Assessment of cell cytotoxicity and comet assay on HER2/neu positive cell line due to 111In Auger electrons as DNA-targeting radioimmunoconjugate

Author:

Piroozfar Behnaz1,Alirezapour Behrouz1,Sedeh Farahnaz Motamedi2,Mirzaii Mohammad1,Jalilian Amir Reza3,Hashemizadeh Miad4,Raisali Gholamreza1

Affiliation:

1. Radiation Applications Research School, Nuclear Science and Technology Research Institute, North Karegar St., P. O. Box 11365-3486, Tehran, Iran

2. Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute, North Karegar St., P. O. Box 11365-3486, Tehran, Iran

3. Radioisotope Products and Radiation Technology Section, Division of Physical and Chemical Sciences, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Vienna. International Centre, PO Box 100, 1400 Vienna, Austria

4. Pars Isotope Company, Tehran, Iran

Abstract

Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab is a high affinity-binding monoclonal antibody against HER2/neu, which is overexpressed in breast tumors and used in radiopharmaceutical development. Objective: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab, and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. Methods: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA-NLS-trastuzumab were labeled with 111In, followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line) was used in the cell viability assessment assay for 11In, 111In-DTPA-trastuzumab, and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT, and comet assay was utilized by DNA damage detection. Results: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1 was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA-NLS-trastuzumab. Conclusion: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate that the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA, leading to cell death.

Publisher

Bentham Science Publishers Ltd.

Subject

Pharmacology,Radiology Nuclear Medicine and imaging

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