Format

Send to

Choose Destination
Mol Oncol. 2016 May;10(5):719-34. doi: 10.1016/j.molonc.2015.12.013. Epub 2016 Jan 2.

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

Author information

1
Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden. Electronic address: kristina.viktorsson@ki.se.
2
Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden; Department of Oncology, Radiumhemmet, Karolinska University Hospital, SE-171 76 Stockholm, Sweden.
3
Karolinska Institutet, Department of Oncology-Pathology, Karolinska Biomics Center, SE-171 76, Stockholm, Sweden.
4
InSpira Medical AB, SE-135 53 Tyresö, Sweden.

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.

KEYWORDS:

Aminopeptidase N; Apoptosis; Cisplatin; Dasatinib; Gemcitabine; Melphalan-flufenamide; Src; Urothelial carcinoma

PMID:
26827254
PMCID:
PMC5423156
DOI:
10.1016/j.molonc.2015.12.013
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Wiley Icon for PubMed Central
Loading ...
Support Center