Feng Hai-Tao, Zhao Wen-Wen, Lu Jin-Jian, Wang Yi-Tao, Chen Xiu-Ping
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China.
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China.
Chin J Nat Med. 2017 Jun;15(6):427-435. doi: 10.1016/S1875-5364(17)30064-X.
Epithelial-mesenchymal transition (EMT) has been implicated in tumor invasion and metastasis and provides novel strategies for cancer therapy. Hypaconitine (HpA), a diester-diterpenoid alkaloid isolated from the root of the Aconitum species, exhibits anti-inflammatory, analgesic, and especially, cardiotoxic activities. Here, we reported the anti-metastatic potentials of HpA in transforming growth factor-β1 (TGF-β1)-induced EMT in lung cancer A549 cells. The cytotoxic effect of HpA was determined by MTT assay. A549 cells were treated with TGF-β1 with or without HpA co-treatment, and the morphological alterations were observed with a microscopy. The expression of E-cadherin, N-cadherin, and NF-κB was determined by both Western blotting and immunofluorescence analyses. The adhesion, migration, and invasion were detected with Matrigel, wound-healing, and transwell assays, respectively. The expression of Snail was determined by Western blotting. The expression of NF-κB p65, IκBα, and p-IκBα in nuclear and cytosolic extracts was assessed by Western blotting. The results showed that low concentration of HpA (<16 μmol·L) had no obvious cytotoxicity to A549 cells. Morphologically, TGF-β1 treatment induced spindle-shaped alteration in the cells. The upregulation of N-cadherin, NF-κB, and Snail and the downregulation of E-cadherin were detected after TGF-β1 treatment. The adhesion, migration and invasion abilities were also increased by TGF-β1. Besides, TGF-β1 induced expression of Snail in a time-dependent manner. Furthermore, TGF-β1 induced nuclear translocation of NF-κB p65. All these alterations were dramatically inhibited by HpA co-treatment. In addition, the NF-κB inhibitor PDTC showed similar inhibitory effect. In conclusion, these results showed that HpA inhibited TGF-β1-induced EMT in A549 cells, which was possibly mediated by the inactivation of the NF-κB signaling pathway, providing an evidence for anti-cancer effect of HpA.
上皮-间质转化（EMT）与肿瘤侵袭和转移有关，并为癌症治疗提供了新策略。次乌头碱（HpA）是从乌头属植物根部分离得到的一种二酯二萜生物碱，具有抗炎、镇痛作用，尤其是心脏毒性作用。在此，我们报道了HpA在转化生长因子-β1（TGF-β1）诱导的肺癌A549细胞上皮-间质转化中的抗转移潜力。通过MTT法测定HpA的细胞毒性作用。用或不用HpA共同处理的情况下，用TGF-β1处理A549细胞，并用显微镜观察形态学改变。通过蛋白质免疫印迹法和免疫荧光分析测定E-钙黏蛋白、N-钙黏蛋白和核因子κB（NF-κB）的表达。分别用基质胶、伤口愈合和Transwell实验检测细胞黏附、迁移和侵袭能力。通过蛋白质免疫印迹法测定Snail的表达。通过蛋白质免疫印迹法评估核提取物和细胞质提取物中NF-κB p65、IκBα和磷酸化IκBα（p-IκBα）的表达。结果表明，低浓度的HpA（<16 μmol·L）对A549细胞无明显细胞毒性。形态学上，TGF-β1处理诱导细胞呈纺锤形改变。TGF-β1处理后检测到N-钙黏蛋白、NF-κB和Snail上调，E-钙黏蛋白下调。TGF-β1还增加了细胞的黏附、迁移和侵袭能力。此外，TGF-β1以时间依赖性方式诱导Snail表达。此外，TGF-β1诱导NF-κB p65核转位。HpA共同处理可显著抑制所有这些改变。此外，NF-κB抑制剂PDTC显示出类似的抑制作用。总之，这些结果表明，HpA抑制TGF-β1诱导的A549细胞上皮-间质转化，这可能是由NF-κB信号通路失活介导的，为HpA的抗癌作用提供了证据。
