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Cui S.-X.,Shandong Medical Biotechnology Center | Cui S.-X.,Shandong Academy of Sciences | Qu X.-J.,Shandong University | Gao Z.-H.,University of Calgary | And 6 more authors.
Cancer Letters | Year: 2010

Aminopeptidase N (APN/CD13) is an essential peptidase involved in the process of tumor growth, metastasis and angiogenesis. Inhibition of APN/CD13 may be an effective strategy for cancer treatment. CIP-13F is a cyclic-imide peptidomimetics compound designed to fit the active pockets S1 and S′1 of APN/CD13 that act in tumor proliferation. Our aim in this study was to evaluate the efficacy of CIP-13F as a candidate compound for cancer treatment. The experiments were performed on the human ovarian carcinoma (OVCA) ES-2 and HRA cell lines, which have high and low levels of APN/CD13 respectively. CIP-13F significantly blocked APN/CD13 activity on the surface of ES-2 cells as measured by quantitating the enzymatic cleavage of the substrate l-leucine-. p-nitroanilide. CIP-13F effectively inhibited ES-2 cell growth and migration without significant cytotoxic effect. In contrast, CIP-13F did not significantly inhibit HRA cell growth, indicating that CIP-13F may inhibit ES-2 cell growth via suppression of APN/CD13. The suppression of APN/CD13 was also observed by using the assays of flow cytometry and Western blot analysis. Further, the inhibitory effects of CIP-13F on APN/CD13 and on ES-2 proliferation were supported by the induction of ES-2 apoptosis. CIP-13F-treated ES-2 cells resulted apoptotic characteristics, such as induction of externalization of phosphatidylserine and DNA laddering fragment. The activation of caspase-3 and poly ADP-ribose polymerase (PARP) was also enhanced. The inhibitory effects of CIP-13F on APN/CD13 expression and on ES-2 proliferation were confirmed in mice bearing ES-2 xenografts. CIP-13F delayed the growth of ES-2 xenografts in mice after 2 weeks of vena caudalis injection. These results suggest that CIP-13F has a high inhibitory effect on the growth of OVCA cells via decreasing the activity and expression of APN/CD13. © 2009 Elsevier Ireland Ltd.

Chai Z.-B.,Shandong Medical Biotechnology Center | Zhang G.-L.,Shandong Medical Biotechnology Center | Han J.-X.,Shandong Medical Biotechnology Center
Chinese Journal of Biologicals | Year: 2014

Protein-protein interaction play an pivotal role in the vital movement of cells. Glutathione-S-transferase (GST)-pull down assay is widely used for the verification of direct protein-protein interaction in vitro,including those between the known proteins and those between known and unknown proteins. Although GST-pull down assay has a strong specificity in verification of direct protein-protein interaction in vitro,the conclusion should be given with caution. This paper reviews the principle and application as well as some problems during application of GST-pull down assay.

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