Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation
Identifieur interne : 000208 ( Istex/Corpus ); précédent : 000207; suivant : 000209Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation
Auteurs : Ljubica Harhaji-Trajkovic ; Katarina Arsikin ; Tamara Kravic-Stevovic ; Sasa Petricevic ; Gordana Tovilovic ; Aleksandar Pantovic ; Nevena Zogovic ; Biljana Ristic ; Kristina Janjetovic ; Vladimir Bumbasirevic ; Vladimir TrajkovicSource :
- Pharmaceutical Research [ 0724-8741 ] ; 2012-08-01.
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Abstract
ABSTRACT: Purpose: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. Results: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. Conclusion: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.
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DOI: 10.1007/s11095-012-0753-1
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Serbia</mods:affiliation></affiliation></author><author><name sortKey="Janjetovic, Kristina" sort="Janjetovic, Kristina" uniqKey="Janjetovic K" first="Kristina" last="Janjetovic">Kristina Janjetovic</name><affiliation><mods:affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</mods:affiliation></affiliation><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Bumbasirevic, Vladimir" sort="Bumbasirevic, Vladimir" uniqKey="Bumbasirevic V" first="Vladimir" last="Bumbasirevic">Vladimir Bumbasirevic</name><affiliation><mods:affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Trajkovic, Vladimir" sort="Trajkovic, Vladimir" uniqKey="Trajkovic V" first="Vladimir" last="Trajkovic">Vladimir Trajkovic</name><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: vtrajkovic@med.bg.ac.rs</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:1512B233B6535036C50CEF13FE47012940AEA0C6</idno><date when="2012" year="2012">2012</date><idno type="doi">10.1007/s11095-012-0753-1</idno><idno type="url">https://api.istex.fr/ark:/67375/VQC-73RDBK3G-K/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">000208</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">000208</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title><author><name sortKey="Harhaji Trajkovic, Ljubica" sort="Harhaji Trajkovic, Ljubica" uniqKey="Harhaji Trajkovic L" first="Ljubica" last="Harhaji-Trajkovic">Ljubica Harhaji-Trajkovic</name><affiliation><mods:affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: buajk@yahoo.com</mods:affiliation></affiliation></author><author><name sortKey="Arsikin, Katarina" sort="Arsikin, Katarina" uniqKey="Arsikin K" first="Katarina" last="Arsikin">Katarina Arsikin</name><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Kravic Stevovic, Tamara" sort="Kravic Stevovic, Tamara" uniqKey="Kravic Stevovic T" first="Tamara" last="Kravic-Stevovic">Tamara Kravic-Stevovic</name><affiliation><mods:affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Petricevic, Sasa" sort="Petricevic, Sasa" uniqKey="Petricevic S" first="Sasa" last="Petricevic">Sasa Petricevic</name><affiliation><mods:affiliation>Institute of Biomedical Research, Galenika a.d., Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Tovilovic, Gordana" sort="Tovilovic, Gordana" uniqKey="Tovilovic G" first="Gordana" last="Tovilovic">Gordana Tovilovic</name><affiliation><mods:affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Pantovic, Aleksandar" sort="Pantovic, Aleksandar" uniqKey="Pantovic A" first="Aleksandar" last="Pantovic">Aleksandar Pantovic</name><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Zogovic, Nevena" sort="Zogovic, Nevena" uniqKey="Zogovic N" first="Nevena" last="Zogovic">Nevena Zogovic</name><affiliation><mods:affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Ristic, Biljana" sort="Ristic, Biljana" uniqKey="Ristic B" first="Biljana" last="Ristic">Biljana Ristic</name><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Janjetovic, Kristina" sort="Janjetovic, Kristina" uniqKey="Janjetovic K" first="Kristina" last="Janjetovic">Kristina Janjetovic</name><affiliation><mods:affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</mods:affiliation></affiliation><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Bumbasirevic, Vladimir" sort="Bumbasirevic, Vladimir" uniqKey="Bumbasirevic V" first="Vladimir" last="Bumbasirevic">Vladimir Bumbasirevic</name><affiliation><mods:affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</mods:affiliation></affiliation></author><author><name sortKey="Trajkovic, Vladimir" sort="Trajkovic, Vladimir" uniqKey="Trajkovic V" first="Vladimir" last="Trajkovic">Vladimir Trajkovic</name><affiliation><mods:affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</mods:affiliation></affiliation><affiliation><mods:affiliation>E-mail: vtrajkovic@med.bg.ac.rs</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Pharmaceutical Research</title><title level="j" type="sub">An Official Journal of the American Association of Pharmaceutical Scientists</title><title level="j" type="abbrev">Pharm Res</title><idno type="ISSN">0724-8741</idno><idno type="eISSN">1573-904X</idno><imprint><publisher>Springer US; http://www.springer-ny.com</publisher><pubPlace>Boston</pubPlace><date type="published" when="2012-08-01">2012-08-01</date><biblScope unit="volume">29</biblScope><biblScope unit="issue">8</biblScope><biblScope unit="page" from="2249">2249</biblScope><biblScope unit="page" to="2263">2263</biblScope></imprint><idno type="ISSN">0724-8741</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0724-8741</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>AMPK</term><term>autophagy</term><term>caloric restriction</term><term>cancer</term><term>chloroquine</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">ABSTRACT: Purpose: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. Results: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. Conclusion: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.</div></front></TEI><istex><corpusName>springer-journals</corpusName><author><json:item><name>Ljubica Harhaji-Trajkovic</name><affiliations><json:string>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</json:string><json:string>E-mail: buajk@yahoo.com</json:string></affiliations></json:item><json:item><name>Katarina Arsikin</name><affiliations><json:string>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Tamara Kravic-Stevovic</name><affiliations><json:string>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Sasa Petricevic</name><affiliations><json:string>Institute of Biomedical Research, Galenika a.d., Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Gordana Tovilovic</name><affiliations><json:string>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Aleksandar Pantovic</name><affiliations><json:string>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Nevena Zogovic</name><affiliations><json:string>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Biljana Ristic</name><affiliations><json:string>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Kristina Janjetovic</name><affiliations><json:string>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</json:string><json:string>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Vladimir Bumbasirevic</name><affiliations><json:string>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</json:string></affiliations></json:item><json:item><name>Vladimir Trajkovic</name><affiliations><json:string>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</json:string><json:string>E-mail: vtrajkovic@med.bg.ac.rs</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>AMPK</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>autophagy</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>caloric restriction</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>cancer</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>chloroquine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AICAR : 5-aminoimidazole-4-carboxamide riboside</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>AMPK : AMP-activated protein kinase</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DHR : dihydrorhodamine</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FCS : fetal calf serum</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>FITC : fluorescein isothyocyanate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>LC3 : microtubule-associated protein 1 light-chain 3</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MEM : Eagle’s Minimum Essential Medium</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PBS : phosphate buffered saline</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>PI : propidium iodide</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ROS : reactive oxygen species</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>shRNA : short hairpin RNA</value></json:item></subject><articleId><json:string>753</json:string><json:string>s11095-012-0753-1</json:string></articleId><arkIstex>ark:/67375/VQC-73RDBK3G-K</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>OriginalPaper</json:string></originalGenre><abstract>ABSTRACT: Purpose: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. Results: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. Conclusion: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.</abstract><qualityIndicators><refBibsNative>false</refBibsNative><abstractWordCount>199</abstractWordCount><abstractCharCount>1604</abstractCharCount><keywordCount>17</keywordCount><score>9.388</score><pdfWordCount>6727</pdfWordCount><pdfCharCount>45583</pdfCharCount><pdfVersion>1.4</pdfVersion><pdfPageCount>15</pdfPageCount><pdfPageSize>595.276 x 790.866 pts</pdfPageSize></qualityIndicators><title>Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title><genre><json:string>research-article</json:string></genre><host><title>Pharmaceutical Research</title><language><json:string>unknown</json:string></language><publicationDate>2012</publicationDate><copyrightDate>2012</copyrightDate><issn><json:string>0724-8741</json:string></issn><eissn><json:string>1573-904X</json:string></eissn><journalId><json:string>11095</json:string></journalId><volume>29</volume><issue>8</issue><pages><first>2249</first><last>2263</last></pages><genre><json:string>journal</json:string></genre><subject><json:item><value>Biomedical Engineering</value></json:item><json:item><value>Medical Law</value></json:item><json:item><value>Biochemistry, general</value></json:item><json:item><value>Pharmacology/Toxicology</value></json:item><json:item><value>Pharmacy</value></json:item></subject></host><ark><json:string>ark:/67375/VQC-73RDBK3G-K</json:string></ark><publicationDate>2012</publicationDate><copyrightDate>2012</copyrightDate><doi><json:string>10.1007/s11095-012-0753-1</json:string></doi><id>1512B233B6535036C50CEF13FE47012940AEA0C6</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/VQC-73RDBK3G-K/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/VQC-73RDBK3G-K/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/VQC-73RDBK3G-K/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">Springer US; http://www.springer-ny.com</publisher><pubPlace>Boston</pubPlace><availability><licence><p>Springer Science+Business Media, LLC, 2012</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-3XSW68JL-F">springer</p></availability><date>2012-02-01</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note>Research Paper</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title><author xml:id="author-0000" corresp="yes"><persName><forename type="first">Ljubica</forename><surname>Harhaji-Trajkovic</surname></persName><email>buajk@yahoo.com</email><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Katarina</forename><surname>Arsikin</surname></persName><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Tamara</forename><surname>Kravic-Stevovic</surname></persName><affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Sasa</forename><surname>Petricevic</surname></persName><affiliation>Institute of Biomedical Research, Galenika a.d., Belgrade, Serbia</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Gordana</forename><surname>Tovilovic</surname></persName><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Aleksandar</forename><surname>Pantovic</surname></persName><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Nevena</forename><surname>Zogovic</surname></persName><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Biljana</forename><surname>Ristic</surname></persName><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation></author><author xml:id="author-0008"><persName><forename type="first">Kristina</forename><surname>Janjetovic</surname></persName><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation></author><author xml:id="author-0009"><persName><forename type="first">Vladimir</forename><surname>Bumbasirevic</surname></persName><affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</affiliation></author><author xml:id="author-0010" corresp="yes"><persName><forename type="first">Vladimir</forename><surname>Trajkovic</surname></persName><email>vtrajkovic@med.bg.ac.rs</email><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation></author><idno type="istex">1512B233B6535036C50CEF13FE47012940AEA0C6</idno><idno type="ark">ark:/67375/VQC-73RDBK3G-K</idno><idno type="DOI">10.1007/s11095-012-0753-1</idno><idno type="article-id">753</idno><idno type="article-id">s11095-012-0753-1</idno></analytic><monogr><title level="j">Pharmaceutical Research</title><title level="j" type="sub">An Official Journal of the American Association of Pharmaceutical Scientists</title><title level="j" type="abbrev">Pharm Res</title><idno type="pISSN">0724-8741</idno><idno type="eISSN">1573-904X</idno><idno type="journal-ID">true</idno><idno type="issue-article-count">26</idno><idno type="volume-issue-count">12</idno><imprint><publisher>Springer US; http://www.springer-ny.com</publisher><pubPlace>Boston</pubPlace><date type="published" when="2012-08-01"></date><biblScope unit="volume">29</biblScope><biblScope unit="issue">8</biblScope><biblScope unit="page" from="2249">2249</biblScope><biblScope unit="page" to="2263">2263</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>2012-02-01</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>ABSTRACT: Purpose: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. Results: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. Conclusion: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.</p></abstract><textClass xml:lang="en"><keywords scheme="keyword"><list><head>KEY WORDS</head><item><term>AMPK</term></item><item><term>autophagy</term></item><item><term>caloric restriction</term></item><item><term>cancer</term></item><item><term>chloroquine</term></item></list></keywords></textClass><textClass><keywords scheme="keyword"><list><head>ABBREVIATIONS</head><item><term>AICAR</term><term>5-aminoimidazole-4-carboxamide riboside</term></item><item><term>AMPK</term><term>AMP-activated protein kinase</term></item><item><term>DHR</term><term>dihydrorhodamine</term></item><item><term>FCS</term><term>fetal calf serum</term></item><item><term>FITC</term><term>fluorescein isothyocyanate</term></item><item><term>LC3</term><term>microtubule-associated protein 1 light-chain 3</term></item><item><term>MEM</term><term>Eagle’s Minimum Essential Medium</term></item><item><term>MTT</term><term>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</term></item><item><term>PBS</term><term>phosphate buffered saline</term></item><item><term>PI</term><term>propidium iodide</term></item><item><term>ROS</term><term>reactive oxygen species</term></item><item><term>shRNA</term><term>short hairpin RNA</term></item></list></keywords></textClass><textClass><keywords scheme="Journal Subject"><list><head>Biomedicine</head><item><term>Biomedical Engineering</term></item><item><term>Medical Law</term></item><item><term>Biochemistry, general</term></item><item><term>Pharmacology/Toxicology</term></item><item><term>Pharmacy</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="2012-02-01">Created</change><change 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TocLevels="0"><ArticleID>753</ArticleID><ArticleDOI>10.1007/s11095-012-0753-1</ArticleDOI><ArticleSequenceNumber>19</ArticleSequenceNumber><ArticleTitle Language="En">Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</ArticleTitle><ArticleCategory>Research Paper</ArticleCategory><ArticleFirstPage>2249</ArticleFirstPage><ArticleLastPage>2263</ArticleLastPage><ArticleHistory><RegistrationDate><Year>2012</Year><Month>4</Month><Day>2</Day></RegistrationDate><Received><Year>2012</Year><Month>2</Month><Day>1</Day></Received><Accepted><Year>2012</Year><Month>4</Month><Day>2</Day></Accepted><OnlineDate><Year>2012</Year><Month>4</Month><Day>27</Day></OnlineDate></ArticleHistory><ArticleCopyright><CopyrightHolderName>Springer Science+Business Media, LLC</CopyrightHolderName><CopyrightYear>2012</CopyrightYear></ArticleCopyright><ArticleGrants Type="Regular"><MetadataGrant Grant="OpenAccess"></MetadataGrant><AbstractGrant Grant="OpenAccess"></AbstractGrant><BodyPDFGrant Grant="Restricted"></BodyPDFGrant><BodyHTMLGrant Grant="Restricted"></BodyHTMLGrant><BibliographyGrant Grant="Restricted"></BibliographyGrant><ESMGrant Grant="Restricted"></ESMGrant></ArticleGrants></ArticleInfo><ArticleHeader><AuthorGroup><Author AffiliationIDS="Aff1" CorrespondingAffiliationID="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Ljubica</GivenName><FamilyName>Harhaji-Trajkovic</FamilyName></AuthorName><Contact><Phone>+381-11-3643233</Phone><Fax>+381-11-3643235</Fax><Email>buajk@yahoo.com</Email></Contact></Author><Author AffiliationIDS="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Katarina</GivenName><FamilyName>Arsikin</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff3"><AuthorName DisplayOrder="Western"><GivenName>Tamara</GivenName><FamilyName>Kravic-Stevovic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff4"><AuthorName DisplayOrder="Western"><GivenName>Sasa</GivenName><FamilyName>Petricevic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Gordana</GivenName><FamilyName>Tovilovic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Aleksandar</GivenName><FamilyName>Pantovic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1"><AuthorName DisplayOrder="Western"><GivenName>Nevena</GivenName><FamilyName>Zogovic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Biljana</GivenName><FamilyName>Ristic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff1 Aff2"><AuthorName DisplayOrder="Western"><GivenName>Kristina</GivenName><FamilyName>Janjetovic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff3"><AuthorName DisplayOrder="Western"><GivenName>Vladimir</GivenName><FamilyName>Bumbasirevic</FamilyName></AuthorName></Author><Author AffiliationIDS="Aff2" CorrespondingAffiliationID="Aff2"><AuthorName DisplayOrder="Western"><GivenName>Vladimir</GivenName><FamilyName>Trajkovic</FamilyName></AuthorName><Contact><Phone>+381-11-3643 233</Phone><Fax>+381-11-3643235</Fax><Email>vtrajkovic@med.bg.ac.rs</Email></Contact></Author><Affiliation ID="Aff1"><OrgDivision>Institute for Biological Research</OrgDivision><OrgName>University of Belgrade</OrgName><OrgAddress><Street>Despot Stefan Blvd. 142</Street><Postcode>11060</Postcode><City>Belgrade</City><Country Code="RS">Serbia</Country></OrgAddress></Affiliation><Affiliation ID="Aff2"><OrgDivision>Institute of Microbiology and Immunology, School of Medicine</OrgDivision><OrgName>University of Belgrade</OrgName><OrgAddress><Street>Dr. Subotica 1</Street><Postcode>11000</Postcode><City>Belgrade</City><Country Code="RS">Serbia</Country></OrgAddress></Affiliation><Affiliation ID="Aff3"><OrgDivision>Institute of Histology and Embryology, School of Medicine</OrgDivision><OrgName>University of Belgrade</OrgName><OrgAddress><City>Belgrade</City><Country Code="RS">Serbia</Country></OrgAddress></Affiliation><Affiliation ID="Aff4"><OrgName>Institute of Biomedical Research, Galenika a.d.</OrgName><OrgAddress><City>Belgrade</City><Country Code="RS">Serbia</Country></OrgAddress></Affiliation></AuthorGroup><Abstract ID="Abs1" Language="En" OutputMedium="All"><Heading>ABSTRACT</Heading><AbstractSection ID="ASec1"><Heading>Purpose</Heading><Para>To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation.</Para></AbstractSection><AbstractSection ID="ASec2"><Heading>Methods</Heading><Para>Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine <Emphasis Type="Italic">in vitro</Emphasis>. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model.</Para></AbstractSection><AbstractSection ID="ASec3"><Heading>Results</Heading><Para>Chloroquine rapidly killed serum-starved cancer cells <Emphasis Type="Italic">in vitro</Emphasis>. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells.</Para></AbstractSection><AbstractSection ID="ASec4"><Heading>Conclusion</Heading><Para>Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.</Para></AbstractSection></Abstract><KeywordGroup Language="En" OutputMedium="All"><Heading>KEY WORDS</Heading><Keyword>AMPK</Keyword><Keyword>autophagy</Keyword><Keyword>caloric restriction</Keyword><Keyword>cancer</Keyword><Keyword>chloroquine</Keyword></KeywordGroup><AbbreviationGroup><Heading>ABBREVIATIONS</Heading><DefinitionList><DefinitionListEntry><Term>AICAR</Term><Description><Para>5-aminoimidazole-4-carboxamide riboside</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>AMPK</Term><Description><Para>AMP-activated protein kinase</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>DHR</Term><Description><Para>dihydrorhodamine</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>FCS</Term><Description><Para>fetal calf serum</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>FITC</Term><Description><Para>fluorescein isothyocyanate</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>LC3</Term><Description><Para>microtubule-associated protein 1 light-chain 3</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>MEM</Term><Description><Para>Eagle’s Minimum Essential Medium</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>MTT</Term><Description><Para>3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>PBS</Term><Description><Para>phosphate buffered saline</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>PI</Term><Description><Para>propidium iodide</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>ROS</Term><Description><Para>reactive oxygen species</Para></Description></DefinitionListEntry><DefinitionListEntry><Term>shRNA</Term><Description><Para>short hairpin RNA</Para></Description></DefinitionListEntry></DefinitionList></AbbreviationGroup><ArticleNote Type="Misc"><SimplePara>Ljubica Harhaji-Trajkovic and Katarina Arsikin contributed equally to the work.</SimplePara></ArticleNote></ArticleHeader><NoBody></NoBody></Article></Issue></Volume></Journal></Publisher></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation</title></titleInfo><name type="personal" displayLabel="corresp"><namePart type="given">Ljubica</namePart><namePart type="family">Harhaji-Trajkovic</namePart><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation><affiliation>E-mail: buajk@yahoo.com</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Katarina</namePart><namePart type="family">Arsikin</namePart><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Tamara</namePart><namePart type="family">Kravic-Stevovic</namePart><affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Sasa</namePart><namePart type="family">Petricevic</namePart><affiliation>Institute of Biomedical Research, Galenika a.d., Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Gordana</namePart><namePart type="family">Tovilovic</namePart><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Aleksandar</namePart><namePart type="family">Pantovic</namePart><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Nevena</namePart><namePart type="family">Zogovic</namePart><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Biljana</namePart><namePart type="family">Ristic</namePart><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Kristina</namePart><namePart type="family">Janjetovic</namePart><affiliation>Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia</affiliation><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Vladimir</namePart><namePart type="family">Bumbasirevic</namePart><affiliation>Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="given">Vladimir</namePart><namePart type="family">Trajkovic</namePart><affiliation>Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia</affiliation><affiliation>E-mail: vtrajkovic@med.bg.ac.rs</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="OriginalPaper" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>Springer US; http://www.springer-ny.com</publisher><place><placeTerm type="text">Boston</placeTerm></place><dateCreated encoding="w3cdtf">2012-02-01</dateCreated><dateIssued encoding="w3cdtf">2012-08-01</dateIssued><copyrightDate encoding="w3cdtf">2012</copyrightDate></originInfo><language><languageTerm type="code" authority="rfc3066">en</languageTerm><languageTerm type="code" authority="iso639-2b">eng</languageTerm></language><abstract lang="en">ABSTRACT: Purpose: To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation. Methods: Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model. Results: Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells. Conclusion: Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.</abstract><note>Research Paper</note><subject lang="en"><genre>KEY WORDS</genre><topic>AMPK</topic><topic>autophagy</topic><topic>caloric restriction</topic><topic>cancer</topic><topic>chloroquine</topic></subject><subject><genre>ABBREVIATIONS</genre><topic>AICAR : 5-aminoimidazole-4-carboxamide riboside</topic><topic>AMPK : AMP-activated protein kinase</topic><topic>DHR : dihydrorhodamine</topic><topic>FCS : fetal calf serum</topic><topic>FITC : fluorescein isothyocyanate</topic><topic>LC3 : microtubule-associated protein 1 light-chain 3</topic><topic>MEM : Eagle’s Minimum Essential Medium</topic><topic>MTT : 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide</topic><topic>PBS : phosphate buffered saline</topic><topic>PI : propidium iodide</topic><topic>ROS : reactive oxygen species</topic><topic>shRNA : short hairpin RNA</topic></subject><relatedItem type="host"><titleInfo><title>Pharmaceutical Research</title><subTitle>An Official Journal of the American Association of Pharmaceutical Scientists</subTitle></titleInfo><titleInfo type="abbreviated"><title>Pharm Res</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>Springer</publisher><dateIssued encoding="w3cdtf">2012-07-18</dateIssued><copyrightDate encoding="w3cdtf">2012</copyrightDate></originInfo><subject><genre>Biomedicine</genre><topic>Biomedical Engineering</topic><topic>Medical Law</topic><topic>Biochemistry, general</topic><topic>Pharmacology/Toxicology</topic><topic>Pharmacy</topic></subject><identifier type="ISSN">0724-8741</identifier><identifier type="eISSN">1573-904X</identifier><identifier type="JournalID">11095</identifier><identifier type="IssueArticleCount">26</identifier><identifier type="VolumeIssueCount">12</identifier><part><date>2012</date><detail type="volume"><number>29</number><caption>vol.</caption></detail><detail type="issue"><number>8</number><caption>no.</caption></detail><extent unit="pages"><start>2249</start><end>2263</end></extent></part><recordInfo><recordOrigin>Springer Science+Business Media, LLC, 2012</recordOrigin></recordInfo></relatedItem><identifier type="istex">1512B233B6535036C50CEF13FE47012940AEA0C6</identifier><identifier type="ark">ark:/67375/VQC-73RDBK3G-K</identifier><identifier type="DOI">10.1007/s11095-012-0753-1</identifier><identifier type="ArticleID">753</identifier><identifier type="ArticleID">s11095-012-0753-1</identifier><accessCondition type="use and reproduction" contentType="copyright">Springer Science+Business Media, LLC, 2012</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-3XSW68JL-F">springer</recordContentSource><recordOrigin>Springer Science+Business Media, LLC, 2012</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/VQC-73RDBK3G-K/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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