calcimycin and ubenimex

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-alpha), IFN-beta and IFN-gamma increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.

Topics: Adjuvants, Immunologic; Aminopeptidases; Calcimycin; Carcinoma, Renal Cell; CD13 Antigens; Cholera Toxin; Colforsin; Cyclic AMP; Cytokines; Dinoprostone; Dipeptidyl Peptidase 4; Epithelial Cells; Glutamyl Aminopeptidase; Humans; Ionophores; Kidney Neoplasms; Kidney Tubules; Leucine; Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of the unstable epoxide intermediate LTA4 into the proinflammatory substance LTB4 and also exhibits an amidase/peptidase activity toward synthetic substrates. Based on proposed reaction mechanisms for other zinc hydrolases, we have synthesized inhibitors of LTA4 hydrolase and evaluated their effects on the formation of LTB4 from LTA4 using both purified enzyme and intact polymorphonuclear leukocytes. The two most effective inhibitors, an alpha-keto-beta-amino ester (compound IV) and a thioamine (compound VIII), exhibited IC50 values of 1.9 +/- 0.9 and 0.19 +/- 0.12 microM (mean +/- SD, n = 4), respectively. Compounds IV and VIII were also potent inhibitors of LTB4 biosynthesis in ionophore stimulated polymorphonuclear leukocytes with IC50 < 200 nM. At higher concentrations, the biosynthesis of 5-hydroxy-eicosatetraenoic acid was also inhibited with IC50 approximately 10 microM for both substances. In contrast, leukocyte 15-lipoxygenase and platelet LTC4 synthase activity were not inhibited by these substances at the highest concentrations tested, 50 and 10 microM, respectively. Compounds IV and VIII thus exhibit selectivity among enzyme activities in the arachidonic acid cascade. In conclusion, we describe two compounds that are among the most potent and selective inhibitors of LTA4 hydrolase and LTB4 biosynthesis by intact polymorphonuclear leukocytes, described thus far.

Topics: Angiotensin-Converting Enzyme Inhibitors; Calcimycin; Captopril; Enzyme Inhibitors; Epoxide Hydrolases; Humans; Ionophores; Kinetics; Leucine; Leukotriene A4; Leukotriene B4; Neutrophils; Protease Inhibitors