{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T16:08:02Z","timestamp":1787069282389,"version":"build-2736575974"},"reference-count":45,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2022,12,7]],"date-time":"2022-12-07T00:00:00Z","timestamp":1670371200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001691","name":"JSPS KAKENHI","doi-asserted-by":"publisher","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"JSPS KAKENHI","doi-asserted-by":"publisher","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"name":"TERUMO LIFE SCIENCE FOUNDATION","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"TERUMO LIFE SCIENCE FOUNDATION","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]},{"name":"Futaba Electronics Memorial Foundation","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"Futaba Electronics Memorial Foundation","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]},{"name":"Ogawa Science and Technology Foundation","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"Ogawa Science and Technology Foundation","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]},{"name":"Takeda Science Foundation","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"Takeda Science Foundation","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]},{"name":"All Japan Coffee Association","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"All Japan Coffee Association","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]},{"name":"Smoking Research Foundation","award":["19H03373"],"award-info":[{"award-number":["19H03373"]}]},{"name":"Smoking Research Foundation","award":["21-III4045"],"award-info":[{"award-number":["21-III4045"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Claudin-2 (CLDN2), a component of tight junctions, is abnormally expressed in human lung adenocarcinoma tissue. CLDN2 contributes to chemoresistance in human lung adenocarcinoma-derived A549 cells, and it may be a target for cancer therapy. Here, we found that coffee ingredients, namely caffeine and theobromine, decreased the protein level of CLDN2 in human lung adenocarcinoma-derived A549 cells. In contrast, other components, such as theophylline and chlorogenic acid, had no effect. These results indicate that the 7-methyl group in methylxanthines may play a key role in the reduction in CLDN2 expression. The caffeine-induced reduction in the CLDN2 protein was inhibited by chloroquine, a lysosome inhibitor. In a protein-stability assay using cycloheximide, CLDN2 protein levels decreased faster in caffeine-treated cells than in vehicle-treated cells. These results suggest that caffeine accelerates the lysosomal degradation of CLDN2. The accumulation and cytotoxicity of doxorubicin were dose-dependently increased, which was exaggerated by caffeine but not by theophylline in spheroids. Caffeine decreased nuclear factor-erythroid 2-related factor 2 (Nrf2) levels without affecting hypoxia-inducible factor-1\u03b1 levels. Furthermore, caffeine decreased the expression of Nrf2-targeted genes. The effects of caffeine on CLDN2 expression and anticancer-drug-induced toxicity were also observed in lung adenocarcinoma RERF-LC-MS cells. We suggest that caffeine enhances doxorubicin-induced toxicity in A549 spheroids mediated by the reduction in CLDN2 and Nrf2 expression.<\/jats:p>","DOI":"10.3390\/ijms232415447","type":"journal-article","created":{"date-parts":[[2022,12,7]],"date-time":"2022-12-07T01:47:46Z","timestamp":1670377666000},"page":"15447","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Elevation of Anticancer Drug Toxicity by Caffeine in Spheroid Model of Human Lung Adenocarcinoma A549 Cells Mediated by Reduction in Claudin-2 and Nrf2 Expression"],"prefix":"10.3390","volume":"23","author":[{"given":"Hiroaki","family":"Eguchi","sequence":"first","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Riho","family":"Kimura","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saki","family":"Onuma","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayaka","family":"Ito","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaqing","family":"Yu","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuta","family":"Yoshino","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshiyuki","family":"Matsunaga","sequence":"additional","affiliation":[{"name":"Laboratory of Bioinformatics, Gifu Pharmaceutical University, Gifu 502-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0578-9672","authenticated-orcid":false,"given":"Satoshi","family":"Endo","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2696-0897","authenticated-orcid":false,"given":"Akira","family":"Ikari","sequence":"additional","affiliation":[{"name":"Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/nrclinonc.2016.144","article-title":"Circulating and disseminated tumour cells - mechanisms of immune surveillance and escape","volume":"14","author":"Mohme","year":"2017","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"646","DOI":"10.1016\/j.cell.2011.02.013","article-title":"Hallmarks of cancer: The next generation","volume":"144","author":"Hanahan","year":"2011","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1016\/S0955-0674(99)00016-2","article-title":"Structural and signalling molecules come together at tight junctions","volume":"11","author":"Tsukita","year":"1999","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1539","DOI":"10.1083\/jcb.141.7.1539","article-title":"Claudin-1 and -2: Novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin","volume":"141","author":"Furuse","year":"1998","journal-title":"J. Cell Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1016\/j.febslet.2011.01.028","article-title":"Predicted expansion of the claudin multigene family","volume":"585","author":"Mineta","year":"2011","journal-title":"FEBS Lett."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2435","DOI":"10.1242\/jcs.01235","article-title":"Barriers built on claudins","volume":"117","author":"Turksen","year":"2004","journal-title":"J. Cell Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1110","DOI":"10.1016\/j.bbamcr.2012.04.005","article-title":"Increase in claudin-2 expression by an EGFR\/MEK\/ERK\/c-Fos pathway in lung adenocarcinoma A549 cells","volume":"1823","author":"Ikari","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_8","first-page":"3729","article-title":"Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer","volume":"27","author":"Kinugasa","year":"2007","journal-title":"Anticancer Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"555","DOI":"10.1016\/j.humpath.2005.12.015","article-title":"Claudin-1 and claudin-2 differentiate fetal and embryonal components in human hepatoblastoma","volume":"37","author":"Halasz","year":"2006","journal-title":"Hum. Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1159\/000135351","article-title":"Expression of Cdx2 and claudin-2 in the multistage tissue of gastric carcinogenesis","volume":"73","author":"Xin","year":"2007","journal-title":"Oncology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"4969","DOI":"10.1242\/jcs.00165","article-title":"Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells","volume":"115","author":"Amasheh","year":"2002","journal-title":"J. Cell Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"7234","DOI":"10.1074\/jbc.M114.597492","article-title":"Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation","volume":"290","author":"Nighot","year":"2015","journal-title":"J. Biol. Chem."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2079","DOI":"10.1016\/j.bbamcr.2014.05.017","article-title":"Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells","volume":"1843","author":"Ikari","year":"2014","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3234","DOI":"10.1038\/onc.2011.43","article-title":"Claudin-2 expression increases tumorigenicity of colon cancer cells: Role of epidermal growth factor receptor activation","volume":"30","author":"Dhawan","year":"2011","journal-title":"Oncogene"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"470","DOI":"10.1016\/j.bbamcr.2017.12.005","article-title":"Elevation of sensitivity to anticancer agents of human lung adenocarcinoma A549 cells by knockdown of claudin-2 expression in monolayer and spheroid culture models","volume":"1865","author":"Maruhashi","year":"2018","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Toth, R.K., and Warfel, N.A. (2017). Strange Bedfellows: Nuclear Factor, Erythroid 2-Like 2 (Nrf2) and Hypoxia-Inducible Factor 1 (HIF-1) in Tumor Hypoxia. Antioxidants, 6.","DOI":"10.3390\/antiox6020027"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4578","DOI":"10.3390\/nu7064578","article-title":"Quercetin Decreases Claudin-2 Expression Mediated by Up-Regulation of microRNA miR-16 in Lung Adenocarcinoma A549 Cells","volume":"7","author":"Sonoki","year":"2015","journal-title":"Nutrients"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Eguchi, H., Matsunaga, T., Endo, S., Ichihara, K., and Ikari, A. (2020). Kaempferide Enhances Chemosensitivity of Human Lung Adenocarcinoma A549 Cells Mediated by the Decrease in Phosphorylation of Akt and Claudin-2 Expression. Nutrients, 12.","DOI":"10.3390\/nu12041190"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Sonoki, H., Tanimae, A., Endo, S., Matsunaga, T., Furuta, T., Ichihara, K., and Ikari, A. (2017). Kaempherol and Luteolin Decrease Claudin-2 Expression Mediated by Inhibition of STAT3 in Lung Adenocarcinoma A549 Cells. Nutrients, 9.","DOI":"10.3390\/nu9060597"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1016\/j.fct.2018.10.005","article-title":"Coffee, caffeine, chlorogenic acid, and the purinergic system","volume":"123","author":"Stefanello","year":"2019","journal-title":"Food Chem. Toxicol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Sartini, M., Bragazzi, N.L., Spagnolo, A.M., Schinca, E., Ottria, G., Dupont, C., and Cristina, M.L. (2019). Coffee Consumption and Risk of Colorectal Cancer: A Systematic Review and Meta-Analysis of Prospective Studies. Nutrients, 11.","DOI":"10.3390\/nu11030694"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"149","DOI":"10.4251\/wjgo.v12.i2.149","article-title":"Caffeine and its main targets of colorectal cancer","volume":"12","author":"Cui","year":"2020","journal-title":"World. J. Gastrointest. Oncol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"172538","DOI":"10.1016\/j.ejphar.2019.172538","article-title":"Caffeine-enhanced anti-tumor immune response through decreased expression of PD1 on infiltrated cytotoxic T lymphocytes","volume":"859","author":"Neogi","year":"2019","journal-title":"Eur. J. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"544","DOI":"10.2174\/1389200219666180302154551","article-title":"An insight into the therapeutic potential of major coffee components","volume":"19","author":"Islam","year":"2018","journal-title":"Curr. Drug Metab."},{"key":"ref_25","first-page":"4902","article-title":"Phosphorylation of claudin-2 on serine 208 promotes membrane retention and reduces trafficking to lysosomes","volume":"125","author":"Tietgens","year":"2012","journal-title":"J. Cell Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1002\/jnr.23350","article-title":"Bradykinin regulates the expression of claudin-5 in brain microvascular endothelial cells via calcium-induced calcium release","volume":"92","author":"Zhou","year":"2014","journal-title":"J. Neurosci. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"7156","DOI":"10.1021\/acs.jmedchem.1c00261","article-title":"The Medicinal Chemistry of Caffeine","volume":"64","author":"Faudone","year":"2021","journal-title":"J. Med. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"54","DOI":"10.3389\/fphar.2018.00054","article-title":"The A2B Adenosine Receptor Modulates the Epithelial- Mesenchymal Transition through the Balance of cAMP\/PKA and MAPK\/ERK Pathway Activation in Human Epithelial Lung Cells","volume":"9","author":"Giacomelli","year":"2018","journal-title":"Front. Pharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"129457","DOI":"10.1016\/j.bbagen.2019.129457","article-title":"Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells","volume":"1864","author":"Kitabatake","year":"2020","journal-title":"Biochim. Biophys. Acta Gen. Subj."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Gao, Z.G., and Jacobson, K.A. (2019). A2B Adenosine Receptor and Cancer. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20205139"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1083\/jcb.201303159","article-title":"The inner and outer compartments of mitochondria are sites of distinct cAMP\/PKA signaling dynamics","volume":"202","author":"Lefkimmiatis","year":"2013","journal-title":"J. Cell Biol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1016\/j.jnutbio.2018.02.016","article-title":"Caffeic acid phenethyl ester down-regulates claudin-2 expression at the transcriptional and post-translational levels and enhances chemosensitivity to doxorubicin in lung adenocarcinoma A549 cells","volume":"56","author":"Sonoki","year":"2018","journal-title":"J. Nutr. Biochem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.ceca.2012.12.001","article-title":"AMPK activity is regulated by calcium-mediated protein phosphatase 2A activity","volume":"53","author":"Park","year":"2013","journal-title":"Cell Calcium"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1002\/jcp.22180","article-title":"Caffeine induces matrix metalloproteinase-2 (MMP-2) and MMP-9 down-regulation in human leukemia U937 cells via Ca2+\/ROS-mediated suppression of ERK\/c-fos pathway and activation of p38 MAPK\/c-jun pathway","volume":"224","author":"Liu","year":"2010","journal-title":"J. Cell. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1007\/s12032-019-1323-8","article-title":"Caffeine enhances the anti-tumor effect of 5-fluorouracil via increasing the production of reactive oxygen species in hepatocellular carcinoma","volume":"36","author":"Wang","year":"2019","journal-title":"Med. Oncol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1186\/2162-3619-4-5","article-title":"The effect of caffeine on cisplatin-induced apoptosis of lung cancer cells","volume":"4","author":"Wang","year":"2015","journal-title":"Exp. Hematol. Oncol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Kryczka, J., Kryczka, J., Czarnecka-Chrebelska, K.H., and Brzezianska-Lasota, E. (2021). Molecular Mechanisms of Chemoresistance Induced by Cisplatin in NSCLC Cancer Therapy. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22168885"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Zimta, A.A., Cenariu, D., Irimie, A., Magdo, L., Nabavi, S.M., Atanasov, A.G., and Berindan-Neagoe, I. (2019). The Role of Nrf2 Activity in Cancer Development and Progression. Cancers, 11.","DOI":"10.3390\/cancers11111755"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1111\/j.1365-2443.2010.01473.x","article-title":"Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution","volume":"16","author":"Taguchi","year":"2011","journal-title":"Genes Cells"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Ito, A., Nasako, H., Akizuki, R., Takashina, Y., Eguchi, H., Matsunaga, T., Yoshino, Y., Endo, S., and Ikari, A. (2021). Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22126582"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1111\/1556-4029.12387","article-title":"Fatal caffeine intoxication: A series of eight cases from 1999 to 2009","volume":"59","author":"Banerjee","year":"2014","journal-title":"J. Forensic Sci."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Cappelletti, S., Piacentino, D., Fineschi, V., Frati, P., Cipolloni, L., and Aromatario, M. (2018). Caffeine-Related Deaths: Manner of Deaths and Categories at Risk. Nutrients, 10.","DOI":"10.3390\/nu10050611"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"118642","DOI":"10.1016\/j.bbamcr.2019.118642","article-title":"Claudin-2 binding peptides, VPDSM and DSMKF, down-regulate claudin-2 expression and anticancer resistance in human lung adenocarcinoma A549 cells","volume":"1867","author":"Nasako","year":"2020","journal-title":"Biochim. Biophys. Acta Mol. Cell Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1248\/bpb.b17-00244","article-title":"Chlorpheniramine Increases Paracellular Permeability to Marker Fluorescein Lucifer Yellow Mediated by Internalization of Occludin in Murine Colonic Epithelial Cells","volume":"40","author":"Manabe","year":"2017","journal-title":"Biol. Pharm. Bull."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"769","DOI":"10.1016\/j.bbamcr.2018.03.001","article-title":"Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells","volume":"1865","author":"Akizuki","year":"2018","journal-title":"Biochim. Biophys. Acta Mol. Cell Res."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15447\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:35:23Z","timestamp":1760146523000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15447"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,7]]},"references-count":45,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2022,12]]}},"alternative-id":["ijms232415447"],"URL":"https:\/\/doi.org\/10.3390\/ijms232415447","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,7]]}}}