{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T11:31:46Z","timestamp":1780486306064,"version":"3.54.1"},"reference-count":49,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2025,12,13]],"date-time":"2025-12-13T00:00:00Z","timestamp":1765584000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100006769","name":"Russian Science Foundation","doi-asserted-by":"publisher","award":["24-25-00344"],"award-info":[{"award-number":["24-25-00344"]}],"id":[{"id":"10.13039\/501100006769","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Cutaneous melanoma is an extremely dangerous tumor disease with poor prognosis at advanced stages. Accounting for a small percentage of all skin tumors, malignant melanoma leads the mortality rate in this group of cancers. Clearly, the search for new drugs and therapeutic approaches for the treatment of cutaneous melanoma is a highly pressing issue in modern medicine. In this study, novel recombinant proteins with anti-melanoma activity, called RGD-apoptins, were produced in an E. coli expression system, and their properties were evaluated in human cell models. These chimeric proteins consist of two parts, each tumor-specific. One part of the chimeric molecule is the RGD peptide, which binds to \u03b1V\u03b23 integrins widely expressed on the surface of malignant melanocytes. The other part is the viral protein apoptin, known to induce programmed cell death in tumor cells but not in normal cells. This molecular design aims to enhance the specificity of potential therapeutic agent toward malignant melanoma cells while reducing cytolytic effects on healthy tissue. In a resazurin assay, RGD-apoptins decreased the viability of MeWo human melanoma cells and did not affect the viability of HaCaT human keratinocyte cell line and primary skin fibroblasts. Using an annexin V assay, we confirmed that malignant melanocytes death occurs via apoptosis. Transcriptomic analysis allowed us to dynamically evaluate the spectrum of differentially expressed genes 24 and 48 h after treating melanoma cells with recombinant RGD-apoptin.<\/jats:p>","DOI":"10.3390\/ijms262412016","type":"journal-article","created":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:36:39Z","timestamp":1765791399000},"page":"12016","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Recombinant RGD-Apoptins Decrease Human Melanoma Cell Viability"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6080-3348","authenticated-orcid":false,"given":"Dmitriy","family":"Shirokov","sequence":"first","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"},{"name":"K.I. Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology, Moscow 109472, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daria","family":"Lepekhina","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2471-0563","authenticated-orcid":false,"given":"Valentin","family":"Manuvera","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"},{"name":"Moscow Center for Advanced Studies, Moscow 123592, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0945-6891","authenticated-orcid":false,"given":"Margarita","family":"Bogomiakova","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9470-7640","authenticated-orcid":false,"given":"Aleksandra","family":"Strokach","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anastasia","family":"Kazakova","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"},{"name":"Moscow Center for Advanced Studies, Moscow 123592, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2323-1859","authenticated-orcid":false,"given":"Georgij","family":"Arapidi","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"},{"name":"Moscow Center for Advanced Studies, Moscow 123592, Russia"},{"name":"Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vassili","family":"Lazarev","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Moscow 119435, Russia"},{"name":"Moscow Center for Advanced Studies, Moscow 123592, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/s41572-025-00603-8","article-title":"Cutaneous melanoma","volume":"11","author":"Tasdogan","year":"2025","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Caraviello, C., Nazzaro, G., Tavoletti, G., Boggio, F., Denaro, N., Murgia, G., Passoni, E., Benzecry Mancin, V., and Marzano, A.V. (2025). Melanoma Skin Cancer: A Comprehensive Review of Current Knowledge. Cancers, 17.","DOI":"10.3390\/cancers17172920"},{"key":"ref_3","first-page":"1554","article-title":"Clinical significance of alpha(v)beta3 integrin and intercellular adhesion molecule-1 expression in cutaneous malignant melanoma lesions","volume":"57","author":"Natali","year":"1997","journal-title":"Cancer Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1038\/309030a0","article-title":"Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule","volume":"309","author":"Pierschbacher","year":"1984","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1038\/nrc2748","article-title":"Integrins in cancer: Biological implications and therapeutic opportunities","volume":"10","author":"Desgrosellier","year":"2010","journal-title":"Nat. Rev. Cancer"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1038\/nrd.2015.10","article-title":"Integrin-based therapeutics: Biological basis, clinical use and new drugs","volume":"15","author":"Ley","year":"2016","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1038\/s41573-021-00284-4","article-title":"Emerging therapeutic opportunities for integrin inhibitors","volume":"21","author":"Slack","year":"2022","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1182\/blood.V80.2.437.437","article-title":"Human endothelial cells express integrin receptors on the luminal aspect of their membrane","volume":"80","author":"Conforti","year":"1992","journal-title":"Blood"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"724","DOI":"10.1016\/S0955-0674(96)80115-3","article-title":"Integrins and cancer","volume":"8","author":"Varner","year":"1996","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"837","DOI":"10.1038\/mt.2009.29","article-title":"Clustering and internalization of integrin alphavbeta3 with a tetrameric RGD-synthetic peptide","volume":"17","author":"Sancey","year":"2009","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1093\/protein\/gzs076","article-title":"Preparation and functional evaluation of RGD-modified streptavidin targeting to integrin-expressing melanoma cells","volume":"26","author":"Syrkina","year":"2013","journal-title":"Protein Eng. Des. Sel. PEDS"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"227","DOI":"10.3103\/S0027131416040076","article-title":"Tetrameric RGD induces clustering of integrin \u03b1v\u03b23 on the melanoma cell surface and decreases cell viability","volume":"71","author":"Rubtsov","year":"2016","journal-title":"Mosc. Univ. Chem. Bull."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1093\/protein\/gzh055","article-title":"Novel RGD lipopeptides for the targeting of liposomes to integrin-expressing endothelial and melanoma cells","volume":"17","author":"Bach","year":"2004","journal-title":"Protein Eng. Des. Sel. PEDS"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5843","DOI":"10.1073\/pnas.94.11.5843","article-title":"Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells","volume":"94","author":"Fischer","year":"1997","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Malla, W.A., Arora, R., Khan, R.I.N., Mahajan, S., and Tiwari, A.K. (2020). Apoptin as a Tumor-Specific Therapeutic Agent: Current Perspective on Mechanism of Action and Delivery Systems. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.00524"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1146\/annurev.pharmtox.48.121806.154910","article-title":"Apoptin: Therapeutic potential of an early sensor of carcinogenic transformation","volume":"48","author":"Backendorf","year":"2008","journal-title":"Annu. Rev. Pharmacol. Toxicol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4485","DOI":"10.1242\/jcs.02580","article-title":"Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway","volume":"118","author":"Maddika","year":"2005","journal-title":"J. Cell Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1952","DOI":"10.1101\/gad.1198404","article-title":"The viral protein Apoptin associates with the anaphase-promoting complex to induce G2\/M arrest and apoptosis in the absence of p53","volume":"18","author":"Teodoro","year":"2004","journal-title":"Genes Dev."},{"key":"ref_19","first-page":"357","article-title":"Genetically engineered fusion proteins for treatment of cancer","volume":"9","author":"Weidle","year":"2012","journal-title":"Cancer Genom. Proteom."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1173","DOI":"10.1126\/science.1683495","article-title":"Recombinant toxins for cancer treatment","volume":"254","author":"Pastan","year":"1991","journal-title":"Science"},{"key":"ref_21","first-page":"44","article-title":"Recombinant toxins for the treatment of cancer","volume":"5","author":"Kreitman","year":"2003","journal-title":"Curr. Opin. Mol. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1153","DOI":"10.1038\/sj.onc.1207224","article-title":"TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells","volume":"23","author":"Guelen","year":"2004","journal-title":"Oncogene"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"49075","DOI":"10.18632\/oncotarget.8682","article-title":"Efficient therapeutic delivery by a novel cell-permeant peptide derived from KDM4A protein for antitumor and antifibrosis","volume":"7","author":"Wang","year":"2016","journal-title":"Oncotarget"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"936","DOI":"10.1002\/prot.26285","article-title":"A novel chimeric protein with enhanced cytotoxic effects on breast cancer in vitro and in vivo","volume":"90","author":"Hazrati","year":"2022","journal-title":"Proteins"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Wyatt, J., M\u00fcller, M.M., and Tavassoli, M. (2019). Cancer Treatment Goes Viral: Using Viral Proteins to Induce Tumour-Specific Cell Death. Cancers, 11.","DOI":"10.3390\/cancers11121975"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"105687","DOI":"10.1016\/j.pep.2020.105687","article-title":"Generation of a soluble and stable apoptin-EGF fusion protein, a targeted viral protein applicable for tumor therapy","volume":"175","author":"Niesler","year":"2020","journal-title":"Protein Expr. Purif."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/1476-4598-9-10","article-title":"Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo","volume":"9","author":"Li","year":"2010","journal-title":"Mol. Cancer"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.peptides.2003.12.002","article-title":"Conformational resemblance between the structures of integrin-activating pentapetides derived from betaig-h3 and RGD peptide analogues in a membrane environment","volume":"25","author":"Park","year":"2004","journal-title":"Peptides"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1535","DOI":"10.1002\/anie.201508709","article-title":"Stable Peptides Instead of Stapled Peptides: Highly Potent \u03b1v\u03b26-Selective Integrin Ligands","volume":"55","author":"Maltsev","year":"2016","journal-title":"Angew. Chem. Int. Ed."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"39805","DOI":"10.1038\/srep39805","article-title":"A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins","volume":"7","author":"Kapp","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3417","DOI":"10.1021\/mp400248f","article-title":"Evaluation of new Tc-99m-labeled Arg-X-Asp-conjugated \u03b1-melanocyte stimulating hormone peptides for melanoma imaging","volume":"10","author":"Flook","year":"2013","journal-title":"Mol. Pharm."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.jmoldx.2012.10.002","article-title":"Frequency and spectrum of BRAF mutations in a retrospective, single-institution study of 1112 cases of melanoma","volume":"15","author":"Greaves","year":"2013","journal-title":"J. Mol. Diagn. JMD"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Castellani, G., Buccarelli, M., Arasi, M.B., Rossi, S., Pisanu, M.E., Bellenghi, M., Lintas, C., and Tabolacci, C. (2023). BRAF Mutations in Melanoma: Biological Aspects, Therapeutic Implications, and Circulating Biomarkers. Cancers, 15.","DOI":"10.3390\/cancers15164026"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1038\/sj.cdd.4402291","article-title":"The mitochondrial serine protease HtrA2\/Omi: An overview","volume":"15","author":"Lamkanfi","year":"2008","journal-title":"Cell Death Differ."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"S84","DOI":"10.1038\/onc.2009.46","article-title":"Noxa: At the tip of the balance between life and death","volume":"27","author":"Ploner","year":"2008","journal-title":"Oncogene"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"939","DOI":"10.1038\/cdd.2009.16","article-title":"Role of BNIP3 and NIX in cell death, autophagy, and mitophagy","volume":"16","author":"Zhang","year":"2009","journal-title":"Cell Death Differ."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1359","DOI":"10.1016\/S0960-9822(00)00781-8","article-title":"ML-IAP, a novel inhibitor of apoptosis that is preferentially expressed in human melanomas","volume":"10","author":"Vucic","year":"2000","journal-title":"Curr. Biol. CB"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"4321","DOI":"10.1073\/pnas.1205575110","article-title":"BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition","volume":"110","author":"Haq","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1087","DOI":"10.1158\/1535-7163.MCT-23-0025","article-title":"Targeting Upregulated cIAP2 in SOX10-Deficient Drug Tolerant Melanoma","volume":"22","author":"Glasheen","year":"2023","journal-title":"Mol. Cancer Ther."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1249","DOI":"10.1007\/s00018-014-1791-0","article-title":"MITF in melanoma: Mechanisms behind its expression and activity","volume":"72","author":"Hartman","year":"2015","journal-title":"Cell. Mol. Life Sci. CMLS"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Singh, M., Cai, H., Bunse, M., Feschotte, C., and Izsv\u00e1k, Z. (2020). Human Endogenous Retrovirus K Rec forms a Regulatory Loop with MITF that Opposes the Progression of Melanoma to an Invasive Stage. Viruses, 12.","DOI":"10.1101\/2020.05.27.120170"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"149834","DOI":"10.1016\/j.bbrc.2024.149834","article-title":"Loss of mutant p53 in HaCaT keratinocytes promotes cadmium-induced keratin 17 expression and cell death","volume":"709","author":"Romashin","year":"2024","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A Flexible Trimmer for Illumina Sequence Data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1038\/nmeth.4197","article-title":"Salmon Provides Fast and Bias-Aware Quantification of Transcript Expression","volume":"14","author":"Patro","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1760","DOI":"10.1101\/gr.135350.111","article-title":"GENCODE: The Reference Human Genome Annotation for The ENCODE Project","volume":"22","author":"Harrow","year":"2012","journal-title":"Genome Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1521","DOI":"10.12688\/f1000research.7563.1","article-title":"Differential Analyses for RNA-Seq: Transcript-Level Estimates Improve Gene-Level Inferences","volume":"4","author":"Soneson","year":"2015","journal-title":"F1000Research"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","article-title":"Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2","volume":"15","author":"Love","year":"2014","journal-title":"Genome Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1089\/omi.2011.0118","article-title":"clusterProfiler: An R Package for Comparing Biological Themes among Gene Clusters","volume":"16","author":"Yu","year":"2012","journal-title":"Omics A J. Integr. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1039\/C5MB00663E","article-title":"ReactomePA: An R\/Bioconductor Package for Reactome Pathway Analysis and Visualization","volume":"12","author":"Yu","year":"2016","journal-title":"Mol. Biosyst."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/12016\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T09:42:03Z","timestamp":1765791723000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/26\/24\/12016"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,13]]},"references-count":49,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["ijms262412016"],"URL":"https:\/\/doi.org\/10.3390\/ijms262412016","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,13]]}}}