槐耳上清对人胃癌HGC-27和MGC-803细胞的抗肿瘤作用研究

Anti-tumor effect of Huaier extract supernatant on human gastric cancer HGC-27 and MGC-803 cells

探讨槐耳上清(Huaier extract supernatant, HES)对人胃癌HGC-27和MGC-803细胞增殖、凋亡、自噬和体外迁移能力的影响及其分子作用机制。采用HPLC-MS对HES中的主要成分进行简单初步分析。通过MTT法、集落形成实验、EdU染色法探究HES对人胃癌HGC-27和MGC-803细胞增殖的影响。利用Hoechst染色法、流式细胞术明确HES对人胃癌HGC-27和MGC-803细胞凋亡的影响。通过吖啶橙染色法和细胞划痕实验分别研究HES对人胃癌HGC-27和MGC-803细胞自噬和体外迁移能力的影响。借助Western blot检测HES对人胃癌HGC-27和MGC-803细胞中与凋亡、上皮-间质转化(epithelial-mesenchymal transition, EMT)以及信号通路相关的蛋白表达水平的调控作用。结果显示,HES中主要含有一些极性较大的成分。HES能够明显降低人胃癌细胞的细胞活力,且具有浓度和时间依赖性,给药处理人胃癌HGC-27和MGC-803细胞48 h时,其半数抑制浓度(IC_(50))分别为7.56、10.77 g·L^(-1)。同时,HES能够显著抑制人胃癌细胞的集落形成能力和短期增殖能力。8 g·L^(-1)的HES作用于2种人胃癌细胞72 h,细胞凋亡率分别为62.13%±8.92%和54.50%±3.26%。HES还能够促进人胃癌细胞发生自噬,并削弱人胃癌细胞的体外迁移能力。此外,HES能够上调人胃癌细胞中凋亡标志物PARP的剪切水平和上皮细胞标志物E-cadherin的蛋白水平,并下调p-mTOR、p-S6和p-ERK的蛋白水平。因此,HES是槐耳发挥抗肿瘤作用的有效成分之一,其能够抑制人胃癌细胞的增殖和迁移,并诱导其发生凋亡和自噬,而且mTOR信号和ERK信号可能参与了HES的抗胃癌作用。该研究能够为槐耳的深入研究和临床应用提供新的参考,对进一步推动槐耳的科学开发利用具有重要意义。

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.