Artesunate inhibits hepatocellular carcinoma cell migration and invasion through OGA-mediated O-GlcNAcylation of ZEB1

Abstract

Metastasis remains a major challenge to improve the survival of patients with hepatocellular carcinoma (HCC). Artesunate is an antimalarial drug that also has anti-cancer properties. Additionally, O-GlcNAcylation has been implicated in cancer progression. In this study, we investigated whether artesunate regulated HCC cell migration and invasion and explored its impact on protein O-GlcNAcylation. Cellular functions, including viability, migration, and invasion, were evaluated using the cell counting kit-8, scratch assay, and Transwell analysis. Molecular docking and biolayer interferometry were employed to assess the binding interaction between artesunate and OGA. Furthermore, the O-GlcNAcylation of ZEB1 was examined using immunoprecipitation, cycloheximide chase assay, and immunoblotting. Our results demonstrated that artesunate significantly inhibited HCC cell viability, migration, and invasion. OGA expression was increased in HCC cells after artesunate treatment. Artesunate directly bound to OGA, and OGA knockdown reversed the inhibition of malignant behaviors induced by artesunate. Additionally, OGA suppressed the O-GlcNAcylation of ZEB1 at the Ser670 site, decreasing protein stability. Knockdown of ZEB1 inhibited HCC cellular behaviors. In conclusion, artesunate inhibits HCC cell migration and invasion by binding to OGA, which removes the O-GlcNAcylation of ZEB1 at the Ser670 site. These findings provide a new action mechanism for artesunate to treat HCC.

Keywords: O-GlcNAcylation; OGA; ZEB1; artesunate; hepatocellular carcinoma; invasion; migration.

Graphical abstract

Figure 1

Artesunate inhibits HCC cell viability. (a) Chemical structure of artemisinin. (b) Normal THLE3, (c) Hep3B, and (d) HCCLM3 cells were treated with 0, 25, 50, 100, and 200 μM artesunate for 48 h, and cell viability was measured using the cell counting kit-8. *P < 0.05, **P < 0.01, and ***P < 0.001 vs the 0 μM group.

Figure 2

Artesunate suppresses the cell migration and invasiveness of HCC cells. Hep3B and HCCLM3 cells were treated with artemisinin, and (a) cell migration was measured using the wound healing assay; the Transwell assay was conducted to determine (b) cell invasion and (c) migration. ***P < 0.001 vs the control group.

Figure 3

Artesunate inhibits OGA-mediated O-GlcNAcylation. (a) Immunoblotting was performed to detect the total O-GlcNAcylation level and protein levels of OGT and OGA. (b) The effect of artesunate on the total O-GlcNAcylation level and protein levels of OGT and OGA was evaluated using immunoblotting. (c) BLI analysis of the binding between artesunate and OGA. (d) The 3D structure of artesunate and the OGA binding complex was acquired using molecular docking. (e) The electrostatic surface of the protein OGA. (f) The detailed binding mode of artesunate and OGA.

Figure 4

Artesunate hampers HCC cell migration and invasion via increasing OGA expression. (a) OGA mRNA expression was measured in Hep3B and HCCLM3 cells transfected with shNC and shOGA using qRT-PCR. (b) HCC cells were treated with artesunate and transfected with shOGA; cell viability was assessed using the cell counting kit-8. (c) Quantification results of the wound healing assay. (d) Represent images of wound healing and Transwell assays. (e) Cell invasion and (f) migration analyzed using the Transwell assay were quantified. ***P < 0.001 vs the shNC group. ^### P < 0.001 vs the artesunate + shNC group.

Figure 5

OGA inhibits O-GlcNAcylation of ZEB1. (a) The effect of OGA on the O-GlcNacylation levels of tumor metastasis-related proteins, including N-cadherin, Vimentin, MMP3, MMP9, ZEB1, E-cadherin, and ZO-1. (b) The interaction between OGA and ZEB1 was assessed using immunoprecipitation. (c) Effect of OGA-mediated O-GlcNAcylation on the stability of ZEB1 protein. (d) Potential O-GlcNAcylation sites in ZEB1 protein. (e) ZEB1 O-GlcNAcylation sites were analyzed using immunoprecipitation and immunoblotting. ***P < 0.001 vs the vector group. ^### P < 0.001 vs the OGA group.

Figure 6

Knockdown of ZEB1 inhibits migration and invasion of HCC cells. (a) ZEB1 mRNA expression was measured in Hep3B and HCCLM3 cells transfected with shNC and shZEB1 using qRT-PCR. After transfection, (b) cell viability was assessed using the cell counting kit-8. (c) Cell migration was evaluated using the wound healing assay, and the percentage of wound healing was quantified. (d) Cell migration was analyzed using. (e) Cell invasion was evaluated using the Transwell assay and quantified. ***P < 0.001 vs the shNC group.