Kristina Viktorsson1, Carl-Henrik Shah2, Therese Juntti3, Petra Hååg3, Katarzyna Zielinska-Chomej3, Adam Sierakowiak3, Karin Holmsten2, Jessica Tu3, Jack Spira4, Lena Kanter3, Rolf Lewensohn2, Anders Ullén2. 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.
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-flufencytotoxicity. 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: Alvin Y Liu; Ricardo Z N Vêncio; Laura S Page; Melissa E Ho; Michelle A Loprieno; Lawrence D True Journal: Cell Tissue Res Date: 2012-03-20 Impact factor: 5.249
Authors: P Surowiak; M Drag; V Materna; S Suchocki; R Grzywa; M Spaczyński; M Dietel; J Oleksyszyn; M Zabel; H Lage Journal: Int J Gynecol Cancer Date: 2006 Sep-Oct Impact factor: 3.437
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: Mol Oncol Date: 2016-01-02 Impact factor: 6.603
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: Mol Oncol Date: 2016-01-02 Impact factor: 6.603
Authors: Malin Wickström; Peter Nygren; Rolf Larsson; Johan Harmenberg; Jakob Lindberg; Per Sjöberg; Markus Jerling; Fredrik Lehmann; Paul Richardson; Kenneth Anderson; Dharminder Chauhan; Joachim Gullbo Journal: Oncotarget Date: 2017-06-08