Behnaz Piroozfar, Behrouz Alirezapour, Farahnaz Motamedi Sedeh, Mohammad Mirzaii, Amir Reza Jalilian, Miad Hashemizadeh, Gholamreza Raisali
{"title":"111In俄格电子作为dna靶向放射免疫偶联物对HER2/neu阳性细胞株的细胞毒性评价及彗星试验","authors":"Behnaz Piroozfar, Behrouz Alirezapour, Farahnaz Motamedi Sedeh, Mohammad Mirzaii, Amir Reza Jalilian, Miad Hashemizadeh, Gholamreza Raisali","doi":"10.2174/1874471014666210625115111","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development.</p><p><strong>Objectives: </strong>In this work, the lethal effects of <sup>111</sup>In<sup>3+</sup>, <sup>111</sup>In-DTPA-trastuzumab and <sup>111</sup>In-trastuzumab coupled-nuclear localizing sequence peptide (<sup>111</sup>In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro.</p><p><strong>Methods: </strong>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 <sup>111</sup>In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for <sup>111</sup>In, <sup>111</sup>In-DTPA-trastuzumab and <sup>111</sup>In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection.</p><p><strong>Results: </strong>A significant radiochemical purity for <sup>111</sup>In-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 <sup>111</sup>In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using <sup>111</sup>In-DTPA- NLS-trastuzumab.</p><p><strong>Conclusion: </strong>The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates <sup>111</sup>In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.</p>","PeriodicalId":10991,"journal":{"name":"Current radiopharmaceuticals","volume":null,"pages":null},"PeriodicalIF":1.5000,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to <sup>111</sup>In Auger Electrons as DNA-Targeting Radioimmunoconjugate.\",\"authors\":\"Behnaz Piroozfar, Behrouz Alirezapour, Farahnaz Motamedi Sedeh, Mohammad Mirzaii, Amir Reza Jalilian, Miad Hashemizadeh, Gholamreza Raisali\",\"doi\":\"10.2174/1874471014666210625115111\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development.</p><p><strong>Objectives: </strong>In this work, the lethal effects of <sup>111</sup>In<sup>3+</sup>, <sup>111</sup>In-DTPA-trastuzumab and <sup>111</sup>In-trastuzumab coupled-nuclear localizing sequence peptide (<sup>111</sup>In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro.</p><p><strong>Methods: </strong>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 <sup>111</sup>In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for <sup>111</sup>In, <sup>111</sup>In-DTPA-trastuzumab and <sup>111</sup>In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection.</p><p><strong>Results: </strong>A significant radiochemical purity for <sup>111</sup>In-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 <sup>111</sup>In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using <sup>111</sup>In-DTPA- NLS-trastuzumab.</p><p><strong>Conclusion: </strong>The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates <sup>111</sup>In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.</p>\",\"PeriodicalId\":10991,\"journal\":{\"name\":\"Current radiopharmaceuticals\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.5000,\"publicationDate\":\"2022-01-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current radiopharmaceuticals\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.2174/1874471014666210625115111\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"PHARMACOLOGY & PHARMACY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current radiopharmaceuticals","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.2174/1874471014666210625115111","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"PHARMACOLOGY & PHARMACY","Score":null,"Total":0}
Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111In Auger Electrons as DNA-Targeting Radioimmunoconjugate.
Background: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development.
Objectives: 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 111In, 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 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 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.