Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX

Vasileios Askoxylakis,Nuh N Rahbari,Susanne Krämer,Uwe Haberkorn,Shoaib Rana,Volker Ehemann,Jürgen Debus

doi:10.3390/ijms131013030

Vasileios Askoxylakis, Nuh N Rahbari + Show 5 more

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https://doi.org/10.3390/ijms131013030

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Abstract

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of 125I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of 125I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.

Highlights

Carbonic anhydrase IX (CAIX) is a membrane-associated zinc metalloenzyme, known to be over-expressed in various human tumors, including renal, ovarian, lung and colorectal carcinomas [1].The protein catalyzes the reversible hydration of carbon dioxide and has a key role in pH regulation, whereas it is involved in various cellular processes including cell proliferation and adhesion
It has been demonstrated that CAIX is a target gene of the hypoxia inducible factor 1α (HIF-1α), a transcription factor that accumulates under conditions of low oxygen concentration and activates a series of genes, which are responsible for cellular response to hypoxia [2]
These results indicate that the novel peptide CaIX-P1 possibly recognizes the extracellular domain of carbonic anhydrase IX in a similar manner like the monoclonal antibody and strengthens the hypothesis that the dodecapeptide might be used as a lead structure for the development of specific molecules targeting CAIX in tumors

Summary

Introduction

Carbonic anhydrase IX (CAIX) is a membrane-associated zinc metalloenzyme, known to be over-expressed in various human tumors, including renal, ovarian, lung and colorectal carcinomas [1].The protein catalyzes the reversible hydration of carbon dioxide and has a key role in pH regulation, whereas it is involved in various cellular processes including cell proliferation and adhesion. The expression of carbonic anhydrase IX in tumors is associated with a poor disease prognosis, most probably due to the correlation of the protein with low oxygen levels [3]. The fact that CAIX is considered to be an intrinsic marker of tumor hypoxia and that its’ expression is associated with an adverse disease prognosis make the protein an attractive candidate for the development of targeting strategies. In this respect, emphasis must be put on the potential of CAIX for non-invasive imaging purposes. In vivo visualization of CAIX expression in tumors could be used for the prediction of treatment response and outcome, which is of high importance, because it might provide a rationale for patient stratification during treatment decision making

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