Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway

Lianfang Liu; Tianya Liu; Weiwei Tao; Naikai Liao; Qiuying Yan; Liu Li; Jiani Tan; Weixing Shen; Haibo Cheng; Dongdong Sun

doi:10.1016/j.phymed.2022.154007

Flavonoids from Scutellaria barbata D. Don exert antitumor activity in colorectal cancer through inhibited autophagy and promoted apoptosis via ATF4/sestrin2 pathway

Phytomedicine. 2022 May:99:154007. doi: 10.1016/j.phymed.2022.154007. Epub 2022 Feb 18.

Authors

Lianfang Liu¹, Tianya Liu², Weiwei Tao², Naikai Liao³, Qiuying Yan⁴, Liu Li⁴, Jiani Tan⁴, Weixing Shen⁴, Haibo Cheng⁵, Dongdong Sun⁶

Affiliations

¹ 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.
² School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China.
³ Department of Urology, the First Affiliated Hospital of Guangxi Medical University, Naning 530021, China.
⁴ Collaborative Innovation Center of Jiangsu Province of Cancer Prevention and Treatment of Chinese Medicine, Nanjing 210023, China.
⁵ Collaborative Innovation Center of Jiangsu Province of Cancer Prevention and Treatment of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory for TCM Formulae Research, Nanjing 210023, China. Electronic address: hbcheng_njucm@163.com.
⁶ School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China; Collaborative Innovation Center of Jiangsu Province of Cancer Prevention and Treatment of Chinese Medicine, Nanjing 210023, China; Jiangsu Key Laboratory for TCM Formulae Research, Nanjing 210023, China. Electronic address: sundd@njucm.edu.cn.

PMID: 35259610
DOI: 10.1016/j.phymed.2022.154007

Abstract

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.

Keywords: ATF4; Autophagy; Colorectal cancer; Scutellaria barbata D. Don; apoptosis; sestrin2.