| Research background and purpose:Extranodal natural killer/T cell lymphoma(ENKTL)is asubtype of non-Hodgkin’s lymphoma,which is derived from mature T cells and NK cells.ENKTL has the characteristics of high invasiveness,rapid progress,easy recurrence,short survival period and poor prognosis.Traditional chemotherapy regimens for lymphoma(such as CHOP)have poor efficacy on ENKTL,and the complete remission rate is less than 40%,which may be related to the expression of multidrug resistance genes.In recent years,the progress of ENKTL in treatment is mainly due to the application of L-asparaginase and gemcitabine.Some studies have shown that L-asparaginase/gemcitabine based chemotherapy scheme significantly improves the prognosis of ENKTL patients compared with the traditional anthracycline based chemotherapy scheme.However,20%-40%of the patients are still difficult to achieve CR or relapse after Cr,and drug resistance is the main reason for treatment failure.Therefore,it is very important to explore the mechanism of drug resistance of ENKTL and find a feasible way to reverse drug resistance to improve the efficacy and prognosis of ENKTL.The mechanism of drug resistance of ENKTL is very complex,and the change of tumor microenvironment is one of them.The abnormal function of tumor microenvironment promotes the proliferation and drug resistance of lymphoma cells through various functions,and the proliferation of lymphoma cells depends on the stimulation of certain signals(antigens,cytokines,etc.)in tumor microenvironment.The abnormal function of tumor microenvironment promotes the proliferation and drug resistance of lymphoma cells through various functions,and the proliferation of lymphoma cells depends on the stimulation of certain signals(antigens,cytokines,etc.)in tumor microenvironment.Under the microscope,a large number of acute and chronic inflammatory cells can be found in ENKTL.Meanwhile,these inflammatory cells can secrete a large number of cytokines,such as TNF-α,IL-4,IL-6,TNF-α,IFN-γ and IL-10.IL-10 is a bi-directional immunoregulatory factor,which has dual effects on tumor.Some researches think that IL-10 can promote the growth of tumor,while others think that IL-10 can inhibit the angiogenesis and metastasis of tumor,so as to play an anti-tumor role.As an immunosuppressive factor,IL-10 is believed to promote tumor immune escape.However,there are still studies that show that IL-10 has strong antitumor effect.The anti-tumor response mediated by IL-10 may be related to the activation of NK cells mediated by IL-10.IL-10 can not only directly stimulate NK cells,but also indirectly stimulate the activation of NK cells in vivo.For example,active oxygen molecules secreted by macrophages can inhibit the activity of NK cells,while IL-10 can inhibit the secretion of active oxygen molecules by macrophages.In general,IL-10 plays a dual role in tumor development.When cancer begins to form,IL-10 may play an important role in stimulating NK and CTL mediated tumor cell killing.However,when tumor cells begin to express IL-10R,IL-10 produced in tumor microenvironment may play an important role in promoting cancer.As an important inflammatory factor,IL-10 is closely related to the occurrence and prognosis of ENKTL,but the relationship between IL-10 and ENKTL resistance has not been reported.This project is to study the molecular mechanism of IL-10 participating in ENKTL multidrug resistance,so as to find a feasible scheme to reverse ENKTL multidrug resistance,and provide new ideas for clinical treatment of ENKTL to improve the prognosis of ENKTL patients.Firstly,the level of IL-10 in the peripheral blood of ENKTL patients before treatment was measured,and the relationship between IL-10 and ENKTL multidrug resistance,as well as the relationship between IL-10 and clinical characteristics of patients were analyzed.In order to further study the role of IL-10 in ENKTL,we stimulated YT AND NK-92 cells or tumor bearing mice with IL-10 in vivo and in vitro,respectively,to detect the biological activities of cells and the growth of tumor in mice.At the same time,we studied the effect of IL-10 on gemcitabine anti-tumor effect,explored the relationship between IL-10 and YT AND NK-92 cell resistance,and looked for the possible role of IL-10 in stimulating YT AND NK-92 resistance White and signal pathway.Chapter I:expression and clinical significance of IL-10 in extranodal NK/T lymphomaObjective:The concentration of IL-10 in serum was measured by enzyme-linked immunosorbent assay,and the relationship with clinical characteristics and efficacy was further analyzed.The expression of P-gp,ABCG2,ABCC4,abcc5 and MRP1 in the ABC membrane transporter family of ENKTL resistant patients was detected by immunohistochemistry to find the resistant protein related to IL-10.Materials and methods:1.50 patients with extranodal NK/T cell lymphoma were collected and divided into two groups according to the therapeutic effect:the effective group and the ineffective group.The serum of healthy volunteers was used as the control,and the concentration of IL-10 in the sera of patients was detected by enzyme-linked immunosorbent assay,and the differences among the three groups were analyzed.2.To analyze the relationship between serum IL-10 concentration and clinical characteristics of patients.3.The expression of ABC superfamily resistance related proteins(P-gp,ABCG2,ABCC4,ABCC5,MRP1)was detected by immunohistochemistry.Result:1.The serum IL-10 levels in ENKTL patients(33.31 ± 12.68)were significantly higher than those in healthy volunteers(14.21 ±6.46)(P<0.001).The serum IL-10 levels in the patients who achieved ineffective treatment(43.28±10.98)with gemcitabine-based chemotherapy were significantly higher than those in the patients who achieved effective treatment(24.01±4.22)(P<0.001).2.There was no correlation between IL-10 concentration and age,gender,Ann Arbor stage and IPI score,but there was significant correlation between IL-10 concentration and PINK score,LDH,B symptom and EBV copy number.3.In 8 cases of extranodal NK/T cell lymphoma,ABCG2、ABCC4、ABCC5、P-gp were positive and MRP1 were negative.In the positive samples.Conclusion:1.The serum level of IL-10 in ENKTL patients was higher than that in healthy volunteers.2.The IL-10 concentration in the serum of ENKTL patients in the ineffective group was higher than that in the effective group.3.The level of serum IL-10 in ENKTL patients was correlated with LDH,B symptoms and EBV copy number.4.ABCG2、ABCC4、ABCC5、P-gp were highly expressed in ENKTL tissues.ChapterⅡ:Study on the resistance and mechanism of IL-10 to extranodal NK/T lymphoma cell line YT AND NK-92Objective:IL-10 stimulated ENKTL cell line YT AND NK-92 in vitro,to study the biological effect of IL-10 on YT AND NK-92 and the resistance to gemcitabine,and verify the correlation between IL-10 and ABC membrane transporter,and to study the molecular mechanism of the effect between them.Materials and methods:1.Different concentrations of IL-10(0,5,10,20,50ng/ml)and gemcitabine(1,5,7.5μg ml)were respectively applied to YT AND NK-92 cells.After 24 hours,CCK-8 method showed the inhibition rate of cells in each group.The IC50 value of gemcitabine was analyzed by Graphpad prism.2.YT and NK-92 cells were treated with different concentrations of IL-10(0,1,5,10,20,50NG/ml)and gemcitabine with a final concentration of 5μg/ml.After 24,48 and 72 hours,the cell activity of each group was measured by CCK-8 method.4.We divided YT AND NK-92 cells into 4 groups,namely negative control group,IL-10 group,IL-10+GEM group and GEM group,IL-10 group was stimulated with IL-10 1ng/ml,GEM group was stimulated with gemcitabine 5ng/ml,IL-10+GEM group was stimulated with both IL-10 and gemcitabine,and cell viability was measured by CCK8 after 24,48 and 72 hours.5.Flow cytometry was used to detect the changes of cell cycle.6.The apoptosis rate changes were detected by flow cytometry.7.The expression of ABCs protein were detected by Western Blot.8.The expression of signaling pathway proteins were detected by Western Blot.Result:1.When the YT and NK-92 cells were treated with IL-10 at 1 ng/ml,5 ng/ml and 1Ong/ml after 24h,the IC50 for gemcitabine were elevated significantly.But the IC50 was not elevated by IL-10 at 20 ng/ml and 50 ng/ml.2.We compared the viability of cells when they treated with different concentrations of IL-10 and a fixed concentration of gemcitabine(5ug/ml).The viability of YT and NK-92 cells was increased significantly when the concentration of IL-10 was 1 ng/ml or 5 ng/ml.The viability of NK-92 cells was also increased by IL-10 at the concentration of 10ng/ml.However,the cell viability was not changed significantly with the higher concentrations of IL-10(20,and 50 ng/ml).3.As shown in the CCK8 assay,compared with control group,the growth of IL-10 group increased not significantly.Gemcitabine suppressed the growth of the cells obviously.By contrast,the viability of cells in IL-10+GEM group was considerably increased compared with that of GEM group.4.Compared with the control group,IL-10 has little effect on the cell cycle.Be treated with gemcitabine,the G0/G1 phase cell ratio increased significantly,and the S phase cell ratio decreased significantly(P<0.05).When IL-10 and gemcitabine acted together,compared with the gemcitabine group,the ratio of G0/G1 phase cells was significantly reduced,and the proportion of S phase cells was significantly increased(P<0.05).The above results suggest that gemcitabine blocked YT AND NK-92 cells in the G0/G1 phase,preventing the progression of the cell cycle,while IL-10 counteracted the gemcitabine block of the cell cycle.5.The apoptosis rate of IL-10 group changed little compared with the control group,the apoptosis rate of GEM group increased significantly,and the apoptosis rate of IL-10+GEM group was significantly lower than that of GEM group,the difference was statistically significant(P<0.05).IL-10 significantly inhibited the pro-apoptotic effect of gemcitabine.6.Both for YT cells and NK-92 cells,the expression of ABCG2,ABCC5 and P-gp did not change in the control group or IL-10 group,but in the GEM group and the IL-10+GEM group,the expression level of ABCG2 and P-gp was significantly reduced.The expression of ABCC5 in the GEM group and the IL-10+GEM group was increased for NK-92 cells,but was reduced for YT cells.Both for YT cells and NK-92 cells,the expression of ABCC4 in the GEM group was reduced,but in the IL-10 group and the IL-10+GEM group,it was significantly increased.7.For both YT and NK-92 cells,the expression of STAT1,p-STAT1,Tyk2 and p-Tyk2 was significantly increased in the IL-10 group but significantly decreased in the GEM group,and the expression of STAT1,p-STAT1,Tyk2 and p-Tyk2 in the IL-10+GEM group was higher than that in the GEM group.The expression of STAT3 was significantly increased in the IL-10 group,the GEM group and the IL-10+GEM group.The expression of p-STAT3 was decreased in the IL-10 and IL-10+GEM groups but increased in the GEM group.The expression of JAK1 and p-JAK1 was decreased in the IL-10 group,GEM group,and IL-10+GEM group,and there was no significant difference among the three groups.Conclusion:1.The low concentration of IL-10 induced the resistance of YT AND NK-92 to gemcitabine.2.IL-10 can antagonize the blockade of gemcitabine on the YT AND NK-92 cell cycle and inhibit the effect of gemcitabine on promoting YT AND NK-92 cell apoptosis.3.The effect of IL-10 on drug resistance of YT AND NK-92 cells is mediated by ABCC4.4.IL-10 induces the resistance of YT AND NK-92 cells to gemcitabine by regulating the JAK/STAT signaling pathway.Chapter Ⅲ:study on the resistance of IL-10 to NK/T cell lymphoma in vivoObjective:To study the effect of IL-10 on gemcitabine resistance of ENKTL and the expression of the ABC proteins in vivo.Materials and methods:1.Construct nude mice model of ENKTL.2.Sixteen nude mice model were divided into four groups.The blank control group was given intraperitoneal injection of normal saline,the IL-10 group was given intraperitoneal injection of IL-10 50μg/kg*2d,the GEM group was given intraperitoneal injection of GEM 15mg/kg,every three days,the IL-10+GEM group was given both IL-10 and GEM.The tumor growth was observed.3.Western Blot was used to detect the expression of ABCC4,ABCC5 and P-gp in the tumor tissue of nude mice.4.The expression of ABCs protein were detected by Western Blot.Result:1.Compared with the control group,there was no significant change in tumor growth in the IL-10 group(P>0.05),and tumor growth in the gem group was significantly slower(P<0.05),while tumor growth in the IL-10+gem group was significantly faster than that in the gem group(P<0.05).2.Compared with the blank control group,the tumor weight of IL-10 group was not significantly reduced(P>0.05),the tumor weight of gem group was significantly reduced(P<0.05),while the tumor weight of IL-10+gem group was significantly increased,but still lower than that of IL-10 group(P<0.05).3.The expression of ABCG2,ABCC5 and P-gp did not change in the negative control group and IL-10 group,whereas it was significantly decreased in the GEM group and IL-10+GEM group;the expression of ABCC4 was decreased in the GEM group,whereas it was significantly increased in the IL-10 group and IL-10+GEM group.Conclusion:1.Under the sustained action of IL-10,gemcitabine resistance occurred in nude mice.2.The effect of IL-10 on gemcitabine resistance may be mediated by ABCC4. |
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