Abstract | BACKGROUND/AIM: 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<0.005). The cell-cycle analysis indicated G2 phase arrest in MCF-7 cells and S phase arrest in WERI-Rb1 cells (p<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<0.005). In MCF-7 and WERI-Rb1 cells, most of the cells underwent necrosis (p<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.
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Authors | Prasanna Kumar Athyala, Jagat Rakesh Kanwar, Srujana Chitipothu, Rupinder Kaur Kanwar, Subramanian Krishnakumar, Jonathan P Watson, Janakiraman Narayanan |
Journal | Anticancer research
(Anticancer Res)
Vol. 37
Issue 7
Pg. 3615-3629
(07 2017)
ISSN: 1791-7530 [Electronic] Greece |
PMID | 28668853
(Publication Type: Journal Article)
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Copyright | Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved. |
Chemical References |
- Antibiotics, Antineoplastic
- EPCAM protein, human
- Epithelial Cell Adhesion Molecule
- Oligonucleotides
- Zinostatin
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Topics |
- Antibiotics, Antineoplastic
(pharmacology)
- Apoptosis
(drug effects)
- Cell Cycle
(drug effects)
- Cell Line, Tumor
- DNA Breaks, Double-Stranded
(drug effects)
- DNA Damage
(drug effects)
- Epithelial Cell Adhesion Molecule
(metabolism)
- Humans
- MCF-7 Cells
- Neoplasms
(drug therapy, metabolism)
- Oligonucleotides
(metabolism)
- Zinostatin
(pharmacology)
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