Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Department of Pharmacognosy, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
BMC Cancer. 2024 Jan 24;24(1):125. doi: 10.1186/s12885-024-11898-3.
T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1.
Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively.
The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001).
Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.
T 细胞免疫球蛋白和粘蛋白结构域蛋白 3（TIM-3）是一种细胞表面分子，最初在 T 细胞中发现。然而，最近的研究表明，它在急性髓系白血病（AML）细胞中也高度表达，与 AML 的进展有关。由于谷氨酰胺似乎在恶性肿瘤的进展中起着突出的作用，尤其是在其髓样细胞群中，因此，在这项研究中，我们旨在评估 TIM-3/半乳糖凝集素-9 轴与两种 AML 细胞系 HL-60 和 THP-1 中的谷氨酰胺代谢之间的关系。
细胞系在补充有 10%胎牛血清和 1%抗生素的 RPMI 1640 中培养。在添加重组半乳糖凝集素-9（Gal-9）后 24、48 和 72 小时，进行 RT-qPCR 分析、反相高效液相色谱法和气相色谱技术，分别评估谷氨酰胺酶（GLS）、谷氨酸脱氢酶（GDH）酶、代谢物浓度；谷氨酸（Glu）和α-酮戊二酸（α-KG）在谷氨酰胺分解途径中的表达。Western blot 和 MTT 测定分别用于检测哺乳动物雷帕霉素靶蛋白复合物（mTORC）作为信号因子、GLS 蛋白和细胞增殖率的表达。
在 Gal-9 处理后 72 小时，HL-60 细胞中 GLS 和 GDH 的 mRNA 表达最为明显（p=0.001，p=0.0001），而在 THP-1 细胞系中则在 Gal-9 加入后 24 小时观察到（p=0.001，p=0.0001）。在 HL-60 和 THP-1 细胞中，mTORC 和 GLS 蛋白的表达在 Gal-9 处理后 72 和 24 小时最为明显（p=0.0001）。MTT 测定表明 Gal-9 可促进两种细胞系的细胞增殖率（p=0.001）。HL-60 细胞中 Glu 浓度和 HL-60（p=0.03）和 THP-1（p=0.0001）细胞系中α-KG 浓度均呈下降趋势。但是，THP-1 细胞系中的 Glu 浓度呈上升趋势（p=0.0001）。
综上所述，本研究表明 TIM-3/半乳糖凝集素-9 相互作用可促进 HL-60 和 THP-1 细胞中的谷氨酰胺代谢，从而导致 AML 的发展。
