Blockade of Asparagine Endopeptidase Inhibits Cancer Metastasis

Asparagine endopeptidase (AEP), also called legumain, is highly expressed in various solid tumors, promoting cancer cell invasion, migration, and metastasis. It has been proposed to be a prognostic marker and therapeutic target for cancer treatment. However, an effective nonpeptide, small-molecule inhibitor against this protease has not yet been identified. Here we show that a family of xanthine derivatives selectively inhibit AEP and suppress matrix metalloproteinase (MMP) cleavage, leading to the inhibition of cancer metastasis. Through structure–activity relationship (SAR) analysis, we obtained an optimized lead compound (<b>38u</b>) that represses breast cancer invasion and migration. Chronic treatment of nude mice, which had been inoculated with MDA-MB-231 cells, with inhibitor <b>38u</b> via oral administration robustly inhibits breast cancer lung metastasis in a dose-dependent manner, associated with blockade of MMP-2 by AEP. Therefore, our study supports that <b>38u</b> might act as a potent and specific AEP inhibitor useful for cancer treatment

Metabolic Consequences of Chronic Alcohol Abuse in Non-Smokers: A Pilot Study

<div><p>An alcohol use disorder (AUD) is associated with an increased susceptibility to respiratory infection and injury and, upon hospitalization, higher mortality rates. Studies in model systems show effects of alcohol on mitochondrial function, lipid metabolism and antioxidant systems. The present study applied high-resolution metabolomics to test for these changes in bronchoalveolar lavage fluid (BALF) of subjects with an AUD. Smokers were excluded to avoid confounding effects and compliance was verified by cotinine measurements. Statistically significant metabolic features, differentially expressed by control and AUD subjects, were identified by statistical and bioinformatic methods. The results show that fatty acid and acylcarnitine concentrations were increased in AUD subjects, consistent with perturbed mitochondrial and lipid metabolism. Decreased concentrations of methyl-donor compounds suggest altered one-carbon metabolism and oxidative stress. An accumulation of peptides suggests proteolytic activity, which could reflect altered epithelial barrier function. Two metabolites of possible microbial origin suggest subclinical bacterial infection. Furthermore, increased diacetylspermine suggests additional metabolic perturbations, which could contribute to dysregulated alveolar macrophage function and vulnerability to infection. Together, the results show an extended metabolic consequence of AUD in the bronchoalveolar space.</p></div

Differentially expressed features were identified by FDR analysis.

<p>FDR analysis was performed on the features identified in the BAL fluid of 10 alcoholic subjects and 10 controls. <b>A.</b> The Manhattan Plot demonstrates that 93 of the 2688 <i>m/z</i> features exceed the significance threshold (q = 0.05), denoted by the dashed line. <b>B.</b> Hierarchical clustering analysis is depicted in a heat map to demonstrate the separation of the two groups by differential expression patterns of 93 features.</p

Significantly perturbed metabolic pathways in AUD subjects.

<p>Pathway analysis identified linoleate metabolism as the most significantly perturbed pathway in the BALF from AUD subjects.</p

Selected m/z features increased in AUD subjects.

<p>Selected m/z features increased in AUD subjects.</p

PLS-DA score plot of group separation along the two principal components.

<p>The results of the PLS-DA analysis separated the two groups by principal components 1 and 2, which are responsible for 30% of the variation. The q² cross-validation was found to be 80%. N = no AUD diagnosis and “E” represents ethanol consumption, i.e. positive for an AUD diagnosis.</p

Box-and-whisker plots of selected significant features.

<p>The plots compare the mean intensity of each feature between the two groups.</p

Subject Demographics.

<p>P-value:</p><p>^b p = 0.01 (compared to nonsmoker control).</p><p>Subject Demographics.</p

Oxidative stress markers are elevated in the BALF of AUD subjects.

<p><b>A.</b> F8-Isoprostane in BALF was quantitated by an ELISA and expressed relative to the BALF protein concentration or on a per mL basis. <b>B.</b> Cystine was measured in the BALF by HPLC following dansylation and the concentration is expressed in μM or as a percentage of the total pool (cysteine + cysteine). <b>C.</b> After dansylation, cysteine was measured in the BALF by HPLC and the concentration expressed as μM. <b>D.</b> Methionine (nmol/mg BALF protein) was measured by HPLC following derivatization with the AccQ.Fluor reagent. <b>E.</b> S-adenosylmethionine (nmol/mg BALF protein) was measured by HPLC following derivatization with the AccQ.Fluor reagent.</p