Alkannin, a bioactive compound from Lithospermum erythrorhizon, demonstrates significant anti-cancer activity against esophageal squamous cell carcinoma (ESCC) by targeting Glycogen Synthase Kinase 3β (GSK3β). Researchers have found that Alkannin induces G2/M phase cell cycle arrest, promotes apoptosis, and inhibits the migration and invasion of ESCC cells both in vitro and in vivo. These findings suggest that Alkannin could be a promising therapeutic agent for ESCC, which has a high incidence and mortality rate worldwide, especially in China where it accounts for approximately 55% of cases.
Alkannin Inhibits ESCC Cell Proliferation, Migration, and Invasion
In vitro experiments using ESCC cell lines KYSE150 and Eca109 showed that Alkannin significantly reduced cell viability in a concentration- and time-dependent manner. The IC50 values for KYSE150 cells after 24, 48, and 72 hours of Alkannin exposure were 4.579 μM, 3.204 μM, and 2.449 μM, respectively, while the values for Eca109 cells were 3.8 μM, 3.589 μM, and 2.615 μM. Colony formation assays further confirmed the inhibitory effects of Alkannin on cell proliferation. Wound healing and transwell assays demonstrated that Alkannin suppresses the migration and invasion of ESCC cells by decreasing the protein levels of key markers such as MMP2, MMP9, Vimentin, and β-catenin.
Alkannin Induces Apoptosis and G2/M Phase Arrest
Hoechst 33342 staining and Annexin V-FITC/PI assays revealed that Alkannin induces apoptosis in ESCC cells in a dose-dependent manner. The proportion of apoptotic cells increased significantly with increasing concentrations of Alkannin. Flow cytometry analysis showed that Alkannin induces G2/M phase arrest in ESCC cells by reducing the levels of CyclinB1 and CDK1. For example, after 24 hours of Alkannin treatment, the percentages of KYSE150 cells in the G2/M phase were 12.9% (0 μM), 15.1% (2 μM), 19.2% (4 μM), and 26.8% (6 μM).
Transcriptomic Analysis Identifies GSK3β as a Key Target
Transcriptomic sequencing of KYSE150 cells treated with Alkannin revealed significant changes in gene expression, with 2534 genes upregulated and 2053 downregulated. Gene Set Enrichment Analysis (GSEA) highlighted the G2/M phase, achieving an Enrichment Score (ES) of 0.5885 and a False Discovery Rate (FDR) of 0. Network pharmacology analysis identified 239 common targets between Alkannin and ESCC, with GSK3β identified as a key upstream regulator of the cell cycle.
Molecular Docking and In Vivo Studies Confirm GSK3β Inhibition
Molecular docking studies showed a strong binding interaction between Alkannin and GSK3β, with a CDOCKER interaction energy of 38.3161 kcal/mol. Immunohistochemistry and Western blot analysis confirmed that Alkannin treatment significantly decreased the expression of p-GSK3β and increased the expression of p21. In vivo experiments using ESCC xenograft models in BALB/c-nu mice demonstrated that Alkannin treatment significantly slowed tumor growth and reduced tumor weight. H&E staining and immunohistochemical staining for Ki67 confirmed suppressed tumor proliferation.
Implications for ESCC Treatment
These findings suggest that Alkannin exerts its anti-ESCC effects by modulating the cell cycle process and targeting GSK3β. "Our study demonstrates the potential of Alkannin as a therapeutic agent for ESCC treatment, particularly as a GSK3β targeting drug," the researchers noted. Future studies will focus on GSK3β inhibition and gene knockout techniques to more precisely determine the anticancer mechanisms of GSK3β. The study provides a new natural anticancer compound as a candidate drug and offers new therapeutic strategies for clinical use.
