Huihui Ma,1– 3,* Peng Xu,1– 3,* Yunchao Jia,1,2 Yehan Zhou,1– 3 Xinzhi Li,1,2,4 Yanming Wang,1– 3 Ketao Ma1– 3 

1Key Laboratory of Xinjiang Endemic and Ethnic Diseases, Ministry of Education, Shihezi University School of Medicine, Shihezi, 832003, People’s Republic of China; 2NHC Key Laboratory of Prevention and Treatment of Central Asia High Incidence Diseases, First Affiliated Hospital, Shihezi University School of Medicine, Shihezi, 832003, People’s Republic of China; 3Department of Physiology, Shihezi University School of Medicine, Shihezi, 832003, People’s Republic of China; 4Department of Pathophysiology, Shihezi University School of Medicine, Shihezi, 832003, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Ketao Ma; Yanming Wang, Department of Physiology, Shihezi University School of Medicine, Shihezi, 832003, People’s Republic of China, Tel +86 150 0164 5180 ; +86 175 9093 3308, Email maketao@hotmail.com; wangyanminglyo@163.com

Purpose: Esophageal squamous cell carcinoma (ESCC) is the most common malignant tumor of the upper gastrointestinal tract, characterized by high mortality and poor prognosis. There is an urgent need for the development of more effective drugs. Alkannin has been shown to inhibit the progression of various cancers, but its inhibitory effects on ESCC remain unclear. This study aims to investigate the therapeutic effects of Alkannin on ESCC and elucidate its potential targets and molecular mechanisms.
Methods: Cell Counting Kit-8 (CCK-8) assays, colony formation assays, Hoechst 33342 staining, wound healing assays, Transwell migration assays, flow cytometry, and Western blotting were used to investigate the therapeutic effects of Alkannin on ESCC in vitro. Transcriptome sequencing and network pharmacology were employed to analyze the potential targets and pathways affected by Alkannin treatment. The anticancer effects of Alkannin in vivo were assessed in a nude mouse model.
Results: Alkannin suppressed cell proliferation, invasion, migration, and induced ESCC cell apoptosis. Mechanistic studies indicated that Alkannin inhibits ESCC by inducing G2/M-phase cell cycle arrest by targeting Glycogen Synthase Kinase 3β (GSK3β). Consistently, in vivo administration of Alkannin significantly reduced the growth of ESCC tumors in nude mice.
Conclusion: This study is the first to demonstrate that Alkannin, by targeting GSK3β, induces G2/M-phase arrest in ESCC cells, thereby inhibiting migration, invasion, and inducing apoptosis, suggesting that Alkannin may be a promising antitumor agent for treating ESCC.

Keywords: esophageal squamous cell carcinoma, Alkannin, network pharmacology, transcriptomics, Glycogen Synthase Kinase 3β