{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:19:34Z","timestamp":1785403174402,"version":"3.56.0"},"reference-count":59,"publisher":"MDPI AG","issue":"6","license":[{"start":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T00:00:00Z","timestamp":1781136000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Indonesian Education Scholarship"},{"name":"Center for Higher Education Funding and Assessment"},{"name":"Ministry of Higher Education, Science, and Technology of the Republic of Indonesia"},{"DOI":"10.13039\/501100014538","name":"Indonesia Endowment Fund for Education","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100014538","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/05p2xef58","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National Institute on Aging, National Institutes of Health","award":["R01-AG082273"],"award-info":[{"award-number":["R01-AG082273"]}]},{"name":"National Institute on Aging, National Institutes of Health","award":["R01-AG071682"],"award-info":[{"award-number":["R01-AG071682"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Life"],"abstract":"<jats:p>The ICAM-1-derived cIBR peptide selectively binds to the I-domain of LFA-1, a receptor highly expressed on leukemia T cells; thus, the MTX-cIBR conjugate can be used to target methotrexate (MTX) to leukemic T cells and reduce its off-target toxicity. However, the uptake, biological mechanism, and selectivity of MTX-cIBR compared with unconjugated MTX remain unclear. Therefore, this study is aimed at evaluating the uptake, cytotoxicity, selectivity, apoptosis, cell cycle effects, and DHFR-related activity of MTX-cIBR in leukemia T cells compared with unconjugated MTX. MTX-cIBR exhibited cytotoxic activity comparable to MTX in LFA-1-expressing MOLT-4 cells but showed lower toxicity toward LFA-1-negative K562 cells, indicating improved selectivity. MTX uptake occurred through RFC and mFBP transport systems, whereas MTX-cIBR no longer depended on these pathways, suggesting altered cellular uptake after conjugation with cIBR by utilizing the LFA-1 receptor. Both compounds predominantly induced apoptosis with minimal necrotic cell populations. MTX induced S-phase arrest at lower concentrations and G2\/M induced arrest at higher concentrations, whereas MTX-cIBR consistently promoted S-phase accumulation. In addition, MTX and MTX-cIBR downregulated the expression of DHFR, FPGS, and TYMS in MOLT-4 cells. Computational analyses further demonstrated that MTX exhibited lower binding free energy (\u0394G) and greater binding stability toward DHFR than MTX-cIBR. These findings suggest that MTX-cIBR retains selective cytotoxic activity toward LFA-1-expressing leukemia T cells through altered cellular uptake and exhibits different interaction characteristics with DHFR compared with unconjugated MTX.<\/jats:p>","DOI":"10.3390\/life16060981","type":"journal-article","created":{"date-parts":[[2026,6,11]],"date-time":"2026-06-11T06:57:17Z","timestamp":1781161037000},"page":"981","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Selectively Targeting Leukemic MOLT-4 Cells by MTX-cIBR Conjugate: Mechanism of Action and Cellular Entry"],"prefix":"10.3390","volume":"16","author":[{"given":"Sista","family":"Werdyani","sequence":"first","affiliation":[{"name":"Study Program of Doctor in Biotechnology, Graduate School, Universitas Gadjah Mada, Jl. Teknika Utara, Kocoran, Caturtunggal, Depok, Sleman, Yogyakarta 55281, Indonesia"},{"name":"Biomedical Laboratory, Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Islam Indonesia, Jalan Kaliurang Km 14.5, Sleman, Yogyakarta 55584, Indonesia"},{"name":"Simons Laboratories, Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meagan E.","family":"Weldele","sequence":"additional","affiliation":[{"name":"Simons Laboratories, Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8344-5587","authenticated-orcid":false,"given":"Enade P.","family":"Istyastono","sequence":"additional","affiliation":[{"name":"Department of Pharmacy, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, Jl. Jenderal Sudirman, Karet Semanggi, Jakarta 12930, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sofia M.","family":"Harjana","sequence":"additional","affiliation":[{"name":"Department of Histology and Cell Biology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jl. Farmako, Sekip Utara, Depok, Sleman, Yogyakarta 55281, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adi","family":"Hermawansyah","sequence":"additional","affiliation":[{"name":"Cell Culture Laboratory, Directorate of Innovation and Downstreaming, Universitas Muhammadiyah Yogyakarta, Jl. Brawijaya, Tamantirto, Kasihan, Bantul, Yogyakarta 55183, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-9833-8170","authenticated-orcid":false,"given":"Wariya","family":"Nirachonkul","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, The University of Kansas, 1251 Wescoe Dr, Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6759-8658","authenticated-orcid":false,"given":"Dewi K.","family":"Paramita","sequence":"additional","affiliation":[{"name":"Department of Histology and Cell Biology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Jl. Farmako, Sekip Utara, Depok, Sleman, Yogyakarta 55281, Indonesia"},{"name":"Research Center for Biotechnology, Universitas Gadjah Mada, Jl. Teknika Utara, Kocoran, Caturtunggal, Depok, Sleman, Yogyakarta 55281, Indonesia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7250-0627","authenticated-orcid":false,"given":"Teruna J.","family":"Siahaan","sequence":"additional","affiliation":[{"name":"Simons Laboratories, Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave., Lawrence, KS 66047, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Huang, J., Chan, S.C., Ngai, C.H., Lok, V., Zhang, L., Lucero-Prisno, D.E., Xu, W., Zheng, Z.J., Elcarte, E., and Withers, M. (2022). Disease Burden, Risk Factors, and Trends of Leukaemia: A Global Analysis. Front. 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