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Abstract
Background: Cholecystokinin (CCK) receptors are overexpressed in numerous human cancers, such as pancreatic, colon and gastric cancers. Previous studies have shown that the specific receptor-binding property of CCK for CCK receptors (CCKRs) can be exploited to produce immunotoxins (ITs) that target cancer cells overexpressing CCK receptors.
Purpose: Construct a new IT-targeting CCKR-overexpressing colon cancers.
Methods: To construct the CCKR-targeted IT, a reverse CCK8 peptide was fused with a modified 38-kDa truncated form of the Pseudomonas exotoxin (PE38KDEL). An efficient immunoaffinity purification procedure was used to produce a PE38-based IT. Several analyses, including CCK8 competition and indirect immunofluorescence assays, were performed to confirm the interaction between rCCK8 and CCKR. After cytotoxic assays on several cell lines, the anti-tumor activity of the new IT was detected in nude mice.
Results: The rCCK8PE38 IT showed specific cytotoxicity for two colon cancer cell lines and one gastric cancer cell line. After purification, 18–26 mg of pure rCCK8PE38 per 1 L of culture was obtained. Purified rCCK8PE38 showed high cytotoxicity in colon cancer cell lines with IC50 values of 0.8–3.5 ng/mL. The results of the CCK8 competition and indirect immunofluorescence assays showed that rCCK8 had a specific interaction with CCKR. Nude mice inoculated with HCT-8 tumor xenografts were treated with rCCK8PE38, which efficiently decreased the tumor size in those mice.
Conclusions and discussion: All of these data suggest that rCCK8PE38 has potential as a new immunotherapy agent. Furthermore, the results of this study further support the high value of the immunoaffinity method for IT purification procedures.
Declaration of interest
This work was supported by the National Natural Science Foundation of China (Grant no. 81401953), the Jilin Province Science & Technology Support (20120966) and the Research Fund for Doctoral Program of Higher Education (2012006111078).