Orexin A induces autophagy in HCT-116 human colon cancer cells through the ERK signaling pathway
Orexin A induces autophagy in HCT-116 human colon cancer cells through the ERK signaling pathway
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Orexin A 通过 ERK 信号通路诱导 HCT-116 人结肠癌细胞自噬
DOI：
10.3892/ijmm.2015.2409
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发表时间：
2016-01-01
影响因子：
5.4
通讯作者：
Guo, Lei
中科院分区：
文献类型：
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作者：
Wen, Jing;Zhao, Yuyan;Guo, Lei
食欲素是一类对多种癌细胞具有有效影响的肽。自噬与肿瘤密切相关;然而，其功能尚未完全了解。在这项研究中，我们的目的是确定食欲素A是否诱导HCT-116人结肠癌细胞的自噬，并阐明所涉及的分子机制。为此目的，HCT-116细胞用食欲素A处理，然后通过MTT法测定细胞活力，并通过流式细胞术测定细胞凋亡。蛋白质印迹法检测自噬相关蛋白的表达水平。吖啶橙子（AO）染色后，使用荧光显微镜进行自噬的定量分析，并在透射电子显微镜下观察细胞形态。此外，HCT-116细胞用细胞外信号调节激酶（ERK）抑制剂U 0126或自噬抑制剂氯喹与食欲素A组合处理，以检查ERK的活化。我们发现，食欲素A显着抑制HCT-116细胞的活力。在食欲素A诱导的HCT-116细胞死亡过程中，自噬和凋亡均被激活。当用食欲素A处理HCT-116细胞24小时时，还检测到点状微管相关蛋白-1轻链3（LC 3）的积累和LC 3-II蛋白水平的增加，表明自噬的激活。此外，增食欲素A上调ERK磷酸化，然而，U 0126或氯喹废除ERK磷酸化和减少自噬，与单独的增食欲素A治疗相比。因此，我们的研究结果表明食欲素A通过ERK途径诱导HCT-116人结肠癌细胞的自噬。因此，自噬的抑制可能被证明是增强食欲素A作为结肠癌治疗的抗肿瘤潜力的有效策略。
Orexins are a class of peptides which have a potent influence on a broad variety of cancer cells. Autophagy is closely associated with tumors; however, its function is not yet completely understood. In this study, we aimed to determine whether orexin A induces autophagy in HCT-116 human colon cancer cells and to elucidate the molecular mechanisms involved. For this purpose, HCT-116 cells were treated with orexin A, and cell viability was then measured by MTT assay, and apoptosis was determined by flow cytometry. The expression levels of autophagy-related proteins were measured by western blot analysis. Quantitative analysis of autophagy following acridine orange (AO) staining was performed using fluorescence microscopy, and cellular morphology was observed under a transmission electron microscope. In addition, the HCT-116 cells were treated with the extracellular signal-regulated kinase (ERK) inhibitor, U0126, or the autophagy inhibitor, chloroquine, in combination with orexin A in order to examine the activation of ERK. We found that orexin A significantly inhibited the viability of the HCT-116 cells. Both autophagy and apoptosis were activated during the orexin A-induced death of HCT-116 cells. When the HCT-116 cells were treated with orexin A for 24 h, an accumulation of punctate microtubule-associated protein-1 light chain 3 (LC3) and an increase in LC3-II protein levels were also detected, indicating the activation of autophagy. Moreover, orexin A upregulated ERK phosphorylation; however, U0126 or chloroquine abrogated ERK phosphorylation and decreased autophagy, compared to treatment with orexin A alone. Therefore, our findings demonstratedm that orexin A induced autophagy through the ERK pathway in HCT-116 human colon cancer cells. The inhibition of autophagy may thus prove to be an effective strategy for enhancing the antitumor potential of orexin A as a treatment for colon cancer.