Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 0 0 2026 02 23 1 7 Amir Kahrizi Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Armin Akbar Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Ahmad Najafi Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Hossein Asgarian-Omran Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran AND Gastrointestinal Cancer Research Center, Mazandaran University of Medical Sciences, Sari, Iran Reza Valadan Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran AND Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran Mohammad Mehri Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran Mohsen Tehrani Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran AND Molecular and Cell-Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran 2025 06 02 2025 11 04 The Warburg effect is one of the most important metabolic alterations in tumor cells. Hypoxia-inducible factor 1-alpha (HIF-1α) targets a broad range of gene promoters in normoxic and hypoxic conditions in cancers. Herein, we investigate the effects of HIF-1α inhibition on cell viability and messenger RNA (mRNA) expression of immune checkpoint receptors (ICRs) in acute myeloid leukemia cell lines. K-562 and HL-60 cells were treated with silibinin as an HIF-1α inhibitor. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, followed by quantification of V-domain immunoglobulin suppressor of T-cell activation (VISTA), T-cell immunoglobulin and mucin domain 3 (TIM3), and Galectin-9 mRNA expression via quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The expression levels of VISTA, TIM3, and Galectin-9 decreased after silibinin treatment within both K-562 and HL-60 cells; however, there were some disparities in gene expression levels between the two cell lines. VISTA and TIM3 expression were reduced by approximately 70% in K-562 at the 40% inhibitory concentration (IC40), while no significant changes were observed in HL-60 cells. Conversely, Galectin-9 expression was decreased significantly at both the IC30 and IC40 in HL-60, whereas it was almost consistent in K-562 cells.  Collectively, we have shown that silibinin could serve as a cytotoxic small-molecule inhibitor and regulate the expression of ICRs, potentially counteracting T-cell exhaustion. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/4476 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/4476/2314