CIP-13F, A Novel Cyclic-imide Peptidomimetics Compound, Inhibits Tumor Growth With Positive APN Expression

Aminopeptidase N (APN/CD13), a zinc-dependent exopeptidase, is highly expressed on the surface of cancer cells and is thought to be involved in the degradation of extracellular matrix barriers and angiogenesis and promotes cancer growth and metastasis. APN has become an attractive target for anti-tumor therapy. We constructed a series of cyclic-imide peptidomimetics compounds, which were designed to fit the active pockets S1and S’l of APN that act in tumor proliferation. We found that the compound CIP-13F displayed a substantial potential inhibitory effect on APN activity.The research of APN inhibitors is considered as a strategy of cancer treatment.OBJECTIVE:We evaluated the efficacy of CIP-13F as a novel APN inhibitor for treatment of cancers with positive APN expression.METHODS:The experiments were performed on cancer cells with positive APN expression. The assay of quantitating the enzymatic cleavage of the substrate L-Leucine p-nitroanilide was employed to evaluate APN activity on the surface of cancer cells with positive APN expression. MTT assay was employed to evaluate the efficacy of CIP-13F in vitro. The transwell chamber assay was also used to measure the inhibitory effect of CIP-13F on the invasion and migration of cancer cells penetrating the Matrigel. Tube formation of HUVEC by matrigel assay was also used to measure the inhibitory effect of CIP-13F on the angiogenesis.The in vivo efficacy of CIP-13F was assessed in an Lewis lung carcinoma (LLC) implantation mouse model. C57BL/6mice were subcutaneously inoculated with LLC cells in anterior flank. Then,0,50and100mg/kg of CIP-13F were injected via vena caudalis. Bestatin was used as the positive control. Administration of CIP-13F or bestatin was performed daily for3consecutive weeks. Mice were killed, and the tumors in anterior flank and metastasis nodules in lungs were examined. The assays of immunohistochemical staining, immunofluorescent flow cytometry and western blotting were performed to estimate the expression of APN in LLC cells. We carried out the experiments of Annexin-V/PI staining, DNA fragmentation analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining to determine apoptotic cells in LLC tissues. Using immunohistochemical staining with CD34, the antiangiogenesis of CIP-13F was evaluated in LLC tissue sections.RESULTS:CIP-13F blocked APN activity on the surface of cancer cells which are positive of APN, as measured by quantitating the enzymatic cleavage of the substrate L-Leucine p-nitroanilide. CIP-13F inhibited ES-2and HT-1080growth and migration. These results indicated that CIP-13F may inhibit cancer cell growth via suppression of APN. Further, we evaluate the inhibitory effect of CIP-13F in mice bearing Lewis lung carcinoma implantation. CIP-13F treatment resulted in a significant delay of LLC growth in anterior flank. Examination of lungs showed that the number of metastatic nodules of LLC was also markedly decreased. The inhibitory effect of CIP-13F on LLC growth was further evidenced by the induction of LLC apoptosis, showing the increases in Annexin-V/PI staining cells, DNA fragmentation and TUNEL staining cells. Molecular analyses of LLC tissues in CIP-13F-treated mice suggested that the decrease in APN expression by CIP-13F might account for its actions of mechanism. The results showed that CIP-13F could suppress the tube formation of the HUVEC in vitro.Further, the inhibition of angiogenesis in LLC tissues was determined, showing the decreases in microvessel density (MVD) and angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-a). These results suggest that CIP-13F has a high inhibitory effect on the growth of cancer cells via decreasing the activity and expression of APN.CONCLUSION:CIP-13F is a novel cyclic-imide peptidomimetics with a great inhibition effect on the activity and expression of APN. Our results showed that CIP-13F effectively inhibited LLC growth and pulmonary metastasis in mice and suggested that CIP-13F is a potential drug for the treatment for cancers with positive APN expression.