Rabbit Anti-ANPEP monoclonal antibody, clone TD81-12

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.

Growth of atherosclerotic plaque requires neovascularization (angiogenesis). To elucidate the involvement of angiotensin II (Ang II) in angiogenesis, we performed gene microarray and microRNA (miRNA) polymerase chain reaction array analyses on human coronary artery endothelial cells exposed to moderate concentration of Ang II for 2 and 12 hours. At 12, but not 2, hours, cultures treated with Ang II exhibited shifts in transcriptional activity involving 267 genes (>1.5-fold difference; P < 0.05). Resulting transcriptome was most significantly enriched for genes associated with blood vessel development, angiogenesis, and regulation of proliferation. Majority of upregulated genes implicated in angiogenesis shared a commonality of being either regulators (HES1, IL-18, and CXCR4) or targets (ADM, ANPEP, HES1, KIT, NOTCH4, PGF, and SOX18) of STAT3. In line with these findings, STAT3 inhibition attenuated Ang II-dependent stimulation of tube formation in Matrigel assay. Expression analysis of miRNAs transcripts revealed that the pattern of differential expression for miRNAs was largely consistent with proangiogenic response with a prominent theme of upregulation of miRs targeting PTEN (miR-19b-3p, miR-21-5p, 23b-3p, and 24-3p), many of which are directly or indirectly STAT3 dependent. We conclude that STAT3 signaling may be an intrinsic part of Ang II-mediated proangiogenic response in human endothelial cells.