Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway
Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway
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DOI：
10.1016/j.phymed.2022.154007
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发表时间：
2022-03-05
期刊：
影响因子：
7.9
通讯作者：
Sun, Dongdong
中科院分区：
文献类型：
--
作者：
Liu, Lianfang;Liu, Tianya;Sun, Dongdong
目的：研究紫菀（Escherichia barbata D.）Don（SB）主要含有黄酮类化合物，常用于癌症治疗。然而，很少有研究从SB黄酮（FSB）的抗肿瘤活性。本研究旨在探讨TFSB的体内、外抗肿瘤作用及其可能的作用机制，研究设计：制备FSB，并采用HPLC-MS分析其化学组成，用不同浓度的FSB处理大肠癌HCT 116细胞。研究了HCT 116细胞的活力、增殖、凋亡、迁移和自噬，如在肿瘤异种移植物中进一步证实的。方法：MTT法检测细胞活力，EdU法检测细胞增殖。DCFH-DA法测定细胞内活性氧（ROS）含量，流式细胞仪检测细胞凋亡。进行Transwell和伤口愈合测定以评价细胞迁移。免疫荧光法检测sestrin 2和ATF 4的表达。Western blot检测p-AMPK、p-ULK 1、p-mTOR、4 E-BP 1、LC 3-I/II、cleaved-caspase-3、Bax、bcl-2蛋白表达。结果：FSB能明显降低HCT 116细胞的存活率，促进ROS的产生，并通过下调Bcl-2的表达，增加Caspase-3和Bax的表达，诱导细胞凋亡。此外，FSB以剂量依赖性方式显著抑制结直肠细胞的迁移。进一步的机制研究表明，FSB上调了p-mTOR蛋白水平，降低了自噬相关基因p-AMPK、p-ULK 1、p-mTOR、p-4 E-BP 1和LC 3-I/II的表达。此外，FSB还可下调内源性mTOR抑制剂sestrin 2和ATF 4的表达。结论：本研究首次揭示FSB通过抑制自噬和诱导凋亡发挥抗肿瘤作用，其机制可能与抑制自噬和诱导凋亡有关。半枝莲[专业]植Don在大肠癌的预防和治疗中具有巨大的应用潜力。
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.