IL-13 Promotes Cell Proliferation In Extranodal Natural Killer/T Cell Lymphoma

ObjectiveExtranodal NK/T cell lymphoma(NKTCL)is a rare but aggressive subtype of non-Hodgkin lymphoma.Multi-agent chemotherapy and involved-field radiotherapy are used to treat this disease,but the prognosis remains poor.It has been identified that 8-17% of cancers are associated with inflammation.Inflammation provides a suitable microenvironment for tumor growth,in which there are various kinds of inflammatory factors.To explore the roles of these factors in NKTCL,we examined their expression profile in NKTCL and found significantly high expression of interleukin 13(IL-13)in the serum of these patients.Interleukin 13 and its receptors(IL-13Rs)were reported correlated with the pathogenesis and progression of various malignances.However,their roles in NKTCL have not been evaluated.In this study,we aim to identify the role of IL-13 in NKTCL and the underlying mechanisms.Methods1.We examined the expression of IL-13 and IL-13 Rs in serum,tumor tissues and cell lines,respectively.(1).In serum: Luminex was used to detect the expression level of IL-13 in 61 patients pathologically diagnosed with NKTCL and 24 healthy individuals.Associations of serum IL-13 expression with clinical characteristics were analyzed.(2).In tumor tissues: Immunohistochemistry(IHC)was used to assess the expression of IL-13 and IL-13 Rs in tumor tissues of 40 NKTCL patients,and tissues from 30 patients with reactive hyperplasia of the lymph node were used for control.(3).In cell lines: Western blotting was used to evaluate the expression of IL-13 Rs in NKTCL cell lines(YT,SNK-6,NKL,NK92)and normal NK cells from peripheral blood.In addition,ELISA was used to identify IL-13 levels in medium from the cultured cell lines and NK cells.2.In vitro,we evaluated the effects of IL-13 on the proliferation or apoptosis of NKTCL cells.(1).CCK-8,EDU assays and Annexin V-APC were used to examine the proliferation or apoptosis of YT and SNK-6 cells induced by IL-13 at various concentrations(0,25,50,100 ng/mL).(2).CCK-8,EDU assays and Annexin V-APC were used to examine the proliferation or apoptosis of YT and SNK-6 cells when IL-13 was blocked by anti-IL-13 antibody(6.5?g/mL).3.The signaling pathway through which IL-13 promotes the proliferation of NKTCL cells.(1).Western blotting was used to examine JAK-STAT pathway in YT cells 2h after treated with IL-13 at various concentrations(0,25,50,100,200ng/mL).(2).YT cells were treated with an anti-IL-13R?1 antibody(6.5?g/mL)in the presence of IL-13 or with an anti-IL-13 antibody(6.5?g/mL)alone for 2h,and then JAK-STAT pathway were determined by western blotting.Results1.IL-13 and IL-13R?1 were overexpressed in NKTCL.(1).We found significantly higher serum IL-13 levels in patients with NKTCL compared to control group.No significant associations were found between clinical characteristics and serum IL-13 expression.(2).IL-13 and IL-13R?1 were observed overexpressed in NKTCL tumor tissues.Specifically,75% and 90% tumor tissues showed strong or medium IL-13(30 of 40,p < 0.001)and IL-13R?1(36/40,p < 0.001)expression,respectively.However,no significant difference was found in IL-13R?2 expression between these two groups.(3).IL-13 Rs expression were found higher in NKTCL cell lines,in comparison with that in normal NK cells.By ELISA,we found that IL-13 could be secreted by all of the four NKTCL cell lines and the levels were all higher than those in normal NK cells.2.IL-13 promotes tumor cell proliferation.(1).IL-13 was found to promote YT and SNK-6 cell proliferation in a dose-dependent manner.But no effect was found about IL-13 on tumor cell apoptosis.(2).Cell proliferation of YT and SNK-6 was inhibited when blocking IL-13 with anti-IL-13 antibody.3.IL-13 activated Stat6 through IL-13R?1.(1).P-Stat6 expression but notp-Stat3 or p-Stat5 was found increased after IL-13 stimulation at various concentrations(0,25,50,100,200 ng/mL),indicating p-Stat6 was induced by IL-13 in a dose-dependent manner.(2).Cells treated with the anti-IL-13 or anti-IL-13R?1antibodies showed much lower levels of p-Stat6 expression than those with IL-13 or untreated cells,indicating that IL-13 activated p-Stat6 via IL-13R?1 in NKTCL.Conclusions1.IL-13 functions as an autocrine growth factor in NKTCL and promotes tumor cell proliferation through IL-13/IL-13R?1/P-Stat6 pathway.2.Blocking IL-13 pathway is a potential therapeutic approach for NKTCL,but further research is needed.