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

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.

Hypoxia-targeted gold nanorods for cancer photothermal therapy

“…So as Figure 2 shows, a much higher uptake of gold was found in HT29 cells treated with GNR/anti-CAIX in contrast with GNR-PEG and medium control, indicating efficient targeting and successful conjugation. Also, hypoxic status did increase intracellular accumulation of GNRs treated with GNR/anti-CAIX at 2 h, which is in agreement with previous studies by showing a positive correlation between CAIX conjugates binding and expression of CAIX in human carcinoma cells [ 29 , 30 ]. Hyperspectral dark field microscopy imaging also confirmed similar findings with the GNR/anti-CAIX group showing strong and specific binding to HT29 cells compared to the control group, while the GNR-PEG group only showing non-specific binding ( Supplementary Figure 3 ).…”

Cancer radiotheranostics targeting carbonic anhydrase-IX with 111In- and 90Y-labeled ureidosulfonamide scaffold for SPECT imaging and radionuclide-based therapy

“…However, the high level of CA-IX expression in MDA-MB-231 cells under normoxic conditions has been observed in several studies 64 . The density-dependent CA-IX up-regulation has been demonstrated for several cell lines including MDA-MB-231 65 - 67 . Culture conditions with a high cell density might create a pericellular hypoxic environment, leading to the accumulation of HIF-1 and subsequently CA-IX expression.…”

“…Culture conditions with a high cell density might create a pericellular hypoxic environment, leading to the accumulation of HIF-1 and subsequently CA-IX expression. It is difficult to control the pericellular microenvironment completely; therefore, the poor reproducibility of CA-IX expression in MDA-MB-231 cells has been demonstrated 65 . VHL binds to HIF-1α hydroxylated by oxygen, leading to the degradation of HIF-1α and the subsequent prevention of CA-IX expression; therefore, under hypoxic conditions, nonhydroxylated HIF-1α is not recognized by the VHL protein, and RCC4-VHL cells should show CA-IX expression.…”

The Potential of Liposomes with Carbonic Anhydrase IX to Deliver Anticancer Ingredients to Cancer Cells in Vivo

“…Furthermore, Askoxylakis et al [ 179 ] suggested that expression of the CA-IX protein is unstable, and microenvironment-dependent. The scientific literature lacks information about the onset of CA-IX expression in the course of carcinogenesis, the percentage of CA-IX-positive cells, and the amount of protein expressed in tumour tissues at different disease stages.…”

“…Thus, more research is needed in order to design a proper treatment regime for patients. The first identified peptide ligand for the CA-IX receptor through phage display technology is CaIX-P1 [ 179 , 180 ]. However, to the best of our knowledge, the endogenous ligand of the CA-IX receptor is unknown.…”