Department of Anatomy, Histology and Embryology, Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan 610075, P.R. China.
Mol Med Rep. 2019 Sep;20(3):2347-2354. doi: 10.3892/mmr.2019.10452. Epub 2019 Jul 1.
The purpose of the present study was to investigate the effect of bufadienolides on the A‑375 melanoma cell line, and to delineate the underlying mechanism. A Cell Counting Kit‑8 assay was used to determine the viability of the cells, and flow cytometry was used to evaluate apoptosis. Western blot analysis was used to evaluate the expression levels of proteins involved in the AKT pathway that are associated with apoptosis and autophagy. The results demonstrated that bufadienolides reduced the viability of A‑375 cells in a dose‑ and a time‑dependent manner. Following treatment with bufadienolides, A‑375 cells exhibited clear properties that were characteristic of apoptosis and autophagy. The expression levels of the pro‑apoptotic proteins Bax and p53 were upregulated, whereas those of the anti‑apoptotic proteins, Bcl‑2 and caspase‑3 were downregulated. In addition, the level of a protein known to be associated with autophagy, microtubule‑associated proteins 1A/1B light chain 3‑II, was increased, whereas that of p62 protein was reduced. Finally, the AKT signaling pathway was blocked in the bufadienolide‑treated A‑375 cells. In conclusion, these results revealed that bufadienolides effectively induced apoptosis and autophagy in A‑375 cells via the AKT pathway, and therefore may be one of the candidate targets for the future development of targeted drugs to treat melanoma.
本研究旨在探讨蟾毒内酯对 A-375 黑素瘤细胞系的影响，并阐明其潜在的作用机制。采用细胞计数试剂盒-8 法检测细胞活力，采用流式细胞术评估细胞凋亡。Western blot 分析用于评估与凋亡和自噬相关的 AKT 通路中参与的蛋白的表达水平。结果表明，蟾毒内酯呈剂量和时间依赖性地降低 A-375 细胞的活力。蟾毒内酯处理后，A-375 细胞表现出明显的凋亡和自噬特征。促凋亡蛋白 Bax 和 p53 的表达上调，而抗凋亡蛋白 Bcl-2 和 caspase-3 的表达下调。此外，与自噬相关的蛋白微管相关蛋白 1A/1B 轻链 3-II 的水平增加，而 p62 蛋白的水平降低。最后，AKT 信号通路在蟾毒内酯处理的 A-375 细胞中被阻断。综上所述，这些结果表明蟾毒内酯通过 AKT 通路有效诱导 A-375 细胞发生凋亡和自噬，因此可能是未来开发治疗黑色素瘤的靶向药物的候选靶点之一。
