Abstract:

Objective To clarify the potential anti-cancer molecular targets and mechanism of grape seed proanthocyanidin extract （GSPE） on oral squamous cell carcinoma （OSCC） with network pharmacology， molecular docking technology and in vitro cell experiments， and to discuss its effects on proliferation， migration and invasion of OSCC cells. Methods Pubchem， Swisstarget Prediction， SuperPred， TargetNet， Genecards and Online Mendelian Inheriatance in Man （OMIM） databases were used to obtain the predicted targets of proanthocyanidins （PACs） and OSCC， respectively； Venn diagram was used to screen the potential target genes of PACs against OSCC； Gene Ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analysis were performed according to David 6.8 database to predict the mechanism of action； String 12.0 was used to construct the protein-protein interaction（PPI） network； Cytoscape software and molecular docking were used to screen and preliminarily verify the binding ability of PACs with the core targets. Human tongue squamous cell carcinoma SCC-15 and CAL-27 cells were cultured in vitro， treated with different concentrations of GSPE， and divided into control group （complete medium） and 5， 10 and 20 mg·L^-1 GSPE groups. CCK-8 method was used to detect the survival rates of the cells in various groups； cell scratch assay was used to detect the scratch healing rates of the cells in various groups； Transwell chamber assay was used to detect the number of invasive cells in various groups； real-time fluorescence quantitative PCR （RT-qPCR） was used to detect the mRNA expression levels of the core targets in the cells in various groups. Results Network pharmacology predicted 50 potential targets of GSPE against OSCC. After screening and preliminary verification by molecular docking， heat shock protein 90 alpha family class A member 1（HSP90AA1）， B-cell lymphoma-2（Bcl-2）， prostaglandin-endoperoxide synthase 2（PTGS2） and nuclear factor kappa B1（NFKB1） were identified as the core targets of GSPE against OSCC. GO function enrichment analysis and KEGG signaling pathway enrichment analysis mainly involved pathways in cancer and microRNAs （miRNAs） in cancer， etc. The CCK-8 assay results showed that compared with control group， the survival rates of the cells in 5， 10 and 20 mg·L^-1 GSPE groups were significantly decreased after treatment for 12， 24， 48 and 72 h （P<0.05 or P<0.01）. The cell scratch assay results showed that compared with control group， the scratch assay rates of the cells in 5， 10 and 20 mg·L^-1 GSPE groups were significantly decreased after treatment for 12 and 24 h （P<0.05 or P<0.01）. The Transwell chamber assay results showed that compared with control group， the numbers of invasion cells in 5， 10 and 20 mg·L?1 GSPE groups were significantly decreased （P<0.01）. The RT-qPCR results showed that compared with HOK cells， the expression levels of HSP90AA1， Bcl-2， PTGS2 and NFKB1 mRNA in SCC-15 and CAL-27 cells were significantly increased （P<0.05 or P<0.01）； compared with control group， the expression levels of HSP90AA1， Bcl-2 and PTGS2 mRNA in SCC-15 cells in 5， 10 and 20 mg·L?1 GSPE groups were significantly decreased （P<0.01）， the expression levels of NFKB1 mRNA in SCC-15 cells in 10 and 20 mg·L?1 GSPE groups were significantly decreased （P<0.01）， the expression levels of HSP90AA1， Bcl-2 and NFKB1 in CAL-27 cells in 5， 10 and 20 mg·L?1 GSPE groups were significantly decreased （P<0.01）， the expression levels of PTGS2 mRNA in CAL-27 cells in 10 and 20 mg·L?1 GSPE groups were significantly decreased （P<0.01）. Conclusion GSPE exerts anti-OSCC effects by regulating pathways in cancer and miRNAs in cancer， etc. through targets such as HSP90AA1， Bcl-2， PTGS2 and NFKB1. In vitro cell experiment validation study shows that GSPE can inhibit the proliferation， migration and invasion of OSCC cells.

Key words: Grape seed proanthocyanidin extract, Oral squamous cell carcinoma, Cell proliferation, Cell migration, Cell invasion