24F(CIP-13F), A Novel Galloyl Cyclic-imide Derivatives, Inhibits Tumor Growth Via Decrease Of APN/CD13

Backgrounds:Aminopeptidase N (APN/CD13) is an essential peptidase involved in the process of tumor growth and metastasis. Inhibition of APN/CD13 may be an effective strategy for cancer treatment. We constructed a series of cyclic-imide peptidomimetics compounds, which were designed to fit the active pockets S1 and S'1 of APN/CD13 that act in tumor proliferation. We found that the compound (S)-2-amino-N-((S)-1-(2-(hydroxyamino)-2-oxoethyl)-2,6-dioxopiperidin-3-yl)-3-phe nylpropanamide hydrochloride (Compound No.24F, also termed as CIP-13F) displayed a substantial potential inhibitory effect on APN/CD13 activity. Herein, we evaluated the efficacy of 24F as a candidate compound for treatment of cancers with positive activity and expression of APN/CD13.Methods:The experiments were performed on several human carcinoma cell lines, which have high and low levels of APN/CD13 respectively. The assay of quantitating the enzymatic cleavage of the substrate L-Leucine p-nitroanilide was employed to evaluate APN/CD13 activity on the surface of cancer cells. An immunofluorescent flow cytometry assay and Western blotting analysis were used to measure the expression level of APN/CD13 after treatment with 24F. MTT assay was employed to evaluate the efficacy of 24F in vitro. The transwell chamber assay was also used to measure the inhibitory effect of 24F on the invasion and migration of cancer cells penetrating the Matrigel. The in vivo efficacy of 24F was assessed in an ES-2 xenograft mouse model. In the assays of apoptosis, hoechst staining analysis was employed to examine DNA fragment after cancer cell apoptosis. Cell surface of phosphatidylserine in apoptotic cells was quantitatively detected by using annexin V/FITC and PI apoptosis detection kit. Western blotting analysis was employed to evaluate the expression levels of apoptosis related proteins in cancer cells exposure to 24F and in ES-2 xenografts in nude mice. In addition, we measured the cell cycle arrest using adridine orange/ethidium bromide staining after 24F treatment.Results:24F significantly blocked APN/CD13 activity on the surface of ES-2 and HL-60 cells, which are high expression levels of APN/CD13, as measured by quantitating the enzymatic cleavage of the substrate L-Leucine p-nitroanilide.24F weakly inhibited APN/CD13 activity on the surface of SKOV3, PLC/PRF/5 and ECV304 cells, which are relatively low levels of APN/CD13.24F effectively inhibited ES-2 and HL60 cell growth and migration without significant cytotoxic effect. These results indicated that 24F may inhibit cancer cell growth via suppression of APN/CD13. Further, we evaluate the inhibitory effect of 24F in mice bearing ES-2 xenografts.24F delayed the growth of ES-2 xenografts in mice after 2 weeks of tail vein injection. At doses of 10 and 50 mg/kg of 24F, the inhibition rates were 31.0% and 38.0%(P＜0.01 vs. control), respectively.24F treatment was generally well tolerated by mice with no significance reduction in body weight. In the assays of Western blotting analysis, we found that the expression levels of APN/CD13 in ES-2 cells were significantly decreased exposure to 24F. The suppression of APN/CD13 was also observed by the assay of flow cytometry. The inhibitory effects of 24F on APN/CD13 expression and on ES-2 proliferation were confirmed in mice bearing ES-2 xenografts. Further, the inhibitory effects of 24F on APN/CD13 and on ES-2 proliferation were supported by the induction of ES-2 apoptosis.24F-treated ES-2 cells resulted apoptotic characteristics, such as induction of externalization of phosphatidylserine. The activation of caspase-3 and poly ADP-ribose polymerase (PARP) was also enhanced. These results suggest that 24F has a high inhibitory effect on the growth of cancer cells via decreasing the activity and expression of APN/CD13. The inhibitory effects of 24F on APN/CD13 expression and induction of apoptosis were confirmed in mice bearing ES-2 xenografts. In addition,24F induced cancer cell cycle and arrested in G0/G1 as examined by flow cytometry analysis. These results suggest that 24F has a high inhibitory effect on the growth of cancer cells via decreasing the activity and expression of APN/CD13.Conclusion:24F is a novel cyclic-imide peptidomimetics with a great inhibition effect on the activity and expression of APN/CD13. Administration of 24F in mice delayed the growth of ES-2 xenografts without significant toxicity. These results suggest that 24F is a potential APN/CD13 inhibitor and a candidate compound for treatment of cancers with positive activity and expression of APN/CD13.