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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Derrone induces autophagic cell death through induction of ROS and ERK in A549 cells</title>
<author><name sortKey="Kang, Myung Ji" sort="Kang, Myung Ji" uniqKey="Kang M" first="Myung-Ji" last="Kang">Myung-Ji Kang</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Soo Yeon" sort="Kim, Soo Yeon" uniqKey="Kim S" first="Soo-Yeon" last="Kim">Soo-Yeon Kim</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kwon, Eun Bin" sort="Kwon, Eun Bin" uniqKey="Kwon E" first="Eun-Bin" last="Kwon">Eun-Bin Kwon</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jo, Yang Hee" sort="Jo, Yang Hee" uniqKey="Jo Y" first="Yang Hee" last="Jo">Yang Hee Jo</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Mi Kyeong" sort="Lee, Mi Kyeong" uniqKey="Lee M" first="Mi Kyeong" last="Lee">Mi Kyeong Lee</name>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Hyun Sun" sort="Lee, Hyun Sun" uniqKey="Lee H" first="Hyun-Sun" last="Lee">Hyun-Sun Lee</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Moon, Dong Oh" sort="Moon, Dong Oh" uniqKey="Moon D" first="Dong-Oh" last="Moon">Dong-Oh Moon</name>
<affiliation><nlm:aff id="aff003"><addr-line>Department of Biology Education, Daegu University, Gyeongsan, Gyeongsangbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Mun Ock" sort="Kim, Mun Ock" uniqKey="Kim M" first="Mun-Ock" last="Kim">Mun-Ock Kim</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">31216334</idno>
<idno type="pmc">6583947</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6583947</idno>
<idno type="RBID">PMC:6583947</idno>
<idno type="doi">10.1371/journal.pone.0218659</idno>
<date when="2019">2019</date>
<idno type="wicri:Area/Pmc/Corpus">000771</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000771</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Derrone induces autophagic cell death through induction of ROS and ERK in A549 cells</title>
<author><name sortKey="Kang, Myung Ji" sort="Kang, Myung Ji" uniqKey="Kang M" first="Myung-Ji" last="Kang">Myung-Ji Kang</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Soo Yeon" sort="Kim, Soo Yeon" uniqKey="Kim S" first="Soo-Yeon" last="Kim">Soo-Yeon Kim</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kwon, Eun Bin" sort="Kwon, Eun Bin" uniqKey="Kwon E" first="Eun-Bin" last="Kwon">Eun-Bin Kwon</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Jo, Yang Hee" sort="Jo, Yang Hee" uniqKey="Jo Y" first="Yang Hee" last="Jo">Yang Hee Jo</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Mi Kyeong" sort="Lee, Mi Kyeong" uniqKey="Lee M" first="Mi Kyeong" last="Lee">Mi Kyeong Lee</name>
<affiliation><nlm:aff id="aff002"><addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lee, Hyun Sun" sort="Lee, Hyun Sun" uniqKey="Lee H" first="Hyun-Sun" last="Lee">Hyun-Sun Lee</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Moon, Dong Oh" sort="Moon, Dong Oh" uniqKey="Moon D" first="Dong-Oh" last="Moon">Dong-Oh Moon</name>
<affiliation><nlm:aff id="aff003"><addr-line>Department of Biology Education, Daegu University, Gyeongsan, Gyeongsangbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kim, Mun Ock" sort="Kim, Mun Ock" uniqKey="Kim M" first="Mun-Ock" last="Kim">Mun-Ock Kim</name>
<affiliation><nlm:aff id="aff001"><addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</idno>
<imprint><date when="2019">2019</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>We studied the effect of derrone (DR), one of the major compounds of unripe fruits of <italic>Cudrania tricuspidata</italic>
, on cancer cell death. DR inhibited cell growth of various cancer cells, and that was partially associated with apoptosis in A549 cells. DR showed the autophagic features, such as the conversion of LC3-I to LC3-II, the formation of autophagosome and the downregulation of SQSTM1/p62 (p62). The treatment of autophagy inhibitor reversed DR-mediated cell death, suggesting that DR induces autophagic cell death. The increase of cytoplasmic Ca<sup>2+</sup>
and ROS by DR treatment significantly influences the formation of autophagosomes; however, only ROS scavengers significantly rescued the reduced cell viability. Additional results revealed that treatment of DR induces sustained phosphorylation of ERK and the inhibition of ERK phosphorylation using U0126 (ERK inhibitor) markedly attenuated DR-induced cell death. Overall, these results suggest that DR induces autophagic cell death through intracellular ROS and sustained ERK phosphorylation in A549 cells.</p>
</div>
</front>
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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id>
<journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id>
<journal-id journal-id-type="publisher-id">plos</journal-id>
<journal-id journal-id-type="pmc">plosone</journal-id>
<journal-title-group><journal-title>PLoS ONE</journal-title>
</journal-title-group>
<issn pub-type="epub">1932-6203</issn>
<publisher><publisher-name>Public Library of Science</publisher-name>
<publisher-loc>San Francisco, CA USA</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31216334</article-id>
<article-id pub-id-type="pmc">6583947</article-id>
<article-id pub-id-type="doi">10.1371/journal.pone.0218659</article-id>
<article-id pub-id-type="publisher-id">PONE-D-18-32405</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Cell Biology</subject>
<subj-group><subject>Cell Processes</subject>
<subj-group><subject>Cell Death</subject>
<subj-group><subject>Autophagic Cell Death</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Cell Biology</subject>
<subj-group><subject>Cell Processes</subject>
<subj-group><subject>Cell Death</subject>
<subj-group><subject>Apoptosis</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject>
<subj-group><subject>Oncology</subject>
<subj-group><subject>Cancers and Neoplasms</subject>
<subj-group><subject>Lung and Intrathoracic Tumors</subject>
<subj-group><subject>Non-Small Cell Lung Cancer</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Cell Biology</subject>
<subj-group><subject>Cell Processes</subject>
<subj-group><subject>Cell Growth</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Medicine and Health Sciences</subject>
<subj-group><subject>Oncology</subject>
<subj-group><subject>Cancer Treatment</subject>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject>
<subj-group><subject>Bioassays and physiological analysis</subject>
<subj-group><subject>Biochemical analysis</subject>
<subj-group><subject>Colorimetric assays</subject>
<subj-group><subject>MTT assay</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Research and analysis methods</subject>
<subj-group><subject>Bioassays and physiological analysis</subject>
<subj-group><subject>Biochemical analysis</subject>
<subj-group><subject>Enzyme assays</subject>
<subj-group><subject>MTT assay</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Cell Biology</subject>
<subj-group><subject>Cellular Structures and Organelles</subject>
<subj-group><subject>Vacuoles</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Biochemistry</subject>
<subj-group><subject>Bioenergetics</subject>
<subj-group><subject>Energy-Producing Organelles</subject>
<subj-group><subject>Mitochondria</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v3"><subject>Biology and Life Sciences</subject>
<subj-group><subject>Cell Biology</subject>
<subj-group><subject>Cellular Structures and Organelles</subject>
<subj-group><subject>Energy-Producing Organelles</subject>
<subj-group><subject>Mitochondria</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</article-categories>
<title-group><article-title>Derrone induces autophagic cell death through induction of ROS and ERK in A549 cells</article-title>
<alt-title alt-title-type="running-head">Derrone induces autophagic cell death</alt-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Kang</surname>
<given-names>Myung-Ji</given-names>
</name>
<role content-type="http://credit.casrai.org/">Conceptualization</role>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Writing – original draft</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff002"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Kim</surname>
<given-names>Soo-Yeon</given-names>
</name>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Kwon</surname>
<given-names>Eun-Bin</given-names>
</name>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Investigation</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff002"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Jo</surname>
<given-names>Yang Hee</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff002"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lee</surname>
<given-names>Mi Kyeong</given-names>
</name>
<role content-type="http://credit.casrai.org/">Investigation</role>
<role content-type="http://credit.casrai.org/">Resources</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<xref ref-type="aff" rid="aff002"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lee</surname>
<given-names>Hyun-Sun</given-names>
</name>
<role content-type="http://credit.casrai.org/">Supervision</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author" equal-contrib="yes"><name><surname>Moon</surname>
<given-names>Dong-Oh</given-names>
</name>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Funding acquisition</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff003"><sup>3</sup>
</xref>
</contrib>
<contrib contrib-type="author" equal-contrib="yes"><contrib-id authenticated="true" contrib-id-type="orcid">http://orcid.org/0000-0003-3722-5724</contrib-id>
<name><surname>Kim</surname>
<given-names>Mun-Ock</given-names>
</name>
<role content-type="http://credit.casrai.org/">Conceptualization</role>
<role content-type="http://credit.casrai.org/">Data curation</role>
<role content-type="http://credit.casrai.org/">Funding acquisition</role>
<role content-type="http://credit.casrai.org/">Supervision</role>
<role content-type="http://credit.casrai.org/">Writing – review & editing</role>
<xref ref-type="aff" rid="aff001"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="cor001">*</xref>
</contrib>
</contrib-group>
<aff id="aff001"><label>1</label>
<addr-line>Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongju, Chungbuk, Republic of Korea</addr-line>
</aff>
<aff id="aff002"><label>2</label>
<addr-line>College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea</addr-line>
</aff>
<aff id="aff003"><label>3</label>
<addr-line>Department of Biology Education, Daegu University, Gyeongsan, Gyeongsangbuk, Republic of Korea</addr-line>
</aff>
<contrib-group><contrib contrib-type="editor"><name><surname>Ulasov</surname>
<given-names>Ilya</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1"><addr-line>Sechenov First Medical University, RUSSIAN FEDERATION</addr-line>
</aff>
<author-notes><fn fn-type="COI-statement" id="coi001"><p><bold>Competing Interests: </bold>
The authors have declared that no competing interests exist.</p>
</fn>
<corresp id="cor001">* E-mail: <email>mokim@kribb.re.kr</email>
</corresp>
</author-notes>
<pub-date pub-type="epub"><day>19</day>
<month>6</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="collection"><year>2019</year>
</pub-date>
<volume>14</volume>
<issue>6</issue>
<elocation-id>e0218659</elocation-id>
<history><date date-type="received"><day>11</day>
<month>11</month>
<year>2018</year>
</date>
<date date-type="accepted"><day>6</day>
<month>6</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>© 2019 Kang et al</copyright-statement>
<copyright-year>2019</copyright-year>
<copyright-holder>Kang et al</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="pone.0218659.pdf"></self-uri>
<abstract><p>We studied the effect of derrone (DR), one of the major compounds of unripe fruits of <italic>Cudrania tricuspidata</italic>
, on cancer cell death. DR inhibited cell growth of various cancer cells, and that was partially associated with apoptosis in A549 cells. DR showed the autophagic features, such as the conversion of LC3-I to LC3-II, the formation of autophagosome and the downregulation of SQSTM1/p62 (p62). The treatment of autophagy inhibitor reversed DR-mediated cell death, suggesting that DR induces autophagic cell death. The increase of cytoplasmic Ca<sup>2+</sup>
and ROS by DR treatment significantly influences the formation of autophagosomes; however, only ROS scavengers significantly rescued the reduced cell viability. Additional results revealed that treatment of DR induces sustained phosphorylation of ERK and the inhibition of ERK phosphorylation using U0126 (ERK inhibitor) markedly attenuated DR-induced cell death. Overall, these results suggest that DR induces autophagic cell death through intracellular ROS and sustained ERK phosphorylation in A549 cells.</p>
</abstract>
<funding-group><award-group id="award001"><funding-source><institution-wrap><institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/501100003715</institution-id>
<institution>Korea Research Institute of Bioscience and Biotechnology</institution>
</institution-wrap>
</funding-source>
</award-group>
<award-group id="award002"><funding-source><institution-wrap><institution-id institution-id-type="funder-id">http://dx.doi.org/10.13039/501100004083</institution-id>
<institution>Ministry of Science ICT and Future Planning</institution>
</institution-wrap>
</funding-source>
<award-id>NRF-2017R1D1A1B03031653</award-id>
<principal-award-recipient><name><surname>Moon</surname>
<given-names>Dong-Oh</given-names>
</name>
</principal-award-recipient>
</award-group>
<funding-statement>This work was supported by a KRIBB Research Initiative Program funded by the Ministry of Science ICT (MSIT) of Republic of Korea. This work was also supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2017R1D1A1B03031653).</funding-statement>
</funding-group>
<counts><fig-count count="6"></fig-count>
<table-count count="0"></table-count>
<page-count count="15"></page-count>
</counts>
<custom-meta-group><custom-meta id="data-availability"><meta-name>Data Availability</meta-name>
<meta-value>All relevant data are within the manuscript.</meta-value>
</custom-meta>
</custom-meta-group>
</article-meta>
<notes><title>Data Availability</title>
<p>All relevant data are within the manuscript.</p>
</notes>
</front>
<body><sec sec-type="intro" id="sec001"><title>Introduction</title>
<p>Despite the improvement in many anticancer drugs, the mortality rate of lung cancer is still increasing [<xref rid="pone.0218659.ref001" ref-type="bibr">1</xref>
]. Lung cancer is classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) according to the size and histology of cancer cells. NSCLC accounts for approximately 80% of the lung cancer [<xref rid="pone.0218659.ref002" ref-type="bibr">2</xref>
]. According to the stage, treatment with NSCLC is attempted with radiation therapy, chemotherapy, target treatment, and bronchoscopy. Since the early 2000s, the development of target therapies that selectively attack cancer cells has been activated, and lung cancer target treatment drugs such as gefitinib (Iressa) and erlotinib (Tarceva) have been used [<xref rid="pone.0218659.ref003" ref-type="bibr">3</xref>
–<xref rid="pone.0218659.ref005" ref-type="bibr">5</xref>
]. Because the target therapies have problems of high cost and low stability, patients receiving treatment were limited. Therefore, many scientists continue to make efforts to develop new lung cancer drugs.</p>
<p>Autophagy and apoptosis play an important role in NSCLC progression. Autophagy is activated in response to several cellular stresses, the process delivers cytoplasmic material and organelles to lysosomes <italic>via</italic>
double membrane vesicles called autophagosomes for degradation. The formation of autophagosomes is controlled by a specific set of autophagy genes called atg genes. ATG8 (in mammals LC3), a well-established marker of autophagy, is covalently linked to phosphatidylethanolamine on the autophagic membrane during autophagosome formation. Apoptosis is usually characterized by membrane blebbing, cytoplasmic shrinkage, mitochondrial depolarization, release of apoptotic factors from the mitochondria, DNA fragmentation and apoptotic body formation [<xref rid="pone.0218659.ref006" ref-type="bibr">6</xref>
].</p>
<p>Autophagy can play a positive and negative role in promoting apoptosis in NSCLC. Autophagy is always suppressed by several oncoproteins, such as PI3K, AKT, Bcl-2 and mutant p53, which may prevent excessive protein degradation in starved or stressed tumor cells [<xref rid="pone.0218659.ref007" ref-type="bibr">7</xref>
, <xref rid="pone.0218659.ref008" ref-type="bibr">8</xref>
]. On the other hand, persistent activation of autophagy causes autophagic programmed cell death or apoptosis [<xref rid="pone.0218659.ref009" ref-type="bibr">9</xref>
, <xref rid="pone.0218659.ref010" ref-type="bibr">10</xref>
].</p>
<p><italic>Cudrania tricuspidata</italic>
(Moraceae) is a deciduous tree which is cultivated in China, Japan and Korea. The roots, stems, barks and fruits of <italic>C</italic>
. <italic>tricuspidata</italic>
have been widely used as traditional medicines and various pharmacological efficacy including anti-atherosclerotic, anti-inflammatory, anti-fungal, anti-lipid peroxidation, anti-oxidant effect have been studied [<xref rid="pone.0218659.ref011" ref-type="bibr">11</xref>
–<xref rid="pone.0218659.ref015" ref-type="bibr">15</xref>
]. Among them, fruits of <italic>C</italic>
. <italic>tricuspidata</italic>
have been reported to contain diverse active constituents such as polyphenols, isoflavonoids and flavonoids [<xref rid="pone.0218659.ref016" ref-type="bibr">16</xref>
, <xref rid="pone.0218659.ref017" ref-type="bibr">17</xref>
], which were affected by environmental conditions including maturation stages. Recently, we investigated the chemical compositions and anti-obesity effects of unripe and ripe fruits of <italic>C</italic>
. <italic>tricuspidata</italic>
[<xref rid="pone.0218659.ref018" ref-type="bibr">18</xref>
]. Further study on the chemical constituents of <italic>C</italic>
. <italic>tricuspiata</italic>
found that derron (DR), an isoflavonoids from unripe fruit, inhibited cell growth of A549 cells (derived from NSCLC). In this study, we investigated molecular mechanisms involved in DR-induced cell death, focusing on autophagy and apoptosis in A549 cells.</p>
</sec>
<sec sec-type="materials|methods" id="sec002"><title>Materials and methods</title>
<sec id="sec003"><title>Reagent and materials</title>
<p>Chloroquine (CQ), <italic>N</italic>
-acetyl-L-cysteine (NAC) and acridine orange hemi (Zinc chloride) salt were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Caspase-8,-9, or -3 colorimetric assay kit and Z-VAD-FMK (a pan-caspase inhibitor) were obtained from R&D systems (MA, USA). Cycletest Plus DNA kit and FITC-Annexin V were purchased from BD bioscience Pharmingen (San Jose, CA, USA). Wortmannin and U0126 were purchased TOCRIS (Bristol, UK). 2’7’-dichlorofluorescein diacetate, BAPTA-AM, Ru360 and JC-1 were obtained from Calbiochem (San Diego, CA, USA). Dihydroethidium, lipofectamine 2000, anti-Alexa Fluor 488, Fluo4-AM and BAPTA were purchased from Invitrogen (Carlsbad, CA, USA). Rhod2 and Ruthenium Red were obtained from Abcam (Cambridge, UK). The antibodies used in this study are as follows; anti-caspase-3, anti-caspase-9, anti-caspase-8, anti-PARP, anti-ATG5, anti-total ERK, anti-p-ERK (Cell signaling Technology, MA, USA), LC3B/MAP1LC3B (NOVUS Biologicals, USA), anti-p62 lck ligand (BD bioscience Pharmingen, CA, USA), HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnologies, CA, USA).</p>
</sec>
<sec id="sec004"><title>Isolation of DR</title>
<p><italic>C</italic>
. <italic>tricuspidata</italic>
unripe fruits were collected from the herb garden at Chungbuk National University from May 2013. A voucher specimen (CBNU2013-CTUF) was deposited at the herbarium of the College of Pharmacy, Chungbuk National University. The unripe fruits (556.0 g) were extracted 2 times with 100% MeOH at room temperature, which yielded the MeOH extract (20.4 g). The MeOH extract was suspended in H<sub>2</sub>
O, then partitioned successively with solvents of rising polarity, to obtain <italic>n</italic>
-hexane (0.8 L), CH<sub>2</sub>
Cl<sub>2</sub>
(0.7 L), EtOAc (0.8 L), and <italic>n</italic>
-BuOH (0.8 L). The CH<sub>2</sub>
Cl<sub>2</sub>
fraction (CTUM, 3.3 g) was subjected to column chromatography over Sephadex LH-20 eluted with 100% MeOH to give four subfractions (CTUM1-CTUM4). Derrone (111.1 mg) was isolated from CTUM3 by semi-preparative HPLC eluted with MeCN-H<sub>2</sub>
O (57:43) [<xref rid="pone.0218659.ref018" ref-type="bibr">18</xref>
]. Derrone is light brown amorphous syrup; UV (MeOH) λ<sub>max</sub>
: 266 nm; ESI-MS <italic>m/z</italic>
337 [M+H]<sup>+</sup>
; <sup>1</sup>
H-NMR (methanol-<italic>d</italic>
<sub>4</sub>
, 500 MHz) (see <xref ref-type="supplementary-material" rid="pone.0218659.s001">S1 Table</xref>
).</p>
</sec>
<sec id="sec005"><title>Cell culture</title>
<p>Human lung cancer A549 cells, human mucoepidermoid pulmonary carcinoma H292 cells and human prostate cancer PC3 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cell were cultured in RPMI-1640 (Welgene, Korea) medium, supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) at 37°C, 100% humidity and 5% CO<sub>2</sub>
. Human colorectal carcinoma HCT116 cells were obtained Bioevaluation Center (Korea Research Institute of Bioscience & Biotechnology, Republic of Korea). HCT116 cells were cultured in high glucose-Dulbecco’s Modified Eagle Medium (DMEM, Welgene, Korea) containing 10% (v/v) heat-inactivated FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO<sub>2</sub>
in air.</p>
</sec>
<sec id="sec006"><title>Cell viability</title>
<p>Cells (1×10<sup>5</sup>
cells/ml) were grown in 24-well plates for 24 h and then were treated with indicated concentrations of DR for designated incubation times. After incubation, cells were treated with MTT solution (final 0.5 mg/ml) for 30 min. Purple formazan was dissolved in dimethyl sulfoxide (DMSO) and absorbance was read at 540 nm using ELISA plate reader (Epoch, Bioteck, USA).</p>
</sec>
<sec id="sec007"><title>Immunoblotting</title>
<p>Cell were washed with ice-cold PBS and lysed with lysis buffer (Pro-Prep, iNtRON) containing protease inhibitor in ice for 30 min. After centrifugation (13,200 rpm, for 25 min at 4°C), collected supernatants were quantified using the Bredford method. The lysates boiling for 5 min at 95°C, separated by SDS-PAGE gels and transferred to a polyvinylidene difluoride membranes (PVDF, Millipore, MA, USA). After blocking nonspecific binding sites for 30 min by Ez-Block Chemi (Amherst, USA), membrane was incubated with specific primary and secondary antibodies. Membrane was then washed three times TBS-T for 30 min. Enhanced chemiluminescence (ECL, Thermo, USA) detection was used to detect immune complexes signal. Equal amount of proteins was assessed by anti-tubulin as internal controls. Protein band intensity was measured by densitometric analysis using the NIH Image J program (National Institutes of Health, Bethesda, MD, USA).</p>
</sec>
<sec id="sec008"><title>Measurement of intracellular calcium</title>
<p>A549 cells were harvested and incubated with 1 μg/ml Fluo-4 AM or 1 μg/ml Rhod 2-AM at 37°C for 15 min, washed with HBSS (without Ca<sup>2+</sup>
or Mg<sup>2+</sup>
). Then immediately analyzed on a flow cytometer (FACSCalibur, Becton Dickinson, CA, USA) using FL-1 or FL-2 channel.</p>
</sec>
<sec id="sec009"><title>Transmission electron microscopy</title>
<p>A549 cells (2×10<sup>5</sup>
cells/ml) were grown in 100 mm plates for 24 h and then were treated with 60 μM DR. Cell were collected and prefixed primary fixing solution (2.5% paraformaldehyde, 2.5% glutaraldehyde). After staining with uranyl acetate and sections were visualized under electron microscope (CM20T, Philips, Netherlands).</p>
</sec>
<sec id="sec010"><title>Analysis of cell cycle and Annexin V staining</title>
<p>A549 cells (2×10<sup>5</sup>
cells/ml) were grown in 12-well plates for 24 h and then were treated with various concentrations of DR for 24 h. Cells were prepared using the Cycletest Plus DNA kit according to the manufacturer's instructions and flow cytometric analysis for cell cycle analysis was performed. Annexin V-FITC staining was performed according to the manufacturer's instructions using the Apoptosis Detection Kit and analyzed via the FL-1 channel of a flow cytometer.</p>
</sec>
<sec id="sec011"><title>Flow cytometric detection of ROS</title>
<p>Intracellular ROS levels were measured flow cytometry in cells loaded with the redox-sensitive dye dihydroethidium (HE) and 2’7’-dichlorofluorescein diacetate (H<sub>2</sub>
DCF-DA). Approximately 5×10<sup>5</sup>
cells were harvested by trypsinization, resuspended in 0.5 ml PBS and incubated with 1 μM HE or 5 μM H<sub>2</sub>
DCF-DA for 20 min in the dark at 37°C. Fluorescence was recorded on FL-2 channel for HE and FL-1 channel for DCF using a flow cytometer.</p>
</sec>
<sec id="sec012"><title>Caspase activity assay</title>
<p>Total colorimetric caspase 3, 8, 9 assay were performed using commercial kit according to the manufacturer’s instructions. Briefly, cell were cultured O/N in 60 mm dishes, and then treated with various concentrations of DR (0, 20, 40, 60 and 80 μM) for 24 h. Cell were lysed with lysis buffer on ice for 30 min and then centrifuged at 13,200 rpm for 15 min. Equal amount of protein (50 μg) from each sample was added to the reaction mixture containing each substrate (DEVE-pNA, IETD-AFC or LEHD-pNA) and incubation for 120 min at 37°C. The absorbance was read at 405 nm in a micro plate reader. Each sample for the assay was taken in duplicates.</p>
</sec>
<sec id="sec013"><title>Immunocytochemistry</title>
<p>A549 cells were cultured on cover slips and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After washing at three times with PBS, the fixed cells were permeabilized with 0.1% Triton-X 100 for 5 min at RT. After three times washes with PBS, the cells were incubated with blocking solution (5% BSA in PBS) for 30 min and then with primary antibody overnight at 4°C. The next day, the cells were washed with PBS and then incubated with secondary antibody for 1h on the rocker. The cells were washed with PBS and Hoechst 33342 stained for 30 min. After washing, the cover slips were mounted on slide using mounting media. Cells were visualized with a fluorescence microscope (ZEISS, Germany).</p>
</sec>
<sec id="sec014"><title>Statistical analysis</title>
<p>Data are presented as mean± standard deviation (SD). Statistical analysis was performed using Student's <italic>t</italic>
-test for the <italic>in vitro</italic>
experiments. Differences were considered significant at <italic>p</italic>
<0.05 (<bold>*</bold>
), <italic>p</italic>
< 0.01 (<bold>**</bold>
), and <italic>p</italic>
< 0.001 (<bold>***</bold>
).</p>
</sec>
</sec>
<sec sec-type="results" id="sec015"><title>Results</title>
<sec id="sec016"><title>DR decreases cell growth of various cancer cells</title>
<p>To investigate the effect of DR on cell growth, DR was treated with different doses in various cancer cell lines (A549, human lung adenocarcinoma epithelial cells; H292, human lung mucoepidermoid cells; PC3, human prostate cancer cells; and HCT116, human colon cancer cells) and analyzed for cell viability using MTT assay (<xref ref-type="fig" rid="pone.0218659.g001">Fig 1B–1E</xref>
). DR dissolved in DMSO and made 1000× stock with 80, 60, 40, and 20 mM in series. The solvent is finally diluted 1000-fold with the cell treatment. In all experiments, we treated all controls with 0.1% DMSO. DR inhibited cell growth in a concentration-dependent manner without any specificity for the four cell lines with different histological origins. The IC<sub>50</sub>
values of DR against A549, H292, PC3 and HCT116 were 42.7 μM, 39.3 μM, 45.0 μM and 42.4 μM, respectively. Collectively, these results indicate that DR reduces cell growth that are not limited to specific cancer cell lines.</p>
<fig id="pone.0218659.g001" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g001</object-id>
<label>Fig 1</label>
<caption><title>The molecular structure of DR and its cytotoxic effects on various cancer cell lines.</title>
<p>(A) The molecular structures of the DR. Non-small cell lung cancer A549 cells (B), mucoepidermoid pulmonary cancer H292 cells (C), prostate cancer PC3 cells (D), and colon cancer HCT116 cells (E) were treated with the DR of indicated concentrations for 24 h or 48 h, and then measured by MTT assay. All data are expressed as the mean ± SD obtained from at least three independent experiments. Differences were considered significant at <italic>p</italic>
<0.05 (*), <italic>p</italic>
< 0.01 (**), and <italic>p</italic>
< 0.001 (***) compared with the DMSO control.</p>
</caption>
<graphic xlink:href="pone.0218659.g001"></graphic>
</fig>
</sec>
<sec id="sec017"><title>DR-mediated cell death is partially involved in apoptosis</title>
<p>To investigate whether the inhibition of cell growth by DR treatment was caused by apoptosis, we examined the changes of apoptotic markers in A549 cells after treatment with DR. Cell cycle analysis showed a gradual increase in the apoptotic sub-G1 phase at a concentration of 60 μM and 80 μM DR for 24 h without arresting at specific cell cycle phases (<xref ref-type="fig" rid="pone.0218659.g002">Fig 2A and 2B</xref>
). There are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Caspases, a family of cysteine proteases, plays an essential role in both pathways. Death receptor pathway is initiated by caspase-8, and activation of the mitochondrial apoptotic pathway leads to activation of caspase-9. The death receptor and the mitochondrial pathway ultimately activate the cell death executor caspase-3. We treated A549 cells with various concentrations of DR for 24 h and confirmed the proteolytic activity of caspase-8, -9 and -3 (<xref ref-type="fig" rid="pone.0218659.g002">Fig 2C</xref>
). Similar to the results of the cell cycle analysis, the activity of caspases was significantly increased in the DR 60 μM and 80 μM treatment groups. Annexin-V staining enables the identification of cells with deteriorated membrane integrity at an early apoptotic stage. As shown in <xref ref-type="fig" rid="pone.0218659.g002">Fig 2D</xref>
, Annexin V-positive cells were markedly increased in cells treated with DR from 60 μM. <xref ref-type="fig" rid="pone.0218659.g002">Fig 2E</xref>
presented the gradual cleavage of Poly(ADP-ribose) polymerase-1 (PARP-1) from 60 μM to 80 μM of DR. PARP-1 is proteolysed by caspases during the execution of the apoptotic process. Finally, pretreatment of z-VAD-fmk, a pan-caspase inhibitor, significantly reversed the cell death by 80 μM DR treatment but did not restored by 60 μM (<xref ref-type="fig" rid="pone.0218659.g002">Fig 2F</xref>
). These results suggest that DR causes apoptotic markers to appear, but based on the fact that cell death is not restored by caspase inhibitor apoptosis, DR-induced A549 cell death is associated with non-apoptotic cell death mode can be involved.</p>
<fig id="pone.0218659.g002" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g002</object-id>
<label>Fig 2</label>
<caption><title>DR induces non-apoptotic cell death at low concentrations.</title>
<p>(A) DR was treated with various concentration for 24 h, and cell cycle analysis was performed using flow cytometry. (B) The cell cycle distribution was quantified and represented as a bar graph. (C) After 24 h incubation with the indicated concentrations of DR, the A549 cells were lysed and aliquots were assayed for <italic>in vitro</italic>
caspase-8, - 9 and -3 activity. (D) After DR treatment for 24 h, the cells were stained with Annexin V. Early apoptotic Annexin V-positive cells were detected by flow cytometry. (E) After treatment of DR with the indicated concentrations, the cells were lysated and analyzed by western blotting. (F) Cells were co-treated with pan caspase inhibitor (<sub>Z</sub>
-VAD-fmk, 20 μM) and cell viability were measured by MTT assay. Statistical differences were presented p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (<sup>##</sup>
) compared with the DMSO control.</p>
</caption>
<graphic xlink:href="pone.0218659.g002"></graphic>
</fig>
</sec>
<sec id="sec018"><title>Autophagy is another cause of DR-induced cell death</title>
<p>After A549 cells were treated with various concentrations of DR, morphological changes were observed under a microscope. Cytoplasmic vacuoles were noticeable from 4 h after treatment of 40 μM DR. In the cells treated with 80 μM, cytoplasmic contraction, a morphological feature of typical apoptosis, was observed at 4 h and most of the cells were floating at 24 h (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3A</xref>
). To determine the origin of cytoplasmic vacuoles, we enlarged the cell using transmission electron microscopy (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3B</xref>
). In the DR-treated group, the intracellular debris in the closed double membrane, which appeared to be autophagosomes were observed (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3B</xref>
, arrow head). In addition, the vacuoles in which all contents are empty are thought to be fused together after autolysosome formation (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3B</xref>
, arrow with dotted line). Immunoblot analysis carried out to confirm the expression of autophagy-related marker proteins such as LC3, ATG5 and p62. The conversion of LC3-I to LC3-II and expression of ATG5 were increased after 6 h of 40 μM DR treatment, whereas p62 was decreased (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3C</xref>
). We further tested whether autophagy inhibitors could blocked the formation of vacuoles. Chloroquine is a lysosomotropic agent that inhibits endosomal acidification and blocks autolysosome formation. Wortmannin is a class III PI3-kinase inhibitor that blocks autophagy at the upstream stage and reduces the conversion of LC3-I to LC3-II. Pretreatment of chloroquine inhibited DR-induced cellular vacuolation, whereas wortmannin did not (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3D</xref>
). Chloroquine significantly rescued the cell viability inhibited by DR (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3E</xref>
). Chloroquine pretreatment also restored DR-induced p62 degradation, while the conversion of LC3-I to LC3-II was more increased in A549 cells (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3F</xref>
). This result shows that DR-induced autophagosomes was inhibited the binding of lysosome by treating chloroquine. Collectively, we suggest that DR induces macroautophagy in A549 cells, which contributes to cell death.</p>
<fig id="pone.0218659.g003" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g003</object-id>
<label>Fig 3</label>
<caption><title>DR induced autophagy in A549 cells.</title>
<p>(A) After A549 cells were treated with DR, the morphological change of cells was observed under the microscope. (B) Cells were treated with 60 μM DR for 6 h and observed under transmission electron microscopy. Arrowheads indicate autophagosome and arrows denote the vacuoles. (C) Cells were treated various concentrations of DR for 24 h before the western blot analysis. (D) Cells were treated 40 μM DR for 24 h with or without 1 h pretreatment with 0.5 μM wortmannin (Wort) or 20 μM chloroquine (CQ) and the morphological change of cells was observed under the microscope. (E) Cell viability was measured by MTT assay. Statistical differences were presented p<0.05 (*) compared with the DR alone; p<0.01 (<sup>##</sup>
) compared with the DMSO control. (F) Cells were treated DR for 24h and immunoblot analysis was performed after cell lysis.</p>
</caption>
<graphic xlink:href="pone.0218659.g003"></graphic>
</fig>
</sec>
<sec id="sec019"><title>DR induces intracellular ROS generation</title>
<p>Mounting evidences suggested that ROS could be a factor in activating the autophagy pathway [<xref rid="pone.0218659.ref019" ref-type="bibr">19</xref>
, <xref rid="pone.0218659.ref020" ref-type="bibr">20</xref>
]. As shown in <xref ref-type="fig" rid="pone.0218659.g004">Fig 4A</xref>
, treatment with 40 μM DR stimulated ROS production, showing a sharp increase up to 1 h, and thereafter decrease. In order to confirm that DR-induced ROS is associated with autophagy induction, the cells were pre-treated with ROS scavenger, an <italic>N</italic>
-acetyl-L-cysteine (NAC), before treatment of DR. The pretreatment of NAC completely abolished the macroautophagic (visible) vacuoles produced by DR (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4C</xref>
). We examined the effect of ROS on DR-mediated cell growth inhibition with NAC, glutathione or MnTBAP. Compared to NAC pretreatment, the glutathione (GSH) pretreatment group was more significantly restored by DR-induced cell death. MnTBAP, a superoxide anion scavenger, also showed a tendency to restore cell death by DR. Based on these results, we confirmed that DR-induced cell death in A549 cells is closely related to intracellular ROS production (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4B</xref>
). In addition, ROS induces autophagy accompanied by a strong mitochondrial dysfunction [<xref rid="pone.0218659.ref021" ref-type="bibr">21</xref>
]. Pretreatment of NAC effectively recovered mitochondrial membrane potential (MMP, <italic>Δ</italic>
Ψm) during DR treatment (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4D</xref>
). Collectively, these results indicate that DR increases intracellular ROS production and mitochondrial dysfunction, and that ROS is a factor in DR-mediated autophagic cell death.</p>
<fig id="pone.0218659.g004" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g004</object-id>
<label>Fig 4</label>
<caption><title>Increased ROS production by DR treatment is related to autophagy.</title>
<p>(A) After A549 cells were treated with DR, the cells were loaded with the hydrogen peroxide sensitive dye H<sub>2</sub>
DCFDA or the superoxide sensitive dye HE at 37°C for 30 min, and ROS generation was then measured using flow cytometry. (B) Cells were treated 40 μM DR for 24 h with or without treatment with NAC, GSH, and MnTBAP. The cell viability was measured by WST assay. (C) The morphological change of cells was observed under the microscope. (D) The cells were incubated with DiOC<sub>6</sub>
(3) for 30 min 37°C in the dark, and analyzed by a flow cytometery. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 40 nM) was used as a positive control. Statistical differences were presented p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (##) compared with the DMSO control.</p>
</caption>
<graphic xlink:href="pone.0218659.g004"></graphic>
</fig>
</sec>
<sec id="sec020"><title>DR increases free cytoplasmic Ca<sup>2+</sup>
concentration</title>
<p>Since intracellular Ca<sup>2+</sup>
is widely recognized as an important modulator of autophagy, we next investigated whether DR perturbs intracellular Ca<sup>2+</sup>
homeostasis. When 40 μM DR treated in Fluo-4 AM-prestained A549 cells, a cell-permeable Ca<sup>2+</sup>
indicator dye, cytoplasmic Ca<sup>2+</sup>
was increased within seconds (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5A and 5B</xref>
). We explored the sources of increased Ca<sup>2+</sup>
after DR treatment using Ca<sup>2+</sup>
chelators including BAPTA (extracellular Ca<sup>2+</sup>
scavenger) and BAPTA-AM (cell-permeable acetoxymethyl ester Ca<sup>2+</sup>
chelator). Flow cytometry analysis presented that the increased cytoplasmic Ca<sup>2+</sup>
by DR was significantly blocked by only BAPTA-AM, indicating that the origin of cytoplasmic Ca<sup>2+</sup>
was derived from an intracellular reservoirs such as ER and mitochondria (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5A</xref>
). The autophagosome formation by DR treatment was greatly inhibited by BAPTA-AM (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5C</xref>
); however, pretreatment of BAPTA or BAPTA-AM did not prevent DR-induced cell death (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5D</xref>
). Many studies established that an abrupt increase in intracellular ROS or Ca<sup>2+</sup>
is one of the causes of MMP reduction. We found that DR-induced MMP loss was significantly restored by BAPTA-AM (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5E</xref>
). Excessive decrease of MMP in the cell leads to mitochondrial permeability transition (MPT) pore opening, we showed that DR-induced cell death was significantly restored by pretreatment with cyclosporine A, which inhibited cyclophillin D, one of the constituent proteins of MPT pore (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5F</xref>
). Cyclosporine A treated alone for 24 hours with the various concentrations showed the decrease the cell growth, and in the group treated with 2 μM, the viability was significantly reduced to about 70%. There is the evidences that cyclosporine A induces apoptosis of cancer cells [<xref rid="pone.0218659.ref022" ref-type="bibr">22</xref>
, <xref rid="pone.0218659.ref023" ref-type="bibr">23</xref>
]. Although 2 μM cyclosporine A alone inhibited cell growth (<xref ref-type="supplementary-material" rid="pone.0218659.s002">S1 Fig</xref>
), pretreatment reduced cell death by inhibiting the DR-induced opening of MPT pore. Collectively, treatment of A549 cells with DR rapidly increases intracellular free calcium concentration in a short period of time, which appears to contribute to cytoplasmic vacuole formation and MMP reduction. However, the calcium chelators could not restore DR-induced cell death.</p>
<fig id="pone.0218659.g005" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g005</object-id>
<label>Fig 5</label>
<caption><title>DR increases cytoplasmic Ca<sup>2+</sup>
concentration.</title>
<p>(A) DR was added to 1 μg/ml Fluo-4 AM preloaded A549 cells and the changes of intracellular Ca<sup>2+</sup>
were examined by flow cytometry. Cells were stained with 1 μg/ml Fluo-4 AM for 30 min and treated 40 μM DR with or without co-treatment with 10 μM BAPTA and/or 10 μM BAPTA-AM for 1 min. (B) Cytoplasmic Ca<sup>2+</sup>
was quantified and represented as a bar graph. (C) The morphological change of cells was observed under the microscope. (D) Cell viability was measured using MTT assay. (E) The cells were stained with 40 nM DiOC<sub>6</sub>
(3) for 30 min at 37°C, and then analyzed by flow cytometry. (F) Cells were treated 40 μM DR for 24 h with or without treatment with 2 μM cyclosporine A. The cell viability was measured by MTT assay. All data are expressed as the mean ± S.D. obtained from at least three independent experiments. Statistical differences were presented p<0.01 (**) and p<0.001 (***) compared with the DR alone; p<0.01 (##) and p<0.001 (###) compared with the DMSO control.</p>
</caption>
<graphic xlink:href="pone.0218659.g005"></graphic>
</fig>
</sec>
<sec id="sec021"><title>DR induces prolonged ERK phosphorylation</title>
<p>Because the ERK plays a crucial roles in modulating autophagy [<xref rid="pone.0218659.ref024" ref-type="bibr">24</xref>
], we have confirmed the increased phosphorylation of ERK by the treatment of DR. A continuous (about 12 h) increased p-ERK was observed when 40 μM DR treated (<xref ref-type="fig" rid="pone.0218659.g006">Fig 6A</xref>
). The pretreatment of U0126, a highly selective inhibitor of both MEK1 and MEK2, markedly reversed the DR-mediated phosphorylation of ERK and degradation of p62 (<xref ref-type="fig" rid="pone.0218659.g006">Fig 6B</xref>
). As illustrated in <xref ref-type="fig" rid="pone.0218659.g006">Fig 6C</xref>
, the cell death induced by DR was effectively restored by U0126. Collectively, these results suggested that DR sustains ERK phosphorylation, which is closely related to DR-induced autophagic cell death.</p>
<fig id="pone.0218659.g006" orientation="portrait" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0218659.g006</object-id>
<label>Fig 6</label>
<caption><title>DR induces autophagy through ERK-mediated pathway.</title>
<p>(A) After treatment of 40 μM DR for the indicated times, the A549 cells were lysated and detected p-ERK by western blotting. (B) Cells were pretreated with U0126 (ERK inhibitor) further treated with DR for 24 h. Western blotting of the indicated proteins was performed, with tubulin detected as a loading control. (C) Cell viability was analyzed MTT assay. All data are expressed as the mean ± S.D. obtained from at least three independent experiments. Statistical differences were considered significant if p<0.05 (*), p<0.01 (**), and p<0.001 (***) compared with the DR alone; p<0.01 (##) compared with the DMSO control.</p>
</caption>
<graphic xlink:href="pone.0218659.g006"></graphic>
</fig>
</sec>
</sec>
<sec sec-type="conclusions" id="sec022"><title>Discussion</title>
<p>In this study, we reported that DR, one of the major constituents of unripe fruits of <italic>C</italic>
. <italic>tricuspidata</italic>
, induces autophagic cell death as well as partial apoptosis in A549 cells.</p>
<p>Our results provide evidence that DR induces autophagy. First, an autophagosome surrounded by a double membrane was observed by electron microscope (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3B</xref>
). Second, the formations of LC3-II and decrease of p62 were confirmed by Western blot (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3C</xref>
). Third, pretreatment of CQ decreased the vacuole of cytoplasm increased by DR and restored decreased p62 expression (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3D and 3F</xref>
). Finally, we can demonstrate that DR induces autophagic cell death by restoring the DR-mediated cell death by CQ (<xref ref-type="fig" rid="pone.0218659.g003">Fig 3E</xref>
).</p>
<p>The molecular mechanisms we have found in relation to DR-induced autophagic cell death in A549 cells are as follows. Treatment with DR increases intracellular ROS levels, which is highest at 1 hour after treatment (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4A</xref>
). Antioxidant agents significantly restored DR-mediated formation of vacuoles as well as cell death (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4B and 4C</xref>
), indicating that ROS production by DR is associated with autophagic cell death. Oxidative stresses caused by ROS can induce rapid depolarization of MMP [<xref rid="pone.0218659.ref025" ref-type="bibr">25</xref>
]. Expectedly, we confirmed that DR induced depolarization of MMP and it was effectively recovered by NAC treatment (<xref ref-type="fig" rid="pone.0218659.g004">Fig 4D</xref>
). In addition, DR increased the Ca<sup>2+</sup>
concentration in the cytoplasm in a second (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5A and 5B</xref>
), and BAPTA-AM, an intracellular Ca<sup>2+</sup>
chelator, significantly inhibited DR-induced formation of cytoplasmic vacuoles as well as reduction of MMP (<xref ref-type="fig" rid="pone.0218659.g005">Fig 5C and 5E</xref>
). Although there have been several previous studies showing that cytoplasmic Ca<sup>2+</sup>
overload induces autophagy [<xref rid="pone.0218659.ref026" ref-type="bibr">26</xref>
, <xref rid="pone.0218659.ref027" ref-type="bibr">27</xref>
], the detailed mechanism of DR-induced autophagy associated with Ca<sup>2+</sup>
need to be further explored. Finally, noteworthy is that prolonged activation of ERK signaling is required for DR-induced autophagy induction in A549 cells (<xref ref-type="fig" rid="pone.0218659.g006">Fig 6A</xref>
). Inhibition of ERK activation by U0126 blocked autophagic cell death (<xref ref-type="fig" rid="pone.0218659.g006">Fig 6C</xref>
), suggesting that ERK may acts as another upstream mediator of DR-induced autophagic cell death.</p>
<p>Constitutive activation of ERK by Raf [<xref rid="pone.0218659.ref028" ref-type="bibr">28</xref>
], cadmium [<xref rid="pone.0218659.ref029" ref-type="bibr">29</xref>
] or IGF-I receptor [<xref rid="pone.0218659.ref030" ref-type="bibr">30</xref>
] has been reported to induce a cell death associated with cytoplasmic massive vacuolization. This morphology could be a sign of autophagic programmed cell death, but also of paraptosis, a form of caspase‐independent cell death associated with cytoplasmic vacuolization [<xref rid="pone.0218659.ref031" ref-type="bibr">31</xref>
]. Therefore, we tried to determine whether the inhibition of ER stress with cycloheximide, which inhibited the protein synthesis by the newly synthesized intracellular proteins, and that it not affected DR-induced cell death (<xref ref-type="supplementary-material" rid="pone.0218659.s003">S2 Fig</xref>
). More specifically, the fate of the cell depends on the degree of activation of ERK. Transient or moderate ERK activation increases cytoprotective autophagy through the increase of Beclin1 by inhibition of mTORC1 or mTORC2, however, sustained or strong ERK activation completely inhibits mTORC1 and mTORC2, leading to an explosive increase of Beclin1 leading to ultimate cytotoxic autophagy [<xref rid="pone.0218659.ref032" ref-type="bibr">32</xref>
, <xref rid="pone.0218659.ref033" ref-type="bibr">33</xref>
]. In addition, in order to examine the effect of ROS scavenger or ERK inhibitor on autophagy, it was confirmed by Western blot that the DR-induced cleavage of LC3-I to LC3-II was suppressed after pretreatment of these compounds. The cleavage induced by DR was moderately reduced by the pretreatment of 20 μM U0126 and almost completely inhibited by pretreatment of NAC and GSH (<xref ref-type="supplementary-material" rid="pone.0218659.s004">S3 Fig</xref>
). Many documents suggest that unusually prolonged ERK activation (between 6 and 72 h) associated with cell death requires the presence of ROS [<xref rid="pone.0218659.ref034" ref-type="bibr">34</xref>
–<xref rid="pone.0218659.ref036" ref-type="bibr">36</xref>
]. Our results also agree with it that the ROS induced by the derrone treatment may be crucial for inducing cell death.</p>
<p>A549 cells are p53 wild-type and therefore are highly resistant to DNA damaging agents such as cisplatin. A549 cells are actively used for studying resistance and recurrence of cancer, and studies using cancer stem cells derived from cisplatin-resistant A549 cells have been actively conducted. Of course, p53 mutations are observed in more than 50% of solid tumors. Therefore, we examined the cell viability of human non-small cell lung carcinoma cell line H1299 (p53-null) by treatment with DR with concentration-dependent manner for 24 h (<xref ref-type="supplementary-material" rid="pone.0218659.s005">S4 Fig</xref>
). As a result, the IC<sub>50</sub>
value of DR in H1299 cells was 27.2 μM, and the IC<sub>50</sub>
value in A549 cells was 42.7 μM, which seems to be no significant difference in IC<sub>50</sub>
values. The results of treatment with DR in the H1299 (p53-null) cells were unexpectedly different from the mode of action in A549 cells. First, a form of DR-mediated cell death in H1299 cells is different from that of A549 cells. In H1299 cells, there was no recovery of DR-induced cell death by pretreatment of the autophagy inhibitors such as chloroquine or wortmannin (<xref ref-type="supplementary-material" rid="pone.0218659.s006">S5A Fig</xref>
). In addition, DR-mediated cell death in H1299 cells was not restored by treatment with the ERK inhibitors U0126 or PD98059 (<xref ref-type="supplementary-material" rid="pone.0218659.s006">S5B Fig</xref>
). Western analysis confirmed that DR-mediated induction of ERK phosphorylation and cleavage from LC3-I to LC3-II in H1299 cells. Pretreatment of U0126 significantly inhibited DR-mediated ERK phosphorylation and cleavage from LC3-I to LC3-II, but ultimately did not inhibit DR-induced autophagic cell death (<xref ref-type="supplementary-material" rid="pone.0218659.s006">S5C Fig</xref>
). Previous evidences have shown that ERK affects p53 stability and activity, and may affect autophagy and cell death modes [<xref rid="pone.0218659.ref031" ref-type="bibr">31</xref>
]. Taken together, DR induces autophagy in p53-null cells but does not contribute to cell death, and whether or not p53-dependent apoptosis contributes to DR-mediated cell death should be clarified in future studies.</p>
<p>As far as cancer is concerned, autophagy can act as a tumor suppressor and tumor promoter [<xref rid="pone.0218659.ref037" ref-type="bibr">37</xref>
–<xref rid="pone.0218659.ref039" ref-type="bibr">39</xref>
]. Decreased autophagy promotes tumorigenesis because monoallelical loss of essential autophagy gene ATG6/BECN1 in 40–75% of in human prostate, breast, and ovarian cancers is observed [<xref rid="pone.0218659.ref039" ref-type="bibr">39</xref>
–<xref rid="pone.0218659.ref041" ref-type="bibr">41</xref>
]. Autophagy inducers may activate apoptosis-independent cell death in apoptosis-resistant cells lacking the expression of the pro-apoptotic proteins such as Bax and Bak [<xref rid="pone.0218659.ref042" ref-type="bibr">42</xref>
, <xref rid="pone.0218659.ref043" ref-type="bibr">43</xref>
]. This suggests that continuous research on the autophagy inducer is necessary from the viewpoint of replacing the apoptosis inducer used as a conventional anticancer chemotherapeutic agent. Indeed, several chemotherapeutic drugs have been reported to engage autophagy [<xref rid="pone.0218659.ref037" ref-type="bibr">37</xref>
, <xref rid="pone.0218659.ref044" ref-type="bibr">44</xref>
, <xref rid="pone.0218659.ref045" ref-type="bibr">45</xref>
].</p>
<p>Our previously research has shown that unripe fruits contains different constituents compared to ripe fruits, which resulted in the excellent biological activities. In this study, we have provided evidence indicating that DR induces autophagic cell death through intracellular ROS and sustained ERK phosphorylation in A549 cells. Therefore, DR from unripe fruits of <italic>C</italic>
. <italic>tricuspidata</italic>
may act as a natural bioactive substance that was chemotherapeutic drugs through the activation of autophagy.</p>
</sec>
<sec sec-type="supplementary-material" id="sec023"><title>Supporting information</title>
<supplementary-material content-type="local-data" id="pone.0218659.s001"><label>S1 Table</label>
<caption><title>1H NMR data of derrone (methanol-<italic>d</italic>
<sub>4</sub>
, 500 MHz).</title>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s001.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0218659.s002"><label>S1 Fig</label>
<caption><title>Effect of cyclosporine A alone on cell viability of A549 cells.</title>
<p>Cells were treated with the cyclosporine A of indicated concentrations for 24 h, and then measured by WST assay. Differences were considered significant at p<0.05 (*) compared with the DMSO control.</p>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s002.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0218659.s003"><label>S2 Fig</label>
<caption><title>Effects of pretreatment of cycloheximide on DR-induced cell death in A549 cells.</title>
<p>Cells were co-treated with CHX and 40 μM DR for 24 h and then measured by WST assay. Statistical differences were presented p<0.001 (###) compared with the DMSO control.</p>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s003.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0218659.s004"><label>S3 Fig</label>
<caption><title>Effect of ROS scavenger or ERK inhibitor on DR-induced autophagy.</title>
<p>Cells were pretreated with U0126, NAC and GSH, and further treated with DR for 24 h. Cells were lysated and detected LC3 by western blotting. The tubulin detected as a loading control.</p>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s004.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0218659.s005"><label>S4 Fig</label>
<caption><title>Effect of DR on cell growth of H1299 cells.</title>
<p>H1299 cells were treated with the DR with indicated concentrations for 24 h, and then measured by MTT assay. Differences were considered significant at p<0.05 (*) and p< 0.01 (**) compared with the DMSO control.</p>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s005.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
<supplementary-material content-type="local-data" id="pone.0218659.s006"><label>S5 Fig</label>
<caption><title>The autophagy and ERK associated with DR-induced cell death in H1299 cells.</title>
<p>(A and B) H1299 cells were pre-treated with chloroquine, wortmannin, U0126 or PD98059, and exposed 40 μM DR further 24 h. Cell viability was measured by MTT assay. (C) H1299 cells were treated with DR with or without U0126 for 24 h. Western blotting was performed to detect p-ERK, ERK, LC3 and Tubulin.</p>
<p>(PDF)</p>
</caption>
<media xlink:href="pone.0218659.s006.pdf"><caption><p>Click here for additional data file.</p>
</caption>
</media>
</supplementary-material>
</sec>
</body>
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