Liu Lianfang, Liu Tianya, Tao Weiwei, Liao Naikai, Yan Qiuying, Li Liu, Tan Jiani, Shen Weixing, Cheng Haibo, Sun Dongdong
Department of Medical Oncology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Suzhou 215600, China; School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
Phytomedicine. 2022 May;99:154007. doi: 10.1016/j.phymed.2022.154007. Epub 2022 Feb 18.
PURPOSE: Scutellaria barbata D. Don (SB), mainly containing flavonoids, has been frequently used for cancer treatment. However, little research has investigated the antitumor activity of flavonoids from SB (FSB). The current study aimed to assess the antitumor effect of TFSB and elucidate the probable underlying mechanism in vivo and in vitro. STUDY DESIGN: FSB was prepared, and its chemical composition was characterized by HPLC-MS. Colorectal HCT116 cells were treated with various concentration of FSB. The viability, proliferation, apoptosis, migration, and autophagy of HCT116 cells were studied, as were further confirmed in tumor xenografts. METHODS: Cell viability and proliferation were respectively examined by MTT and EdU staining. ROS was determined with DCFH-DA, and cell apoptosis was detected using flow cytometry. Transwell and wound-healing assays were performed to evaluate cell migration. Immunofluorescence was employed to evaluate sestrin2 and ATF4 level. The protein expressions of p-AMPK, p-ULK1, p-mTOR, 4E-BP1, LC3-I/II, cleaved-caspase-3, Bax, and bcl-2 were investigated by western blot. ATF4 was overexpressed in experiments to explore the role of ATF4/sestrin2 pathway in FSB-mediated efficacy. RESULTS: FSB clearly reduced the cell viability, promoted ROS generation, and induced apoptosis in HCT116 cells by down-regulated Bcl-2, and increased cleaved-caspase-3 and Bax. Furthermore, FSB significantly inhibited migration of colorectal cells in a dose-dependent manner. Further mechanistic study indicated that FSB upregulated p-mTOR protein level, and reduced p-AMPK, p-ULK1, p-mTOR, p-4E-BP1 and LC3-I/II expression, which were major autophagy-related genes. In addition, FSB could cause downregulation of endogenous mTOR inhibitor sestrin2 and ATF4 expression. Transient overexpression of ATF4 resulted in mTOR and sestrin2 inhibition, and significantly compromised the effects of FSB on apoptosis and autophagy in HCT116 cells. CONCLUSION: Our results reveal, for the first time, that FSB exerts antitumor activity through autophagy inhibition and apoptosis induction via ATF4/sestrin2 pathway in colorectal cancer cells. Scutellaria barbata D. Don may have great potential in the application for the prevention and treatment of human colorectal cancer.
目的：半枝莲（SB）主要含有黄酮类化合物，常用于癌症治疗。然而，关于半枝莲中黄酮类化合物（FSB）的抗肿瘤活性的研究较少。本研究旨在评估总黄酮半枝莲（TFSB）的抗肿瘤作用，并阐明其体内外可能的潜在机制。 研究设计：制备FSB，并通过高效液相色谱 - 质谱联用（HPLC-MS）对其化学成分进行表征。用不同浓度的FSB处理结肠直肠癌HCT116细胞。研究了HCT116细胞的活力、增殖、凋亡、迁移和自噬，并在肿瘤异种移植模型中进一步得到证实。 方法：通过MTT和EdU染色分别检测细胞活力和增殖。用2',7'-二氯二氢荧光素二乙酸酯（DCFH-DA）测定活性氧（ROS），并使用流式细胞术检测细胞凋亡。进行Transwell和伤口愈合试验以评估细胞迁移。采用免疫荧光法评估 sestrin2和激活转录因子4（ATF4）水平。通过蛋白质印迹法研究磷酸化腺苷酸活化蛋白激酶（p-AMPK）、磷酸化Unc-51样自噬激活激酶1（p-ULK1）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）、真核细胞起始因子4E结合蛋白1（4E-BP1）、微管相关蛋白1轻链3-I/II（LC3-I/II）、裂解的半胱天冬酶-3（cleaved-caspase-3）、促凋亡蛋白Bax和抗凋亡蛋白bcl-2的蛋白表达。在实验中过表达ATF4以探讨ATF4/sestrin2途径在FSB介导的效应中的作用。 结果：FSB明显降低HCT116细胞的活力，促进ROS生成，并通过下调Bcl-2诱导细胞凋亡，增加裂解的caspase-3和Bax。此外，FSB以剂量依赖性方式显著抑制结肠直肠癌细胞的迁移。进一步的机制研究表明，FSB上调p-mTOR蛋白水平，并降低p-AMPK、p-ULK1、p-mTOR、p-4E-BP1和LC3-I/II的表达（这些是主要的自噬相关基因）。此外，FSB可导致内源性mTOR抑制剂sestrin2和ATF4表达下调。ATF4的瞬时过表达导致mTOR和sestrin2抑制，并显著削弱FSB对HCT116细胞凋亡和自噬的影响。 结论：我们的结果首次揭示，FSB通过抑制自噬和经由ATF4/sestrin2途径诱导结肠癌细胞凋亡发挥抗肿瘤活性。半枝莲在人类结肠直肠癌的预防和治疗应用中可能具有巨大潜力。
Zhongguo Zhong Yao Za Zhi. 2017-4
Front Pharmacol. 2022-12-16
Front Pharmacol. 2020-3-20
Molecules. 2023-11-21
Pharmaceuticals (Basel). 2023-2-15
Zotero 插件