{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T15:48:13Z","timestamp":1791388093159,"version":"4.3.3"},"reference-count":40,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100007783","name":"Hanyang University","doi-asserted-by":"publisher","award":["HY-202200000003365"],"award-info":[{"award-number":["HY-202200000003365"]}],"id":[{"id":"10.13039\/501100007783","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003725","name":"National Research Foundation of Korea","doi-asserted-by":"publisher","award":["RS-2023-00302489"],"award-info":[{"award-number":["RS-2023-00302489"]}],"id":[{"id":"10.13039\/501100003725","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of targetable receptors, leading to limited treatment options and a critical need for novel therapeutic strategies. This study aimed to evaluate the potential of azelastine, a clinically approved H1-antihistamine, for drug repositioning against TNBC and to elucidate its underlying HRH1-independent mechanism of action. Cell viability assays (CCK-8) were performed on TNBC cell lines (MDA-MB-231 and BT-549) following treatment with azelastine and its major metabolite, desmethyl azelastine. After observing ambiguous clinical associations between HRH1 expression and patient prognosis, HRH1 dependency was assessed through histamine stimulation and HRH1 knockdown (siRNA). Subsequently, the role of ADP-ribosylation factor 1 (ARF1), found to be overexpressed in TNBC and linked to poor prognosis, was investigated using ARF1 knockdown (siRNA), co-treatment with the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) inhibitor golgicide A (GCA), and co-treatment with the Drp1 inhibitor M-divi 1. Azelastine and desmethyl azelastine potently reduced MDA-MB-231 cell viability in a dose- and time-dependent manner, achieving cell survivals of 61.3 \u00b1 6.1% (30 \u00b5M) and 34.9 \u00b1 3.7% (50 \u00b5M) for azelastine, and 52.4 \u00b1 12.5% (30 \u00b5M) for desmethyl azelastine, respectively, after 72 h, with an IC50 of 35.93 \u00b5M determined for azelastine in MDA-MB-231 cells. Additionally, azelastine significantly reduced the viability of BT-549 cells. Bioinformatic analysis of clinical datasets revealed HRH1 downregulation in tumors and, functionally, neither histamine stimulation nor HRH1 knockdown mediated azelastine cytotoxicity in cell culture. Importantly, ARF1 expression was significantly upregulated in TNBC and associated with poor prognosis. Co-treatment with GCA, preventing ARF1 activation, restored viability to near-control levels, supporting dependence on the GBF1\u2013ARF1 activation axis of azelastine, whereas the Dynamic-related protein 1 (Drp1) inhibitor M-divi 1 not only partially rescued CCK-8-based cell viability but also normalized azelastine-induced loss of MitoTracker\u2122 Red CMXRos signal and partially preserved (4\u2032,6-diamidino-2-phenylindole) DAPI-based cell density, indicating Drp1-dependent mitochondrial dysfunction. Furthermore, azelastine selectively reduced p-ERK phosphorylation in the cell signaling pathway. Azelastine exerts potent anticancer effects in TNBC cells via an HRH1-independent, ARF1-dependent mechanism that attenuates the Extracellular signal-regulated kinase (ERK)\u2013Drp1 axis, and induces Drp1-dependent mitochondrial dysfunction, independent of its canonical HRH1 receptor function. This ARF1-dependent mechanism provides strong scientific rationale for the drug repositioning of azelastine as an effective therapeutic agent for ARF1-driven TNBC.<\/jats:p>","DOI":"10.3390\/ijms262411849","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T16:20:29Z","timestamp":1765210829000},"page":"11849","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Azelastine Inhibits Triple-Negative Breast Cancer Cell Viability via an ARF1-Dependent Mechanism"],"prefix":"10.3390","volume":"26","author":[{"given":"Seon Uk","family":"Park","sequence":"first","affiliation":[{"name":"Graduate School of Biomedical Science and Engineering, Hanyang Biomedical Research Institute, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gi Ung","family":"Jung","sequence":"additional","affiliation":[{"name":"Graduate School of Biomedical Science and Engineering, Hanyang Biomedical Research Institute, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3135-7219","authenticated-orcid":false,"given":"Eun Kyung","family":"Paik","sequence":"additional","affiliation":[{"name":"Department of Biomedical Informatics, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeong-Yeon","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Pathology, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong Charn","family":"Cho","sequence":"additional","affiliation":[{"name":"Hanyang Intercollege, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2599-8941","authenticated-orcid":false,"given":"Hee Kyoung","family":"Chung","sequence":"additional","affiliation":[{"name":"Department of Pathology, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hang Joon","family":"Jo","sequence":"additional","affiliation":[{"name":"Department of Biomedical Informatics, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea"},{"name":"Department of Biomedical Engineering, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea"},{"name":"Department of Physiology, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1051-6495","authenticated-orcid":false,"given":"Sung Jun","family":"Jung","sequence":"additional","affiliation":[{"name":"Graduate School of Biomedical Science and Engineering, Hanyang Biomedical Research Institute, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"},{"name":"Department of Physiology, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1186\/s13058-020-01296-5","article-title":"Triple-negative breast cancer molecular subtyping and treatment progress","volume":"22","author":"Yin","year":"2020","journal-title":"Breast Cancer Res."},{"key":"ref_2","first-page":"47","article-title":"Integrating Immunotherapy into the Treatment Landscape for Patients with Triple-Negative Breast Cancer","volume":"42","author":"Loibl","year":"2022","journal-title":"Am. 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