Melphalan-flufenamide is cytotoxic and potentiates treatment with chemotherapy and the Src inhibitor dasatinib in urothelial carcinoma.

Abstract

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.

• Authors: Kristina Viktorsson, Carl-Henrik Shah, Therese Juntti, Petra Hååg, Katarzyna Zielinska-Chomej, Adam Sierakowiak, Karin Holmsten, Jessica Tu, Jack Spira, Lena Kanter, Rolf Lewensohn, Anders Ullén
• Journal: Molecular oncology (Mol Oncol) Vol. 10 Issue 5 Pg. 719-34 (05 2016) ISSN: 1878-0261 [Electronic] United States
• PMID: 26827254 (Publication Type: Journal Article)
• Copyright: Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Chemical References

• Antineoplastic Agents, Alkylating
• BAK1 protein, human
• Protein Kinase Inhibitors
• bcl-2 Homologous Antagonist-Killer Protein
• bcl-2-Associated X Protein
• melflufen
• Phenylalanine
• src-Family Kinases
• Melphalan
• Dasatinib

Topics

• Antineoplastic Agents, Alkylating (pharmacology)
• Antineoplastic Combined Chemotherapy Protocols (pharmacology)
• Apoptosis (drug effects)
• Cell Line, Tumor
• Dasatinib (pharmacology)
• Humans
• Melphalan (analogs & derivatives, pharmacology)
• Phenylalanine (analogs & derivatives, pharmacology)
• Protein Kinase Inhibitors (pharmacology)
• Urologic Neoplasms (drug therapy, pathology)
• Urothelium (drug effects, pathology)
• bcl-2 Homologous Antagonist-Killer Protein (metabolism)
• bcl-2-Associated X Protein (metabolism)
• src-Family Kinases (antagonists & inhibitors)