Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran.
Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran.
Hum Exp Toxicol. 2020 Aug;39(8):1031-1045. doi: 10.1177/0960327120909530. Epub 2020 Mar 13.
Calprotectin is a heterodimeric EF-hand Ca binding protein that is typically released by infiltrating polymorphonuclear leukocytes and macrophages. This protein is a key player linking inflammation and cancer. Due to the increased levels of calprotectin in different inflammatory diseases and cancer, it is considered as a marker for diagnostic purposes. In this study, we evaluated the mechanism of cell viability and apoptotic-inducing effects of recombinant human calprotectin (rhS100A8/S100A9) on the gastric adenocarcinoma (AGS), the most common type of gastric cancer cell line. AGS cells were exposed to the different concentrations (5-100 μg/ml) of calprotectin for 24, 48, and 72 h, and cell viability was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptotic-inducing effects of calprotectin were evaluated by sub-G1 cell cycle assay and Annexin V/propidium iodide double staining. Furthermore, real-time polymerase chain reaction and Western blot analysis were performed to evaluate the mechanism of action of calprotectin. Our findings indicated that calprotectin inhibits growth and viability of AGS cells in a time- and dose-dependent manner. The half-maximal inhibitory concentration values were measured as 85.77, 79.14, and 65.39 μg/ml for 24, 48, and 72 h, respectively. Additionally, we found that calprotectin downregulated the expression of antiapoptotic protein Bcl-2 and upregulated proapoptotic protein Bax in a time- and concentration-dependent fashion. Calprotectin also slightly upregulated the expression of extracellular signal-regulated protein kinase 2 (ERK2), while it significantly decreased the levels of phospho-ERK in a time-dependent manner. Overall, these findings indicated that calprotectin has cytotoxicity and apoptosis-inducing effects on AGS cell lines in high concentration by modulating Bax/Bcl-2 expression ratio accompanied by inhibition of ERK activation.
钙卫蛋白是一种异二聚体 EF 手钙结合蛋白，通常由浸润的多形核白细胞和巨噬细胞释放。这种蛋白质是连接炎症和癌症的关键因素。由于不同炎症性疾病和癌症中钙卫蛋白水平的升高，它被认为是一种用于诊断目的的标志物。在这项研究中，我们评估了重组人钙卫蛋白（rhS100A8/S100A9）对胃腺癌（AGS）-最常见的胃癌细胞系-细胞活力和诱导凋亡的作用机制。AGS 细胞暴露于不同浓度（5-100μg/ml）的钙卫蛋白 24、48 和 72 小时，通过 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐测定法评估细胞活力。通过亚 G1 细胞周期测定法和 Annexin V/碘化丙啶双重染色法评估钙卫蛋白诱导凋亡的作用。此外，进行实时聚合酶链反应和 Western blot 分析以评估钙卫蛋白的作用机制。我们的研究结果表明，钙卫蛋白以时间和剂量依赖的方式抑制 AGS 细胞的生长和活力。半最大抑制浓度值分别为 24、48 和 72 小时时的 85.77、79.14 和 65.39μg/ml。此外，我们发现钙卫蛋白以时间和浓度依赖的方式下调抗凋亡蛋白 Bcl-2 的表达，上调促凋亡蛋白 Bax 的表达。钙卫蛋白还略微上调细胞外信号调节激酶 2（ERK2）的表达，同时显著降低磷酸化 ERK 的水平，呈时间依赖性。总体而言，这些发现表明钙卫蛋白在高浓度下通过调节 Bax/Bcl-2 表达比值并抑制 ERK 激活对 AGS 细胞系具有细胞毒性和诱导凋亡作用。
