| BackgroundLymphoma is a group of lymphoid hematopoietic malignant tumor that derive from lymphocytes,which can be divided into two main categories—Hodgkin's lymphoma(HL)and the non-Hodgkin's lymphoma(NHL).As a specific subtype of NHL,extranodal NK/T cell lymphoma(ENKTCL)has its characteristic feature of epidemiology,pathology and clinical manifestation.The disease is rare in Europe and America,but common with young and middle-aged men in east Asia and Latin America.Tumors are generally involving the extranodal site—nasopharynx is common,other upper respiratory or digestive tract sites like palate,tonsil and throat can be seen,but it is rare in lung,skin and testis.It shows high aggressive progression,easy to recurrence and poor prognosis.Recent studies show its etiology is associated with epstein-barr virus infection.Internationally,there is no definite standard therapeutic regimen.The common NHL therapeutic regimens,like CHOP regimen containing adriamycin,show poor outcome owing to drug resistant.Over the past few years,therapeutic regimens based on gemcitabine and L-asparaginase,such as modified SMILE,P-Gemox and DDGP chemotherapy,show their better efficacy,but there are still some refractory or recurrent patients.It is high time to explore the drug resistance mechanisms of ENKTCL,which is of importance to enhance the overall curative property and improve the life quality.In the past decades,the theory of “Seed(tumor)and soil(microenvironment)” has drawn greater attention.Microenvironment interact with tumor cells,promoting cell proliferation,avoiding and tolerating harmful stimulation,which may cause the tumour formation and drug resistance.Tumor necrosis factor-?(TNF-?),an important member of cytokines in microenvironment,can act as both a promoter and inhibitor in the development of tumor.TNF-? — the earliest discovered cytokine,which is secreted by activated macrophage,plays an important role in inflammation.Nowadays,the study of TNF-? limited in the field of autoimmune diseases like rheumatoid arthritis,mandatory spondylitis and inflammatory bowel disease,rare in cancer.The latest research shows that TNF-? can reverse the resistance to adriamycin in adriamycin resistant cell line MCF-7/Adr and promote the sensitivity of chemotherapy and radiotherapy in breast cancer.The role of TNF-? in ENKTCL and its drug resistance is not reported yet.NF-?B pathway is the research hotspot,whose activation correlates with inflammation and tumor.It activates the NF-?B protein mainly through classic pathway and alternative pathway,entering the nucleus,combining the promoter or enhancer on targeted genes and inducing the related genes' transcription and expression.The object of this study is NK/T cell lymphoma cell line YTS,using low concentration gradually increasing and high dose intermittently impact method to build a gemcitabine(Gem)resistant cell line YTS/Gem.Under the treatment of Gem and/or TNF-?,this study adopt the method of CCK8,Flow Cytometer(FCM)and Western blot to test cell proliferation,cell cycle and protein expression in order to find the role of TNF-? in ENKTCL and its drug resistance.ObjectiveTake YTS,the NK/T cell lymphoma cell line,as the study object,building the Gemcitabine-resistant cell line of YTS(YTS/Gem).Then compare the cell proliferation,cell cycle changes,and the expression of associated protein between YTS and YTS/Gem under the treatment of Gem and/or TNF-?,and investigate the role of TNF-? in the drug-resistant mechanism of ENKTCL.Method1.Cell culture in vitro: YTS cells were cultured in 1640 culture medium containing 10% fetal bovine serum and YTS/Gem cells in 1640 culture medium containing 10% fetal bovine serum and a certain concentration of Gem.Both were placed in the incubator under appropriate condition(37 ?,5% CO2)and replaced or added appropriate volume of culture medium every 2-3 days.2.Built the YTS/Gem cell line: YTS was exposed to gemcitabine,using low concentration gradually increasing and high dose intermittently impact method to obtain a stable gemcitabine-resistant cell line(YTS/Gem).3.Determination of drug resistance index: YTS cells and YTS/Gem cells were treated with different concentrations of Gem(0,5,10,20,40,80nM)in 96-well plates,CCK8 method was used to measure the absorbance OD value at 450 nm by the microplate reader.The IC50 values of YTS cells and YTS/Gem cells were calculated and repeated three times to obtain the drug resistance index of YTS / Gem cells.4.Experimental grouping:(1)Control group: YTS cells or YTS/Gem cells were cultured in medium for 48 hours.(2)TNF-? group: YTS cells or YTS/Gem cells were cultured in medium with TNF-? 20ng/ml for 48 h.(3)Gem group: YTS cells or YTS/Gem cells were cultured in medium with Gem 20 nM for 48h;(4)Gem+TNF-? group: YTS cells or YTS/Gem cells were cultured in medium with TNF-? 20ng/ml and Gem 20 nM for 48 h.5.Detection of cell proliferation: CCK8 method was used to measure the OD value at 450 nm by the microplate reader.The changes of proliferation of YTS cells were compared with YTS/Gem cells in TNF-? group,Gem group and Gem+TNF-? group.6.Protein detection: Western blot was used to detect the expression level of phospho-NF-?B(p-p65),total NF-?B(p65)of NF-?B pathway in control group,TNF-? group,Gem group and Gem+TNF-? group.7.Detection of cell cycle: Flow cytometry was used to compare the cell cycles in TNF-? group,Gem group and Gem+TNF-? group.8.Statistical methods: SPSS 17.0 software was used for data analysis and drawing.The data were shown as means ± standard deviation(sx ±).Comparison between two samples were conducted by using t-test for homoscedasticity and t'-test for heterogeneity of variance.Multiple sample means were compared by using analyzing of variance.Comparison between any two means was analyzed by using LSD-t-test.p<0.05 indicated that the difference is statistically significant.Result1.A stable gemcitabine-resistant cell line(YTS/Gem)was successfully built.The IC50 values of Gem to YTS and YTS/Gem were(18.09±1.67)nM and(314.90±5.62)nM,respectively.The resistance index of YTS/Gem reached 17.41.2.TNF-? could promote the proliferation of YTS/Gem cell:(1)in the effect of TNF-?: comparing TNF-? group with the control group,TNF-? had no obvious effect on YTS cells(p>0.05),while the survival rate of YTS/Gem cells was increased(p<0.05).(2)in the effect of TNF-? and Gem: comparing Gem+TNF-? group with Gem group,TNF-? had no obvious effect on YTS cells(p>0.05),while the survival rate of YTS/Gem cells was increased(p<0.05).3.TNF-? was capable of activating NF-?B pathway in YTS/Gem cells: TNF-? increased the expression of phospho-NF-?B(p-p65)in TNF-? group compared with control group in YTS/Gem cells,while the total expression of NF-?B(p65)protein had no obviously change.In the Gem+TNF-? group,the expression of phospho-NF-?B(p-p65)caused by TNF-? was significantly higher than that in the Gem group,while the total expression of NF-?B(p65)had no obviously change.4.TNF-? had no influence on YTS/Gem cell cycle: TNF-? decreased the percentage of G0/G1 phase cells and increased the proportion of S phase cells in YTS/Gem cells compared with the control group without the statistically significant difference(p>0.05).Compared with Gem group,TNF-? decreased the percentage of G0/G1 phase cells and activated the proportion of S phase cells in Gem+TNF-? group,and the difference was not statistically significant(p>0.05).Conclusion:1.The gemcitabine-resistance cell line of NK/T cell lymphoma YTS/Gem is successfully built.2.TNF-? can promote the proliferation of NK/T cell lymphoma gemcitabine-resistance cell line YTS/Gem by up-regulating NF-?B pathway and play a role in the mechanism of its drug resistance. |
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