Antitumor effects of isoliquiritin on oral squamous cell carcinoma by VEGFC/FLT4-PI3K/AKT signaling axis.

OBJECTIVES: As a principal flavonoid glycoside derived from Glycyrrhiza uralensis, isoliquiritin (ISLT) has exerted notable anti-tumor activity in multiple solid malignancies including lung and liver cancers. This study aims to investigate the therapeutic effect and mechanism of ISLT on oral squamous cell carcinoma (OSCC). METHODS: CCK-8, colony formation and Ki67 were used to detect the effect on cell proliferation. The cytotoxic effects were detected by flow cytometry and Live/Dead cell staining. The effects on cell migration and invasion were detected by scratch and invasion assays. Further, the key molecule of ISLT in the treatment of OSCC, Fms Related Receptor Tyrosine Kinase 4 (FLT4), commonly termed vascular endothelial growth factor receptor 3 (VEGFR3), was further identified by transcriptome sequencing combined with molecular docking. Subsequently, FLT4 knockdown cells were constructed, and the regulatory effects and downstream molecular mechanisms of FLT4 on OSCC cells were determined by cell phenotype detection and protein expression level changes. Finally, OSCC mouse models were constructed to demonstrate the therapeutic efficacy and biological safety of ISLT in vivo. RESULTS: The results demonstrated that ISLT inhibited the proliferation of OSCC cells in a dose-dependent manner, with a significant inhibitory effect observed at 20 μg/mL. ISLT increased the apoptosis rate and exerted a marked killing effect on OSCC cells, also significantly suppressed the migration and invasion of OSCC cells with high metastatic and invasive potential. At the gene expression level, treatment with ISLT significantly upregulated the mRNA levels of P53, Bax and Caspase-3, while downregulating Bcl-2 expression, leading to an elevated Bax/Bcl-2 ratio. FLT4, the target of ISLT, was screened by transcriptome sequencing and molecular docking. Research has found that FLT4 knockdown can also significantly inhibit the proliferation, migration and invasion of OSCC cells, and can also promote apoptosis. At the protein level, the changes in the expression levels of apoptosis-related proteins after FLT4 knockdown were consistent with the trend caused by ISLT. Furthermore, studies on downstream molecular mechanisms showed that after FLT4 knockdown, the phosphorylation levels of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) are significantly downregulated, resulting in decreased phosphorylated PI3K (p-PI3K)/PI3K and phosphorylated AKT (p-AKT)/AKT ratios. Finally, in vivo studies have shown that ISLT can significantly reduce the tumor volume of OSCC without causing any organ toxicity. CONCLUSION: This study reveals that ISLT has anti-tumor activity against OSCC, providing new insights into the traditional Chinese medicine treatment of human cancers.