Wei Xue-Jiao, Liu Ya-Xin, Huang Hui-Ming, Ouyang Li-Shan, Xie Jin-Xin, Wang Long-Yan, Liu Dong-Xiao, Tu Peng-Fei, Hu Zhong-Dong
Modern Research Center for Traditional Chinese Medicine, School of Chinese Materia Medica, Beijing University of Chinese Medicine Beijing 100029, China.
Zhongguo Zhong Yao Za Zhi. 2022 Dec;47(23):6457-6465. doi: 10.19540/j.cnki.cjcmm.20220830.401.
The purpose of this study was to investigate the effect of Huaier extract supernatant(HES) on the proliferation, apoptosis, autophagy, and migration of human gastric cancer HGC-27 and MGC-803 cells and its molecular mechanisms. The main components in HES were preliminarily analyzed by high-performance liquid chromatography-mass spectrometry(HPLC-MS). Methyl thiazolyl tetrazolium(MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine(EdU) staining assay were used to explore the effect of HES on the proliferation of human gastric cancer HGC-27 and MGC-803 cells. Hoechst staining and flow cytometry assay were used to determine the effect of HES on apoptosis of human gastric cancer HGC-27 and MGC-803 cells. Acridine orange staining and cell scratch assay were used to determine the effect of HES on autophagy and migration of human gastric cancer HGC-27 and MGC-803 cells, respectively. Western blot was used to investigate the regulatory effect of HES on the expression levels of proteins related to apoptosis, epithelial-mesenchymal transition(EMT), and signaling pathways in human gastric cancer HGC-27 and MGC-803 cells. The results showed that HES mainly contained some components with high polarities. HES significantly reduced the cell viability of human gastric cancer cells in a dose-and time-dependent manner. The IC_(50 )values after 48 h of HES treatment in human gastric cancer HGC-27 and MGC-803 cells were 7.56 and 10.77 g·L(-1), respectively. Meanwhile, HES inhibited the colony-forming ability and short-term proliferation of human gastric cancer cells. The apoptosis rates of HGC-27 and MGC-803 cells treated with 8 g·L(-1) HES for 72 h were 62.13%±8.92% and 54.50%±3.26%, respectively. HES also promoted autophagy in human gastric cancer cells and impaired their migration ability in vitro. Moreover, HES up-regulated the cleavage of the apoptosis marker poly ADP-ribose polymerase(PARP) and the protein expression level of the epithelial cell marker E-cadherin, and down-regulated the protein levels of phosphorylated-mammalian target of rapamycin(p-mTOR), phosphorylated-S6(p-S6), and phosphorylated-extracellular signal-regulated kinase(p-ERK) in human gastric cancer cells. Therefore, HES is one of the effective anti-tumor components of Huaier, which inhibits the proliferation and migration of human gastric cancer cells, and induces apoptosis and autophagy. Moreover, the mTOR signal and ERK signal may be involved in the anti-gastric cancer effect of HES. This study provides novel references for the in-depth research and clinical application of Huaier. It is also of great significance to promote the scientific development and utilization of Huaier.
本研究旨在探讨槐耳提取物上清液（HES）对人胃癌HGC-27和MGC-803细胞增殖、凋亡、自噬及迁移的影响及其分子机制。采用高效液相色谱-质谱联用（HPLC-MS）对HES中的主要成分进行了初步分析。运用甲基噻唑基四氮唑（MTT）法、集落形成试验和5-乙炔基-2'-脱氧尿苷（EdU）染色试验，探究HES对人胃癌HGC-27和MGC-803细胞增殖的影响。采用Hoechst染色和流式细胞术检测HES对人胃癌HGC-27和MGC-803细胞凋亡的影响。分别用吖啶橙染色和细胞划痕试验检测HES对人胃癌HGC-27和MGC-803细胞自噬和迁移的影响。通过蛋白质免疫印迹法研究HES对人胃癌HGC-27和MGC-803细胞中凋亡、上皮-间质转化（EMT）相关蛋白表达水平及信号通路的调控作用。结果表明，HES主要含有一些高极性成分。HES能以剂量和时间依赖性方式显著降低人胃癌细胞的活力。HES处理人胃癌HGC-27和MGC-803细胞48 h后的半数抑制浓度（IC50）值分别为7.56和10.77 g·L-1。同时，HES抑制人胃癌细胞的集落形成能力和短期增殖。用8 g·L-1 HES处理HGC-27和MGC-803细胞72 h后的凋亡率分别为62.13%±8.92%和54.50%±3.26%。HES还能促进人胃癌细胞的自噬，并在体外削弱其迁移能力。此外，HES上调了凋亡标志物聚ADP-核糖聚合酶（PARP）的裂解及上皮细胞标志物E-钙黏蛋白的蛋白表达水平，下调了人胃癌细胞中磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、磷酸化核糖体蛋白S6（p-S6）和磷酸化细胞外信号调节激酶（p-ERK）的蛋白水平。因此，HES是槐耳有效的抗肿瘤成分之一，可抑制人胃癌细胞的增殖和迁移，并诱导凋亡和自噬。此外，mTOR信号和ERK信号可能参与了HES的抗胃癌作用。本研究为槐耳的深入研究和临床应用提供了新的参考依据。对推动槐耳的科学开发利用也具有重要意义。
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