Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Nov 25:18:5377-5395.
doi: 10.2147/DDDT.S470061. eCollection 2024.

Alkannin Induces G2/M-Phase Arrest, Apoptosis, and Inhibition of Invasion by Targeting GSK3β in Esophageal Squamous Cell Carcinoma

Huihui Ma #  1   2   3 , Peng Xu #  1   2   3 , Yunchao Jia  1   2 , Yehan Zhou  1   2   3 , Xinzhi Li  1   2   4 , Yanming Wang  1   2   3 , Ketao Ma  1   2   3
Affiliations

Alkannin Induces G2/M-Phase Arrest, Apoptosis, and Inhibition of Invasion by Targeting GSK3β in Esophageal Squamous Cell Carcinoma

Huihui Ma et al. Drug Des Devel Ther. .

Abstract

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: Alkannin; Glycogen Synthase Kinase 3β; esophageal squamous cell carcinoma; network pharmacology; transcriptomics.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

Figures

None
Graphical abstract
Figure 1
Figure 1
Alkannin inhibits the proliferation of ESCC cells. (A) Chemical structure of Alkannin. (B) Cell viability of KYSE150 and Eca109 cells treated with different concentrations of Alkannin for 24, 48, and 72 h. (C) Colony formation assay of KYSE150 and Eca109 cells after treatment with Alkannin for 24 h. (D) Western blot detects the PCNA protein levels of KYSE150 and Eca109 cells after treatment with Alkannin for 24 h. Data are expressed as means ± SD from five independent experiments, *P < 0.05; ***P < 0.001 compared to control by one-way ANOVA.
Figure 2
Figure 2
Alkannin inhibits the migration and invasion of ESCC cells. (A) Wound healing assay shows the mobility of KYSE150 and Eca109 cells after treatment with Alkannin. (B) Transwell assay detects the invasion ability of KYSE150 and Eca109 cells after treatment with Alkannin. (C) Western blot analyzes the protein expression of MMP2, MMP9, Vimentin, and β-Catenin in KYSE150 and Eca109 cells after treatment with Alkannin. Data are expressed as means ± SD from five independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001 compared to control by one-way ANOVA.
Figure 3
Figure 3
Alkannin induces apoptosis in ESCC cells. (A) Hoechst 33342 staining shows the extent of nuclear morphology of KYSE150 and Eca109 cells after treatment with different concentrations of Alkannin for 24 h. (B) Annexin V-FITC/PI assay is used to evaluate the apoptotic ratio of KYSE150 and Eca109 cells after treatment with Alkannin for 24 h. (C) Western blot analyzes the protein levels of BAX and Bcl-2 in KYSE150 and Eca109 cells. Data are expressed as means ± SD from five independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001 compared to control by one-way ANOVA.
Figure 4
Figure 4
Alkannin induces G2/M phase arrest in ESCC cells. (A) Flow cytometry analysis shows cell cycle distribution of KYSE150 and Eca109 cells after treatment with Alkannin for 24 h. (B) Western blot analyzes the protein levels of CyclinB1 and CDK1 in KYSE150 and Eca109 cells after treatment with Alkannin for 24 h. Data are expressed as means ± SD from five independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001 compared to control by one-way ANOVA.
Figure 5
Figure 5
Transcriptomic analysis predicts the signal pathway of Alkannin anti-ESCC. (A) Volcano plot of differential gene expression (n = 4). Blue: downregulated, red: upregulated, gray: non-significant. (B) Heat map of differentially expressed genes. (C) Gene Set Enrichment Analysis (GSEA) map of the G2/M phase gene sets. (D) Gene Ontology (GO) enrichment analysis of pathways in the top 20. The red boxes indicate the most significantly enriched pathways for the G2/M phase and cell cycle that appear frequently. (E) KEGG enrichment analysis of the top 20 signaling pathways, with the red boxes highlighting the highest-ranked pathways and the cell cycle pathway ranked first.
Figure 6
Figure 6
Network pharmacology and transcriptomics predict GSK3β as a key G2/M phase regulator. (A) Protein-protein interaction (PPI) network analysis shows GSK3β is closely associated with the G2/M phase related targets. (B) Transcriptomic and network pharmacology analyses show GSK3β is the key upstream regulator of the cell cycle. Red labels: network pharmacology targets; Green labels: transcriptome related differential genes; Yellow labels: both involved in.
Figure 7
Figure 7
Alkannin has anti-ESCC effect via targeting GSK3β. (A) Molecular docking of Alkannin with GSK3β. (B) Immunohistochemistry analyzes the protein levels of p-GSK3β in xenograft tumor tissues. (C) Western blot analyzes the protein levels of GSK3β, p-GSK3β, and p21 in KYSE150 and Eca109 cells treated with Alkannin. Data are expressed as means ± SD from five independent experiments, *P < 0.05; **P < 0.01; ***P < 0.001 compared to control by one-way ANOVA.
Figure 8
Figure 8
Alkannin inhibits the growth of ESCC xenograft tumors. (A) The flowchart of the ESCC xenograft tumor establishment and Alkannin intervention. (B) The changes of the tumor volume after treatment with Alkannin. (C) Representative images of tumors in different groups (n = 6). (D) The weight of xenograft tumors in different groups. (E) HE staining and immunohistochemistry analyzes the Ki67 and p-GSK3β protein expression. Data are expressed as means ± SD from six independent experiments, ***P < 0.001 compared to control by one-way ANOVA.
See this image and copyright information in PMC

References

    1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71(3):209–249. doi:10.3322/caac.21660 - DOI - PubMed
    1. Ma R, Liu Q, Zheng S, et al. Pkm2-regulated stat3 promotes esophageal squamous cell carcinoma progression via tgf-β1-induced emt. J Cell Biochem. 2019;120(7):11539–11550. doi:10.1002/jcb.28434 - DOI - PubMed
    1. Chen R, Zheng R, Zhang S, et al. Patterns and trends in esophageal cancer incidence and mortality in China: an analysis based on cancer registry data. J Natl Cancer Cent. 2023;3(1):21–27. doi:10.1016/j.jncc.2023.01.002 - DOI - PMC - PubMed
    1. Abnet CC, Arnold M, Wei W-Q. Epidemiology of esophageal squamous cell carcinoma. Gastroenterology. 2018;154(2):360–373. doi:10.1053/j.gastro.2017.08.023 - DOI - PMC - PubMed
    1. Yang H, Liu H, Chen Y, et al. Neoadjuvant chemoradiotherapy followed by surgery versus surgery alone for locally advanced squamous cell carcinoma of the esophagus (neocrtec5010): a Phase III multicenter, randomized, open-label clinical trial. J Clin Oncol. 2018;36(27):2796–2803. doi:10.1200/jco.2018.79.1483 - DOI - PMC - PubMed

MeSH terms