Remdesivir Inhibits The Proliferation,Migration And Invasion Of Glioblastoma Cells

Glioblastoma(GBM)is a common neuroepithelial solid tumor in the central nervous system.GBM is classified as a grade IV glioma by the World Health Organization(WHO),which is also the most malignant and lethal primary brain tumor.In addition,GBM is the most aggressive type of brain cancer,and its aggressive character is at least in part due to the high migratory potential to invade surrounding tissues.The prognosis of GBM patients is very poor.The median survival time after diagnosis is only 14 months,and the 1-year survival rate is 39.5%,and the 5-year survival rate is only 5.4%.The global annual incidence of GBM is about 3 to 4 out of100,000 people,and it is very common in developing countries.The conventional clinical treatment for GBM is surgical resection followed by radiotherapy and temozolomide(TMZ)treatment,but GBM is prone to develop TMZ resistance,and most patients relapse within 6 months after treatment.Therefore,there is an urgent need to explore new therapeutic drugs for GBM.Drug reuse(also known as re-use of old drugs)is the application of approved drugs for another indication,which has the advantages of reducing the risk of drug development failure,shortening the research and development period,and reducing research and development costs.Our previous drug screening found that remdesivir(RDS)had strong anti-GBM cell activity.RDS is a new type of phosphorylated predrug of 1 ’-cyanosubstituted nucleoside analogue,which is a broad-spectrum antiviral drug and has a good inhibitory effect on a variety of RNA retroviruses,including Ebola virus and coronavirus.RDS plays an antiviral role by inhibiting RNA-dependent RNA polymerase(Rd Rp)in RNA retroviruses.Its triphosphate metabolite NTP can be incorporated into viral RNA as a substrate for RNA synthesis to compete with endogenous nucleotides and lead to early termination of replication,thus inhibiting viral proliferation.The permeable blood-brain barrier formed between the blood and the brain is one of the reasons reducing the therapeutic effect of treatment for GBM,and RDS has a good blood-brain barrier permeability.In recent years,a large amount of evidence has shown that some antiviral drugs have antitumor activity,and it has also been found that antiviral drugs combined with commonly used chemotherapy drugs can enhance the antitumor effect.However,the effects of RDS on GBM cells and the molecular mechanism of action are still unclear,and relevant studies are lacking.In this paper,we conducted in vitro cytological and in vivo animal experiments to investigate the role of RDS in inhibiting GBM cell proliferation,migration and invasion,and further to explore its molecular mechanism.The main research results included in this paper are described as follows:1.RDS inhibits the proliferation and clonogenesis of GBM cellsIn order to explore the effect of RDS on the proliferation of GBM cells,we treated five GBM cell lines with different concentrations of RDS,and found that with the increase of drug concentration,the cell viability gradually decreased,and showed an obvious concentration-dependent effect.The inhibitory effect of RDS on U87-MG and LN229 GBM cells was the most significant,with the semi-inhibitory concentration(IC50 value)of 11.88μM and 5.418μM,respectively.We found that RDS significantly inhibited GBM cell proliferation and clonogenesis through MTT assay,cell count assay,Edu assay and plate clonogenesis assay.The effect of RDS on GBM cell cycle was detected by flow cytometry,and it was found that RDS could block cells in G2/M phase.Western blotting assay showed that the G2/M phase related proteins cyclin B1,cyclin A2 and CDK1 were significantly down-regulated.RDS significantly inhibited the tumorigenesis of GBM cells in vitro and in vivo by soft agar and subcutaneous xenograft experiments.2.RDS inhibits migration and invasion of GBM cellsIn order to explore the effect of RDS on the migration and invasion ability of GBM cells,we found that RDS could significantly inhibit the migration ability of GBM cells through cell scratch healing and cell migration ability analysis experiments.It was found that RDS significantly inhibited GBM cell invasion ability by cell invasion ability analysis using a cell chamber with matrix gel.Finally,Western blotting was used to detect the effect of RDS on migration and invasion related proteins.The results showed that N-cadherin and vimentin expression were down-regulated,while E-cadherin expression was up-regulated.3.RDS inhibits FAK/PI3K/AKT signaling pathway in GBM cellsIn order to explore the mechanism of RDS inhibiting the proliferation,migration and invasion ability of GBM cells,RNA transcriptome sequencing was performed on GBM cells treated with RDS.Sequencing results showed that the extracellular matrix(ECM),ECM receptor interaction and epithelial-to-mesenchymal transition(EMT)related genes were significantly down-regulated.In addition,the FAK/PI3K/AKT signaling pathway,which positively regulates the proliferation,migration and invasion of tumor cells,was also significantly inhibited.Western blotting was used to verify the expression of proteins related to the FAK/PI3K/AKT signaling pathway after RDS treatment.It was found that the phosphorylation levels of FAK,PI3 K and AKT were significantly decreased,which confirmed that this signaling pathway was significantly inhibited by RDS.4.RDS inhibits proliferation,migration and invasion of GBM cells by inhibiting FAK/PI3K/AKT signaling pathwayIn order to further investigate the relationship between the inhibitory effect of RDS on proliferation,migration and invasion of GBM cells and the FAK/PI3K/AKT signaling pathway,we tested the effects of FAK inhibitor PF573228 or PI3 K inhibitor LY294002 combined with RDS on the proliferation,migration and invasion of GBM cells.The results showed that the proliferation,migration and invasion ability of GBM cells were further inhibited,and the downregulation of FAK/PI3K/AKT signaling pathway related proteins were verified by western blotting assay.These results suggest that RDS can inhibit the proliferation,migration and invasion of GBM cells by regulating the FAK/PI3K/AKT signaling pathway.In summary,the results of this study suggest that RDS can significantly inhibit the proliferation,migration and invasion of GBM cells,and reveal that the FAK/PI3K/AKT signaling pathway plays an important regulatory role.Therefore,this study identifies RDS as a new small-molecule drug with anti-GBM activity,which establishes a relevant theoretical foundation for the future clinical use of RDS in the treatment of GBM.