Study On The Effect And Mechanism Of 4’-azid-2’-deoxy-2’-fluorarabinoside On Extranodal NK/T-cell Lymphoma

Objective:Extranodal NK/T-cell lymphoma(ENKTL)is an aggressive lymphoma derived from NK cells or cytotoxic T cells,which is highly associated with Epstein-Barr virus(EBV)infection and poor prognosis.Although L-asparaginase-based chemotherapy is effective in improving response rates,approximately 50 % of patients relapse after treatment,and there is an urgent need to develop new therapeutic agents.4’-azid-2’-deoxy-2’-fluorarabinoside(FNC)is a new nucleoside drug with independent intellectual property rights.Previous studies have shown that FNC has the effect on certain types of B-cell lymphoma,but whether FNC is effective against ENKTL remains unknown.In this study,human ENKTL cells(YT cells)were selected to study the effect of FNC on ENKTL and its mechanism,to provide theoretical basis and experimental basis for the development of FNC into antilymphoma drugs.Methods:(1)The proliferation inhibition rate of FNC on YT cells was detected by CCK-8 method,and the IC50 was calculated;(2)Annexin V-FITC/PI double fluorescence staining and PI staining were used to detect the effect of FNC on the apoptosis rate and cell cycle distribution of YT cells by flow cytometry;(3)The effect of FNC on the apoptotic morphology of YT cells was observed by fluorescence microscopy using AO/EB double fluorescence staining;(4)Transwell assay was used to detect the effect of FNC on YT cell invasion;(5)The effect of FNC on the structure of YT cells was observed by transmission electron microscopy(TEM);(6)The effect of FNC on the gene expression profile(GEP)of YT cells was detected by m RNA-seq,and the differential genes were screened for bioinformatics analysis;(7)The effect of FNC on the genes expression levels of HSPA8,HSP90AB1,PLK1,PLK2,TP53INP2 and TP53INP1 was detected by q PCR;(8)The effects of FNC on the protein expression levels of HSPA8,HSP90AB1,PLK1 and TP53 were detected by Western Blot;(9)The effects of FNC on the autophagy related m RNA expression levels of TP53,m TOR,LC3 and Beclin-1 were detected by q PCR;(10)Human ENKTL cells(YT cells)were used to construct a BALB/c nude xenograft mouse model for drug intervention.The body weight and tumor volume of the mice were measured and recorded.After administration,tumor tissue was collected,weighed,and fixed.The liver,spleen,kidney,and tumor tissue of the mice were taken for pathological examination.The expression levels of m TOR,LC3-Ⅱ,and Beclin-1 in tumor tissues were detected by immunohistochemistry(IHC).Results:(1)FNC significantly inhibited the proliferation of YT cells(P<0.01)in a doseand time-dependent manner.The IC50 values at 24,48 and 72 h were 10.70 ± 0.11,1.95 ± 0.03 and 0.37 ± 0.01 μM,respectively;(2)FNC blocked YT cell cycle in G0/G1 phase(P<0.01);(3)FNC induced YT cell apoptosis in a dose-dependent manner(P<0.01);(4)FNC significantly inhibited the invasion of YT cells in a dose-dependent manner(P<0.01);(5)FNC induced autophagy-lysosome formation and caused mitochondrial damage;(6)A total of 1573 differential genes were screened out in the FNC treatment group,including 1364 up-regulated genes and 209 down-regulated genes.Differential genes are involved in many biological processes such as cell proliferation,cell cycle,autophagy,and cytoskeleton,as well as adhesion,PI3 KAkt,MAPK signaling pathways.Differential alternative splicing genes are involved in many biological processes such as cell cycle,autophagy,and cell proliferation,as well as cell cycle and AMPK signaling pathway;(7)FNC significantly down-regulated the expressions of HSPA8,HSP90AB1,and PLK1 genes in YT cell,and up-regulated the gene expressions of PLK2,TP53INP2,and TP53INP1 in a dose-dependent manner(P<0.01);(8)FNC significantly down-regulated the protein expressions of HSPA8,HSP90AB1 and PLK1,and up-regulated the protein expressions of TP53 in a dosedependent manner(P<0.01);(9)FNC down-regulated m TOR gene expression in YT cells,and up-regulated TP53,Beclin-1 and LC3 genes expression(P<0.01);(10)The tumor inhibition rates of positive control cisplatin,FNC(3 mg/kg)and FNC(6 mg/kg)groups were 57.67 %,28.47 % and 68.21 %,respectively.Compared with the negative control group,the body weight of mice in the FNC(3 mg/kg)group increased slowly,the body weight of mice in the FNC(6 mg/kg)group decreased slightly but there was no statistical difference,and the body weight loss of mice in the positive drug cisplatin(2 mg/kg)group was significantly different(P<0.01).The pathological examination of liver,spleen,and kidney in FNC(6 mg/kg)group showed no abnormality.Immunohistochemical(IHC)results showed that compared with the negative control group,the expression levels of m TOR protein in FNC(6 mg/kg)group was decreased,and the expression levels of LC3-Ⅱ and Beclin-1 protein was increased(P<0.01).Conclusions:(1)FNC significantly inhibits the proliferation and invasion of ENKTL cell line YT cells.(2)FNC inhibits cell proliferation by regulating the cell cycle and inducing apoptosis.(3)FNC plays an anti-tumor role by regulating multiple targets in ENKTL.(4)Autophagy may be one of the mechanisms of FNC treatment of ENKTL.