{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,18]],"date-time":"2025-12-18T14:01:54Z","timestamp":1766066514553,"version":"3.37.3"},"reference-count":38,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2016,1,2]],"date-time":"2016-01-02T00:00:00Z","timestamp":1451692800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"name":"Swedish Cancer Society","award":["130277","130550"],"award-info":[{"award-number":["130277","130550"]}]},{"name":"Stockholm Cancer Society","award":["141433","144233","131253","131102","111213"],"award-info":[{"award-number":["141433","144233","131253","131102","111213"]}]},{"name":"Swedish National Board of Health and Welfare"},{"DOI":"10.13039\/501100004348","name":"Stockholm County Council","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100004348","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Karolinska Institutet Research Fund"}],"content-domain":{"domain":["febs.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Molecular Oncology"],"published-print":{"date-parts":[[2016,5]]},"abstract":"<jats:p><jats:italic>Background:<\/jats:italic> Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide\u2010based alkylating agent melphalan\u2010flufenamide (mel\u2010flufen) for UC.<\/jats:p><jats:p><jats:italic>Methods:<\/jats:italic> UC cell lines J82, RT4, TCCsup and 5637 were treated with mel\u2010flufen, alone or combined with cisplatin, gemcitabine, dasatinib or bestatin. Cell viability (MTT assay), intracellular drug accumulation (liquid chromatography) apoptosis induction (apoptotic cell nuclei morphology, western blot analysis of PARP\u20101\/caspase\u20109 cleavage and Bak\/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho\u2010Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome.<\/jats:p><jats:p><jats:italic>Results:<\/jats:italic> In J82, RT4, TCCsup and 5637 UC cells, mel\u2010flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel\u2010flufen induced apoptosis seen as activation of Bak\/Bax, cleavage of caspase\u20109\/PARP\u20101 and induction of apoptotic cell nuclei morphology. Combining mel\u2010flufen with cisplatin or gemcitabine in J82\u00a0cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of mel\u2010flufen\u2010induced kinome alterations in J82\u00a0cells revealed that mel\u2010flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for mel\u2010flufen cytotoxicity. Immunohistochemical analysis of the putative mel\u2010flufen biomarker ANPEP demonstrated prominent expression levels in tumours from 82 of 83 cystectomy patients. Significantly longer median overall survival was found in patients with high ANPEP expression (P\u00a0=\u00a00.02).<\/jats:p><jats:p><jats:italic>Conclusion:<\/jats:italic> Mel\u2010flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC.<\/jats:p>","DOI":"10.1016\/j.molonc.2015.12.013","type":"journal-article","created":{"date-parts":[[2016,1,2]],"date-time":"2016-01-02T16:47:03Z","timestamp":1451753223000},"page":"719-734","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Melphalan\u2010flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma"],"prefix":"10.1002","volume":"10","author":[{"given":"Kristina","family":"Viktorsson","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carl-Henrik","family":"Shah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Therese","family":"Juntti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Petra","family":"H\u00e5\u00e5g","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Katarzyna","family":"Zielinska-Chomej","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adam","family":"Sierakowiak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Karin","family":"Holmsten","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jessica","family":"Tu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jack","family":"Spira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lena","family":"Kanter","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rolf","family":"Lewensohn","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anders","family":"Ull\u00e9n","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2016,1,2]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.3389\/fphar.2013.00003"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2008.20.5534"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/annonc\/mdt007"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10637-015-0299-2"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-12-3752"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142735.im0507s60"},{"key":"e_1_2_10_8_1","article-title":"Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012","author":"Ferlay J.","year":"2014","journal-title":"Int. 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