Sharma Jiten R, Agraval Hina, Yadav Umesh C S
School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat 382030, India.
Department of Medicine, National Jewish Health, Denver, CO 80206, USA.
Life Sci. 2023 Apr 1;318:121480. doi: 10.1016/j.lfs.2023.121480. Epub 2023 Feb 10.
An elevated level of galectin-3, a carbohydrate-binding lectin implicated in tumorigenesis, metastasis, and epithelial-mesenchymal transition (EMT), has been found in cigarette smokers. However, the regulation of its expression and role in the pathogenesis of CS-induced EMT and lung cancer metastasis is unclear. Here, we have investigated the mechanism of CS-induced and galectin-3-mediated EMT in airway epithelial cells (AECs).
A549 adenocarcinoma cells and primary small airway epithelial cells cultured on an air-liquid interface (ALI) were exposed to cigarette smoke extract (CSE), and MTT, trypan blue, migration, invasion, tumor spheroid and colony formation assays were performed to assess EMT phenotype. Immunoblotting was performed to assess EMT and stemness markers and other regulatory proteins.
CSE exposure affected cell survival and morphology, migration, invasion, and clonogenicity of AECs, which were concomitant with an increase in the expression of EMT markers, galectin-3, and runt-related transcription factor-2 (RUNX-2), an osteogenic transcription factor and upstream regulator of galectin-3. Chemical inhibition or silencing of RUNX-2 downregulated galectin-3 and modulated EMT marker expression, migration, invasion, and clonogenicity in CSE-exposed AECs. Recombinant human galectin-3 also induced EMT and stemness-associated changes in the AECs, and GB1107, a galectin-3 inhibitor, ameliorated these changes. Further, CSE-induced intracellular ROS enabled an increase in RUNX-2 and galectin-3 expression, which were reversed by n-acetyl-cysteine.
These results provide a novel mechanistic insight into CSE-induced EMT via RUNX-2/galectin-3 axis mediated through ROS, which promoted EMT-associated changes, including invasion, migration, and stemness in AECs, which could be implicated in CS-induced lung cancer progression.
在吸烟者中发现，半乳糖凝集素-3（一种与肿瘤发生、转移及上皮-间质转化（EMT）相关的碳水化合物结合凝集素）水平升高。然而，其表达调控及其在香烟烟雾（CS）诱导的EMT和肺癌转移发病机制中的作用尚不清楚。在此，我们研究了CS诱导的以及半乳糖凝集素-3介导的气道上皮细胞（AECs）EMT机制。
将在气液界面（ALI）培养的A549腺癌细胞和原代小气道上皮细胞暴露于香烟烟雾提取物（CSE），并进行MTT、台盼蓝、迁移、侵袭、肿瘤球和集落形成试验以评估EMT表型。进行免疫印迹以评估EMT和干性标志物及其他调节蛋白。
CSE暴露影响AECs的细胞存活和形态、迁移、侵袭及克隆形成能力，这与EMT标志物、半乳糖凝集素-3和 runt相关转录因子2（RUNX-2，一种成骨转录因子及半乳糖凝集素-3的上游调节因子）表达增加相关。RUNX-2的化学抑制或沉默下调了半乳糖凝集素-3，并调节了CSE暴露的AECs中EMT标志物的表达、迁移、侵袭及克隆形成能力。重组人半乳糖凝集素-3也诱导了AECs中的EMT和干性相关变化，而半乳糖凝集素-3抑制剂GB1107改善了这些变化。此外，CSE诱导的细胞内活性氧增加了RUNX-2和半乳糖凝集素-3的表达，而N-乙酰半胱氨酸可使其逆转。
这些结果为CS通过ROS介导的RUNX-2/半乳糖凝集素-3轴诱导EMT提供了新的机制见解，该轴促进了EMT相关变化，包括AECs中的侵袭、迁移和干性，这可能与CS诱导的肺癌进展有关。
