Gentiopicroside-Induced gastric cancer necroptosis via the HIF-1 signaling pathway: A study involving molecular docking and experimental validation

Abstract

Objectives: Gentiopicroside is an effective treatment for several types of cancer, inducing numerous forms of programmed cancer cell death. However, there are few investigations into the role of necroptosis. By utilizing molecular docking, and experimental validation, this study aims to investigate whether gentiopicroside elicits necroptosis in gastric cancer.

Methods: Using software PyMOL and AutoDock, gentiopicroside was docked with RIPK1, RIPK3, MLKL and HIF-1α proteins. And a cell study was performed based on SGC7901 cells. The necroptosis-related proteins and HIF-1 signaling pathways were explored using western blot (WB) analysis. Finally, an animal study was performed to test the inhibitory effect in vivo.

Results: Docking studies indicated that the docking energies of gentiopicroside to necroptosis-related proteins and necroptosis-characteristic proteins are all below -5 kcal/mol. Additionally, gentiopicroside cells reduce gastric cancer viability and inhibit proliferation. Results from the animal experiments indicated that gentiopicroside inhibits the growth of the gastric cancer xenograft tumor. Western blot and immunohistochemistry (IHC) staining demonstrated that gentiopicroside higher p-receptor-interacting protein kinase 3(p-RIPK3) levels in vitro and in vivo.

Conclusion: The findings of this study revealed that necroptosis is involved in the inhibitory effect of gentiopicroside toward gastric cancer.

Fig 1

Two and three-dimensional images of compound Gentiopicroside docking with RIPK1, RIPK3, MLKL and HIF-1α protein: (A) Gentiopicroside docking with HIF-1α; (B) Gentiopicroside docking with RIPK1; (C) Gentiopicroside docking with RIPK3; (D) Gentiopicroside docking with MLKL.

Fig 2. Cell experiments.

(A) Cell viability assay using CCK-8; (B) Colony assay and quantification; (C) Flow cytometry assay; (D) Western blot experiments and quantification. Experiments were repeated three times. Statistical differences compared with the model group were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 3. Animal experiments (# 6 in each group).

(A) Tumor sizes; (B) Tumor weights; (C) Tumor properties at the time of euthanization; (D) Representative HIF-1 IHC and quantification. Magnification = 200; Scale bar: 200μm. (E) Representative p-RIPK3 IHC and quantification. Magnification = 200. Scale bar: 200μm. Statistical differences compared with the model group were considered significant at * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig 4. Workflow of Gentiopicroside-Induced necroptosis in gastric cancer via the HIF-1 pathway.