Vortioxetine Inhibits The Proliferation Of Esophageal Squamous Cell Carcinoma(ESCC) By Downregulating CDK4/6-RB1 Signaling Pathway

Background and purposeEsophageal squamous cell carcinoma(ESCC)accounts for 90% of esophageal cancer cases worldwide.ESCC patients in China account for about half of all ESCC patients.The main clinical treatment of ESCC is surgical resection,followed by radiotherapy and chemotherapy.ESCC is easy to be ignored because of its hidden early symptoms and low specificity.Most patients are already in advanced stages of cancer when they are diagnosed,so the prognosis of patients is poor and the recurrence rate is high.Currently,Currently,effective drug for the treatment of ESCC is scarce.It is of great clinical significance to find new targeted drugs for the prevention and treatment of ESCC.Cell cycle dependent kinase 4/6(CDK4/6)plays a key role in mammalian Cell cycle regulation.CDK4/6 promotes the passage of cells through the G1 phase to the S phase by forming complexes with Cyclin D1.Previous studies have shown abnormal activation of the CDK4/6-Rb signaling pathway in a variety of solid tumors,including breast,ovarian,non-small cell lung cancer,melanoma,glioma,and esophageal cancer.Due to the importance of CDK4/6 activity in cancer cells,CDK4/6 inhibitors have become a promising candidate for the treatment of cancer.Drug repurposing refers to the use of existing approved drugs to treat new medical indications,which reduces the capital and time cost of drug research.It is of great clinical significance to find new and affordable anticancer drugs through drug repurposing.In a screening of 1600 FDA-approved clinical drugs,we found that the antidepressant vortioxetine has a significant toxic effect on ESCC cells.Antidepressants have been used clinically for a long time,because most patients need long-term medication,so most antidepressants are less toxic and highly safe.Antidepressants also play an important role in the treatment of cancer patients,half of whom suffer from major depression or depression.This suggests that antidepressants are valuable for drug repurposing.The purpose of this study was to explore the molecular mechanism of vortioxetine inhibiting proliferation of ESCC cells.Methods1.Screening of 1600 FDA-approved clinical drugs was conducted through cytotoxicity test;Cell proliferation assay was conducted to study the effect of vortioxetine on the proliferation of ESCC cells;The effect of vortioxetine on the clone formation of ESCC cells was observed by anchorage-independent growth assay.2.The molecular mechanism of vortioxetine inhibiting proliferation of ESCC cells was studied by phosphoproteomics.3.The cell cycle experiment was conducted to detect the effect of vortioxetine on the cell cycle of ESCC cells.4.Auto Dock 4.0 software was used to simulate molecular docking to explore the binding sites of vortioxetine and CDK4/6 kinases;Pull down assays were performed to verify the ability of vortioxetine to bind to CDK4 and CDK6 kinases in KYSE150 and KYSE450 cells.5.In vitro kinase assay was used to investigate whether vortioxetine would affect the function of CDK4/6 kinase.6.The expression level of CDK4/6 gene in ESCC patients were investigated by TCGA database.7.The PDX model of ESCC was established to observe the anti-tumor effect of vortioxetine in vivo;The expression levels of Ki67 and p-RB1 S788 in tumor tissues were detected by immunohistochemical staining.Results1.Through screening of clinical drugs,it was found that vortioxetine had obvious cytotoxicity to ESCC cells;The results of cell proliferation assay showed that vortioxetine could inhibit the proliferation of ESCC cells;The results of the anchorage-independent growth assay showed that vortioxetine inhibited the clone formation of ESCC cells.2.Phosphoproteomics showed that p-RB1 S788 was a key phosphorylation site of vortioxetine in the inhibition of proliferation of ESCC cells.3.The cell cycle experiment showed vortioxetine induced cell cycle G1 phase arrest of KYSE150 and KYSE450,and the effect of the block was apparent as the concentration of drugs increased.4.Molecular docking simulation results showed that vortioxetine could bind to Glu56 of CDK4 and Val101 of CDK6,respectively;The pull down assays verified that vortioxetine can bind to CDK4 and CDK6 in KYSE150 and KYSE450.5.Through in vitro kinase assay,we demonstrated that vortioxetine inhibits the catalytic function of CDK4/6.Western blot results showed that votioxetine significantly downregulated the expression levels of p-RB1 S788 and p-RB1 T826.6.The TCGA database showed that the CDK4/6 was highly expressed in patients with ESCC.7.The results of ESCC PDX model experiments indicate that vortioxetine can significantly inhibit the growth of ESCC in vivo;Immunohistochemical staining of tumor tissues showed that the expression levels of Ki67 and p-RB1 S788 were significantly decreased in the drug-treated group.Conclusions1.The antidepressant drug vortioxetine can inhibit the cell proliferation in vitro and the tumor growth in vivo of ESCC.2.The antidepressant vortioxetine is a CDK4/6 inhibitor that inhibits the phosphorylation of RB1 by the CDK4/6 kinase.