Neocarzinostatin, Aptamer Conjugates for Targeting EpCAM-positive Tumor Cells
Authors: Srujana Chitipothu
Subramanian Krishnakumar
Jonathan P Watson
Jagat Rakesh Kanwar
Rupinder Kaur Kanwar
Janakiraman Narayanan
Prasanna Kumar Athyala
Journal: Anticancer research (2017)
Discipline: CLINICAL MEDICINE
Keywords: BrDU; DNA-damage; Epithelial cell adhesion molecule (EpCAM); H2AX; Neocarzinostatin (NCS)
Affiliations: Department of Nanobiotechnology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Chennai, India., Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Burwood, Australia., Central Research Instrumentation Facility, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Chennai, India., Department of Nanobiotechnology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Chennai, India .

Abstract:  Background/aim: The aim of this study was to investigate the role of Neocarzinostatin (NCS) conjugated with epithelial cell adhesion molecule (EpCAM) aptamer in EpCAM-positive cancer cells. NCS is an antitumor antibiotic protein chromophore that has the ability to cleave double stranded DNA and can be used as a potential drug for the treatment of EpCAM-positive cancers. EpCAM aptamer is an oligonucleotide ligand that binds specifically to EpCAM, a protein overexpressed in tumor cells. Materials and methods: NCS was conjugated with EpCAM aptamer using Sulfo-Succinimidyl 6-(3-(2-pyridyldithio) - propionamide hexanoate) LC-(SPDP) cross-linker to deliver it to EpCAM-positive tumor cells. The conjugates were characterized using polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC). Flow cytometry was used to study the binding efficiency of the aptamer and the conjugates in cancer cells. The effect of the conjugate on cancer cells was studied using propidium iodide (PI) to analyze the cell cycle phase changes. The apoptosis assay was performed using the IC50 concentration of NCS. Microarrays were performed to study the gene level changes in cancer cells upon treatment with NCS and the conjugate. Results: Flow cytometry revealed significant binding of aptamer and conjugate in the MCF-7 and WERI-Rb1 cell lines. Briefly, 62% in MCF and 30% in WERI-Rb1 cells with conjugate treated cells (p&lt;0.005). The cell-cycle analysis indicated G2 phase arrest in MCF-7 cells and S phase arrest in WERI-Rb1 cells (p&lt;0.005). Microarray analysis showed differentially expressed genes involved in cell cycle, DNA damage, and apoptosis. The BrDU assay and the apoptosis assay showed that the expression of BrDU was reduced in conjugate-treated cells and the PARP levels were increased confirming the double stranded DNA breaks (p&lt;0.005). In MCF-7 and WERI-Rb1 cells, most of the cells underwent necrosis (p&lt;0.005). Conclusion: The EpCAM aptamer conjugated NCS showed specificity to EpCAM-positive cells. The effect of the conjugates on cancer cells were impressive as the conjugate arrested the cell cycle and promoted apoptosis and necrosis. The high levels of PARP expression confirmed the DNA breaks upon conjugate treatment. Our study demonstrates that the NCS conjugated with EpCAM can be targeted to cancer cells sparing normal cells. 

Retracted: No

PubPeer comments:
 There is an instance of image duplication in this paper. The aptamer and conjugate central panels of Figure 3 have been duplicated across all of the imaging technologies. This is shown in the attached figures in the orange boxes for the phase contrast image. ![file] ,