Study On The Effect And Molecular Mechanism Of Gamabufotalin On Autophagy In Nasopharyngeal Carcinoma Cells

Object:To investigate the effects of Gamabufotalin（CS-6）on the proliferation of HK-1 and 5-8F cells by using human nasopharyngeal carcinoma（NPC）cell models,and the molecular mechanisms of CS-6-induced HK-1 and 5-8F cells proliferation inhibition were explored in terms of cell cycle arrest and autophagy,providing a strong basis for CS-6 as a potential therapeutic agent for NPC.Methods:In this project,the inhibitory effect of CS-6 on the proliferation in HK-1 and 5-8F cells was verified firstly by CCK-8 assay and clone formation assay.In the second place,the effect of CS-6 on cell cycle was detected by flow cytometry,and the expressions of CDK1 and Cyclin B1 were detected by western blot.Then,the effect of CS-6 on apoptosis was detected by flow cytometry.Next,RNA-Seq transcriptome sequencing and ingenuity pathway analysis（IPA）software revealed that the significantly different genes were mainly enriched in PFKFB4,and the literature revealed that PFKFB4 can be involved in autophagy regulation as an autophagy regulator.The expression of PFKFB4 in HK-1 and5-8F cells was detected by q RT-PCR and western blot.At the same time,the effect of CS-6 on ultrastructure of autophagic vesicles was observed by transmission electron microscopy,and the expressions of LC3 and p62 were detected by western blot,and the changes of autophagic flux were observed by laser confocal microscopy after transient transfection of m Cherry-EGFP-LC3 plasmid.Moreover,to investigate the role of PFKFB4 in proliferation,cycle arrest and autophagy by CS-6regulated in HK-1 and 5-8F cells,lentiviral was used to overexpress PFKFB4 in HK-1 and 5-8F cells,and then the changes of cell viability and colony formation were detected,the expression changes of Cyclin B1,CDK1,LC3 and p62 were detected by western blot,and the changes of autophagic flux were observed by laser confocal microscopy.Finally,the expressions of autophagy-related pathway protein GSK3βand mTOR（GSK3β,p-GSK3β,mTOR,p-mTOR）and the expression changes in autophagy-related pathway proteins after overexpression of PFKFB4 were detected by western blot after CS-6 treatment on HK-1 and 5-8F cells.Results:1.After treatment of 48 h,CS-6 significantly inhibited the proliferation of HK-1 and 5-8F cells with IC₅₀of 8.684 n M and 14.32 n M,respectively.CS-6 induced the G2/M phase arrest and markedly increased the expression levels of CDK1 and Cyclin B1 in HK-1 and 5-8F cells.CS-6 did not induce apoptosis in HK-1 and 5-8F cells.2.After treatment of 48 h,CS-6 significantly increased the expression of PFKFB4 at the m RNA and protein levels in HK-1 and 5-8F cells.At the same time,the classic bilayer membrane structure and autophagic lysosomes were observed in HK-1 and 5-8F cells,and CS-6increased the expression level of LC3II/LC3I and downregulated the expression level of p62,and promoted the fusion of autophagosomes with lysosomes.3.Overexpression of PFKFB4 reversed the inhibitory effect of CS-6 on HK-1 and 5-8F cell proliferation and colony formation ability,and restored the increase in Cyclin B1 and CDK1.In addition,overexpression of PFKFB4 reversed the increase in LC3II/LC3I and the decrease in p62,and the fusion of autophagosome with lysosome after CS-6treatment.4.CS-6 significantly downregulated p-mTOR and upregulated p-GSK3β.And further studies revealed that overexpression of PFKFB4 reversed the increase in p-GSK3βand the decrease in p-mTOR.Conclusion:CS-6 may induce autophagy through regulating the expression of PFKFB4 and autophagy-relevant mTOR signaling pathway,thus inhibiting the proliferation in NPC cells.