Journal
MOLECULAR ONCOLOGY
Volume 10, Issue 5, Pages 719-734Publisher
WILEY
DOI: 10.1016/j.molonc.2015.12.013
Keywords
Urothelial carcinoma; Melphalan-flufenamide; Aminopeptidase N; Apoptosis; Src; Cisplatin; Gemcitabine; Dasatinib
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Funding
- Swedish Cancer Society [130277, 130550]
- Stockholm Cancer Society [141433, 144233, 131253, 131102, 111213]
- Swedish National Board of Health and Welfare
- Stockholm County Council
- Karolinska Institutet Research Fund
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Background: Chemotherapy options in advanced urothelial carcinoma (UC) remain limited. Here we evaluated the peptide-based alkylating agent melphalan-flufenamide (mel-flufen) for UC. Methods: UC cell lines J82; RT4, TCCsup and 5637 were treated with mel-flufen, 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-1/caspase-9 cleavage and Bak/Bax activation) were evaluated. Kinome alterations were characterized by PathScan array and phospho-Src validated by western blotting. Aminopeptidase N (ANPEP) expression was evaluated in UC clinical specimens in relation to patient outcome. Results: In J82, RT4, TCCsup and 5637 UC cells, mel-flufen amplified the intracellular loading of melphalan in part via aminopeptidase N (ANPEP), resulting in increased cytotoxicity compared to melphalan alone. Mel-flufen induced apoptosis seen as activation of Bak/Bax, cleavage of caspase-9/PARP-1 and induction of apoptotic cell nuclei morphology. Combining mel-flufen with cisplatin or gemcitabine in J82 cells resulted in additive cytotoxic effects and for gemcitabine also increased apoptosis induction. Profiling of melflufen-induced kinome alterations in J82 cells revealed that mel-flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for melflufen cytotoxicity. Immunohistochemical analysis of the putative mel-flufen 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 = 0.02). Conclusion: Mel-flufen alone or in combination with cisplatin, gemcitabine or Src inhibition holds promise as a novel treatment for UC. (C) 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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