies will be needed to elucidate the comprehensive mechanisms underlying the cytoprotective effect of <italic>MT3</italic> on glioma resistance.</p><p id="Par19">In summary, we demonstrated the roles of <italic>MT3</italic> and zinc in radiation-induced autophagy and radioresistance in glioma cells. <italic>MT3</italic> appears to be a key regulator for autophagy flux via zinc-dependent lysosomal acidification, and hence contributes to resistance of glioma cells to irradiation treatment. As such, <italic>MT3</italic> may prove to be a suitable target in improving the efficacy of irradiation treatment in gliomas.</p></sec><sec id="Sec10" sec-type="materials|methods"><title>Materials and Methods</title><sec id="Sec11"><title>Cell lines and cell culture</title><p id="Par20">The mouse glioma cell line GL261, recognized as recapitulating many of the features of human glioblastoma<sup><xref ref-type="bibr" rid="CR42">42</xref></sup>, was obtained from the Tumor Bank Repository at the National Cancer Institute (Frederick, MD, USA). The human glioma cell line U251 and SF295 was obtained from the Tumor Bank Repository at the National Cancer Institute (Frederick, MD, USA). The GL261 and U251 cells were cultured in Dulbecco’s modified eagle’s medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin; Lonza, Allendale, NJ, USA) at 37 °C with 5% CO<sub>2</sub>. The SF295 cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin; Lonza, Allendale, NJ, USA) at 37 °C with 5% CO<sub>2</sub>.</p></sec><sec id="Sec12"><title>Irradiation</title><p id="Par21">Cells were irradiated using a 6-MV photon beam linear accelerator system (Varian Medical Systems, Palo Alto, CA, USA). For radiation dose titration, a single-dose of 2, 5 or 10 Gy was tested for autophagy induction and cell survival. Having identified that 2 Gy of radiation was sufficient for autophagy induction and optimal for cell survival analysis (data not presented), 2 Gy was used for experiments.</p></sec><sec id="Sec13"><title>Reagents and antibodies</title><p id="Par22">3-methyladenine (3MA), bafilomycin A1 (BA), tetrakis [(2-pyridylmethyl)ethylenediamine] (TPEN), zinc chloride (Zn), ammonium chloride (NH<sub>4</sub>Cl), leupeptin, and pepstatin A were purchased from Sigma (St. Louis, MO, USA). Anti-microtubule-associated protein light chain 3 (LC3) and anti-p62 antibodies were obtained from Novus (Littleton, CO, USA) and MBL (Nagoya, Japan), respectively. Anti-β-actin and anti-beclin1 antibodies were from Cell Signaling (Beverly, MA, USA).</p></sec><sec id="Sec14"><title>Plate colony forming assay</title><p id="Par23">Cells were seeded in six-well plates at a density of 100 (for controls) or 200 cells (for irradiated cells) per well and allowed to attach overnight before irradiation. Fourteen days after irradiation, cells were fixed with 4% paraformaldehyde and stained with Giemsa staining reagent (Sigma). The number of colonies with > 50 cells was counted under a dissecting microscope. The cell survival fraction or relative survival rate was expressed in terms of the plating efficiency in control groups: the number of colonies formed after treatment divided by both the number of cells seeded and the plating efficiency. Each assay was performed in triplicate.</p></sec><sec id="Sec15"><title>LDH efflux assay for cell death</title><p id="Par24">Cells death was quantitatively assessed by measuring lactate dehydrogenase (LDH) activity released into the culture medium from damaged cells, as described previously<sup><xref ref-type="bibr" rid="CR43">43</xref></sup>. LDH concentrations were normalized to the mean concentration of cells exposed to 400 μM H<sub>2</sub>O<sub>2</sub> (100%) after subtracting the mean value of sham-washed control cells (0%).</p></sec><sec id="Sec16"><title>Western blot analysis</title><p id="Par25">Cells were washed with ice-cold PBS and lysed in PRO-PREP protein extraction solution (iNtRON, Sungnam, Korea). Cell lysates were separated on 12–15% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked and probed with antibodies against LC3, p62, beclin1 or β-actin for 12 h at 4 °C. After washing, blots were incubated with appropriate HRP-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature, and were washed. The blots were developed with SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Scientific, Waltham, MA, USA). The band intensity was analyzed using ImageJ (NIH, Bethesda, MD, USA). All experiments were triplicated.</p></sec><sec id="Sec17"><title>Real-Time reverse transcriptase PCR</title><p id="Par26">Total RNA was extracted from GL261, SH295 and U251 cells with Qiazol Reagent (Qiagen, Hilden, Germany). ImProm-2 reverse transcriptase kit (Promega, Madison, WI, USA) was used to generate cDNA according to the manufacturer’s instructions. Samples were subjected to real time PCR amplification using forward and reverse primers for <italic>MT3</italic>, LightCycler 480 SYBR Green 1 Master and LightCycler 480 machine (Roche, Mannheim, Germany). The following thermocycle conditions were used: an initial cycle at 95 °C for 5 min; 50 cycles, each at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s; and a final cycle at 95 °C for 15 min. Melting curve analysis was performed on all PCR products to ensure that specific PCR products were generated. Data were analyzed using the comparative cycle threshold method. The levels of mRNA were normalized to those of the housekeeping gene Gapdh (glyceraldehyde-3-dehydrogenase), and expressed as relative fold changes.</p></sec><sec id="Sec18"><title>Confocal microscopy</title><p id="Par27">1 × 10<sup>5</sup> GL261 cells were seeded onto a 0.1 mg/mL poly-L-lysine-coated coverslip in 4-well plates and incubated for 12 h. After irradiation, cells were stained with 100 nM Lysotracker Red DND-99 or Lysotracker Green DND-26 (Invitrogen, Carlsbad, CA, USA) for 1 h or 10 μM FluoZin-3 (Invitrogen) for 30 min before fixation. For detection of changes in lysosomal pH, cells were stained with 0.5 mg/mL Lysosensor Yellow/Blue dextran (Invitrogen), a fluorescent lysosomal pH indicator dye, for 1 h. Then, cells were washed with PBS twice and were fixed with 4% paraformaldehyde. For counterstain, the fixed cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were mounted with Fluoromount G (Southern Biotech, Birmingham, AL, USA), and were examined by LSM 710 confocal microscope (Carl Zeiss, Dublin, CA, USA).</p></sec><sec id="Sec19"><title>RFP-LC3 plasmid transfection</title><p id="Par28">The RFP-LC3 expression plasmid was a generous gift from Drs. Maria Colombo and Michel Rabinovitch (Universidad Nacional de Cuyo, Mendoza, Argentina). Plasmid transfection was performed using Effectene (Qiagen) according to the manufacturer’s instructions. After 24 h transfection, cellular expression of RFP-LC3 was confirmed by Western blot analysis.</p></sec><sec id="Sec20"><title>Silencing of <italic>beclin</italic> and <italic>MT3</italic> genes</title><p id="Par29">Small interfering RNA (siRNA) targeting for <italic>beclin</italic> was purchased from Thermo Scientific. Negative control siRNA and siRNAs targeting for <italic>MT3</italic> were from Genolution Pharmaceuticals Inc. (Seoul, Korea). Transient siRNA transfection into glioma cells was carried out using TransIT-TKO reagent (Mirus, Madison, WI, USA) according to the manufacturer’s instructions. After 24 h transfection with siRNA, the specific silencing was confirmed by real-time PCR or Western blot analysis as described elsewhere. Total RNA was reverse transcribed and the prepared cDNA was then subjected to PCR analysis using the following primer sets: Control siRNA (5′-CCUCGUGCCGUUCCAUCAGGUAGUU-3′, 5′-CUACCUGAUGGAACGGCACG-AGGUU-3′), siRNA targeting for <italic>MT3</italic> (GL261 cell) (5′-UAAAUCCCAUGCACAACAUUU-3′, 5′-AUGUUGUGCAUGGGAUUUAUU-3′), <italic>MT3</italic> #1 (5′-UAAAUCCCAUGAACAGCAUU-3′, 5′-UGCUGUUCAUGGGAUUUAUU-3′), <italic>MT3</italic> #2 (5′-GUGUGGCUGGUGUCCCCUU-3′, 5′-GGGGACACCAGCCACACUU-3′). Human cell line using MT3 #1 and #2 primer sequence.</p></sec><sec id="Sec21"><title>Statistics</title><p id="Par30">All data presented are representative from at least 3 independent experiments. The numeric data are presented as means ± SEMs and evaluated using the Student <italic>t</italic> tests. <italic>P</italic> < 0.05 was considered statistically significant.</p></sec></sec><sec sec-type="supplementary-material"><title>Supplementary information</title><sec id="Sec22"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="41598_2020_58237_MOESM1_ESM.docx"><caption><p>supplementary figures and legends.</p></caption></media></supplementary-material></p></sec></sec></body><back><fn-group><fn><p><bold>Publisher’s note</bold> Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn></fn-group><sec><title>Supplementary information</title><p>is available for this paper at 10.1038/s41598-020-58237-7.</p></sec><ack><title>Acknowledgements</title><p>This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT (NRF-2016R1E1A1A01941212, and NRF-2017M3C7A1028949), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Walfare, Republic of Korea (HI14C1913), the Asan Institute for Lift Science, Asan Medical Center, Republic of Korea (2009-466) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B04934383) and Jeonbuk National University.</p></ack><notes notes-type="author-contribution"><title>Author contributions</title><p>J.Y.K. supervised the project. Y.H.C. designed the experiments. S.H.L. and H.N.K. collected data and carried out the data analysis. S.J.L. interpreted the data. All authors are involved in manuscript completion.</p></notes><notes notes-type="COI-statement"><title>Competing interests</title><p id="Par31">The authors declare no competing interests.</p></notes><ref-list id="Bib1"><title>References</title><ref id="CR1"><label>1.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stupp</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial</article-title><source>Lancet Oncol.</source><year>2009</year><volume>10</volume><issue>5</issue><fpage>459</fpage><lpage>66</lpage><pub-id pub-id-type="doi">10.1016/S1470-2045(09)70025-7</pub-id><pub-id pub-id-type="pmid">19269895</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wu</surname><given-names>WK</given-names></name><etal></etal></person-group><article-title>The autophagic paradox in cancer therapy</article-title><source>Oncogene</source><year>2012</year><volume>31</volume><fpage>939</fpage><lpage>53</lpage><pub-id pub-id-type="doi">10.1038/onc.2011.295</pub-id><pub-id pub-id-type="pmid">21765470</pub-id></element-citation></ref><ref id="CR3"><label>3.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dalby</surname><given-names>KN</given-names></name><name><surname>Tekedereli</surname><given-names>I</given-names></name><name><surname>Lopez-Berestein</surname><given-names>G</given-names></name><name><surname>Ozpolat</surname><given-names>B</given-names></name></person-group><article-title>Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer</article-title><source>Autophagy</source><year>2010</year><volume>6</volume><fpage>322</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.4161/auto.6.3.11625</pub-id><pub-id pub-id-type="pmid">20224296</pub-id></element-citation></ref><ref id="CR4"><label>4.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Amaravadi</surname><given-names>RK</given-names></name><etal></etal></person-group><article-title>Principles and current strategies for targeting autophagy for cancer treatment</article-title><source>Clin. cancer res.</source><year>2011</year><volume>17</volume><issue>4</issue><fpage>654</fpage><lpage>66</lpage><pub-id pub-id-type="doi">10.1158/1078-0432.CCR-10-2634</pub-id><pub-id pub-id-type="pmid">21325294</pub-id></element-citation></ref><ref id="CR5"><label>5.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Janku</surname><given-names>F</given-names></name><name><surname>McConkey</surname><given-names>DJ</given-names></name><name><surname>Hong</surname><given-names>DS</given-names></name><name><surname>Kurzrock</surname><given-names>R</given-names></name></person-group><article-title>Autophagy as a target for anticancer therapy</article-title><source>Nat. Rev. Clin. Oncol.</source><year>2011</year><volume>8</volume><fpage>528</fpage><lpage>39</lpage><pub-id pub-id-type="doi">10.1038/nrclinonc.2011.71</pub-id><pub-id pub-id-type="pmid">21587219</pub-id></element-citation></ref><ref id="CR6"><label>6.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krizkova</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>Metallothioneins and zinc in cancer diagnosis and therapy</article-title><source>Drug. Metab. Rev.</source><year>2012</year><volume>44</volume><fpage>287</fpage><lpage>301</lpage><pub-id pub-id-type="doi">10.3109/03602532.2012.725414</pub-id><pub-id pub-id-type="pmid">23050852</pub-id></element-citation></ref><ref id="CR7"><label>7.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pedersen</surname><given-names>MO</given-names></name><name><surname>Larsen</surname><given-names>A</given-names></name><name><surname>Stoltenberg</surname><given-names>M</given-names></name><name><surname>Penkowa</surname><given-names>M</given-names></name></person-group><article-title>The role of metallothionein in oncogenesis and cancer prognosis</article-title><source>Prog. Histochem. Cytochem.</source><year>2009</year><volume>44</volume><fpage>29</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1016/j.proghi.2008.10.001</pub-id><pub-id pub-id-type="pmid">19348910</pub-id></element-citation></ref><ref id="CR8"><label>8.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sens</surname><given-names>MA</given-names></name><etal></etal></person-group><article-title>Metallothionein isoform 3 as a potential biomarker for human bladder cancer</article-title><source>Env. Health Perspect.</source><year>2000</year><volume>108</volume><fpage>413</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1289/ehp.00108413</pub-id></element-citation></ref><ref id="CR9"><label>9.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Somji</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>Absence of Metallothionein 3 Expression in Breast Cancer is a Rare, But Favorable Marker of Outcome that is Under Epigenetic Control</article-title><source>Toxicol. Env. Chem.</source><year>2010</year><volume>92</volume><fpage>1673</fpage><lpage>95</lpage><pub-id pub-id-type="doi">10.1080/02772241003711274</pub-id><pub-id pub-id-type="pmid">21170156</pub-id></element-citation></ref><ref id="CR10"><label>10.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Werynska</surname><given-names>B</given-names></name><etal></etal></person-group><article-title>Expression of metallothionein-III in patients with non-small cell lung cancer</article-title><source>Anticancer. Res.</source><year>2013</year><volume>33</volume><fpage>965</fpage><lpage>74</lpage><pub-id pub-id-type="pmid">23482768</pub-id></element-citation></ref><ref id="CR11"><label>11.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Juang</surname><given-names>HH</given-names></name><etal></etal></person-group><article-title>Metallothionein 3: an androgen-upregulated gene enhances cell invasion and tumorigenesis of prostate carcinoma cells</article-title><source>Prostate</source><year>2013</year><volume>73</volume><fpage>1495</fpage><lpage>506</lpage><pub-id pub-id-type="doi">10.1002/pros.22697</pub-id><pub-id pub-id-type="pmid">23794209</pub-id></element-citation></ref><ref id="CR12"><label>12.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kmiecik</surname><given-names>AM</given-names></name><etal></etal></person-group><article-title>Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression</article-title><source>PLoS One</source><year>2015</year><volume>10</volume><issue>(5)</issue><fpage>e0124865</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0124865</pub-id><pub-id pub-id-type="pmid">25933064</pub-id></element-citation></ref><ref id="CR13"><label>13.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Deng</surname><given-names>D</given-names></name><etal></etal></person-group><article-title>Hypermethylation of metallothionein-3 CpG island in gastric carcinoma</article-title><source>Carcinogenesis</source><year>2003</year><volume>24</volume><fpage>25</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1093/carcin/24.1.25</pub-id><pub-id pub-id-type="pmid">12538345</pub-id></element-citation></ref><ref id="CR14"><label>14.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peng</surname><given-names>D</given-names></name><etal></etal></person-group><article-title>Location-specific epigenetic regulation of the metallothionein 3 gene in esophageal adenocarcinomas</article-title><source>PLoS One</source><year>2011</year><volume>6</volume><fpage>e22009</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0022009</pub-id><pub-id pub-id-type="pmid">21818286</pub-id></element-citation></ref><ref id="CR15"><label>15.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tao</surname><given-names>YF</given-names></name><etal></etal></person-group><article-title>Metallothionein III (MT3) is a putative tumor suppressor gene that is frequently inactivated in pediatric acute myeloid leukemia by promoter hypermethylation</article-title><source>J. Transl. Med.</source><year>2014</year><volume>12</volume><fpage>182</fpage><pub-id pub-id-type="doi">10.1186/1479-5876-12-182</pub-id><pub-id pub-id-type="pmid">24962166</pub-id></element-citation></ref><ref id="CR16"><label>16.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Falnoga</surname><given-names>I</given-names></name><etal></etal></person-group><article-title>Arsenic trioxide (ATO) influences the gene expression of metallothioneins in human glioblastoma cells</article-title><source>Biol. Trace Elem. Res.</source><year>2012</year><volume>149</volume><fpage>331</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1007/s12011-012-9431-8</pub-id><pub-id pub-id-type="pmid">22555517</pub-id></element-citation></ref><ref id="CR17"><label>17.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mehrian-Shai</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>High metallothionein predicts poor survival in glioblastoma multiforme</article-title><source>BMC Med. Genomics</source><year>2015</year><volume>8</volume><fpage>68</fpage><pub-id pub-id-type="doi">10.1186/s12920-015-0137-6</pub-id><pub-id pub-id-type="pmid">26493598</pub-id></element-citation></ref><ref id="CR18"><label>18.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Uchida</surname><given-names>Y</given-names></name><name><surname>Takio</surname><given-names>K</given-names></name><name><surname>Titani</surname><given-names>K</given-names></name><name><surname>Ihara</surname><given-names>Y</given-names></name><name><surname>Tomonaga</surname><given-names>M</given-names></name></person-group><article-title>The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein</article-title><source>Neuron</source><year>1991</year><volume>7</volume><fpage>337</fpage><lpage>47</lpage><pub-id pub-id-type="doi">10.1016/0896-6273(91)90272-2</pub-id><pub-id pub-id-type="pmid">1873033</pub-id></element-citation></ref><ref id="CR19"><label>19.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes</article-title><source>Mol. Brain</source><year>2010</year><volume>3</volume><fpage>30</fpage><pub-id pub-id-type="doi">10.1186/1756-6606-3-30</pub-id><pub-id pub-id-type="pmid">20974010</pub-id></element-citation></ref><ref id="CR20"><label>20.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Erickson</surname><given-names>JC</given-names></name><name><surname>Hollopeter</surname><given-names>G</given-names></name><name><surname>Thomas</surname><given-names>SA</given-names></name><name><surname>Froelick</surname><given-names>GJ</given-names></name><name><surname>Palmiter</surname><given-names>RD</given-names></name></person-group><article-title>Disruption of the metallothionein-III gene in mice: analysis of brain zinc, behavior, and neuron vulnerability to metals, aging, and seizures</article-title><source>J. Neurosci.</source><year>1997</year><volume>17</volume><fpage>1271</fpage><lpage>81</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.17-04-01271.1997</pub-id><pub-id pub-id-type="pmid">9006971</pub-id></element-citation></ref><ref id="CR21"><label>21.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Carrasco</surname><given-names>J</given-names></name><etal></etal></person-group><article-title>Role of metallothionein-III following central nervous system damage</article-title><source>Neurobiol. Dis.</source><year>2003</year><volume>13</volume><fpage>22</fpage><lpage>36</lpage><pub-id pub-id-type="doi">10.1016/S0969-9961(03)00015-9</pub-id><pub-id pub-id-type="pmid">12758064</pub-id></element-citation></ref><ref id="CR22"><label>22.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Puttaparthi</surname><given-names>K</given-names></name><etal></etal></person-group><article-title>Disease progression in a transgenic model of familial amyotrophic lateral sclerosis is dependent on both neuronal and non-neuronal zinc binding proteins</article-title><source>J. Neurosci.</source><year>2002</year><volume>22</volume><fpage>8790</fpage><lpage>6</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.22-20-08790.2002</pub-id><pub-id pub-id-type="pmid">12388585</pub-id></element-citation></ref><ref id="CR23"><label>23.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Park</surname><given-names>MH</given-names></name><name><surname>Kim</surname><given-names>HJ</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions</article-title><source>Glia</source><year>2010</year><volume>58</volume><fpage>1186</fpage><lpage>96</lpage><pub-id pub-id-type="doi">10.1002/glia.20998</pub-id><pub-id pub-id-type="pmid">20544854</pub-id></element-citation></ref><ref id="CR24"><label>24.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>West</surname><given-names>AK</given-names></name><name><surname>Hidalgo</surname><given-names>J</given-names></name><name><surname>Eddins</surname><given-names>D</given-names></name><name><surname>Levin</surname><given-names>ED</given-names></name><name><surname>Aschner</surname><given-names>M</given-names></name></person-group><article-title>Metallothionein in the central nervous system: Roles in protection, regeneration and cognition</article-title><source>Neurotoxicol</source><year>2008</year><volume>29</volume><fpage>489</fpage><lpage>503</lpage><pub-id pub-id-type="doi">10.1016/j.neuro.2007.12.006</pub-id></element-citation></ref><ref id="CR25"><label>25.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>JY</given-names></name><name><surname>Kim</surname><given-names>JH</given-names></name><name><surname>Palmiter</surname><given-names>RD</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Zinc released from metallothionein-iii may contribute to hippocampal CA1 and thalamic neuronal death following acute brain injury</article-title><source>Exp. Neurol.</source><year>2003</year><volume>184</volume><fpage>337</fpage><lpage>47</lpage><pub-id pub-id-type="doi">10.1016/S0014-4886(03)00382-0</pub-id><pub-id pub-id-type="pmid">14637104</pub-id></element-citation></ref><ref id="CR26"><label>26.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hwang</surname><given-names>JJ</given-names></name><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Kim</surname><given-names>TY</given-names></name><name><surname>Cho</surname><given-names>JH</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Zinc and 4-hydroxy-2-nonenal mediate lysosomal membrane permeabilization induced by H2O2 in cultured hippocampal neurons</article-title><source>J. Neurosci.</source><year>2008</year><volume>28</volume><fpage>3114</fpage><lpage>22</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.0199-08.2008</pub-id><pub-id pub-id-type="pmid">18354014</pub-id></element-citation></ref><ref id="CR27"><label>27.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Cho</surname><given-names>KS</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Oxidative injury triggers autophagy in astrocytes: the role of endogenous zinc</article-title><source>Glia</source><year>2009</year><volume>57</volume><fpage>1351</fpage><lpage>61</lpage><pub-id pub-id-type="doi">10.1002/glia.20854</pub-id><pub-id pub-id-type="pmid">19229997</pub-id></element-citation></ref><ref id="CR28"><label>28.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Seo</surname><given-names>BR</given-names></name><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Cho</surname><given-names>KS</given-names></name><name><surname>Yoon</surname><given-names>YH</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>The zinc ionophore clioquinol reverses autophagy arrest in chloroquine-treated ARPE-19 cells and in APP/mutant presenilin-1-transfected Chinese hamster ovary cells</article-title><source>Neurobiol. Aging</source><year>2015</year><volume>36</volume><fpage>3228</fpage><lpage>38</lpage><pub-id pub-id-type="doi">10.1016/j.neurobiolaging.2015.09.006</pub-id><pub-id pub-id-type="pmid">26453000</pub-id></element-citation></ref><ref id="CR29"><label>29.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>JH</given-names></name><etal></etal></person-group><article-title>Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations</article-title><source>Cell</source><year>2010</year><volume>141</volume><fpage>1146</fpage><lpage>58</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2010.05.008</pub-id><pub-id pub-id-type="pmid">20541250</pub-id></element-citation></ref><ref id="CR30"><label>30.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Poole</surname><given-names>B</given-names></name><name><surname>Ohkuma</surname><given-names>S</given-names></name></person-group><article-title>Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages</article-title><source>J. Cell Biol.</source><year>1981</year><volume>90</volume><fpage>665</fpage><lpage>9</lpage><pub-id pub-id-type="doi">10.1083/jcb.90.3.665</pub-id><pub-id pub-id-type="pmid">6169733</pub-id></element-citation></ref><ref id="CR31"><label>31.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rangwala</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>Combined MTOR and autophagy inhibition: phase I trial of hydroxychloroquine and temsirolimus in patients with advanced solid tumors and melanoma</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><fpage>1391</fpage><lpage>402</lpage><pub-id pub-id-type="doi">10.4161/auto.29119</pub-id><pub-id pub-id-type="pmid">24991838</pub-id></element-citation></ref><ref id="CR32"><label>32.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mahalingam</surname><given-names>D</given-names></name><etal></etal></person-group><article-title>Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><fpage>1403</fpage><lpage>14</lpage><pub-id pub-id-type="doi">10.4161/auto.29231</pub-id><pub-id pub-id-type="pmid">24991835</pub-id></element-citation></ref><ref id="CR33"><label>33.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rosenfeld</surname><given-names>MR</given-names></name><etal></etal></person-group><article-title>A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><fpage>1359</fpage><lpage>68</lpage><pub-id pub-id-type="doi">10.4161/auto.28984</pub-id><pub-id pub-id-type="pmid">24991840</pub-id></element-citation></ref><ref id="CR34"><label>34.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rangwala</surname><given-names>R</given-names></name><etal></etal></person-group><article-title>Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><fpage>1369</fpage><lpage>79</lpage><pub-id pub-id-type="doi">10.4161/auto.29118</pub-id><pub-id pub-id-type="pmid">24991839</pub-id></element-citation></ref><ref id="CR35"><label>35.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vogl</surname><given-names>DT</given-names></name><etal></etal></person-group><article-title>Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma</article-title><source>Autophagy</source><year>2014</year><volume>10</volume><fpage>1380</fpage><lpage>90</lpage><pub-id pub-id-type="doi">10.4161/auto.29264</pub-id><pub-id pub-id-type="pmid">24991834</pub-id></element-citation></ref><ref id="CR36"><label>36.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Knipp</surname><given-names>M</given-names></name></person-group><article-title>Metallothioneins and platinum(II) anti-tumor compounds</article-title><source>Curr. Med. Chem.</source><year>2009</year><volume>16</volume><fpage>522</fpage><lpage>37</lpage><pub-id pub-id-type="doi">10.2174/092986709787458452</pub-id><pub-id pub-id-type="pmid">19199919</pub-id></element-citation></ref><ref id="CR37"><label>37.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cai</surname><given-names>L</given-names></name><name><surname>Satoh</surname><given-names>M</given-names></name><name><surname>Tohyama</surname><given-names>C</given-names></name><name><surname>Cherian</surname><given-names>MG</given-names></name></person-group><article-title>Metallothionein in radiation exposure: its induction and protective role</article-title><source>Toxicol.</source><year>1999</year><volume>132</volume><fpage>85</fpage><lpage>98</lpage><pub-id pub-id-type="doi">10.1016/S0300-483X(98)00150-4</pub-id></element-citation></ref><ref id="CR38"><label>38.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ding</surname><given-names>ZC</given-names></name><name><surname>Ni</surname><given-names>FY</given-names></name><name><surname>Huang</surname><given-names>ZX</given-names></name></person-group><article-title>Neuronal growth-inhibitory factor (metallothionein-3): structure-function relationships</article-title><source>FEBS J.</source><year>2010</year><volume>277</volume><fpage>2912</fpage><lpage>20</lpage><pub-id pub-id-type="doi">10.1111/j.1742-4658.2010.07716.x</pub-id><pub-id pub-id-type="pmid">20561055</pub-id></element-citation></ref><ref id="CR39"><label>39.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>SJ</given-names></name><name><surname>Seo</surname><given-names>BR</given-names></name><name><surname>Koh</surname><given-names>JY</given-names></name></person-group><article-title>Metallothionein-3 modulates the amyloid beta endocytosis of astrocytes through its effects on actin polymerization</article-title><source>Mol. Brain</source><year>2015</year><volume>8</volume><fpage>84</fpage><pub-id pub-id-type="doi">10.1186/s13041-015-0173-3</pub-id><pub-id pub-id-type="pmid">26637294</pub-id></element-citation></ref><ref id="CR40"><label>40.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>El Ghazi</surname><given-names>I</given-names></name><name><surname>Martin</surname><given-names>BL</given-names></name><name><surname>Armitage</surname><given-names>IM</given-names></name></person-group><article-title>New proteins found interacting with brain metallothionein-3 are linked to secretion</article-title><source>Int. J. Alzheimers Dis.</source><year>2010</year><volume>2011</volume><fpage>208634</fpage><pub-id pub-id-type="pmid">21234102</pub-id></element-citation></ref><ref id="CR41"><label>41.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bell</surname><given-names>SG</given-names></name><name><surname>Vallee</surname><given-names>BL</given-names></name></person-group><article-title>The metallothionein/thionein system: an oxidoreductive metabolic zinc link</article-title><source>Chembiochem</source><year>2009</year><volume>10</volume><fpage>55</fpage><lpage>62</lpage><pub-id pub-id-type="doi">10.1002/cbic.200800511</pub-id><pub-id pub-id-type="pmid">19089881</pub-id></element-citation></ref><ref id="CR42"><label>42.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jacobs</surname><given-names>VL</given-names></name><name><surname>Valdes</surname><given-names>PA</given-names></name><name><surname>Hickey</surname><given-names>WF</given-names></name><name><surname>De Leo</surname><given-names>JA</given-names></name></person-group><article-title>Current review of <italic>in vivo</italic> GBM rodent models: emphasis on the CNS-1 tumour model</article-title><source>ASN neuro</source><year>2011</year><volume>3</volume><fpage>e00063</fpage><pub-id pub-id-type="doi">10.1042/AN20110014</pub-id><pub-id pub-id-type="pmid">21740400</pub-id></element-citation></ref><ref id="CR43"><label>43.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Koh</surname><given-names>JY</given-names></name><name><surname>Choi</surname><given-names>DW</given-names></name></person-group><article-title>Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay</article-title><source>J. Neurosci. Methods</source><year>1987</year><volume>20</volume><fpage>83</fpage><lpage>90</lpage><pub-id pub-id-type="doi">10.1016/0165-0270(87)90041-0</pub-id><pub-id pub-id-type="pmid">2884353</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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|texte= A role of metallothionein-3 in radiation-induced autophagy in glioma cells
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HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:32029749" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a ChloroquineV1
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