LYP, a novel bestatin derivative, inhibits cell growth and suppresses APN/CD13 activity in human ovarian carcinoma cells more potently than bestatin

Abstract

LYP is a bestatin dimethylaminoethyl ester which inhibits aminopeptidase N (APN/CD13). Our goal in this study was to evaluate LYP as a candidate compound for cancer treatment, beginning by studying its inhibitory effects on tumors and then comparing it to bestatin. Experiments were performed on human ovarian carcinoma (OVCA) ES-2 and SKOV-3 cell lines, which have high and low levels of APN/CD13 respectively. LYP effectively inhibited ES-2 cell growth as estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue dye-exclusion test. LYP significantly suppressed APN/CD13 activity on the surface of ES-2 cells as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The inhibitory effects of LYP were greater than those of bestatin at the same concentrations. In contrast, LYP was a weak inhibitor of SKOV-3 cell growth, suggesting that LYP may inhibit ES-2 cell growth via suppression of APN/CD13. Inhibition of APN/CD13 expression was also demonstrated with immunofluorescent flow cytometry and Western blot analysis. Inhibitory effects of LYP were confirmed by using a mouse model in which LYP delayed the growth of ES-2 xenografts in mice after 2 weeks of LYP injections. Inhibition of APN/CD13 expression was demonstrated in the ES-2 xenografts using Western blot analysis. The inhibitory effects of LYP on the ES-2 xenografts were stronger than those of bestatin. These results suggest that LYP has a powerful inhibitory effect on the growth of OVCA cells and that the mechanism may be via a decrease in the expression of APN/CD13.

Proteomic identification of galectin-3 binding ligands and characterization of galectin-3 proteolytic cleavage in human prostasomes

“…Significantly, the CD13 immunoblots had multiple bands, with the most intense band at ~150 kDa. This is a novel finding because previous laboratories only showed a portion of the Western blot corresponding to the intense ~150 kDa band (Alponti et al ., ; Gao et al ., ). Both intact CD13 and fragments of the protein may be present in prostasomes.…”

Aminopeptidase N (CD13) as a target for cancer chemotherapy

“…Other APN inhibitors Several other compounds inhibiting APN are under preclinical development, including novel synthetic compounds or structure analogs, for example, the bestatin dimethylaminoethyl ester, LYP. ( 54 ) Others, like curcumin, a phenolic natural product, has, in addition to other effects, been described as an irreversible inhibitor of APN. Curcumin is currently being investigated for its effects on cancer patients in several clinical trials.…”

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

“…15 For the in vitro assay, A549 and NCI-H292 cells (3×10 5 ) seeded in six-well plates were treated with increasing concentrations of HSS for 24 h. The carcinoma cells were harvested and cell lysates (30 μg of protein per lane) were fractionated by 10% SDS-PAGE as described below. In the A549 xenograft assay, the tumor tissues were dispersed mechanically with PBS and then the supernatants were collected and the protein concentrations determined using the bicinchoninic acid protein assay kit (Pierce, Rockford, USA).…”