华蟾毒精通过调节肿瘤相关巨噬细胞中的糖代谢重编程抑制非小细胞肺癌的侵袭和迁移。

Cinobufagin Inhibits Invasion and Migration of Non-Small Cell Lung Cancer via Regulating Glucose Metabolism Reprogramming in Tumor-Associated Macrophages.

作者信息

Sun Ying, Yang Huitong, Mei Xue, Xia Jinchan, Feng Long, Gao Jianfeng, Jiang Wei, Jiang Min, Hao Xu, Feng Yilin, Lian Yunfeng

机构信息

Medical College, Henan University of Chinese Medicine, Zhengzhou, Henan, 450046, People's Republic of China.

出版信息

Drug Des Devel Ther. 2025 Aug 2;19:6647-6664. doi: 10.2147/DDDT.S531190. eCollection 2025.

DOI:10.2147/DDDT.S531190

PMID:40771860

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12327427/

Abstract

BACKGROUND: The immunosuppressive tumor microenvironment (TME) in lung cancer, driven in part by M2-polarized tumor-associated macrophages (TAMs), contributes to worse prognosis and supports tumor progression. Cinobufagin (CB), an active compound in cinobufotalin injections, has demonstrated potential antitumor effects by modulating macrophage activity. This study investigated the mechanism by which CB influences glucose metabolism and polarization in M2 TAMs by focusing on the regulation of HIF-1α. METHODS: Human THP-1 monocytes were differentiated into M2 macrophages by stimulation with interleukin-4 at 20 ng/mL and then treated with cinobufagin at 2 μM, either alone or together with the HIF-1α activator DMOG at 1 mM. HIF-1α hydroxylation and ubiquitination were evaluated by Western blot and co-immunoprecipitation. Glycolytic activity was determined by measuring uptake of the glucose analogue 2-NBDG, extracellular lactate levels and expression of GLUT1, PKM2, LDHA and MCT1. M2 polarization markers CD206, Arg-1 and IL-10 were quantified by qRT-PCR, and TGF-β and IL-10 secretion was measured by ELISA. PD-L1 expression was assessed by Western blot, immunofluorescence and chromatin immunoprecipitation. Finally, conditioned media from treated macrophages were applied to A549 cells to evaluate migration through wound-healing assays and invasion using Transwell inserts, and to HUVECs to quantify tube formation. RESULTS: Using DMOG, an HIF-1α activator, we stimulated glycolysis in M2 macrophages, promoting their immunosuppressive polarization and elevating PD-L1 expression, a checkpoint protein associated with immune evasion. CB treatment reversed this effect by increasing HIF-1α hydroxylation and ubiquitination, leading to decreased HIF-1α stability, glucose uptake, and lactate production in M2 macrophages. Additionally, CB pre-treatment of M2 macrophages reduced the secretion of the cytokines TGF-β and IL-10, thereby limiting lung cancer cell migration, invasion, and angiogenesis. CONCLUSION: These findings suggest that CB suppresses M2 macrophage-mediated tumor support by targeting HIF-1α and glycolysis, thereby reprogramming the TME toward an anti-tumor state. This highlights CB's potential of CB in the treatment of lung cancer by countering immunosuppressive macrophage activity.

摘要

背景：肺癌中的免疫抑制性肿瘤微环境（TME）部分由M2极化的肿瘤相关巨噬细胞（TAM）驱动，其会导致预后更差并促进肿瘤进展。华蟾毒精（CB）是蟾酥注射液中的一种活性化合物，已证明其可通过调节巨噬细胞活性发挥潜在的抗肿瘤作用。本研究通过聚焦于缺氧诱导因子-1α（HIF-1α）的调节，探讨了CB影响M2 TAM中葡萄糖代谢和极化的机制。方法：用人白细胞介素-4以20 ng/mL刺激人THP-1单核细胞，使其分化为M2巨噬细胞，然后用2 μM华蟾毒精单独处理，或与1 mM的HIF-1α激活剂二甲基乙二酰甘氨酸（DMOG）联合处理。通过蛋白质免疫印迹法和免疫共沉淀法评估HIF-1α的羟基化和泛素化。通过测量葡萄糖类似物2-脱氧葡萄糖（2-NBDG）摄取、细胞外乳酸水平以及葡萄糖转运蛋白1（GLUT1）、丙酮酸激酶M2（PKM2）、乳酸脱氢酶A（LDHA）和单羧酸转运蛋白1（MCT1）的表达来测定糖酵解活性。通过实时定量聚合酶链反应（qRT-PCR）对M2极化标志物CD206、精氨酸酶1（Arg-1）和白细胞介素-10（IL-10）进行定量，并通过酶联免疫吸附测定（ELISA）测量转化生长因子-β（TGF-β）和IL-10的分泌。通过蛋白质免疫印迹法、免疫荧光法和染色质免疫沉淀法评估程序性死亡受体配体1（PD-L1）表达。最后，将处理过的巨噬细胞的条件培养基应用于A549细胞，通过伤口愈合试验评估迁移能力，使用Transwell小室评估侵袭能力，并应用于人类脐静脉内皮细胞（HUVEC）以定量血管生成。结果：使用HIF-1α激活剂DMOG刺激M2巨噬细胞中的糖酵解，促进其免疫抑制性极化并提高PD-L1表达，PD-L1是一种与免疫逃逸相关的检查点蛋白。CB处理通过增加HIF-1α的羟基化和泛素化来逆转这种作用，导致M2巨噬细胞中HIF-1α稳定性降低、葡萄糖摄取和乳酸产生减少。此外，对M2巨噬细胞进行CB预处理可减少细胞因子TGF-β和IL-10的分泌，从而限制肺癌细胞的迁移、侵袭和血管生成。结论：这些发现表明，CB通过靶向HIF-1α和糖酵解来抑制M2巨噬细胞介导的肿瘤支持作用，从而将TME重编程为抗肿瘤状态。这突出了CB通过对抗免疫抑制性巨噬细胞活性在肺癌治疗中的潜力。