The Study Of Mechanisms Of ABC Family In Regulating Drug Resistance In Nasal NK/T-cell Lymphoma Mediated By IL-6

NK/T cell lymphoma(NKTCL)is one type of Non-Hodgkin’s lymphoma(NHL),and the most common subtype of T cell lymphoma.Epidemically,the incidence of of NKTCL is higher in Latin America and East Asia than European and American countries.However,the specific cause of the disease is still not clear.At present,it is generally thought that the morbidity of NKTCL is associated with epstein-barr virus infection and chronic rhinitis,while some researchers believe that genetic factors may play a role in genesis and development of NKTCL,which made many researchers focus on lymphoma in recent years.The complexity of the cause of NKTCL caused great difficulties in treatment and prognosis of patients with this disease.In the tumor treatment at an early stage,chemotherapy is more traditional and important method in clinical.However,multi-resistant(MDR)tend to occur in the cancer patients,which is a major obstacle to the successful chemotherapy.Therefore,further study of the molecular mechanism of tumor resistance,will provide essential theoretical basis for designing reverse multidrug resistance in tumor cells.Excessive secretion of interleukin 6(IL-6),plays an important role in many chronic inflammation.IL-6 binding to IL-6R can activate 3 signaling pathways: JAK/STAT,ERK1/2/MAPK and PI3-K signaling pathways.Some reports suggest that the expression of IL-6R participates in tumor growth and metastasis,which suggested that IL-6 signaling pathways were related to tumor growth and development.IL-6 mediated JAK1/STAT3 or the NF-κB pathway play an important role in drug resistance of cancers,such as lung cancer cells,and ovarian cancer cells and prostate cancer cells.However,because of the complexity of the IL-6 regulation network,the relationship between IL-6 and NK/T lymphoma of growth and development are still unclear.The molecular mechanism of the effect of IL-6 and its mediated pathway on NK/T cell lymphoma remains to be a further research.In this study,the level of IL-6 in peripheral blood of patients with nasal type NK/T cell lymphoma and chronic rhinitis was detected by enzyme linked immunosorbent assay(ELISA),and we also analysed the relationship among the level of IL-6,clinical pathological parameters and the curative effect.Western blot was used to detected the expression of the ABC superfamily drug-resistant related proteins(ABCG2,ABCC4,P-gp and MRP1).In vitro,the effects of IL-6 on cell proliferation,cell cycle,apoptosis and the chemotherapy drug sensitivity of NK/T cell lymphoma were measured by flow cytometry and CCK-8 assays repectively.Further,western blot was used to detect the expression of multi-drug resistant proteins and IL-6 mediated signaling pathway-related proteins.In vivo,by animal experiment,we explored the effect of IL-6 on ABC membrane transport protein mediated drug resistance in NK/T cell lymphoma.Exploring the effect of IL-6 on ABC membrane transport proteins mediated drug resistance is conducive to providing experimental and theoretical reference for exploring the NK/T cell lymphoma drug resistance reversal drugs.This topic includes the following three parts:1.Expression of IL-6 in NK/T cell lymphoma tissue and its significance;2.The study of molecular mechanism of the effect of exogenous IL-6 on SNK cell resistance;3.The study of the effect of exogenous IL-6 on drug resistance of NK/T cell lymphoma in vivo.The first chapter: Expression and significance of IL-6 in NK/T cell lymphoma Methods1.HE staining(hematoxylin-eosin staining): HE staining was used to detect the cell morphology and histological features of this NK/ T cell lymphoma tumor.2.Enzyme-linked immunosorbent assay(ELISA): according to the instructions,ELISA was used to detect the level of IL-6 in peripheral serum of patients with NK/ T cell lymphoma.3.We analyzed the relationship between the expression of IL-6 in peripheral serum of patients with NK/T cell lymphoma with clinical factors.4.Immunohistochemistry was used to detect the levels of ABCG2、ABCC4、P-gP and MRP1 in NK/T cell lymphoma tissue.Results1.HE staining revealed that the tumor grew with the diffuse and infiltrative characteristics,tissue necrosis,surface of evident mucosal ulcerate.Tumor cells form a single,medium-sized,the cytoplasm stained lightly,and has invasion and destruction of the vessel wall.2.ELISA showed that the level of IL-6 in peripheral serum was significantly reduced,compared with the control group.3.The results showed that the expression change of IL-6 in peripheral serum of patients with NK/T-cell lymphoma has no obvious relationship with patients’ age,gender,location,clinical stage and the levels of lactate dehydrogenase(LDH).We found the level of IL-6 in peripheral serum of patients with Ki-67 > 80% was significantly lower than that in peripheral serum of patients with Ki-67 < 80% and the rate of the decreased expression of IL-6 in the effective treatment(CR+PR)group was higher than that in the ineffective treatment group(SD+PD).4.Immunohistological study showed that the levels of ABCG2,ABCC4 and P-gp in NK/T cell lymphoma tumor tissue were significantly increased compared with the control group.MRP1 was weakly positive in NK/T cell lymphoma tumor tissue.The second chapter: The study of molecular mechanism of the effect of exogenous IL-6 on SNK cell resistance Methods1.We established human multidrug-resistant NK/T cell lymphoma cell line(SNK-6/ADM)by low doses of intermittent induction.2.CCK-8 assay was used to detect the effects of IL-6 on growth,proliferation and multidrug resistance of SNK-6/ADM cells.3.Flow cytomerty was used to detect the effects of IL-6 on cell cycle and apoptosis of SNK-6/ADM cells.4.The expression of ABCC4,P-gp,JAK2,STAT3 and NF-κB was examined by western blot.Results1.CCK-8 assays showed that 50% inhibitive concentration(IC50)for adriamycin was 35.54 μg/mL in SNK-6/ADM cells and SNK-6/ADM cells had a resistance index of 5.61 μg/mL.The results indicated that we successfully obtained multidrug-resistant NK/T cell lymphoma cell line(SNK-6/ADM).2.CCK-8 assays showed that IL-6 had no obvious effect on cell proliferation SNK-6 and SNK-6/ADM cells,whereas IL-6 significantly inhibited the resistance of SNK-6/ADM cells to adriamycin.3.Flow cytomerty analysis showed that the IL-6 in combination with adriamycin treatment significantly inhibited the cell cycle of SNK-6/ADM cells and promoted apoptosis in SNK-6/ADM cells.4.Western Blot analysis revealed that compared with control group,the levels of ABCC4 and P-gp in SNK-6/ADM cells were significantly increased.IL-6 in combination with adriamycin treatment significantly inhibited the ABCC4 protein expression,whereas the level of P-gp had no obvious change.In addition,the levels of p-JAK2,p-STAT3 and NF-κB in SNK-6/ADM cells treated with IL-6 in combination with adriamycin were significantly decreased.The third chapter: The study of the effect of exogenous IL-6 on drug resistance of NK/T cell lymphoma in vivo Methods1.The mice models bearing MDR solid tumors were established by injecting SNK-6/ADM cells subcutaneously in the left side of the nipple of mice.2.The intravenous injection administration of IL-6 or adriamycin,we analyzed tumor growth and the survival rate of mice bearing MDR solid tumors.3.Immunohistochemistry was used to detect the levels of ABCC4 and P-gP in NK/T cell lymphoma tissue of mice.Results1.Compared with the control group,IL-6 in combination with adriamycin treatment significantly inhibited growth of tumor,and survival rate of SNK-6/ADM mice was significantly increased.2.Immunohistological study showed that the level of ABCC4 in NK/T cell lymphoma tumor tissue of mice treated with IL-6 in combination with adriamycin was significantly increased compared with the control group,whereas P-gp expression had no obvious change.Conclusion1.The change in level of IL-6 has a close relationship with Ki-67 expression and treatment efficacy in patients with NK/T cell lymphoma.In addition,in tumor tissue of patients with NK/T cell lymphoma the levels of ABCG2,ABCC4 and P-gp were significantly increased.These results suggested that IL-6 may have a close relation to NK/T cell lymphoma multi-resistants,and function as a potential drug reversing MDR with high efficiency and low toxicity.2.IL-6 had no significant effect on cell proliferation of SNK-6 and SNK-6/ADM cells.IL-6 in combination with adriamycin treatment had obvious inhibitory effects on cell growth and cell cycle process and positive effect on apoptosis of SNK-6/ADM.IL-6 in combination with adriamycin had obvious inhibitory effect on expression of ABCC4,p-JAK2,p-STAT3 and NF-κB.IL-6 may inhibit drug resistance mediated by ABCC4 in NK/T cell lymphocytes through the JAK2/ STAT3 and the NF-κB signaling pathways.3.IL-6 in combination with adriamycin can inhibit the tumor growth in SNK-6/ADM mice,and significantly increase the survival rate of SNK-6/ADM mice.IL-6 in combination with adriamycin treatment decreased expression of ABCC4 in tumor tissues of SNK-6/ADM mice.We speculate that IL-6 has a reverse effect on drug resistance in NK/T cell lymphoma.Therefore,IL-6 can be used as a potential target.