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Recombinant ADI (ADI_br, 5 µg/mL) expressed in E. coli BL21 (DE3) suppressed AGS cell growth, causing a 65.32% reduction in viability. ADI_br increased apoptosis-related gene expression (bax, bad, caspase-7, caspase-3) and caspase-9 activity versus untreated cells, with flow cytometry showing rising apoptotic rates after 24–28 h. The findings support ADI_br as a candidate enzyme for anticancer applications.",{"@graph":14,"@context":73},[15,34,56],{"@type":16,"itemListElement":17},"BreadcrumbList",[18,23,27,31],{"item":19,"name":20,"@type":21,"position":22},"https://docshare.wps.com","Home","ListItem",1,{"item":24,"name":25,"@type":21,"position":26},"https://docshare.wps.com/document/","Document",2,{"item":28,"name":29,"@type":21,"position":30},"https://docshare.wps.com/document/research-report/","Research & 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Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway  \nRemilyn M. Mendoza 1, Ji Hoon Song 1, Yong Tae Jung 2, Hyun-Dong Paik 3, Young-Seo Park 4 and Dae-Kyung Kang 1, *  \nCitation: Mendoza, R.M.; Song, J.H.; Jung, Y.T.; Paik, H.-D.; Park, Y.-S.; Kang, D.-K. Recombinant Arginine Deiminase from Levilactobacillus brevis Inhibits the Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway. Int.  \nJ. Mol. Sci. 2024, 25, 4163. [https://](https://)[ ](https://)[doi.org/10.3390/ijms25084163](doi.org/10.3390/ijms25084163)  \nAcademic Editors: Djuro Josic,  \nMauro Coluccia and Daniela Fiocco  \nReceived: 13 February 2024  \nRevised: 25 March 2024  \nAccepted: 8 April 2024  \nPublished: 9 April 2024  \nCopyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ([https://](https://)[ ](https://)[creativecommons.org/licenses/by/](creativecommons.org/licenses/by/)[ ](creativecommons.org/licenses/by/)[4.0/](4.0/)) .  \n1 Department of Animal Biotechnology, Dankook University, Cheonan 31116, Republic of Korea; [rmmendoza02@dankook.ac.kr](rmmendoza02@dankook.ac.kr) (R.M.M.); [sr9412@naver.com](sr9412@naver.com) (J.H.S.)  \n2 Department of Microbiology, Dankook University, Cheonan 31116, Republic of Korea; [yjung@dankook.ac.kr](yjung@dankook.ac.kr)  \n3 Department of Food Science and Biotechnology of Animal Resource, Konkuk University, Seoul 05029, Republic of Korea; [hdpaik@konkuk.ac.kr](hdpaik@konkuk.ac.kr)  \n4 Department of Food Science and Biotechnology, Gachon University, Seongnam 13120, Republic of Korea; [ypark@gachon.ac.kr](ypark@gachon.ac.kr)  \n* Correspondence: [dkkang@dankook.ac.kr](dkkang@dankook.ac.kr); Tel.: +82-41-550-3655  \nAbstract: The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications.  \nKeywords: lactic acid bacteria; anticancer; arginine deiminase; apoptosis; genome analysis  \n1. Introduction  \nCancer, characterized by the uncontrolled proliferation of abnormal cells with the potential to invade adjacent tissues and metastasize to other organs, ranks as the second leading cause of death worldwide [1]. Current cancer management strategies, such as surgery, irradiation, and chemotherapy, commonly cause undesirable side effects [1,2] . Therefore, there is a continuous pursuit for safer and more effective alternatives to these conventional cancer treatments [3]. Amino acid deprivation therapy (AADT) is one of the most promising alternative cancer treatments [4]. The metabolic alterations in cancer cells result in an increased demand for energy to sus","cbCaigTnZPPe1iRl","https://ap.wps.com/l/cbCaigTnZPPe1iRl","pdf",3979170,13,"English","# Abstract\n# Keywords\n# 1. Introduction\n## Amino acid deprivation therapy and cancer vulnerability\n## Arginine metabolism and arginine deiminase mechanism\n## Lactic acid bacteria and anticancer mechanisms","[{\"question\":\"What is the main goal of the study involving Levilactobacillus brevis?\",\"answer\":\"The study analyzes the genome of L. brevis KU15176 to identify key genes that may confer anticancer properties, with emphasis on arginine deiminase (ADI).\"},{\"question\":\"How was recombinant arginine deiminase produced and tested against stomach cancer cells?\",\"answer\":\"Recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL) was overexpressed in E. coli BL21 (DE3) and tested in vitro against AGS cells to assess effects on cell growth and viability.\"},{\"question\":\"What evidence indicates that ADI_br induces apoptosis in AGS cells?\",\"answer\":\"ADI_br-treated cells showed increased expression of apoptosis-related genes (bax, bad, caspase-7, caspase-3), higher caspase-9 activity, apoptosis-like cell behavior, and increased apoptotic rates measured by flow cytometry at 24 and 28 hours.\"}]","Recombinant Arginine Deiminase from Levilactobacillus brevis Inhibits the Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway | PDF",1789996321,33]