Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes
Identifieur interne : 000762 ( Pmc/Corpus ); précédent : 000761; suivant : 000763Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes
Auteurs : Xiafang Wu ; Ru Sun ; Huihui Wang ; Bei Yang ; Fang Wang ; Hongtao Xu ; Shimin Chen ; Rui Zhao ; Jingbo Pi ; Yuanyuan XuSource :
- Oxidative Medicine and Cellular Longevity [ 1942-0900 ] ; 2019.
Abstract
Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing
Url:
DOI: 10.1155/2019/1038932
PubMed: 31781319
PubMed Central: 6875345
Links to Exploration step
PMC:6875345Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes</title><author><name sortKey="Wu, Xiafang" sort="Wu, Xiafang" uniqKey="Wu X" first="Xiafang" last="Wu">Xiafang Wu</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation><affiliation><nlm:aff id="I2">The First Hospital of China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Sun, Ru" sort="Sun, Ru" uniqKey="Sun R" first="Ru" last="Sun">Ru Sun</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Huihui" sort="Wang, Huihui" uniqKey="Wang H" first="Huihui" last="Wang">Huihui Wang</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Bei" sort="Yang, Bei" uniqKey="Yang B" first="Bei" last="Yang">Bei Yang</name><affiliation><nlm:aff id="I3">College of Basic Medical Sciences, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Fang" sort="Wang, Fang" uniqKey="Wang F" first="Fang" last="Wang">Fang Wang</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Hongtao" sort="Xu, Hongtao" uniqKey="Xu H" first="Hongtao" last="Xu">Hongtao Xu</name><affiliation><nlm:aff id="I3">College of Basic Medical Sciences, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Shimin" sort="Chen, Shimin" uniqKey="Chen S" first="Shimin" last="Chen">Shimin Chen</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Rui" sort="Zhao, Rui" uniqKey="Zhao R" first="Rui" last="Zhao">Rui Zhao</name><affiliation><nlm:aff id="I4">School of Forensic Medicine, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Pi, Jingbo" sort="Pi, Jingbo" uniqKey="Pi J" first="Jingbo" last="Pi">Jingbo Pi</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Yuanyuan" sort="Xu, Yuanyuan" uniqKey="Xu Y" first="Yuanyuan" last="Xu">Yuanyuan Xu</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31781319</idno><idno type="pmc">6875345</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6875345</idno><idno type="RBID">PMC:6875345</idno><idno type="doi">10.1155/2019/1038932</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000762</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000762</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes</title><author><name sortKey="Wu, Xiafang" sort="Wu, Xiafang" uniqKey="Wu X" first="Xiafang" last="Wu">Xiafang Wu</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation><affiliation><nlm:aff id="I2">The First Hospital of China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Sun, Ru" sort="Sun, Ru" uniqKey="Sun R" first="Ru" last="Sun">Ru Sun</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Huihui" sort="Wang, Huihui" uniqKey="Wang H" first="Huihui" last="Wang">Huihui Wang</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Yang, Bei" sort="Yang, Bei" uniqKey="Yang B" first="Bei" last="Yang">Bei Yang</name><affiliation><nlm:aff id="I3">College of Basic Medical Sciences, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Wang, Fang" sort="Wang, Fang" uniqKey="Wang F" first="Fang" last="Wang">Fang Wang</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Hongtao" sort="Xu, Hongtao" uniqKey="Xu H" first="Hongtao" last="Xu">Hongtao Xu</name><affiliation><nlm:aff id="I3">College of Basic Medical Sciences, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Chen, Shimin" sort="Chen, Shimin" uniqKey="Chen S" first="Shimin" last="Chen">Shimin Chen</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Rui" sort="Zhao, Rui" uniqKey="Zhao R" first="Rui" last="Zhao">Rui Zhao</name><affiliation><nlm:aff id="I4">School of Forensic Medicine, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Pi, Jingbo" sort="Pi, Jingbo" uniqKey="Pi J" first="Jingbo" last="Pi">Jingbo Pi</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author><author><name sortKey="Xu, Yuanyuan" sort="Xu, Yuanyuan" uniqKey="Xu Y" first="Yuanyuan" last="Xu">Yuanyuan Xu</name><affiliation><nlm:aff id="I1">School of Public Health, China Medical University, China</nlm:aff></affiliation></author></analytic><series><title level="j">Oxidative Medicine and Cellular Longevity</title><idno type="ISSN">1942-0900</idno><idno type="eISSN">1942-0994</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>Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing <italic>p62</italic> ameliorated elevation in mRNA levels of NRF2 downstream genes (<italic>AKR1C1</italic> and <italic>NQO1</italic>) and malignant phenotypes (acquired invasiveness and anchor-independent growth) induced by chronic arsenite exposure. On the other hand, silencing <italic>NRF2</italic> abrogated the increase in mRNA and protein levels of p62 and malignant phenotypes induced by arsenite. In response to acute arsenite exposure, impaired autophagic flux with an increase in p62 protein level and interrupted autophagosome-lysosome fusion was observed. The increase in p62 protein levels in response to arsenite was not completely dependent on NRF2 activation and at least partially attributed to protein degradation. Our data indicate that accumulation of p62 by impaired autophagic flux is involved in the activation of NRF2 and contributes to skin tumorigenesis due to chronic arsenite exposure.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Platanias, L C" uniqKey="Platanias L">L. C. Platanias</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alain, G" uniqKey="Alain G">G. Alain</name></author><author><name sortKey="Tousignant, J" uniqKey="Tousignant J">J. Tousignant</name></author><author><name sortKey="Rozenfarb, E" uniqKey="Rozenfarb E">E. Rozenfarb</name></author></analytic></biblStruct><biblStruct></biblStruct><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Degenhardt, K" uniqKey="Degenhardt K">K. Degenhardt</name></author><author><name sortKey="Mathew, R" uniqKey="Mathew R">R. Mathew</name></author><author><name sortKey="Beaudoin, B" uniqKey="Beaudoin B">B. Beaudoin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="White, E" uniqKey="White E">E. White</name></author><author><name sortKey="Karp, C" uniqKey="Karp C">C. Karp</name></author><author><name sortKey="Strohecker, A M" uniqKey="Strohecker A">A. M. Strohecker</name></author><author><name sortKey="Guo, Y" uniqKey="Guo Y">Y. Guo</name></author><author><name sortKey="Mathew, R" uniqKey="Mathew R">R. Mathew</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rybstein, M D" uniqKey="Rybstein M">M. D. Rybstein</name></author><author><name sortKey="Bravo San Pedro, J M" uniqKey="Bravo San Pedro J">J. M. Bravo-San Pedro</name></author><author><name sortKey="Kroemer, G" uniqKey="Kroemer G">G. Kroemer</name></author><author><name sortKey="Galluzzi, L" uniqKey="Galluzzi L">L. Galluzzi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Amaravadi, R K" uniqKey="Amaravadi R">R. K. Amaravadi</name></author><author><name sortKey="Thompson, C B" uniqKey="Thompson C">C. B. Thompson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rao, S" uniqKey="Rao S">S. Rao</name></author><author><name sortKey="Tortola, L" uniqKey="Tortola L">L. Tortola</name></author><author><name sortKey="Perlot, T" uniqKey="Perlot T">T. Perlot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rogov, V" uniqKey="Rogov V">V. Rogov</name></author><author><name sortKey="Dotsch, V" uniqKey="Dotsch V">V. Dotsch</name></author><author><name sortKey="Johansen, T" uniqKey="Johansen T">T. Johansen</name></author><author><name sortKey="Kirkin, V" uniqKey="Kirkin V">V. Kirkin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, H G R" uniqKey="Thompson H">H. G. R. Thompson</name></author><author><name sortKey="Harris, J W" uniqKey="Harris J">J. W. Harris</name></author><author><name sortKey="Wold, B J" uniqKey="Wold B">B. J. Wold</name></author><author><name sortKey="Lin, F" uniqKey="Lin F">F. Lin</name></author><author><name sortKey="Brody, J P" uniqKey="Brody J">J. P. Brody</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Inoue, D" uniqKey="Inoue D">D. Inoue</name></author><author><name sortKey="Suzuki, T" uniqKey="Suzuki T">T. Suzuki</name></author><author><name sortKey="Mitsuishi, Y" uniqKey="Mitsuishi Y">Y. Mitsuishi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Umemura, A" uniqKey="Umemura A">A. Umemura</name></author><author><name sortKey="He, F" uniqKey="He F">F. He</name></author><author><name sortKey="Taniguchi, K" uniqKey="Taniguchi K">K. Taniguchi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qiang, L" uniqKey="Qiang L">L. Qiang</name></author><author><name sortKey="Zhao, B" uniqKey="Zhao B">B. Zhao</name></author><author><name sortKey="Ming, M" uniqKey="Ming M">M. Ming</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Moscat, J" uniqKey="Moscat J">J. Moscat</name></author><author><name sortKey="Diaz Meco, M T" uniqKey="Diaz Meco M">M. T. Diaz-Meco</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jain, A" uniqKey="Jain A">A. Jain</name></author><author><name sortKey="Lamark, T" uniqKey="Lamark T">T. Lamark</name></author><author><name sortKey="Sj Ttem, E" uniqKey="Sj Ttem E">E. Sjøttem</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yamamoto, M" uniqKey="Yamamoto M">M. Yamamoto</name></author><author><name sortKey="Kensler, T W" uniqKey="Kensler T">T. W. Kensler</name></author><author><name sortKey="Motohashi, H" uniqKey="Motohashi H">H. Motohashi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Komatsu, M" uniqKey="Komatsu M">M. Komatsu</name></author><author><name sortKey="Kurokawa, H" uniqKey="Kurokawa H">H. Kurokawa</name></author><author><name sortKey="Waguri, S" uniqKey="Waguri S">S. Waguri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, A" uniqKey="Lau A">A. Lau</name></author><author><name sortKey="Wang, X J" uniqKey="Wang X">X. J. Wang</name></author><author><name sortKey="Zhao, F" uniqKey="Zhao F">F. Zhao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pi, J" uniqKey="Pi J">J. Pi</name></author><author><name sortKey="Diwan, B A" uniqKey="Diwan B">B. A. Diwan</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y. Sun</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, A" uniqKey="Lau A">A. Lau</name></author><author><name sortKey="Zheng, Y" uniqKey="Zheng Y">Y. Zheng</name></author><author><name sortKey="Tao, S" uniqKey="Tao S">S. Tao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Qi, Y" uniqKey="Qi Y">Y. Qi</name></author><author><name sortKey="Zhang, M" uniqKey="Zhang M">M. Zhang</name></author><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Liu, X" uniqKey="Liu X">X. Liu</name></author><author><name sortKey="Ling, M" uniqKey="Ling M">M. Ling</name></author><author><name sortKey="Chen, C" uniqKey="Chen C">C. Chen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shah, P" uniqKey="Shah P">P. Shah</name></author><author><name sortKey="Trinh, E" uniqKey="Trinh E">E. Trinh</name></author><author><name sortKey="Qiang, L" uniqKey="Qiang L">L. Qiang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Dodson, M" uniqKey="Dodson M">M. Dodson</name></author><author><name sortKey="De La Vega, M R" uniqKey="De La Vega M">M. R. de la Vega</name></author><author><name sortKey="Harder, B" uniqKey="Harder B">B. Harder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Leone, A" uniqKey="Leone A">A. Leone</name></author><author><name sortKey="Flatow, U" uniqKey="Flatow U">U. Flatow</name></author><author><name sortKey="King, C R" uniqKey="King C">C. R. King</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Valko, M" uniqKey="Valko M">M. Valko</name></author><author><name sortKey="Morris, H" uniqKey="Morris H">H. Morris</name></author><author><name sortKey="Cronin, M T" uniqKey="Cronin M">M. T. Cronin</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bj Rk Y, G" uniqKey="Bj Rk Y G">G. Bjørkøy</name></author><author><name sortKey="Lamark, T" uniqKey="Lamark T">T. Lamark</name></author><author><name sortKey="Pankiv, S" uniqKey="Pankiv S">S. Pankiv</name></author><author><name sortKey=" Vervatn, A" uniqKey=" Vervatn A">A. Øvervatn</name></author><author><name sortKey="Brech, A" uniqKey="Brech A">A. Brech</name></author><author><name sortKey="Johansen, T" uniqKey="Johansen T">T. Johansen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mizushima, N" uniqKey="Mizushima N">N. Mizushima</name></author><author><name sortKey="Yoshimori, T" uniqKey="Yoshimori T">T. Yoshimori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Shintani, T" uniqKey="Shintani T">T. Shintani</name></author><author><name sortKey="Klionsky, D J" uniqKey="Klionsky D">D. J. Klionsky</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pajares, M" uniqKey="Pajares M">M. Pajares</name></author><author><name sortKey="Rojo, A I" uniqKey="Rojo A">A. I. Rojo</name></author><author><name sortKey="Arias, E" uniqKey="Arias E">E. Arias</name></author><author><name sortKey="Diaz Carretero, A" uniqKey="Diaz Carretero A">A. Diaz-Carretero</name></author><author><name sortKey="Cuervo, A M" uniqKey="Cuervo A">A. M. Cuervo</name></author><author><name sortKey="Cuadrado, A" uniqKey="Cuadrado A">A. Cuadrado</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pi, J" uniqKey="Pi J">J. Pi</name></author><author><name sortKey="He, Y" uniqKey="He Y">Y. He</name></author><author><name sortKey="Bortner, C" uniqKey="Bortner C">C. Bortner</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, R" uniqKey="Zhao R">R. Zhao</name></author><author><name sortKey="Hou, Y" uniqKey="Hou Y">Y. Hou</name></author><author><name sortKey="Zhang, Q" uniqKey="Zhang Q">Q. Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, R" uniqKey="Zhao R">R. Zhao</name></author><author><name sortKey="Yang, B" uniqKey="Yang B">B. Yang</name></author><author><name sortKey="Wang, L" uniqKey="Wang L">L. Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ke, N" uniqKey="Ke N">N. Ke</name></author><author><name sortKey="Wang, X" uniqKey="Wang X">X. Wang</name></author><author><name sortKey="Xu, X" uniqKey="Xu X">X. Xu</name></author><author><name sortKey="Abassi, Y A" uniqKey="Abassi Y">Y. A. Abassi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Xu, Y" uniqKey="Xu Y">Y. Xu</name></author><author><name sortKey="Tokar, E J" uniqKey="Tokar E">E. J. Tokar</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y. Sun</name></author><author><name sortKey="Waalkes, M P" uniqKey="Waalkes M">M. P. Waalkes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ghosh, P" uniqKey="Ghosh P">P. Ghosh</name></author><author><name sortKey="Banerjee, M" uniqKey="Banerjee M">M. Banerjee</name></author><author><name sortKey="De Chaudhuri, S" uniqKey="De Chaudhuri S">S. de Chaudhuri</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tseng, W P" uniqKey="Tseng W">W. P. Tseng</name></author><author><name sortKey="Chu, H M" uniqKey="Chu H">H. M. Chu</name></author><author><name sortKey="How, S W" uniqKey="How S">S. W. How</name></author><author><name sortKey="Fong, J M" uniqKey="Fong J">J. M. Fong</name></author><author><name sortKey="Lin, C S" uniqKey="Lin C">C. S. Lin</name></author><author><name sortKey="Yeh, S" uniqKey="Yeh S">S. Yeh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Edwards, D H" uniqKey="Edwards D">D. H. Edwards</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y. Li</name></author><author><name sortKey="Ellinsworth, D C" uniqKey="Ellinsworth D">D. C. Ellinsworth</name></author><author><name sortKey="Griffith, T M" uniqKey="Griffith T">T. M. Griffith</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snow, E T" uniqKey="Snow E">E. T. Snow</name></author><author><name sortKey="Sykora, P" uniqKey="Sykora P">P. Sykora</name></author><author><name sortKey="Durham, T" uniqKey="Durham T">T. Durham</name></author><author><name sortKey="Klein, C" uniqKey="Klein C">C. Klein</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhang, D D" uniqKey="Zhang D">D. D. Zhang</name></author><author><name sortKey="Hannink, M" uniqKey="Hannink M">M. Hannink</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Son, Y O" uniqKey="Son Y">Y. O. Son</name></author><author><name sortKey="Pratheeshkumar, P" uniqKey="Pratheeshkumar P">P. Pratheeshkumar</name></author><author><name sortKey="Roy, R V" uniqKey="Roy R">R. V. Roy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ramos Gomez, M" uniqKey="Ramos Gomez M">M. Ramos-Gomez</name></author><author><name sortKey="Kwak, M K" uniqKey="Kwak M">M. K. Kwak</name></author><author><name sortKey="Dolan, P M" uniqKey="Dolan P">P. M. Dolan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ramos Gomez, M" uniqKey="Ramos Gomez M">M. Ramos-Gomez</name></author><author><name sortKey="Dolan, P M" uniqKey="Dolan P">P. M. Dolan</name></author><author><name sortKey="Itoh, K" uniqKey="Itoh K">K. Itoh</name></author><author><name sortKey="Yamamoto, M" uniqKey="Yamamoto M">M. Yamamoto</name></author><author><name sortKey="Kensler, T W" uniqKey="Kensler T">T. W. Kensler</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rolfs, F" uniqKey="Rolfs F">F. Rolfs</name></author><author><name sortKey="Huber, M" uniqKey="Huber M">M. Huber</name></author><author><name sortKey="Kuehne, A" uniqKey="Kuehne A">A. Kuehne</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ni, H M" uniqKey="Ni H">H. M. Ni</name></author><author><name sortKey="Woolbright, B L" uniqKey="Woolbright B">B. L. Woolbright</name></author><author><name sortKey="Williams, J" uniqKey="Williams J">J. Williams</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ngo, H K C" uniqKey="Ngo H">H. K. C. Ngo</name></author><author><name sortKey="Kim, D H" uniqKey="Kim D">D. H. Kim</name></author><author><name sortKey="Cha, Y N" uniqKey="Cha Y">Y. N. Cha</name></author><author><name sortKey="Na, H K" uniqKey="Na H">H. K. Na</name></author><author><name sortKey="Surh, Y J" uniqKey="Surh Y">Y. J. Surh</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fu, J" uniqKey="Fu J">J. Fu</name></author><author><name sortKey="Woods, C G" uniqKey="Woods C">C. G. Woods</name></author><author><name sortKey="Yehuda Shnaidman, E" uniqKey="Yehuda Shnaidman E">E. Yehuda-Shnaidman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Copple, I M" uniqKey="Copple I">I. M. Copple</name></author><author><name sortKey="Lister, A" uniqKey="Lister A">A. Lister</name></author><author><name sortKey="Obeng, A D" uniqKey="Obeng A">A. D. Obeng</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taguchi, K" uniqKey="Taguchi K">K. Taguchi</name></author><author><name sortKey="Fujikawa, N" uniqKey="Fujikawa N">N. Fujikawa</name></author><author><name sortKey="Komatsu, M" uniqKey="Komatsu M">M. Komatsu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Komatsu, M" uniqKey="Komatsu M">M. Komatsu</name></author><author><name sortKey="Kageyama, S" uniqKey="Kageyama S">S. Kageyama</name></author><author><name sortKey="Ichimura, Y" uniqKey="Ichimura Y">Y. Ichimura</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Linares, J F" uniqKey="Linares J">J. F. Linares</name></author><author><name sortKey="Duran, A" uniqKey="Duran A">A. Duran</name></author><author><name sortKey="Reina Campos, M" uniqKey="Reina Campos M">M. Reina-Campos</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eskelinen, E L" uniqKey="Eskelinen E">E. L. Eskelinen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Issa, A R" uniqKey="Issa A">A. R. Issa</name></author><author><name sortKey="Sun, J" uniqKey="Sun J">J. Sun</name></author><author><name sortKey="Petitgas, C" uniqKey="Petitgas C">C. Petitgas</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Oxid Med Cell Longev</journal-id><journal-id journal-id-type="iso-abbrev">Oxid Med Cell Longev</journal-id><journal-id journal-id-type="publisher-id">OMCL</journal-id><journal-title-group><journal-title>Oxidative Medicine and Cellular Longevity</journal-title></journal-title-group><issn pub-type="ppub">1942-0900</issn><issn pub-type="epub">1942-0994</issn><publisher><publisher-name>Hindawi</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31781319</article-id><article-id pub-id-type="pmc">6875345</article-id><article-id pub-id-type="doi">10.1155/2019/1038932</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group></article-categories><title-group><article-title>Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Xiafang</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref><xref ref-type="aff" rid="I2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Ru</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Huihui</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Bei</given-names></name><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Fang</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Xu</surname><given-names>Hongtao</given-names></name><xref ref-type="aff" rid="I3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Shimin</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Rui</given-names></name><xref ref-type="aff" rid="I4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><contrib-id contrib-id-type="orcid" authenticated="false">https://orcid.org/0000-0003-0227-8041</contrib-id><name><surname>Pi</surname><given-names>Jingbo</given-names></name><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid" authenticated="false">https://orcid.org/0000-0003-2354-9453</contrib-id><name><surname>Xu</surname><given-names>Yuanyuan</given-names></name><email>yyxu@cmu.edu.cn</email><xref ref-type="aff" rid="I1"><sup>1</sup></xref></contrib></contrib-group><aff id="I1"><sup>1</sup>School of Public Health, China Medical University, China</aff><aff id="I2"><sup>2</sup>The First Hospital of China Medical University, China</aff><aff id="I3"><sup>3</sup>College of Basic Medical Sciences, China Medical University, China</aff><aff id="I4"><sup>4</sup>School of Forensic Medicine, China Medical University, China</aff><author-notes><fn fn-type="other"><p>Academic Editor: Aldrin V. Gomes</p></fn></author-notes><pub-date pub-type="collection"><year>2019</year></pub-date><pub-date pub-type="epub"><day>30</day><month>10</month><year>2019</year></pub-date><volume>2019</volume><elocation-id>1038932</elocation-id><history><date date-type="received"><day>13</day><month>3</month><year>2019</year></date><date date-type="rev-recd"><day>16</day><month>8</month><year>2019</year></date><date date-type="accepted"><day>29</day><month>8</month><year>2019</year></date></history><permissions><copyright-statement>Copyright © 2019 Xiafang Wu et al.</copyright-statement><copyright-year>2019</copyright-year><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>Chronic exposure to arsenic induces a variety of cancers, particularly in the skin. Autophagy is a highly conserved process which plays a dual role in tumorigenesis. In the present study, we found that chronic exposure to an environmentally relevant dose of arsenite induced malignant transformation of human keratinocytes (HaCaT) with dysregulated autophagy as indicated by an increased number of autophagosomes, activation of mTORC1 pathway, and elevated protein levels of p62 and LC3II. Meanwhile, arsenite-transformed cells showed lower intracellular levels of reactive oxygen species compared with control. Silencing <italic>p62</italic> ameliorated elevation in mRNA levels of NRF2 downstream genes (<italic>AKR1C1</italic> and <italic>NQO1</italic>) and malignant phenotypes (acquired invasiveness and anchor-independent growth) induced by chronic arsenite exposure. On the other hand, silencing <italic>NRF2</italic> abrogated the increase in mRNA and protein levels of p62 and malignant phenotypes induced by arsenite. In response to acute arsenite exposure, impaired autophagic flux with an increase in p62 protein level and interrupted autophagosome-lysosome fusion was observed. The increase in p62 protein levels in response to arsenite was not completely dependent on NRF2 activation and at least partially attributed to protein degradation. Our data indicate that accumulation of p62 by impaired autophagic flux is involved in the activation of NRF2 and contributes to skin tumorigenesis due to chronic arsenite exposure.</p></abstract><funding-group><award-group><funding-source>Department of Science and Technology of Liaoning Province</funding-source></award-group><award-group><funding-source>Liaoning Revitalization Talents Program</funding-source><award-id>XLYC1807225</award-id></award-group><award-group><funding-source>National Natural Science Foundation of China</funding-source><award-id>81302391</award-id><award-id>81573187</award-id></award-group></funding-group></article-meta></front><body><sec id="sec1"><title>1. Introduction</title><p>Arsenic is a metalloid ubiquitously distributed in the environment. Chronic exposure to excessive levels of arsenic usually occurs through consumption of drinking water and contaminated food. Arsenic and arsenic compounds are identified as human carcinogens by the International Agency for Research on Cancer (IARC) [<xref rid="B1" ref-type="bibr">1</xref>]. Chronic exposure to arsenic induces a variety of cancers, particularly in the skin, lung, bladder, liver, and kidney [<xref rid="B2" ref-type="bibr">2</xref>]. However, the exact molecular mechanism of arsenic carcinogenicity is not well understood. The skin is one of the most sensitive tissues to chronic arsenic exposure. In humans, chronic exposure to arsenic results in various skin lesions, including hyperpigmentation, hyperkeratosis, and Bowen's disease, which are considered as precancerous lesions [<xref rid="B3" ref-type="bibr">3</xref>]. The characteristic arsenic-associated skin cancers include squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) [<xref rid="B4" ref-type="bibr">4</xref>, <xref rid="B5" ref-type="bibr">5</xref>].</p><p>Autophagy, an evolutionarily conserved cellular catabolic mechanism in eukaryotes, has vital roles in maintaining protein homeostasis and is essential to cell fate in response to stress [<xref rid="B6" ref-type="bibr">6</xref>]. Defects of autophagy lead to accumulation of dysfunctional organelles, damaged proteins, etc., which increase the risk of cancer [<xref rid="B7" ref-type="bibr">7</xref>, <xref rid="B8" ref-type="bibr">8</xref>]. On the other hand, autophagy facilitates drug resistance and stress adaptation of cancer cells [<xref rid="B9" ref-type="bibr">9</xref>]. Thus, it is considered that autophagy suppresses tumor formation and growth in the early stage of cancer but promotes cancer in the later stage. p62 acts as an autophagy receptor and is usually degraded after autophagy with the use of lysosomal proteases [<xref rid="B10" ref-type="bibr">10</xref>, <xref rid="B11" ref-type="bibr">11</xref>]. Elevated expression of p62 has been found in liver cancer, lung cancer, breast cancer, and skin cancer [<xref rid="B1" ref-type="bibr">1</xref>, <xref rid="B12" ref-type="bibr">12</xref>–<xref rid="B15" ref-type="bibr">15</xref>]. Impaired autophagy resulting in p62 accumulation is reported to promote tumorigenesis [<xref rid="B16" ref-type="bibr">16</xref>]. Consistently, deficiency in <italic>p62</italic> diminishes chemical-induced hepatocarcinogenesis in the mouse model [<xref rid="B14" ref-type="bibr">14</xref>]. In skin tumors, p62 is upregulated and promotes cell proliferation and migration by stabilizing the oncogenic factor TWSIT1 [<xref rid="B15" ref-type="bibr">15</xref>].</p><p>It is interesting that p62 is able to form a positive feedback loop with nuclear factor erythroid 2-related factor 2 (NRF2) [<xref rid="B17" ref-type="bibr">17</xref>], a key transcription factor in antioxidative defense [<xref rid="B18" ref-type="bibr">18</xref>]. Accumulation of p62 inhibits Keap1-mediated NRF2 protein degradation by competing with NRF2 for the binding site of Keap1, resulting in transcriptional upregulation of NRF2 downstream genes [<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B20" ref-type="bibr">20</xref>]. On the other hand, NRF2 regulates the expression of p62 by direct binding to the antioxidant response element on its promotor region. Our previous study has shown that NRF2 is constitutively activated in arsenic-transformed human keratinocytes (HaCaT cells) [<xref rid="B21" ref-type="bibr">21</xref>]. Recently, chronic exposure to low levels of arsenite has been found to inhibit autophagy [<xref rid="B22" ref-type="bibr">22</xref>–<xref rid="B25" ref-type="bibr">25</xref>], which is attributed to overproduction of interleukin 6 [<xref rid="B23" ref-type="bibr">23</xref>]. Moreover, NRF2 activation in the scenario of low-level arsenic exposure is indicated to be dependent on p62 accumulation due to blockage of autophagic flux rather than reactive oxygen species (ROS) [<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B25" ref-type="bibr">25</xref>, <xref rid="B26" ref-type="bibr">26</xref>]. However, the role of this p62-NRF2 feedback loop in arsenic carcinogenesis has not been clearly identified.</p><p>In the present study, we found that arsenite-transformed human keratinocytes showed dysregulated autophagy with enhanced p62-NRF2 feedback loop and decreased intracellular ROS levels. Acute exposure to the environmentally relevant dose of arsenite blocked autophagic flux by interfering autophagosome-lysosome fusion, which contributed to the accumulation of p62. Silencing <italic>p62</italic> or <italic>NRF2</italic> ameliorated the arsenite-induced enhancement of p62-NRF2 feedback loop and furthermore the acquisition of malignant phenotypes. Our data suggest the important role of p62-NRF2 feedback loop in arsenite-induced skin tumorigenesis. This loop may be a target in prevention and therapy of arsenite-induced skin cancer.</p></sec><sec id="sec2"><title>2. Results</title><sec id="sec2.1"><title>2.1. Chronic Exposure to an Environmentally Relevant Dose of Arsenite Induces Malignant Transformation and Dysregulated Autophagy in Human Keratinocytes</title><p>After a 30-week continuous arsenite exposure, HaCaT cells exhibited increased invasion capacity (<xref ref-type="fig" rid="fig1">Figure 1(a)</xref>) and anchorage-independent growth as illustrated by the formation of bigger colonies in soft agar (<xref ref-type="fig" rid="fig1">Figure 1(b)</xref>) compared with control (Con), all indicative of malignant transformation [<xref rid="B27" ref-type="bibr">27</xref>]. Thus, the 30-week arsenite-exposed cells were named as arsenite-transformed (As-TM) cells thereafter. Transmission electron microscopy (TEM) analysis of As-TM cells showed the massive accumulation of autophagosomes (Figures <xref ref-type="fig" rid="fig1">1(c)</xref> and <xref ref-type="fig" rid="fig1">1(d)</xref>), recognized as double-membrane vesicles engulfing cytosolic contents or organelles. The ratio of LC3II to LC3I (LC3II/I) and protein levels of p62 were markedly increased in As-TM cells compared with the control (<xref ref-type="fig" rid="fig1">Figure 1(e)</xref>). Upstream signaling pathways of autophagy, such as mTORC1 and BECN1, were also determined. The levels of phosphorylated mTOR (p-mTOR) and phosphorylated P70S6K (p-P70S6K) were higher in As-TM cells than control (<xref ref-type="fig" rid="fig1">Figure 1(f)</xref>). No alteration was found in protein levels of BECN1 or RAPTOR in As-TM cells (<xref ref-type="fig" rid="fig1">Figure 1(f)</xref>). Collectively, these data indicate that chronic exposure to an environmentally relevant dose of arsenite induces malignant transformation of HaCaT cells with dysregulated autophagy.</p></sec><sec id="sec2.2"><title>2.2. Amplified p62-NRF2 Autoregulatory Loop Is Required for Arsenite-Induced Malignant Transformation of Human Keratinocytes</title><p>Arsenite is a well-known oxidative stressor [<xref rid="B28" ref-type="bibr">28</xref>]. However, in As-TM cells, intracellular ROS levels were only 50% of the control (Figures <xref ref-type="fig" rid="fig2">2(a)</xref> and <xref ref-type="fig" rid="fig2">2(b)</xref>). When cells were acutely challenged by a relatively high dose (10 <italic>μ</italic>M) of arsenite, intracellular ROS levels were increased to 2.8-fold of Veh in control cells and 1.8-fold of Veh in As-TM cells, respectively (Figures <xref ref-type="fig" rid="fig2">2(a)</xref> and <xref ref-type="fig" rid="fig2">2(b)</xref>). The increase in ROS levels in As-TM cells was not as much as the control (<italic>p</italic> < 0.05) (Figures <xref ref-type="fig" rid="fig2">2(a)</xref> and <xref ref-type="fig" rid="fig2">2(b)</xref>). NRF2 and p62 were increased in both mRNA levels (<xref ref-type="fig" rid="fig2">Figure 2(c)</xref>) and protein levels (Figures <xref ref-type="fig" rid="fig1">1(e)</xref> and <xref ref-type="fig" rid="fig2">2(d)</xref>) in As-TM cells compared with the control (<italic>p</italic> < 0.05). This is consistent with our previous finding that NRF2 is activated in As-TM cells [<xref rid="B21" ref-type="bibr">21</xref>]. These results also indicate that adaptive antioxidative response instead of oxidative stress occurs in chronic arsenite-exposed cells.</p><p>Amplified p62-NRF2 autoregulatory loop has been observed in several arsenic-exposed cells [<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B25" ref-type="bibr">25</xref>]. However, the role of this loop in arsenic carcinogenesis is not fully defined. Thus, we silenced <italic>p62</italic> and <italic>NRF2</italic>, respectively, to determine whether suppression of this loop affects arsenic-induced malignant transformation. Successful knockdown of <italic>p62</italic> was verified at mRNA and protein levels (Figures <xref ref-type="fig" rid="fig3">3(a)</xref> and <xref ref-type="fig" rid="fig3">3(b)</xref>). The protein expression of NRF2 in total cell lysis, as well as downstream genes of <italic>NRF2</italic>, <italic>AKR1C1</italic>, and <italic>NQO</italic>1, was significantly suppressed by <italic>p62</italic> silencing in arsenite-exposed cells (Figures <xref ref-type="fig" rid="fig3">3(b)</xref> and <xref ref-type="fig" rid="fig3">3(c)</xref>). Effective <italic>NRF2</italic> silencing was verified by mRNA levels of itself and its downstream genes (<xref ref-type="fig" rid="fig3">Figure 3(d)</xref>). Silencing <italic>NRF2</italic> abolished arsenite-induced upregulation of p62 in both mRNA and protein levels (<xref ref-type="fig" rid="fig3">Figure 3(e)</xref>). Moreover, when cells with silenced <italic>NRF2</italic> or <italic>p62</italic> were exposed to arsenite for 30 weeks, the invasion capacity (<xref ref-type="fig" rid="fig3">Figure 3(f)</xref>) and colony-forming ability (<xref ref-type="fig" rid="fig3">Figure 3(g)</xref>) were significantly decreased compared with scramble.</p></sec><sec id="sec2.3"><title>2.3. Arsenite Exposure Blocks Autophagic Flux by Interfering Autophagosome-Lysosome Fusion</title><p>Amplified p62-NRF2 autoregulatory loop can be attributed to impaired autophagy flux [<xref rid="B22" ref-type="bibr">22</xref>]. To further investigate the specific alteration of autophagy in response to arsenite, HaCaT cells were treated with arsenite at the concentrations of 100 nM or 200 nM for 6 h. Consistent with the results in As-TM cells, accumulation of autolysosomes in arsenite-treated cells was observed (Figures <xref ref-type="fig" rid="fig4">4(a)</xref> and <xref ref-type="fig" rid="fig4">4(b)</xref>). Though arsenite led to a concentration-dependent increase in LC3II/I and p62 protein levels within 200 nM at 6 h, 500 nM arsenite did not show such an effect (<xref ref-type="fig" rid="fig4">Figure 4(c)</xref>). In the time-course analysis, protein levels of LC3II/I and p62 were significantly increased in cells treated with 100 nM of arsenite after 3 h (<xref ref-type="fig" rid="fig4">Figure 4(d)</xref>). Accumulation of p62 is an indicator for defects in autophagic degradation [<xref rid="B29" ref-type="bibr">29</xref>, <xref rid="B30" ref-type="bibr">30</xref>]. To assess how arsenite interferes with autophagic flux, lysosomal protease inhibitor chloroquine (CQ) was used. The protein levels of p62 were not significantly changed in cells treated with CQ plus arsenite compared to cells treated with CQ alone (<xref ref-type="fig" rid="fig4">Figure 4(e)</xref>). These results indicate that the accumulation of p62 induced by arsenite is due to the blockage of autophagosome degradation.</p><p>We next compared the location of a tandem mouse red/green fluorescent protein- (mRFP-) GFP-LC3 signals to the lysosome, to determine whether arsenite impaired autophagosome-lysosome fusion. HaCaT cells transfected with mRFP-GFP-LC3 showed yellow/orange puncta due to CQ treatment (Figures <xref ref-type="fig" rid="fig4">4(f)</xref> and <xref ref-type="fig" rid="fig4">4(g)</xref>), which is known to inhibit autophagosome-lysosome fusion [<xref rid="B31" ref-type="bibr">31</xref>]. We found similar results in cells exposed to arsenite for 4 h with varying degrees of yellow/orange convergence (Figures <xref ref-type="fig" rid="fig4">4(f)</xref> and <xref ref-type="fig" rid="fig4">4(g)</xref>), indicating that arsenite impairs autophagic flux by preventing the fusion of autophagosomes and lysosomes. Lysosome-associated membrane protein (LAMP) 1 and LAMP2 are major protein components of the lysosomal membrane, which mediate a number of essential functions of this compartment [<xref rid="B32" ref-type="bibr">32</xref>]. A concentration (100 nM to 500 nM at 6 h)- and time (100nM within 12 h)- dependent reduction in protein levels of LAMP1 and LAMP2 in response to arsenite was observed (Figures <xref ref-type="fig" rid="fig4">4(g)</xref> and <xref ref-type="fig" rid="fig4">4(h)</xref>, respectively).</p></sec><sec id="sec2.4"><title>2.4. P62 Overexpression Induced by Environmentally Relevant Dose of Arsenite Is Attributed to Enhanced Transcription and Reduced Protein Degradation</title><p>Arsenite has been shown to activate NRF2, which transcriptionally regulates p62 expression. No alteration was found in NRF2 protein expression in total cell lysis at 6 h with 100 nM of arsenite exposure, while p62 protein levels were increased (<xref ref-type="fig" rid="fig5">Figure 5(a)</xref>). Analysis of <italic>NRF2</italic> downstream genes showed that mRNA levels of <italic>p62</italic> were significantly increased at 6 h, but mRNA levels of NQO1 and GCLC were upregulated at 12 h (<xref ref-type="fig" rid="fig5">Figure 5(b)</xref>). <italic>NRF2</italic> silencing suppressed arsenite-induced p62 overexpression (<xref ref-type="fig" rid="fig5">Figure 5(a)</xref>). As expected, arsenite, even at such a low nontoxic level, induces NRF2 activation which contributes to increased p62 protein levels. Further, we assessed the effect of arsenite on p62 turnover. As shown in <xref ref-type="fig" rid="fig5">Figure 5(c)</xref>, inhibition of protein synthesis by cycloheximide (CHX) decreased p62 protein levels. Under such a condition, arsenite treatment significantly attenuated decrease of p62 protein levels (<xref ref-type="fig" rid="fig5">Figure 5(c)</xref>), indicating that the inhibition of protein degradation contributes to p62 accumulation by arsenite.</p></sec></sec><sec id="sec3"><title>3. Materials and Methods</title><sec id="sec3.1"><title>3.1. Cell Culture and Arsenite Exposure</title><p>HaCaT human keratinocytes (N.E. Fusening, German Cancer Research Center, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific, Beijing, China) supplemented with 10% fetal bovine serum (FBS) (Biological Industries (BI), Hazafon, Israel) and 1% penicillin-streptomycin solution (BI) at 37°C in a humidified 5% CO<sub>2</sub> atmosphere. Cells at 80% confluence were passaged according to 1 : 5 proportion. The culture medium was refreshed every 2 days. For chronic arsenite exposure, HaCaT cells were maintained continuously in a medium containing 100 nM of sodium arsenite (NaAsO<sub>2</sub>) (Sigma-Aldrich, St. Louis, USA) for 30 weeks. Passage-matched nontreated cells were used as the control. This arsenite concentration is comparable to the blood arsenite level of chronic arsenicosis patients [<xref rid="B21" ref-type="bibr">21</xref>, <xref rid="B33" ref-type="bibr">33</xref>].</p></sec><sec id="sec3.2"><title>3.2. Lentiviral-Based shRNA Transduction</title><p>Transduction of HaCaT cells with lentiviral-based shRNAs targeting <italic>NRF2</italic> (SHVRS-NM 010902, Sigma-Aldrich), <italic>p62</italic> (GeneChem, Shanghai, China), or scrambled nontarget negative control was performed as described previously [<xref rid="B34" ref-type="bibr">34</xref>, <xref rid="B35" ref-type="bibr">35</xref>]. The selection media for HaCaT cells contained 1 <italic>μ</italic>g/mL of puromycin (Thermo Fisher Scientific).</p></sec><sec id="sec3.3"><title>3.3. Kinetics of Cell Invasion</title><p>A cell invasion test was performed on the RTCA xCELLigence system (ACEA Biosciences Inc., CA, USA) equipped with a CIM-plate 16. The plate is composed of upper and lower chambers separated by a microporous metallic membrane with a thin layer of Matrigel basement membrane. In each well, 4 × 10<sup>4</sup> cells were added in the upper chamber. The xCELLigence system based on electrical impedance measurement allows for the dynamic monitoring [<xref rid="B36" ref-type="bibr">36</xref>]. Electronic signals at the lower chamber as invasion cell indexes were monitored every min for up to 50 h. The cell index was calculated as follows: (impedance at time point <italic>n</italic>‐impedance in the absence of cells)/nominal impedance value [<xref rid="B36" ref-type="bibr">36</xref>].</p></sec><sec id="sec3.4"><title>3.4. Colony Formation in Soft Agar</title><p>We used a colony formation assay to assess anchorage-independent growth. 2 mL of 0.5% agar (Sigma-Aldrich) in complete growth media was used to cover the bottom of each 35 mm dish. 1.25 × 10<sup>4</sup> cells suspended with 1 mL of 0.33% agar in complete growth media were overlaid onto base agar. Cells in agar medium were cultured in a CO<sub>2</sub> incubator. The colonies were identified with iodonitrotetrazolium chloride (INT) (Sigma-Aldrich) staining after a 4-week incubation.</p></sec><sec id="sec3.5"><title>3.5. Transmission Electron Microscopy</title><p>Cell pellets were fixed in 2.5% glutaraldehyde solution (Sigma-Aldrich) in 0.1 M phosphate buffer for 24 h at 4°C, dehydrated using a gradient series of ethanol, and infiltrated with Epon 812 (Structure Probe, Inc., West Chester, USA). Finally, the specimens were cut into 1 <italic>μ</italic>m sections with LKB-V ultramicrotome (Bromma, Stockholm, Sweden) and stained with uranyl acetate and lead citrate. Sections were examined using a transmission electron microscope (Hitachi, Tokyo, Japan).</p></sec><sec id="sec3.6"><title>3.6. Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR)</title><p>RT-qPCR was conducted as previously described [<xref rid="B37" ref-type="bibr">37</xref>]. Total RNA was isolated using a TRIzol reagent (Thermo Fisher Scientific) and reverse transcribed to cDNA with a Prime Script RT reagent Kit (TaKaRa, Dalian, China). A SYBR Premix Ex Taq Kit (TaKaRa) and QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, USA) were used to assess cDNA amplifications. Data were analyzed using the delta-delta cycle time (CT) method. All primers were designed with Primer-BLAST online (<ext-link ext-link-type="uri" xlink:href="https://www.ncbi.nlm.nih.gov/tools/primer-blast">https://www.ncbi.nlm.nih.gov/tools/primer-blast</ext-link>) and obtained from Sigma-Aldrich. Primer sequences are provided in supplemental material <xref ref-type="supplementary-material" rid="supplementary-material-1"></xref>. The average CT value of <italic>β</italic>-actin and GAPDH was used as the value for internal control. The assays were conducted in triplicate for each sample, and three experiments from separately generated samples were performed.</p></sec><sec id="sec3.7"><title>3.7. Western Blot</title><p>Cells were incubated with cell lysis buffer (Cell Signaling, Danvers, USA) containing inhibitor cocktail of protease and phosphatase (Sigma-Aldrich) on ice for 30 min. The protein sample (25-40 <italic>μ</italic>g) was run on 6%, 10%, or 15% Tris-glycine gels, transferred to polyvinylidene fluoride (PVDF) membrane, blocked in 5% nonfat dry milk at room temperature (RT) for 2 h, and incubated with the primary antibody at 4°C overnight and secondary antibody at RT for 1 h. Membranes were developed with electrochemiluminescence (Tanon, Shanghai, China) and subsequently autoradiographied (Tanon). Quantification of the results adjusted to internal reference protein (<italic>β</italic>-actin) was performed by ImageJ (Standard Edition, Bethesda, USA). Primary antibodies for phospho-mTOR (#s6448, 1 : 1000), mTOR (#2983P, 1 : 1000), phospho-P70S6K (#9234, 1 : 1000), RAPTOR (#2280, 1 : 1500), P70S6K (#2708, 1 : 1000), and LAMP2 (#49067, 1 : 1000) were from Cell Signaling Technology. Primary antibodies for LC3 (#12135-1-AP, 1 : 1000), p62/SQSTM1 (#18420-1-AP, 1 : 10000), LAMP1 (#21997-1-AP, 1 : 1000), and BECN1 (#11306-1-AP, 1 : 1000) were from Proteintech Group (Wuhan, China). Primary antibodies for NRF2 (#SC13032, 1 : 500) and <italic>β</italic>-actin (#SC1616, 1 : 5000) were from Santa Cruz Biotechnology (Santa Cruz, USA). Secondary antibodies were from Thermo Fisher Scientific.</p></sec><sec id="sec3.8"><title>3.8. Intracellular ROS Measurement</title><p>ROS levels were measured by flow cytometry using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) (Thermo Fisher Scientific). Briefly, cells in each group were washed with PBS and incubated with 5 <italic>μ</italic>M of CM-H2DCFDA at RM in dark for 30 min. The fluorescence of dichlorofluorescein was measured using Canto II flow cytometry (Becton Dickinson, San Jose, USA) with an excitation wavelength of 488 nm and emission wavelength of 525 nm.</p></sec><sec id="sec3.9"><title>3.9. Autophagosome-Lysosome Fusion Detection</title><p>HaCaT cells were incubated with HBLV-mRFP-GFP-LC3 adenovirus (Hanhbio, Shanghai, China) for 24 h and screened with puromycin. The transduced cells were cultured in glass bottom cell culture dishes (NEST, Wuxi, China) and treated with 100 nM of sodium arsenite for 4 h or 30 <italic>μ</italic>M of CQ for 6 h. Pictures were acquired with AI<sup>+</sup> confocal microscopy (Nikon, Tokyo, Japan).</p></sec><sec id="sec3.10"><title>3.10. Statistical Analysis</title><p>All statistical analyses were performed by using GraphPad Prism 5 software (La Jolla, USA). For comparison between two groups, a <italic>t</italic>-test was performed. For comparison among multiple groups, one-way ANOVA with Tukey's multiple comparison test or two-way ANOVA with the Bonferroni post hoc test was performed. <italic>p</italic> < 0.05 was considered significant. Data were expressed as mean ± standard deviation (SD). Each experiment was independently repeated for at least three times.</p></sec></sec><sec id="sec4"><title>4. Discussion</title><p>Environmental arsenic exposure has been known as a risk factor for skin cancer according to a number of epidemiological studies [<xref rid="B13" ref-type="bibr">13</xref>, <xref rid="B38" ref-type="bibr">38</xref>, <xref rid="B39" ref-type="bibr">39</xref>]. So, it is important to elucidate the intracellular target in response to arsenic at low, environmentally relevant doses. Arsenic is well known to increase intracellular ROS levels when the exposure dose is high [<xref rid="B40" ref-type="bibr">40</xref>]. However, at a relatively low dose, arsenic may not significantly increase ROS levels. When human fibroblast cells were exposed to 500 nM of arsenite for 24 h, ROS levels significantly decreased compared with control due to induction of stress-responsive genes [<xref rid="B41" ref-type="bibr">41</xref>]. In the present study, treatment with 100 nM of arsenite did not significantly change ROS levels within 24 h (data not shown), but decreased ROS levels in the long term with enhanced p62-NRF2 loop. Similarly, acute exposure to arsenite (2 h or 4 h) below 10 <italic>μ</italic>M did not alter intracellular ROS amount in various cell lines, as detected with the same method in the present study or even with a more sensitive ROS detection method (electron paramagnetic resonance spectroscopy) [<xref rid="B20" ref-type="bibr">20</xref>, <xref rid="B26" ref-type="bibr">26</xref>]. In this scenario, arsenic does not appear to activate NRF2 via excessive generation of ROS, though the subtle ROS changes beyond sensitivity of the detection method cannot be excluded. It has been demonstrated that arsenic does not activate NRF2 in the traditional Keap1-C151-depedent manner [<xref rid="B42" ref-type="bibr">42</xref>], which is common for transient NRF2 activation by sepharophone or tBHQ [<xref rid="B20" ref-type="bibr">20</xref>]. Earlier studies have established a noncanonical pathway for NRF2 activation mediated by p62 in arsenic-exposed cells [<xref rid="B17" ref-type="bibr">17</xref>, <xref rid="B20" ref-type="bibr">20</xref>–<xref rid="B22" ref-type="bibr">22</xref>, <xref rid="B43" ref-type="bibr">43</xref>]. Our data provide further evidence that ROS-independent noncanonical NRF2 activation may be involved in carcinogenesis caused by the environmentally relevant dose of arsenite.</p><p>Clearly, NRF2 protects cells from harmful effects of electrophilic and oxidative stressors [<xref rid="B18" ref-type="bibr">18</xref>]. It is considered that some NRF2 inducers, such as natural small molecules and food antioxidants, can reduce the risk of cancer. This concept is further verified in rodent experiments. <italic>Nrf2</italic>-deficient mice are more susceptible to chemical carcinogens [<xref rid="B44" ref-type="bibr">44</xref>, <xref rid="B45" ref-type="bibr">45</xref>]. Although a transient increase in NRF2 levels is able to achieve protective antioxidant effects, constitutive NRF2 activation is very common in many epithelial cancers. <italic>In vivo</italic> study found an unexpected tumor-promoting role of NRF2 during early stage of skin tumorigenesis that was induced by virus [<xref rid="B46" ref-type="bibr">46</xref>]. NRF2 and its downstream genes were activated in the major precancerous human skin lesion [<xref rid="B46" ref-type="bibr">46</xref>]. Meanwhile, NRF2 overactivation is indicated to promote tumorigenesis in the liver of mice with loss of hepatic autophagy [<xref rid="B47" ref-type="bibr">47</xref>] or diethylnitrosamine administration [<xref rid="B48" ref-type="bibr">48</xref>]. Constitutive NRF2 activation promotes cancer initiation by conferring keratinocyte survival advantage (such as antiapoptosis) in adverse conditions [<xref rid="B38" ref-type="bibr">38</xref>, <xref rid="B49" ref-type="bibr">49</xref>], as well as protumorigenic metabolic reprogramming toward anabolic glucose metabolism via the pentose phosphate pathway [<xref rid="B46" ref-type="bibr">46</xref>, <xref rid="B48" ref-type="bibr">48</xref>]. The drug detoxifying function may sometimes mask the oncogenic role of NRF2 in cases of chemical carcinogenesis. The present study provides a direct proof that inhibiting Nrf2 expression during long-term exposure to a low-level arsenite prevents from malignant transformation. Thus, the role of NRF2 in homeostatic response and in the pathogenesis of cancer is more complex than expected from the basic concepts.</p><p>The oncogenic role of p62 has been reported in several cancers [<xref rid="B12" ref-type="bibr">12</xref>–<xref rid="B14" ref-type="bibr">14</xref>], including in the skin [<xref rid="B15" ref-type="bibr">15</xref>]. Silencing <italic>p62</italic> weakens malignant phenotypes of arsenite-transformed human lung bronchial epithelial BEAS-2B cells and human keratinocyte HaCaT cells, such as proliferation, migration, and clonogenicity [<xref rid="B25" ref-type="bibr">25</xref>, <xref rid="B43" ref-type="bibr">43</xref>]. It is suggested that arsenic induces p62 accumulation due to disrupted autophagy. Once phosphorylated, p62 competes with NRF2 for binding to Keap1 [<xref rid="B19" ref-type="bibr">19</xref>], resulting in stabilization of NRF2 protein and subsequent activation of NRF2 downstream genes. Furthermore, p62 controls autophagy-induced degradation of Keap1 [<xref rid="B50" ref-type="bibr">50</xref>, <xref rid="B51" ref-type="bibr">51</xref>] and is transcriptionally regulated by NRF2, thus forming a positive feedback loop with the NRF2 pathway. In the present study, we observed the augmented p62-NRF2 feedback loop in the arsenite-transformed keratinocyte model. The data suggest that protein degradation contributes to p62 protein accumulation in arsenite-exposed cells. This is consistent with the previous report that increased p62 mRNA level is insufficient for p62 protein aggravation, because the determinant factor of p62 protein level is autophagosomal-lysosomal proteolysis [<xref rid="B52" ref-type="bibr">52</xref>]. Upstream signaling of autophagy was also determined in this study. Activation of the mTORC1 pathway was observed in As-TM cells but not in acute arsenic-exposed cells (data not shown), which may be attributed to p62 accumulation [<xref rid="B52" ref-type="bibr">52</xref>, <xref rid="B53" ref-type="bibr">53</xref>]. Data from acute exposure also suggest that arsenite inhibits autophagy efflux by interfering lysosome dysfunction, such as decreased expression of the key lysosome membrane proteins, LAMP1 and LAMP2 [<xref rid="B54" ref-type="bibr">54</xref>, <xref rid="B55" ref-type="bibr">55</xref>]. The lysosome is a potential target organelle for arsenite toxicity and should be investigated in the future. Though alteration in response to acute arsenic exposure may not fully reflect mechanism underlying chronic exposure, determining the acute cellular response will greatly enhance our understanding of the early stages of arsenic carcinogenesis.</p><p>In summary, we have demonstrated that p62 accumulation resulting from inhibition of autophagic flux forms a positive feedback loop with the master regulator of antioxidative defense, NRF2, in response to the environmentally relevant dose of arsenite. Intervention of this loop may have significance in prophylactic strategy of arsenite-induced skin cancer and probably have implications in other arsenic-related cancers.</p></sec></body><back><ack><title>Acknowledgments</title><p>This work was supported by the National Natural Science Foundation of China (81573187 and 81302391), the Liaoning Revitalization Talents Program (XLYC1807225), and the Key R&D Plan Guidance Project (to Y.X.) from the Department of Science and Technology of Liaoning Province, China.</p></ack><sec sec-type="data-availability"><title>Data Availability</title><p>The data used to support the findings of this study are included within the article.</p></sec><sec sec-type="COI-statement"><title>Conflicts of Interest</title><p>No potential conflicts of interest were disclosed.</p></sec><sec><title>Authors' Contributions</title><p>Xiafang Wu and Ru Sun contributed equally to this work.</p></sec><sec sec-type="supplementary-material" id="supplementary-material-1"><title>Supplementary Materials</title><supplementary-material content-type="local-data" id="supp-1"><label>Supplementary Materials</label><caption><p>Table S1: genes and primers for real-time RT-PCR.</p></caption><media xlink:href="1038932.f1.pdf"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec><ref-list><ref id="B1"><label>1</label><element-citation publication-type="journal"><collab>IARC Working Group on the Evaluation of Carcinogenic Risks to Humans</collab><article-title>Arsenic, metals, fibres, and dusts</article-title><source><italic toggle="yes">IARC Monographs on the Evaluation of Carcinogenic Risks to Humans</italic></source><year>2012</year><volume>100</volume><fpage>11</fpage><lpage>465</lpage><pub-id pub-id-type="pmid">23189751</pub-id></element-citation></ref><ref id="B2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Platanias</surname><given-names>L. C.</given-names></name></person-group><article-title>Biological responses to arsenic compounds</article-title><source><italic toggle="yes">The Journal of Biological Chemistry</italic></source><year>2009</year><volume>284</volume><issue>28</issue><fpage>18583</fpage><lpage>18587</lpage><pub-id pub-id-type="doi">10.1074/jbc.R900003200</pub-id><pub-id pub-id-type="other">2-s2.0-67650514338</pub-id><pub-id pub-id-type="pmid">19363033</pub-id></element-citation></ref><ref id="B3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alain</surname><given-names>G.</given-names></name><name><surname>Tousignant</surname><given-names>J.</given-names></name><name><surname>Rozenfarb</surname><given-names>E.</given-names></name></person-group><article-title>Chronic arsenic toxicity</article-title><source><italic toggle="yes">International Journal of Dermatology</italic></source><year>1993</year><volume>32</volume><issue>12</issue><fpage>899</fpage><lpage>901</lpage><pub-id pub-id-type="doi">10.1111/j.1365-4362.1993.tb01413.x</pub-id><pub-id pub-id-type="other">2-s2.0-0027772330</pub-id><pub-id pub-id-type="pmid">8125698</pub-id></element-citation></ref><ref id="B4"><label>4</label><element-citation publication-type="journal"><collab>IARC Working Group on the Evaluation of Carcinogenic Risks to Humans</collab><article-title>Some drinking-water disinfectants and contaminants, including arsenic. Monographs on chloramine, chloral and chloral hydrate, dichloroacetic acid, trichloroacetic acid and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone</article-title><source><italic toggle="yes">IARC Monographs on the Evaluation of Carcinogenic Risks to Humans</italic></source><year>2004</year><volume>84</volume><fpage>269</fpage><lpage>477</lpage><pub-id pub-id-type="pmid">15645578</pub-id></element-citation></ref><ref id="B5"><label>5</label><element-citation publication-type="book"><collab>National Research Council Subcommittee to Update the Arsenic in Drinking Water R</collab><source><italic toggle="yes">Arsenic in Drinking Water: 2001 Update</italic></source><year>2001</year><publisher-loc>Washington (DC)</publisher-loc><publisher-name>National Academies Press (US) Copyright 2001 by the National Academy of Sciences</publisher-name><comment>All rights reserved</comment></element-citation></ref><ref id="B6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Degenhardt</surname><given-names>K.</given-names></name><name><surname>Mathew</surname><given-names>R.</given-names></name><name><surname>Beaudoin</surname><given-names>B.</given-names></name><etal></etal></person-group><article-title>Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis</article-title><source><italic toggle="yes">Cancer Cell</italic></source><year>2006</year><volume>10</volume><issue>1</issue><fpage>51</fpage><lpage>64</lpage><pub-id pub-id-type="doi">10.1016/j.ccr.2006.06.001</pub-id><pub-id pub-id-type="other">2-s2.0-33745713171</pub-id><pub-id pub-id-type="pmid">16843265</pub-id></element-citation></ref><ref id="B7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>White</surname><given-names>E.</given-names></name><name><surname>Karp</surname><given-names>C.</given-names></name><name><surname>Strohecker</surname><given-names>A. M.</given-names></name><name><surname>Guo</surname><given-names>Y.</given-names></name><name><surname>Mathew</surname><given-names>R.</given-names></name></person-group><article-title>Role of autophagy in suppression of inflammation and cancer</article-title><source><italic toggle="yes">Current Opinion in Cell Biology</italic></source><year>2010</year><volume>22</volume><issue>2</issue><fpage>212</fpage><lpage>217</lpage><pub-id pub-id-type="doi">10.1016/j.ceb.2009.12.008</pub-id><pub-id pub-id-type="other">2-s2.0-77951220669</pub-id><pub-id pub-id-type="pmid">20056400</pub-id></element-citation></ref><ref id="B8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rybstein</surname><given-names>M. D.</given-names></name><name><surname>Bravo-San Pedro</surname><given-names>J. M.</given-names></name><name><surname>Kroemer</surname><given-names>G.</given-names></name><name><surname>Galluzzi</surname><given-names>L.</given-names></name></person-group><article-title>The autophagic network and cancer</article-title><source><italic toggle="yes">Nature Cell Biology</italic></source><year>2018</year><volume>20</volume><issue>3</issue><fpage>243</fpage><lpage>251</lpage><pub-id pub-id-type="doi">10.1038/s41556-018-0042-2</pub-id><pub-id pub-id-type="other">2-s2.0-85042586202</pub-id><pub-id pub-id-type="pmid">29476153</pub-id></element-citation></ref><ref id="B9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Amaravadi</surname><given-names>R. K.</given-names></name><name><surname>Thompson</surname><given-names>C. B.</given-names></name></person-group><article-title>The roles of therapy-induced autophagy and necrosis in cancer treatment</article-title><source><italic toggle="yes">Clinical Cancer Research: An Official Journal of the American Association for Cancer Research</italic></source><year>2007</year><volume>13</volume><issue>24</issue><fpage>7271</fpage><lpage>7279</lpage><pub-id pub-id-type="doi">10.1158/1078-0432.CCR-07-1595</pub-id><pub-id pub-id-type="other">2-s2.0-37549056216</pub-id><pub-id pub-id-type="pmid">18094407</pub-id></element-citation></ref><ref id="B10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rao</surname><given-names>S.</given-names></name><name><surname>Tortola</surname><given-names>L.</given-names></name><name><surname>Perlot</surname><given-names>T.</given-names></name><etal></etal></person-group><article-title>A dual role for autophagy in a murine model of lung cancer</article-title><source><italic toggle="yes">Nature Communications</italic></source><year>2014</year><volume>5</volume><issue>1, article 3056</issue><pub-id pub-id-type="doi">10.1038/ncomms4056</pub-id><pub-id pub-id-type="other">2-s2.0-84892882660</pub-id></element-citation></ref><ref id="B11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rogov</surname><given-names>V.</given-names></name><name><surname>Dotsch</surname><given-names>V.</given-names></name><name><surname>Johansen</surname><given-names>T.</given-names></name><name><surname>Kirkin</surname><given-names>V.</given-names></name></person-group><article-title>Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy</article-title><source><italic toggle="yes">Molecular Cell</italic></source><year>2014</year><volume>53</volume><issue>2</issue><fpage>167</fpage><lpage>178</lpage><pub-id pub-id-type="doi">10.1016/j.molcel.2013.12.014</pub-id><pub-id pub-id-type="other">2-s2.0-84892859905</pub-id><pub-id pub-id-type="pmid">24462201</pub-id></element-citation></ref><ref id="B12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thompson</surname><given-names>H. G. R.</given-names></name><name><surname>Harris</surname><given-names>J. W.</given-names></name><name><surname>Wold</surname><given-names>B. J.</given-names></name><name><surname>Lin</surname><given-names>F.</given-names></name><name><surname>Brody</surname><given-names>J. P.</given-names></name></person-group><article-title>p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells</article-title><source><italic toggle="yes">Oncogene</italic></source><year>2003</year><volume>22</volume><issue>15</issue><fpage>2322</fpage><lpage>2333</lpage><pub-id pub-id-type="doi">10.1038/sj.onc.1206325</pub-id><pub-id pub-id-type="other">2-s2.0-0037994340</pub-id><pub-id pub-id-type="pmid">12700667</pub-id></element-citation></ref><ref id="B13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Inoue</surname><given-names>D.</given-names></name><name><surname>Suzuki</surname><given-names>T.</given-names></name><name><surname>Mitsuishi</surname><given-names>Y.</given-names></name><etal></etal></person-group><article-title>Accumulation of p62/SQSTM1 is associated with poor prognosis in patients with lung adenocarcinoma</article-title><source><italic toggle="yes">Cancer Science</italic></source><year>2012</year><volume>103</volume><issue>4</issue><fpage>760</fpage><lpage>766</lpage><pub-id pub-id-type="doi">10.1111/j.1349-7006.2012.02216.x</pub-id><pub-id pub-id-type="other">2-s2.0-84862811895</pub-id><pub-id pub-id-type="pmid">22320446</pub-id></element-citation></ref><ref id="B14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Umemura</surname><given-names>A.</given-names></name><name><surname>He</surname><given-names>F.</given-names></name><name><surname>Taniguchi</surname><given-names>K.</given-names></name><etal></etal></person-group><article-title>p62, upregulated during preneoplasia, induces hepatocellular carcinogenesis by maintaining survival of stressed HCC-initiating cells</article-title><source><italic toggle="yes">Cancer Cell</italic></source><year>2016</year><volume>29</volume><issue>6</issue><fpage>935</fpage><lpage>948</lpage><pub-id pub-id-type="doi">10.1016/j.ccell.2016.04.006</pub-id><pub-id pub-id-type="other">2-s2.0-84975468197</pub-id><pub-id pub-id-type="pmid">27211490</pub-id></element-citation></ref><ref id="B15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qiang</surname><given-names>L.</given-names></name><name><surname>Zhao</surname><given-names>B.</given-names></name><name><surname>Ming</surname><given-names>M.</given-names></name><etal></etal></person-group><article-title>Regulation of cell proliferation and migration by p62 through stabilization of Twist 1</article-title><source><italic toggle="yes">Proceedings of the National Academy of Sciences of the United States of America</italic></source><year>2014</year><volume>111</volume><issue>25</issue><fpage>9241</fpage><lpage>9246</lpage><pub-id pub-id-type="doi">10.1073/pnas.1322913111</pub-id><pub-id pub-id-type="other">2-s2.0-84903481496</pub-id><pub-id pub-id-type="pmid">24927592</pub-id></element-citation></ref><ref id="B16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Moscat</surname><given-names>J.</given-names></name><name><surname>Diaz-Meco</surname><given-names>M. T.</given-names></name></person-group><article-title>p62 at the crossroads of autophagy, apoptosis, and cancer</article-title><source><italic toggle="yes">Cell</italic></source><year>2009</year><volume>137</volume><issue>6</issue><fpage>1001</fpage><lpage>1004</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.05.023</pub-id><pub-id pub-id-type="other">2-s2.0-66449114033</pub-id><pub-id pub-id-type="pmid">19524504</pub-id></element-citation></ref><ref id="B17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jain</surname><given-names>A.</given-names></name><name><surname>Lamark</surname><given-names>T.</given-names></name><name><surname>Sjøttem</surname><given-names>E.</given-names></name><etal></etal></person-group><article-title><italic>p62/SQSTM1</italic> is a target gene for transcription factor NRF2 and creates a positive feedback loop by inducing antioxidant response element-driven gene transcription</article-title><source><italic toggle="yes">The Journal of Biological Chemistry</italic></source><year>2010</year><volume>285</volume><issue>29</issue><fpage>22576</fpage><lpage>22591</lpage><pub-id pub-id-type="doi">10.1074/jbc.M110.118976</pub-id><pub-id pub-id-type="other">2-s2.0-77954599053</pub-id><pub-id pub-id-type="pmid">20452972</pub-id></element-citation></ref><ref id="B18"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yamamoto</surname><given-names>M.</given-names></name><name><surname>Kensler</surname><given-names>T. W.</given-names></name><name><surname>Motohashi</surname><given-names>H.</given-names></name></person-group><article-title>The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis</article-title><source><italic toggle="yes">Physiological Reviews</italic></source><year>2018</year><volume>98</volume><issue>3</issue><fpage>1169</fpage><lpage>1203</lpage><pub-id pub-id-type="doi">10.1152/physrev.00023.2017</pub-id><pub-id pub-id-type="other">2-s2.0-85046763273</pub-id><pub-id pub-id-type="pmid">29717933</pub-id></element-citation></ref><ref id="B19"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Komatsu</surname><given-names>M.</given-names></name><name><surname>Kurokawa</surname><given-names>H.</given-names></name><name><surname>Waguri</surname><given-names>S.</given-names></name><etal></etal></person-group><article-title>The selective autophagy substrate p62 activates the stress responsive transcription factor Nrf 2 through inactivation of Keap 1</article-title><source><italic toggle="yes">Nature Cell Biology</italic></source><year>2010</year><volume>12</volume><issue>3</issue><fpage>213</fpage><lpage>223</lpage><pub-id pub-id-type="doi">10.1038/ncb2021</pub-id><pub-id pub-id-type="other">2-s2.0-77649265091</pub-id><pub-id pub-id-type="pmid">20173742</pub-id></element-citation></ref><ref id="B20"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lau</surname><given-names>A.</given-names></name><name><surname>Wang</surname><given-names>X. J.</given-names></name><name><surname>Zhao</surname><given-names>F.</given-names></name><etal></etal></person-group><article-title>A noncanonical mechanism of Nrf 2 activation by autophagy deficiency: direct interaction between Keap 1 and p62</article-title><source><italic toggle="yes">Molecular and Cellular Biology</italic></source><year>2010</year><volume>30</volume><issue>13</issue><fpage>3275</fpage><lpage>3285</lpage><pub-id pub-id-type="doi">10.1128/MCB.00248-10</pub-id><pub-id pub-id-type="other">2-s2.0-77953366801</pub-id><pub-id pub-id-type="pmid">20421418</pub-id></element-citation></ref><ref id="B21"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pi</surname><given-names>J.</given-names></name><name><surname>Diwan</surname><given-names>B. A.</given-names></name><name><surname>Sun</surname><given-names>Y.</given-names></name><etal></etal></person-group><article-title>Arsenic-induced malignant transformation of human keratinocytes: involvement of Nrf 2</article-title><source><italic toggle="yes">Free Radical Biology & Medicine</italic></source><year>2008</year><volume>45</volume><issue>5</issue><fpage>651</fpage><lpage>658</lpage><pub-id pub-id-type="doi">10.1016/j.freeradbiomed.2008.05.020</pub-id><pub-id pub-id-type="other">2-s2.0-48449084354</pub-id><pub-id pub-id-type="pmid">18572023</pub-id></element-citation></ref><ref id="B22"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lau</surname><given-names>A.</given-names></name><name><surname>Zheng</surname><given-names>Y.</given-names></name><name><surname>Tao</surname><given-names>S.</given-names></name><etal></etal></person-group><article-title>Arsenic inhibits autophagic flux, activating the Nrf2-Keap1 pathway in a p62-dependent manner</article-title><source><italic toggle="yes">Molecular and Cellular Biology</italic></source><year>2013</year><volume>33</volume><issue>12</issue><fpage>2436</fpage><lpage>2446</lpage><pub-id pub-id-type="doi">10.1128/MCB.01748-12</pub-id><pub-id pub-id-type="other">2-s2.0-84878963658</pub-id><pub-id pub-id-type="pmid">23589329</pub-id></element-citation></ref><ref id="B23"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Qi</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>M.</given-names></name><name><surname>Li</surname><given-names>H.</given-names></name><etal></etal></person-group><article-title>Autophagy inhibition by sustained overproduction of IL6 contributes to arsenic carcinogenesis</article-title><source><italic toggle="yes">Cancer Research</italic></source><year>2014</year><volume>74</volume><issue>14</issue><fpage>3740</fpage><lpage>3752</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-13-3182</pub-id><pub-id pub-id-type="other">2-s2.0-84904268484</pub-id><pub-id pub-id-type="pmid">24830721</pub-id></element-citation></ref><ref id="B24"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liu</surname><given-names>X.</given-names></name><name><surname>Ling</surname><given-names>M.</given-names></name><name><surname>Chen</surname><given-names>C.</given-names></name><etal></etal></person-group><article-title>Impaired autophagic flux and p62-mediated EMT are involved in arsenite-induced transformation of L-02 cells</article-title><source><italic toggle="yes">Toxicology and Applied Pharmacology</italic></source><year>2017</year><volume>334</volume><fpage>75</fpage><lpage>87</lpage><pub-id pub-id-type="doi">10.1016/j.taap.2017.09.004</pub-id><pub-id pub-id-type="other">2-s2.0-85029093414</pub-id><pub-id pub-id-type="pmid">28888487</pub-id></element-citation></ref><ref id="B25"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shah</surname><given-names>P.</given-names></name><name><surname>Trinh</surname><given-names>E.</given-names></name><name><surname>Qiang</surname><given-names>L.</given-names></name><etal></etal></person-group><article-title>Arsenic induces p62 expression to form a positive feedback loop with Nrf2 in human epidermal keratinocytes: implications for preventing arsenic-induced skin cancer</article-title><source><italic toggle="yes">Molecules</italic></source><year>2017</year><volume>22</volume><issue>2</issue><fpage>p. 194</fpage><pub-id pub-id-type="doi">10.3390/molecules22020194</pub-id><pub-id pub-id-type="other">2-s2.0-85014031599</pub-id></element-citation></ref><ref id="B26"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dodson</surname><given-names>M.</given-names></name><name><surname>de la Vega</surname><given-names>M. R.</given-names></name><name><surname>Harder</surname><given-names>B.</given-names></name><etal></etal></person-group><article-title>Low-level arsenic causes proteotoxic stress and not oxidative stress</article-title><source><italic toggle="yes">Toxicology and Applied Pharmacology</italic></source><year>2018</year><volume>341</volume><fpage>106</fpage><lpage>113</lpage><pub-id pub-id-type="doi">10.1016/j.taap.2018.01.014</pub-id><pub-id pub-id-type="other">2-s2.0-85041399858</pub-id><pub-id pub-id-type="pmid">29408041</pub-id></element-citation></ref><ref id="B27"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Leone</surname><given-names>A.</given-names></name><name><surname>Flatow</surname><given-names>U.</given-names></name><name><surname>King</surname><given-names>C. R.</given-names></name><etal></etal></person-group><article-title>Reduced tumor incidence, metastatic potential, and cytokine responsiveness of <italic>nm23</italic>-transfected melanoma cells</article-title><source><italic toggle="yes">Cell</italic></source><year>1991</year><volume>65</volume><issue>1</issue><fpage>25</fpage><lpage>35</lpage><pub-id pub-id-type="doi">10.1016/0092-8674(91)90404-M</pub-id><pub-id pub-id-type="other">2-s2.0-0025753514</pub-id><pub-id pub-id-type="pmid">2013093</pub-id></element-citation></ref><ref id="B28"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Valko</surname><given-names>M.</given-names></name><name><surname>Morris</surname><given-names>H.</given-names></name><name><surname>Cronin</surname><given-names>M. T.</given-names></name></person-group><article-title>Metals, toxicity and oxidative stress</article-title><source><italic toggle="yes">Current Medicinal Chemistry</italic></source><year>2005</year><volume>12</volume><issue>10</issue><fpage>1161</fpage><lpage>1208</lpage><pub-id pub-id-type="doi">10.2174/0929867053764635</pub-id><pub-id pub-id-type="other">2-s2.0-18544371009</pub-id><pub-id pub-id-type="pmid">15892631</pub-id></element-citation></ref><ref id="B29"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bjørkøy</surname><given-names>G.</given-names></name><name><surname>Lamark</surname><given-names>T.</given-names></name><name><surname>Pankiv</surname><given-names>S.</given-names></name><name><surname>Øvervatn</surname><given-names>A.</given-names></name><name><surname>Brech</surname><given-names>A.</given-names></name><name><surname>Johansen</surname><given-names>T.</given-names></name></person-group><article-title>Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1</article-title><source><italic toggle="yes">Methods in Enzymology</italic></source><year>2009</year><volume>452</volume><fpage>181</fpage><lpage>197</lpage><pub-id pub-id-type="doi">10.1016/S0076-6879(08)03612-4</pub-id><pub-id pub-id-type="other">2-s2.0-59249105964</pub-id><pub-id pub-id-type="pmid">19200883</pub-id></element-citation></ref><ref id="B30"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mizushima</surname><given-names>N.</given-names></name><name><surname>Yoshimori</surname><given-names>T.</given-names></name></person-group><article-title>How to interpret LC3 immunoblotting</article-title><source><italic toggle="yes">Autophagy</italic></source><year>2007</year><volume>3</volume><issue>6</issue><fpage>542</fpage><lpage>545</lpage><pub-id pub-id-type="doi">10.4161/auto.4600</pub-id><pub-id pub-id-type="other">2-s2.0-35848967804</pub-id><pub-id pub-id-type="pmid">17611390</pub-id></element-citation></ref><ref id="B31"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shintani</surname><given-names>T.</given-names></name><name><surname>Klionsky</surname><given-names>D. J.</given-names></name></person-group><article-title>Autophagy in health and disease: a double-edged sword</article-title><source><italic toggle="yes">Science</italic></source><year>2004</year><volume>306</volume><issue>5698</issue><fpage>990</fpage><lpage>995</lpage><pub-id pub-id-type="doi">10.1126/science.1099993</pub-id><pub-id pub-id-type="other">2-s2.0-8344242220</pub-id><pub-id pub-id-type="pmid">15528435</pub-id></element-citation></ref><ref id="B32"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pajares</surname><given-names>M.</given-names></name><name><surname>Rojo</surname><given-names>A. I.</given-names></name><name><surname>Arias</surname><given-names>E.</given-names></name><name><surname>Diaz-Carretero</surname><given-names>A.</given-names></name><name><surname>Cuervo</surname><given-names>A. M.</given-names></name><name><surname>Cuadrado</surname><given-names>A.</given-names></name></person-group><article-title>Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A</article-title><source><italic toggle="yes">Autophagy</italic></source><year>2018</year><volume>14</volume><issue>8</issue><fpage>1310</fpage><lpage>1322</lpage><pub-id pub-id-type="doi">10.1080/15548627.2018.1474992</pub-id><pub-id pub-id-type="other">2-s2.0-85050949042</pub-id><pub-id pub-id-type="pmid">29950142</pub-id></element-citation></ref><ref id="B33"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pi</surname><given-names>J.</given-names></name><name><surname>He</surname><given-names>Y.</given-names></name><name><surname>Bortner</surname><given-names>C.</given-names></name><etal></etal></person-group><article-title>Low level, long-term inorganic arsenite exposure causes generalized resistance to apoptosis in cultured human keratinocytes: potential role in skin co-carcinogenesis</article-title><source><italic toggle="yes">International Journal of Cancer</italic></source><year>2005</year><volume>116</volume><issue>1</issue><fpage>20</fpage><lpage>26</lpage><pub-id pub-id-type="doi">10.1002/ijc.20990</pub-id><pub-id pub-id-type="other">2-s2.0-20444450161</pub-id><pub-id pub-id-type="pmid">15756686</pub-id></element-citation></ref><ref id="B34"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>R.</given-names></name><name><surname>Hou</surname><given-names>Y.</given-names></name><name><surname>Zhang</surname><given-names>Q.</given-names></name><etal></etal></person-group><article-title>Cross-regulations among NRFs and KEAP1 and effects of their silencing on arsenic-induced antioxidant response and cytotoxicity in human keratinocytes</article-title><source><italic toggle="yes">Environmental Health Perspectives</italic></source><year>2012</year><volume>120</volume><issue>4</issue><fpage>583</fpage><lpage>589</lpage><pub-id pub-id-type="doi">10.1289/ehp.1104580</pub-id><pub-id pub-id-type="other">2-s2.0-84859395513</pub-id><pub-id pub-id-type="pmid">22476201</pub-id></element-citation></ref><ref id="B35"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>R.</given-names></name><name><surname>Yang</surname><given-names>B.</given-names></name><name><surname>Wang</surname><given-names>L.</given-names></name><etal></etal></person-group><article-title>Curcumin protects human keratinocytes against inorganic arsenite-induced acute cytotoxicity through an NRF2-dependent mechanism</article-title><source><italic toggle="yes">Oxidative Medicine and Cellular Longevity</italic></source><year>2013</year><volume>2013</volume><fpage>11</fpage><pub-id pub-id-type="publisher-id">412576</pub-id><pub-id pub-id-type="doi">10.1155/2013/412576</pub-id><pub-id pub-id-type="other">2-s2.0-84878343958</pub-id><pub-id pub-id-type="pmid">23710286</pub-id></element-citation></ref><ref id="B36"><label>36</label><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Ke</surname><given-names>N.</given-names></name><name><surname>Wang</surname><given-names>X.</given-names></name><name><surname>Xu</surname><given-names>X.</given-names></name><name><surname>Abassi</surname><given-names>Y. A.</given-names></name></person-group><person-group person-group-type="editor"><name><surname>Stoddart</surname><given-names>M.</given-names></name></person-group><article-title>The xCELLigence system for real-time and label-free monitoring of cell viability</article-title><source><italic toggle="yes">Mammalian Cell Viability</italic></source><year>2011</year><volume>740</volume><publisher-name>Humana Press</publisher-name><fpage>33</fpage><lpage>43</lpage><series>Methods in Molecular Biology</series><pub-id pub-id-type="doi">10.1007/978-1-61779-108-6_6</pub-id><pub-id pub-id-type="other">2-s2.0-79960133978</pub-id></element-citation></ref><ref id="B37"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Xu</surname><given-names>Y.</given-names></name><name><surname>Tokar</surname><given-names>E. J.</given-names></name><name><surname>Sun</surname><given-names>Y.</given-names></name><name><surname>Waalkes</surname><given-names>M. P.</given-names></name></person-group><article-title>Arsenic-transformed malignant prostate epithelia can convert noncontiguous normal stem cells into an oncogenic phenotype</article-title><source><italic toggle="yes">Environmental Health Perspectives</italic></source><year>2012</year><volume>120</volume><issue>6</issue><fpage>865</fpage><lpage>871</lpage><pub-id pub-id-type="doi">10.1289/ehp.1204987</pub-id><pub-id pub-id-type="other">2-s2.0-84862001935</pub-id><pub-id pub-id-type="pmid">22472196</pub-id></element-citation></ref><ref id="B38"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ghosh</surname><given-names>P.</given-names></name><name><surname>Banerjee</surname><given-names>M.</given-names></name><name><surname>de Chaudhuri</surname><given-names>S.</given-names></name><etal></etal></person-group><article-title>Comparison of health effects between individuals with and without skin lesions in the population exposed to arsenic through drinking water in West Bengal, India</article-title><source><italic toggle="yes">Journal of Exposure Science & Environmental Epidemiology</italic></source><year>2007</year><volume>17</volume><issue>3</issue><fpage>215</fpage><lpage>223</lpage><pub-id pub-id-type="doi">10.1038/sj.jes.7500510</pub-id><pub-id pub-id-type="other">2-s2.0-34249039336</pub-id><pub-id pub-id-type="pmid">16835595</pub-id></element-citation></ref><ref id="B39"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tseng</surname><given-names>W. P.</given-names></name><name><surname>Chu</surname><given-names>H. M.</given-names></name><name><surname>How</surname><given-names>S. W.</given-names></name><name><surname>Fong</surname><given-names>J. M.</given-names></name><name><surname>Lin</surname><given-names>C. S.</given-names></name><name><surname>Yeh</surname><given-names>S.</given-names></name></person-group><article-title>Prevalence of skin cancer in an endemic area of chronic arsenicism in Taiwan</article-title><source><italic toggle="yes">JNCI: Journal of the National Cancer Institute</italic></source><year>1968</year><volume>40</volume><issue>3</issue><fpage>453</fpage><lpage>463</lpage><pub-id pub-id-type="doi">10.1093/jnci/40.3.453</pub-id><pub-id pub-id-type="other">2-s2.0-0014265743</pub-id><pub-id pub-id-type="pmid">5644201</pub-id></element-citation></ref><ref id="B40"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Edwards</surname><given-names>D. H.</given-names></name><name><surname>Li</surname><given-names>Y.</given-names></name><name><surname>Ellinsworth</surname><given-names>D. C.</given-names></name><name><surname>Griffith</surname><given-names>T. M.</given-names></name></person-group><article-title>The effect of inorganic arsenic on endothelium-dependent relaxation: role of NADPH oxidase and hydrogen peroxide</article-title><source><italic toggle="yes">Toxicology</italic></source><year>2013</year><volume>306</volume><fpage>50</fpage><lpage>58</lpage><pub-id pub-id-type="doi">10.1016/j.tox.2013.01.019</pub-id><pub-id pub-id-type="other">2-s2.0-84874557671</pub-id><pub-id pub-id-type="pmid">23384446</pub-id></element-citation></ref><ref id="B41"><label>41</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snow</surname><given-names>E. T.</given-names></name><name><surname>Sykora</surname><given-names>P.</given-names></name><name><surname>Durham</surname><given-names>T.</given-names></name><name><surname>Klein</surname><given-names>C.</given-names></name></person-group><article-title>Arsenic, mode of action at biologically plausible low doses: what are the implications for low dose cancer risk?</article-title><source><italic toggle="yes">Toxicology and Applied Pharmacology</italic></source><year>2005</year><volume>207</volume><issue>2</issue><supplement>Supplement</supplement><fpage>557</fpage><lpage>564</lpage><pub-id pub-id-type="doi">10.1016/j.taap.2005.01.048</pub-id><pub-id pub-id-type="other">2-s2.0-24644459501</pub-id><pub-id pub-id-type="pmid">15996700</pub-id></element-citation></ref><ref id="B42"><label>42</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhang</surname><given-names>D. D.</given-names></name><name><surname>Hannink</surname><given-names>M.</given-names></name></person-group><article-title>Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress</article-title><source><italic toggle="yes">Molecular and Cellular Biology</italic></source><year>2003</year><volume>23</volume><issue>22</issue><fpage>8137</fpage><lpage>8151</lpage><pub-id pub-id-type="doi">10.1128/MCB.23.22.8137-8151.2003</pub-id><pub-id pub-id-type="other">2-s2.0-0242580049</pub-id><pub-id pub-id-type="pmid">14585973</pub-id></element-citation></ref><ref id="B43"><label>43</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Son</surname><given-names>Y. O.</given-names></name><name><surname>Pratheeshkumar</surname><given-names>P.</given-names></name><name><surname>Roy</surname><given-names>R. V.</given-names></name><etal></etal></person-group><article-title>Antioncogenic and oncogenic properties of Nrf2 in arsenic-induced carcinogenesis</article-title><source><italic toggle="yes">The Journal of Biological Chemistry</italic></source><year>2015</year><volume>290</volume><issue>45</issue><fpage>27090</fpage><lpage>27100</lpage><pub-id pub-id-type="doi">10.1074/jbc.M115.675371</pub-id><pub-id pub-id-type="other">2-s2.0-84946781660</pub-id><pub-id pub-id-type="pmid">26385919</pub-id></element-citation></ref><ref id="B44"><label>44</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ramos-Gomez</surname><given-names>M.</given-names></name><name><surname>Kwak</surname><given-names>M. K.</given-names></name><name><surname>Dolan</surname><given-names>P. M.</given-names></name><etal></etal></person-group><article-title>Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in <italic>nrf2</italic> transcription factor-deficient mice</article-title><source><italic toggle="yes">Proceedings of the National Academy of Sciences of the United States of America</italic></source><year>2001</year><volume>98</volume><issue>6</issue><fpage>3410</fpage><lpage>3415</lpage><pub-id pub-id-type="doi">10.1073/pnas.051618798</pub-id><pub-id pub-id-type="other">2-s2.0-0035853157</pub-id><pub-id pub-id-type="pmid">11248092</pub-id></element-citation></ref><ref id="B45"><label>45</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ramos-Gomez</surname><given-names>M.</given-names></name><name><surname>Dolan</surname><given-names>P. M.</given-names></name><name><surname>Itoh</surname><given-names>K.</given-names></name><name><surname>Yamamoto</surname><given-names>M.</given-names></name><name><surname>Kensler</surname><given-names>T. W.</given-names></name></person-group><article-title>Interactive effects of <italic>nrf2</italic> genotype and oltipraz on benzo[a]pyrene-DNA adducts and tumor yield in mice</article-title><source><italic toggle="yes">Carcinogenesis</italic></source><year>2003</year><volume>24</volume><issue>3</issue><fpage>461</fpage><lpage>467</lpage><pub-id pub-id-type="doi">10.1093/carcin/24.3.461</pub-id><pub-id pub-id-type="other">2-s2.0-0037356451</pub-id><pub-id pub-id-type="pmid">12663505</pub-id></element-citation></ref><ref id="B46"><label>46</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rolfs</surname><given-names>F.</given-names></name><name><surname>Huber</surname><given-names>M.</given-names></name><name><surname>Kuehne</surname><given-names>A.</given-names></name><etal></etal></person-group><article-title>Nrf2 activation promotes keratinocyte survival during early skin carcinogenesis via metabolic alterations</article-title><source><italic toggle="yes">Cancer Research</italic></source><year>2015</year><volume>75</volume><issue>22</issue><fpage>4817</fpage><lpage>4829</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-15-0614</pub-id><pub-id pub-id-type="other">2-s2.0-84955071911</pub-id><pub-id pub-id-type="pmid">26530903</pub-id></element-citation></ref><ref id="B47"><label>47</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ni</surname><given-names>H. M.</given-names></name><name><surname>Woolbright</surname><given-names>B. L.</given-names></name><name><surname>Williams</surname><given-names>J.</given-names></name><etal></etal></person-group><article-title>Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy</article-title><source><italic toggle="yes">Journal of Hepatology</italic></source><year>2014</year><volume>61</volume><issue>3</issue><fpage>617</fpage><lpage>625</lpage><pub-id pub-id-type="doi">10.1016/j.jhep.2014.04.043</pub-id><pub-id pub-id-type="other">2-s2.0-84906315540</pub-id><pub-id pub-id-type="pmid">24815875</pub-id></element-citation></ref><ref id="B48"><label>48</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ngo</surname><given-names>H. K. C.</given-names></name><name><surname>Kim</surname><given-names>D. H.</given-names></name><name><surname>Cha</surname><given-names>Y. N.</given-names></name><name><surname>Na</surname><given-names>H. K.</given-names></name><name><surname>Surh</surname><given-names>Y. J.</given-names></name></person-group><article-title>Nrf2 mutagenic activation drives hepatocarcinogenesis</article-title><source><italic toggle="yes">Cancer Research</italic></source><year>2017</year><volume>77</volume><issue>18</issue><fpage>4797</fpage><lpage>4808</lpage><pub-id pub-id-type="doi">10.1158/0008-5472.CAN-16-3538</pub-id><pub-id pub-id-type="other">2-s2.0-85031424914</pub-id><pub-id pub-id-type="pmid">28655791</pub-id></element-citation></ref><ref id="B49"><label>49</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fu</surname><given-names>J.</given-names></name><name><surname>Woods</surname><given-names>C. G.</given-names></name><name><surname>Yehuda-Shnaidman</surname><given-names>E.</given-names></name><etal></etal></person-group><article-title>Low-level arsenic impairs glucose-stimulated insulin secretion in pancreatic beta cells: involvement of cellular adaptive response to oxidative stress</article-title><source><italic toggle="yes">Environmental Health Perspectives</italic></source><year>2010</year><volume>118</volume><issue>6</issue><fpage>864</fpage><lpage>870</lpage><pub-id pub-id-type="doi">10.1289/ehp.0901608</pub-id><pub-id pub-id-type="other">2-s2.0-77953255611</pub-id><pub-id pub-id-type="pmid">20100676</pub-id></element-citation></ref><ref id="B50"><label>50</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Copple</surname><given-names>I. M.</given-names></name><name><surname>Lister</surname><given-names>A.</given-names></name><name><surname>Obeng</surname><given-names>A. D.</given-names></name><etal></etal></person-group><article-title>Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway</article-title><source><italic toggle="yes">The Journal of Biological Chemistry</italic></source><year>2010</year><volume>285</volume><issue>22</issue><fpage>16782</fpage><lpage>16788</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.096545</pub-id><pub-id pub-id-type="other">2-s2.0-77952781968</pub-id><pub-id pub-id-type="pmid">20378532</pub-id></element-citation></ref><ref id="B51"><label>51</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taguchi</surname><given-names>K.</given-names></name><name><surname>Fujikawa</surname><given-names>N.</given-names></name><name><surname>Komatsu</surname><given-names>M.</given-names></name><etal></etal></person-group><article-title>Keap1 degradation by autophagy for the maintenance of redox homeostasis</article-title><source><italic toggle="yes">Proceedings of the National Academy of Sciences of the United States of America</italic></source><year>2012</year><volume>109</volume><issue>34</issue><fpage>13561</fpage><lpage>13566</lpage><pub-id pub-id-type="doi">10.1073/pnas.1121572109</pub-id><pub-id pub-id-type="other">2-s2.0-84865287281</pub-id><pub-id pub-id-type="pmid">22872865</pub-id></element-citation></ref><ref id="B52"><label>52</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Komatsu</surname><given-names>M.</given-names></name><name><surname>Kageyama</surname><given-names>S.</given-names></name><name><surname>Ichimura</surname><given-names>Y.</given-names></name></person-group><article-title>p62/SQSTM1/A170: physiology and pathology</article-title><source><italic toggle="yes">Pharmacological Research</italic></source><year>2012</year><volume>66</volume><issue>6</issue><fpage>457</fpage><lpage>462</lpage><pub-id pub-id-type="doi">10.1016/j.phrs.2012.07.004</pub-id><pub-id pub-id-type="other">2-s2.0-84869495080</pub-id><pub-id pub-id-type="pmid">22841931</pub-id></element-citation></ref><ref id="B53"><label>53</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Linares</surname><given-names>J. F.</given-names></name><name><surname>Duran</surname><given-names>A.</given-names></name><name><surname>Reina-Campos</surname><given-names>M.</given-names></name><etal></etal></person-group><article-title>Amino acid activation of mTORC1 by a PB1-domain-driven kinase complex cascade</article-title><source><italic toggle="yes">Cell Reports</italic></source><year>2015</year><volume>12</volume><issue>8</issue><fpage>1339</fpage><lpage>1352</lpage><pub-id pub-id-type="doi">10.1016/j.celrep.2015.07.045</pub-id><pub-id pub-id-type="other">2-s2.0-84939800232</pub-id><pub-id pub-id-type="pmid">26279575</pub-id></element-citation></ref><ref id="B54"><label>54</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eskelinen</surname><given-names>E. L.</given-names></name></person-group><article-title>Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy</article-title><source><italic toggle="yes">Molecular Aspects of Medicine</italic></source><year>2006</year><volume>27</volume><issue>5-6</issue><fpage>495</fpage><lpage>502</lpage><pub-id pub-id-type="doi">10.1016/j.mam.2006.08.005</pub-id><pub-id pub-id-type="other">2-s2.0-33749041268</pub-id><pub-id pub-id-type="pmid">16973206</pub-id></element-citation></ref><ref id="B55"><label>55</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Issa</surname><given-names>A. R.</given-names></name><name><surname>Sun</surname><given-names>J.</given-names></name><name><surname>Petitgas</surname><given-names>C.</given-names></name><etal></etal></person-group><article-title>The lysosomal membrane protein LAMP2A promotes autophagic flux and prevents SNCA-induced Parkinson disease-like symptoms in the <italic>Drosophila</italic> brain</article-title><source><italic toggle="yes">Autophagy</italic></source><year>2018</year><volume>14</volume><issue>11</issue><fpage>1898</fpage><lpage>1910</lpage><pub-id pub-id-type="doi">10.1080/15548627.2018.1491489</pub-id><pub-id pub-id-type="other">2-s2.0-85053553734</pub-id><pub-id pub-id-type="pmid">29989488</pub-id></element-citation></ref></ref-list></back><floats-group><fig id="fig1" orientation="portrait" position="float"><label>Figure 1</label><caption><p>Alterations of autophagy markers and upstream signaling pathways in arsenite-transformed (As-TM) cells. HaCaT cells were treated with 100 nM of sodium arsenite (As) for 30 weeks. Passage-matched nontreated cells were used as the control (Con). (a) Cell invasion analyzed with real-time cell analysis (RTCA) xCELLigence system. The left chart shows the kinetic analysis of cell invasion. The right panel shows the average index reflecting cell invasion capacity at 45 h. (b) Colony formation in soft agar. Representative images of the control (upper left panel), As-TM cells (upper right panel), and quantification of colonies (lower panel) are shown. Scale bar is 100 <italic>μ</italic>m. For quantification, three fields of view were randomly selected from each 35 mm culture dish. The number of clones with a diameter greater than 100 <italic>μ</italic>m was counted. (c) Increase in number of autophagosomes in As-TM cells. Autophagosomes were observed with transmission electron microscopy (TEM). Scale bar is 2 <italic>μ</italic>m (up) and 0.5 <italic>μ</italic>m (down). Arrows indicate autophagosomes. (d) Number of autophagosomes per cell according to TEM. (e) Western blot for autophagy markers, LC3 and p62. Upper: representative image; lower: quantification of protein levels of LC3II/I and p62 determined with Western blot. (f) Western blot for BECN1 and proteins in the mTORC1 pathway, including p-mTOR, mTOR, RAPTOR, p-P70S6K, and P70S6K. Upper: representative image; lower: quantification of p-mTOR/mTOR and p-P70S6K/P70S6K determined with Western blot. <italic>n</italic> = 3 except for colony formation, in which <italic>n</italic> = 6. <sup>∗</sup><italic>p</italic> < 0.05, compared with Con.</p></caption><graphic xlink:href="OMCL2019-1038932.001"></graphic></fig><fig id="fig2" orientation="portrait" position="float"><label>Figure 2</label><caption><p>Long-term exposure to low-level arsenite induced adaptive antioxidative response in HaCaT cells. (a) Representative histogram for intracellular ROS levels detected by flow cytometer. As-TM cells or passage-matched nontreated control (Con) was challenged with 10 <italic>μ</italic>M of sodium arsenite (As) or equal volume of PBS (Veh) for 24 h. (b) Quantification of intracellular ROS levels determined by flow cytometer. (c) mRNA levels of NRF2 and p62 in As-TM and control cells. (d) Western blot of NRF2 in As-TM and control cells. Upper: representative image; lower: quantification of NRF2 protein levels determined with Western blot. <italic>n</italic> = 3. <sup>∗</sup><italic>p</italic> < 0.05, compared with Con compartment. <sup>#</sup><italic>p</italic> < 0.05, compared with Veh compartment.</p></caption><graphic xlink:href="OMCL2019-1038932.002"></graphic></fig><fig id="fig3" orientation="portrait" position="float"><label>Figure 3</label><caption><p>Amplification of p62-NRF2 feedback loop is required for the acquisition of arsenite-induced malignant phenotypes. (a) mRNA levels of <italic>p62</italic> in HaCaT cells infected with lentiviral vector expressing shRNA targeting <italic>p62</italic> (<italic>p62</italic>-KD) or scrambled nontarget negative control (SCR). (b) Protein levels of p62 and NRF2 in <italic>p62</italic>-KD and SCR cells. Left: representative image; right: quantification of protein levels of p62 and NRF2 determined with Western blot. (c) mRNA levels of <italic>NRF2</italic> downstream genes, <italic>AKR1C1</italic>, <italic>GCLC</italic>, and <italic>NQO1</italic>, in <italic>p62</italic>-KD and SCR cells. (d) mRNA levels of <italic>NRF2</italic> and its downstream genes in chronic arsenite-exposed cells with <italic>NRF2</italic> knockdown (<italic>NRF2</italic>-KD). (e) mRNA and protein levels of p62 in <italic>NRF2</italic>-KD cells analyzed with RT-PCR (left) and Western blot (right), respectively. Upper right: representative image for Western blot; lower right: quantification of p62 protein levels determined with Western blot. (f) Invasion capacity determined by xCELLigence RTCA. Cell index at 45 h after seeding was used to assess invasion capacity. (g) Colony formation in soft agar. Representative image (upper) and quantification of the colonies (lower). Scale bar is 100 <italic>μ</italic>m. As (As+): cells were chronically exposed to 100 nM of sodium arsenite for 30 weeks. Con: passage-matched nontreated cells. <italic>n</italic> = 3 except for colony formation assay, in which <italic>n</italic> = 6. <sup>∗</sup><italic>p</italic> < 0.05, compared with control (As-) compartment. <sup>#</sup><italic>p</italic> < 0.05, compared with SCR (<italic>NRF2</italic>-KD- or <italic>p62</italic>-KD-) compartment.</p></caption><graphic xlink:href="OMCL2019-1038932.003"></graphic></fig><fig id="fig4" orientation="portrait" position="float"><label>Figure 4</label><caption><p>Arsenite inhibits autophagosome-lysosome fusion. (a) Autophagosomes observed with TEM in cells nontreated (Con) or treated with 100 nM or 200 nM sodium arsenite for 4 h. Arrows indicate autophagosomes. Scale bar is 2 <italic>μ</italic>m (up) and 0.5 <italic>μ</italic>m (down). (b) Number of autophagosomes per cell according to TEM. (c) Western blot for LC3 and p62 in HaCaT cells treated with 100 nM, 200 nM, or 500 nM arsenite for 6 h. (d) Western blot for LC3 and p62 in HaCaT cells treated with 100 nM arsenite at different time points. (e) Protein levels of p62 detected with Western blot. Arsenite-induced inhibition of autophagic flux was tested with chloroquine (CQ, 30 <italic>μ</italic>M) pretreatment and in the absence (-) or presence (+) of 100 nM arsenite for 6 h. (f) Quantification of orange/yellow LC3 puncta in the cell. HaCaT cells were transfected with a tandem mRFP-GFP-LC3 and then treated with 100 nM arsenite for 4 h or 30 <italic>μ</italic>M CQ for 6 h. The number of puncta in cells was counted using ImageJ software. Average number of orange/yellow puncta per cell from 16 randomly selected cells in each group was shown. (g) Representative image of LC3 fluorescence observed by a confocal microscope. Scale bar is 50 <italic>μ</italic>m. (h) Western blot for LAMP1 and LAMP2 in HaCaT cells treated with 100 nM, 200 nM, or 500 nM arsenite for 6 h. (i) Western blot for LAMP1 and LAMP2 in HaCaT cells exposed to 100 nM arsenite at different time points. For Western blot, upper: representative image; lower: quantification of protein levels determined with Western blot. <italic>n</italic> = 3. <sup>∗</sup><italic>p</italic> < 0.05, compared with Con (or As- and CQ-).</p></caption><graphic xlink:href="OMCL2019-1038932.004"></graphic></fig><fig id="fig5" orientation="portrait" position="float"><label>Figure 5</label><caption><p>Enhanced transcription and decreased protein turnover contribute to accumulation of p62 protein in response to arsenite exposure. (a) Western blot of NRF2 and p62 under basal and arsenite-treated conditions in SCR and <italic>NRF2</italic>-KD cells. As: cells were treated with 100 nM sodium arsenite for 6 h. PC: positive control, cells were treated with 20 <italic>μ</italic>M sodium arsenite for 6 h. (b) mRNA levels of <italic>p62</italic>, <italic>NQO1</italic>, and <italic>GCLC</italic> in HaCaT cells treated with 100 nM sodium arsenite at different time points. (c) Exposure to arsenite inhibited p62 degradation in HaCaT cells. Cells were treated with CHX (10 <italic>μ</italic>g/mL) or CHX+ As (100 nM) at different time points, followed by Western blot analysis. For Western blot, left: representative image; right: quantification of p62 protein levels. <italic>n</italic> = 3. <sup>∗</sup><italic>p</italic> < 0.05, compared with Con compartment. <sup>#</sup><italic>p</italic> < 0.05, compared with SCR compartment or CHX-treated compartment.</p></caption><graphic xlink:href="OMCL2019-1038932.005"></graphic></fig></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/ChloroquineV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000762 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000762 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= ChloroquineV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:6875345
|texte= Enhanced p62-NRF2 Feedback Loop due to Impaired Autophagic Flux Contributes to Arsenic-Induced Malignant Transformation of Human Keratinocytes
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:31781319" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a ChloroquineV1
| This area was generated with Dilib version V0.6.33. |  |