Open AccessMelphalan‐flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma
Author(s) -
Viktorsson Kristina,
Shah Carl-Henrik,
Juntti Therese,
Hååg Petra,
Zielinska-Chomej Katarzyna,
Sierakowiak Adam,
Holmsten Karin,
Tu Jessica,
Spira Jack,
Kanter Lena,
Lewensohn Rolf,
Ullén Anders
Publication year - 2016
Publication title -
molecular oncology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.332
H-Index - 88
eISSN - 1878-0261
pISSN - 1574-7891
DOI - 10.1016/j.molonc.2015.12.013
Subject(s) - dasatinib , cancer research , apoptosis , doxorubicin , viability assay , gemcitabine , cytotoxicity , cisplatin , mtt assay , biology , chemistry , medicine , cancer , chemotherapy , imatinib , biochemistry , in vitro , myeloid leukemia
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 mel‐flufen‐induced kinome alterations in J82 cells revealed that mel‐flufen alone did not inhibit Src phosphorylation. Accordingly, the Src inhibitor dasatinib sensitized for mel‐flufen 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.