Mechanism Of Curcumol Inhibits EBV-positive Nasopharyngeal Carcinoma Invasion And Metastasis Via Down-regulating Nucleolin

Tumor metastasis is the main reason for the recurrence and death of Epstein-Barr virus(EBV)positive nasopharyngeal carcinoma(NPC).Previous studies showed that curcumol has both anti-tumor and anti-viral effects,but the mechanism of curcumol on EBV-positive tumors is still unclear.Previously,we reported that nucleolin(NCL,also known as C23)is the target protein of curcumol in CNE2,a kind of NPC cell line,and the expression level of NCL is related to the anti-tumor effect of curcumol.Here,we found that the expression of NCL in C666-1 cell line,a kind of NPC cell line positive for Epstein-Barr virus,was higher than that in CNE2 cells.C666-1 cell line is a highly invasive NPC cell line,and whether its strong migration and invasion ability and the inhibition of curcumol on this cell line are related to the target protein NCL deserves further discussion.Therefore,this study aims to explore the effect and mechanism of curcumol on EBV-positive NPC cells,and reveal the relationship between this effect and NCL.Objective: To clarify the inhibitory effect of curcumol on the migration and invasion of EBV-positive NPC cells,and explore its related mechanisms.Methods: The inhibitory effect of Curcumol on EBV-positive NPC was determined by scratch,Transwell,and Western Blot assays;The key amino acid sites involved in NCL binding to Curcumol were detected using overlap PCR,cell heat transfer and other experiments;Lentiviral infection was used to construct NCL overexpression,silencing or mutation cell lines,and the effect of curcumol on down-regulating NCL and inhibiting migration and invasion of nasopharyngeal carcinoma cells was analyzed in vivo and in vitro.The expression of Epstein-Barr Virus Nuclear Antigen 1(EBNA1)and NCL was detected in different NPC models and clinical NPC patient tumor tissues.Co-immunoprecipitation was used to detect the interaction between NCL and EBNA1,and the effects of differential expression of NCL and curcumol intervention on EBNA1 and PI3K/AKT signaling pathway were analyzed.The differential expression of NCL and the effect of curcumol intervention on the expression of VEGFA/VEGFR1 were analyzed by Western Blot,and the interaction between NCL and VEGFR1 was detected by Co-immunoprecipitation.Cellular immunofluorescence and fractionation extractions were performed to analyze the effect of curcumol on the nucleocytoplasmic distribution of VEGFR1,and to analyze whether these effects are associated with NCL expression.Results: Curcumol inhibited the migration and invasion abilities of EBV-positive C666-1 cells,and downregulated the expression of NCL.NCL overexpression promoted invasion and metastasis of C666-1 cells,while curcumol intervention inhibits the invasion and metastasis promoting ability of NCL,and this function is related to the binding site R457 of NCL and curcumol.NCL is highly expressed in EBV-positive NPC cells and tumor tissues from NPC patients and interacts with EBNA1.NCL overexpression upregulated EBNA1 and PI3K/AKT signaling pathway,while NCL silencing did the opposite.After Curcumol intervention,it can inhibit the expression of EBNA1 and PI3K/AKT signaling pathway by down regulating NCL,and it is related to NCL R457.Curcumol inhibited the expression of VEGFA/VEGFR1 by down regulating NCL,and inhibited the nuclear accumulation of VEGFR1.Conclusion: Curcumol can inhibit the expression of EBNA1,inactivate PI3K/AKT signal pathway,and inhibit the accumulation of VEGFR1 nucleus by down-regulation of NCL,thus exerting the function of anti invasion and metastasis of EBV-positive NPC.