{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T09:51:38Z","timestamp":1784886698686,"version":"3.55.0"},"reference-count":49,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2021,10,22]],"date-time":"2021-10-22T00:00:00Z","timestamp":1634860800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Hepatocellular carcinoma (HCC) is one of the high-metastatic types of cancer, and metastasis occurs in one-third of patients with HCC. To maintain the effectiveness of drug compounds on cancer cells and minimize their side effects on normal cells, it is important to use new approaches for overcoming malignancies. Immunotoxins (ITs), an example of such a new approach, are protein-structured compounds consisting of toxic and binding moieties which can specifically bind to cancer cells and efficiently induce cell death. Here, we design and scrutinize a novel immunotoxin against an oncofetal marker on HCC cells. We applied a truncated diphtheria toxin (DT389) without binding domain as a toxin moiety to be fused with a humanized YP7 scFv against a high-expressed Glypican-3 (GPC3) antigen on the surface of HCC cells. Cytotoxic effects of this IT were investigated on HepG2 (GPC3+) and SkBr3 (GPC3\u2212) cell lines as positive- and negative-expressed GPC3 antigens. The dissociation constant (Kd) was calculated 11.39 nM and 18.02 nM for IT and YP7 scfv, respectively, whereas only IT showed toxic effects on the HepG2 cell line, and decreased cell viability (IC50 = 848.2 ng\/mL). Changing morphology (up to 85%), cell cycle arrest at G2 phase (up to 13%), increasing intracellular reactive oxygen species (ROSs) (up to 50%), inducing apoptosis (up to 38% for apoptosis and 23% for necrosis), and an almost complete inhibition of cell movement were other effects of immunotoxin treatment on HepG2 cells, not on SkBr3 cell line. These promising results reveal that this new recombinant immunotoxin can be considered as an option as an HCC inhibitor. However, more extensive studies are needed to accomplish this concept.<\/jats:p>","DOI":"10.3390\/toxins13110749","type":"journal-article","created":{"date-parts":[[2021,10,25]],"date-time":"2021-10-25T21:42:05Z","timestamp":1635198125000},"page":"749","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":23,"title":["DT389-YP7, a Recombinant Immunotoxin against Glypican-3 That Inhibits Hepatocellular Cancer Cells: An In Vitro Study"],"prefix":"10.3390","volume":"13","author":[{"given":"Hamid","family":"Hashemi Yeganeh","sequence":"first","affiliation":[{"name":"Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran 1435916471, Iran"},{"name":"Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran 1435916471, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9982-2773","authenticated-orcid":false,"given":"Mohammad","family":"Heiat","sequence":"additional","affiliation":[{"name":"Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran 1435916471, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5836-4865","authenticated-orcid":false,"given":"Marek","family":"Kieliszek","sequence":"additional","affiliation":[{"name":"Department of Food Biotechnology and Microbiology, Institute of Food Sciences, Warsaw University of Life Sciences\u2014SGGW, Nowoursynowska 159 C, 02\u2013776 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Seyed Moayed","family":"Alavian","sequence":"additional","affiliation":[{"name":"Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran 1435916471, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4420-0173","authenticated-orcid":false,"given":"Ehsan","family":"Rezaie","sequence":"additional","affiliation":[{"name":"Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran 1435916471, Iran"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/S1470-2045(18)30059-7","article-title":"Oncological management and obstetric and neonatal outcomes for women diagnosed with cancer during pregnancy: A 20-year international cohort study of 1170 patients","volume":"19","author":"Verheecke","year":"2018","journal-title":"Lancet Oncol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"407","DOI":"10.3892\/ijo.2018.4661","article-title":"From chemotherapy to biological therapy: A review of novel concepts to reduce the side effects of systemic cancer treatment","volume":"54","author":"Schirrmacher","year":"2019","journal-title":"Int. J. Oncol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.5761\/atcs.ra.16-00162","article-title":"Current status and future prospects for esophageal cancer treatment","volume":"23","author":"Sohda","year":"2017","journal-title":"Ann. Thorac. Cardiovasc. Surg."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1010428317692226","DOI":"10.1177\/1010428317692226","article-title":"Immunotoxin: A new tool for cancer therapy","volume":"39","author":"Allahyari","year":"2017","journal-title":"Tumor Biol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1055","DOI":"10.1158\/1078-0432.CCR-15-1623","article-title":"New life for immunotoxin cancer therapy","volume":"22","author":"Hassan","year":"2016","journal-title":"Clin. Cancer Res."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Dandawate, P.R., Subramaniam, D., Jensen, R.A., and Anant, S. (2016). Targeting cancer stem cells and signaling pathways by phytochemicals: Novel approach for breast cancer therapy. Seminars in Cancer Biology, Elsevier.","DOI":"10.1016\/j.semcancer.2016.09.001"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"El Hout, M., Dos Santos, L., Hama\u00ef, A., and Mehrpour, M. (2018). A promising new approach to cancer therapy: Targeting iron metabolism in cancer stem cells. Seminars in Cancer Biology, Elsevier.","DOI":"10.1016\/j.semcancer.2018.07.009"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Mohammadi, M., Rezaie, E., Sakhteman, A., and Zarei, N. (2020). A highly potential cleavable linker for tumor targeting antibody-chemokines. J. Biomol. Struct. Dyn., 1\u201311.","DOI":"10.1080\/07391102.2020.1841025"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1065","DOI":"10.1007\/s13258-021-01116-w","article-title":"The tissue expression of MCT3, MCT8, and MCT9 genes in women with breast cancer","volume":"43","author":"Sohrabi","year":"2021","journal-title":"Genes Genom."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1326","DOI":"10.1007\/s10528-021-10062-x","article-title":"An Integrated Data Analysis of mRNA, miRNA and Signaling Pathways in Pancreatic Cancer","volume":"59","author":"Sohrabi","year":"2021","journal-title":"Biochem. Genet."},{"key":"ref_11","first-page":"1333","article-title":"Expression pattern of glypican-3-GPC3-during human embryonic and fetal development","volume":"23","author":"Iglesias","year":"2008","journal-title":"Histol. Histopathol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"626","DOI":"10.1038\/modpathol.2008.26","article-title":"Utility of glypican-3 in differentiating hepatocellular carcinoma from other primary and metastatic lesions in FNA of the liver: An immunocytochemical study","volume":"21","author":"Ligato","year":"2008","journal-title":"Mod. Pathol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"576","DOI":"10.1002\/hep.26996","article-title":"Inactivation of Wnt signaling by a human antibody that recognizes the heparan sulfate chains of glypican-3 for liver cancer therapy","volume":"60","author":"Gao","year":"2014","journal-title":"Hepatology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"6536","DOI":"10.1038\/ncomms7536","article-title":"Immunotoxin targeting glypican-3 regresses liver cancer via dual inhibition of Wnt signalling and protein synthesis","volume":"6","author":"Gao","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"E1083","DOI":"10.1073\/pnas.1217868110","article-title":"Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma","volume":"110","author":"Feng","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"735-IN25","DOI":"10.1593\/neo.11664","article-title":"Suppression of glypican 3 inhibits growth of hepatocellular carcinoma cells through up-regulation of TGF-\u03b22","volume":"13","author":"Sun","year":"2011","journal-title":"Neoplasia"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1016\/j.humpath.2012.06.016","article-title":"Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms","volume":"44","author":"Mounajjed","year":"2013","journal-title":"Hum. Pathol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1973","DOI":"10.1016\/j.ajpath.2018.05.014","article-title":"Glypican 3 overexpression across a broad spectrum of tumor types discovered with functional genomic mRNA profiling of a large cancer database","volume":"188","author":"Moek","year":"2018","journal-title":"Am. J. Pathol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"387","DOI":"10.2217\/imt-2017-0008","article-title":"Bioinformatic prediction and experimental validation of a PE38-based recombinant immunotoxin targeting the Fn14 receptor in cancer cells","volume":"9","author":"Keshtvarz","year":"2017","journal-title":"Immunotherapy"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"172912","DOI":"10.1016\/j.ejphar.2020.172912","article-title":"A new scfv-based recombinant immunotoxin against EPHA2-overexpressing breast cancer cells; High in vitro anti-cancer potency","volume":"870","author":"Rezaie","year":"2020","journal-title":"Eur. J. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1007\/s00894-018-3846-x","article-title":"Application of molecular dynamics simulations to design a dual-purpose oligopeptide linker sequence for fusion proteins","volume":"24","author":"Rezaie","year":"2018","journal-title":"J. Mol. Modeling"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.micpath.2018.12.003","article-title":"Different frequencies of memory B-cells induced by tetanus, botulinum, and heat-labile toxin binding domains","volume":"127","author":"Rezaie","year":"2019","journal-title":"Microb. Pathog."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1007\/s40203-021-00079-w","article-title":"In silico analysis of STX2a-PE15-P4A8 chimeric protein as a novel immunotoxin for cancer therapy","volume":"9","author":"Keshtvarz","year":"2021","journal-title":"Silico Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1007\/s10989-019-09901-8","article-title":"Bioinformatics Predictions, Expression, Purification and Structural Analysis of the PE38KDEL-scfv Immunotoxin against EPHA2 Receptor","volume":"26","author":"Rezaie","year":"2020","journal-title":"Int. J. Pept. Res. Ther."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2199","DOI":"10.1126\/science.1076071","article-title":"Maintenance of serological memory by polyclonal activation of human memory B cells","volume":"298","author":"Bernasconi","year":"2002","journal-title":"Science"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Fleming, B.D., and Ho, M. (2016). Glypican-3 targeting immunotoxins for the treatment of liver cancer. Toxins, 8.","DOI":"10.3390\/toxins8100274"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"32450","DOI":"10.18632\/oncotarget.10592","article-title":"Construction of an immunotoxin, HN3-mPE24, targeting glypican-3 for liver cancer therapy","volume":"8","author":"Wang","year":"2017","journal-title":"Oncotarget"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"33878","DOI":"10.1038\/srep33878","article-title":"Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma","volume":"6","author":"Zhang","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Heiat, M., Hashemi Yeganeh, H., Alavian, S.M., and Rezaie, E. (2021). Immunotoxins Immunotherapy against Hepatocellular Carcinoma: A Promising Prospect. Toxins, 13.","DOI":"10.3390\/toxins13100719"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1314","DOI":"10.1245\/s10434-013-3430-7","article-title":"EpCAM-Targeted Therapy for Human Hepatocellular Carcinoma","volume":"21","author":"Ogawa","year":"2014","journal-title":"Ann. Surg. Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.imlet.2012.09.006","article-title":"Construction, expression, and characterization of an anti-tumor immunotoxin containing the human anti-c-Met single-chain antibody and PE38KDEL","volume":"149","author":"Liu","year":"2013","journal-title":"Immunol. Lett."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"460264","DOI":"10.1155\/2015\/460264","article-title":"Construction, expression, and characterization of a recombinant immunotoxin targeting EpCAM","volume":"2015","author":"Lv","year":"2015","journal-title":"Mediat. Inflamm."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1038\/ng.3148","article-title":"Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis","volume":"47","author":"Marvig","year":"2015","journal-title":"Nat. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"963","DOI":"10.3389\/fmicb.2015.00963","article-title":"Pseudomonas Exotoxin A: Optimized by evolution for effective killing","volume":"6","author":"Michalska","year":"2015","journal-title":"Front. Microbiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1469","DOI":"10.1007\/s11033-018-4258-9","article-title":"An overview of apoptosis assays detecting DNA fragmentation","volume":"45","year":"2018","journal-title":"Mol. Biol. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2718","DOI":"10.1182\/blood-2003-04-1317","article-title":"Induction of caspase-dependent programmed cell death in B-cell chronic lymphocytic leukemia by anti-CD22 immunotoxins","volume":"103","author":"Decker","year":"2004","journal-title":"Blood"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"16934","DOI":"10.1021\/bi980995m","article-title":"Role of caspases in immunotoxin-induced apoptosis of cancer cells","volume":"37","author":"Brinkmann","year":"1998","journal-title":"Biochemistry"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"882","DOI":"10.1007\/s10495-013-0840-8","article-title":"GM-CSF-DFF40: A novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells","volume":"18","author":"Mathew","year":"2013","journal-title":"Apoptosis"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1090287","DOI":"10.1155\/2018\/1090287","article-title":"EGFR-targeted immunotoxin exerts antitumor effects on esophageal cancers by increasing ROS accumulation and inducing apoptosis via inhibition of the Nrf2-Keap1 pathway","volume":"2018","author":"Yang","year":"2018","journal-title":"J. Immunol. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1023\/A:1009616228304","article-title":"Role of reactive oxygen species (ROS) in apoptosis induction","volume":"5","author":"Simon","year":"2000","journal-title":"Apoptosis"},{"key":"ref_41","first-page":"982","article-title":"Reactive oxygen species-induced apoptosis in PC12 cells and protective effect of bilobalide","volume":"293","author":"Zhou","year":"2000","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1329","DOI":"10.1182\/blood.V83.5.1329.1329","article-title":"Anti-CD19 inhibits the growth of human B-cell tumor lines in vitro and of Daudi cells in SCID mice by inducing cell cycle arrest","volume":"83","author":"Ghetie","year":"1994","journal-title":"Blood"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"e13339","DOI":"10.1111\/cmi.13339","article-title":"Pseudomonas aeruginosa ExoT induces G1 cell cycle arrest in melanoma cells","volume":"23","author":"Mohamed","year":"2021","journal-title":"Cell. Microbiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"493","DOI":"10.3892\/or.2020.7878","article-title":"Novel EGFR-bispecific recombinant immunotoxin based on cucurmosin shows potent anti-tumor efficiency in vitro","volume":"45","author":"Zhang","year":"2021","journal-title":"Oncol. Rep."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1093\/molehr\/4.12.1099","article-title":"The relationship between BcI2, Bax and p53: Consequences for cell cycle progression and cell death","volume":"4","author":"Basu","year":"1998","journal-title":"Mol. Hum. Reprod."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.molcel.2005.05.023","article-title":"Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis","volume":"19","author":"Yuan","year":"2005","journal-title":"Mol. Cell"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1146\/annurev.pathmechdis.3.121806.151523","article-title":"Metastatic cancer cell","volume":"3","author":"Bacac","year":"2008","journal-title":"Annu. Rev. Pathol."},{"key":"ref_48","first-page":"1179","article-title":"The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA\u2019 suppresses growth of a highly metastatic pancreatic carcinoma cell line","volume":"23","author":"Bruell","year":"2003","journal-title":"Int. J. Oncol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"2091","DOI":"10.1007\/s40995-019-00676-7","article-title":"Co-solvents effects on the stability of recombinant immunotoxin denileukin diftitox: Structure and function assessment","volume":"43","author":"Bayat","year":"2019","journal-title":"Iran. J. Sci. Technol. Trans. A Sci."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/11\/749\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:21:28Z","timestamp":1760167288000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/11\/749"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,22]]},"references-count":49,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2021,11]]}},"alternative-id":["toxins13110749"],"URL":"https:\/\/doi.org\/10.3390\/toxins13110749","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,22]]}}}