The Mechanism Of Facarinediol Induced Apoptosis In Human Gastric Carcinoma Cells

Objective:Gastric cancer（GC）is a malignant disease with high incidence,which is difficult to find and easy to relapse,seriously endangering human life safety.At present,anti-tumor therapy has entered a new era of interdisciplinary,through the combination of traditional Chinese herbal medicine and modern surgery,radiotherapy,chemotherapy,immunotherapy,and other methods can play a role in“reducing toxicity and increasing effectiveness”,so it has been widely concerned by scholars.Falcarindiol（FAD）is a natural alkyne compound,which is mainly widespread in Angelica dahurica,Glehnia littoralis,Saposhnikovia divaricata,Peucedanum praeruptorum Dunn and Notopterygium incisum.In addition,FAD had shown satisfactory effects on anti-bacterial,anti-inflammatory and anti-tumor aspects,but its efficacy and pharmacological activity in gastric cancer are still unknown.In this study,GC cell lines were used as an experimental model to explore the killing effect of FAD,further study its induced apoptosis and inhibitory migration effect on AGS cells,and predict the binding site targeted by FAD-GC.Methods:In this study,the CCK-8 assay was used to determine the killing effect of FAD on twelve GC lines and fourth normal cells.Hoechst/PI staining,Annexin V/PI staining and flow cytometry were used to detect the apoptosis in gastric cancer cells at the cellular level.Moreover,Western blot was used to evaluate the regulation of FAD on downstream apoptotic proteins.The regulation of FAD on MAPK/STAT3/NF-κB pathway and the relationship between MAPK and STAT3 pathway in GC cells were investigated through Western blot.DCFH-DA staining fluorescent probe was used to test the regulation of FAD on ROS levels and related signaling pathways in GC cells.Cell scratch assay,Transwell assay and Western blot were used to determine the migration effects and the changes in migration proteins.Furthermore,the 2D structure of FAD molecule was used to predict the active target information by Swiss Target Prediction,and the key target information of GC known in the literature was analyzed by using Gene Cards and OMIM disease database.Venn diagram to obtain the intersection of FAD target and GC target:2 proteins（PLA2G2A and HDAC1）.PDB database obtain the structures of PLA2G2A and HDAC1 proteins,Py MOL software to remove water molecules to obtain protein receptors,and CB-Dock online molecular docking to obtain PLA2G2A-FAD and HDAC1-FAD3D pattern maps.Results:1、FAD suppressed the viability of twelve human GC lines in a dose-dependent manner,and AGS cells have the strongest drug sensitivity based on IC₅₀ data.Similarly,FAD had no significant Toxicity effects on human lung cell line IMR-90,human liver cell line THLE-2,gastric cell line GES-1,and human kidney cell line 293T.2、FAD promotes the release of pro-apoptotic proteins from the mitochondrial permeability transformation pore（MPTP）,so the expression of released Bad proteins in the cytoplasm is upregulated,while the expression of the cytoplasmic anti-apoptotic protein Bcl-2 is down-regulated.In addition,a large numbers of open MPTP would destabilize the dynamics of multivalent ion channels,leading to a rapid decrease in mitochondrial membrane potential,release of activated Cytochrome C proteins into the cytoplasm,regulate the elevated expression of cle-caspase-3 and cle-PARP proteins,and induce mitochondria-dependent apoptosis in gastric cancer AGS cells.3、FAD can increase the ROS level in AGS cells,down-regulate the expression of p-ERK pathway proteins and up-regulate the expression of p-p38 and p-JNK pathway proteins,which in turn regulate the expression of downstream p-STAT3 and NF-κB pathway proteins,and inhibit the expression of the migration protein SNAI 1 through theβ-catenin pathway and increases the protein expression of downstream E-cadherin,finally inducing mitochondria-dependent apoptosis and inhibiting cell migration in gastric cancer AGS cells.4、The binding pattern between FAD molecules and gastric cancer receptor proteins was predicted by database,and the binding sites FAD-PLA2G2A and FAD-HDAC1 were further identified by molecular docking simulations.Conclusion:FAD accumulates ROS levels in AGS cells,regulates MAPK signaling pathway and downstream key proteins of STAT3,NF-κB,andβ-catenin signaling pathway,inhibits cell migration and induces mitochondria-dependent apoptosis,thus showing a good anti-gastric cancer effect.It is predicted that FAD has two binding sites targeting gastric cancer,PLA2G2A and HDAC1,providing experimental data and a theoretical basis for clinical treatment of gastric cancer and the development of new drugs.