Effect Of Cisplatin On IL-6 Expression In Tumor Mesenchymal Stem Cells And Underlying Mechanism

Lung cancer is not only one of the most common malignant tumors all over the world,but also the malignant tumor with the highest morbidity and mortality in China.According to pathological types,lung cancer can be divided into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC),and NSCLC accounts for about 85%of lung cancer.Because the clinical symptoms of early stage lung cancer are not obvious,most patients have progressed to advanced lung cancer at the time of diagnosis.In recent years,although immunotherapy,targeted therapy and other treatments are constantly evolving,chemotherapy is still one of the main treatments for patients in advanced stage,or without driven gene mutations.And the two-drug combination chemotherapy based on platinum drugs is the main treatment for advanced NSCLC.Among them,cisplatin is one of the most commonly used platinum drugs in clinical practice.Previous studies have confirmed that cisplatin can directly kill tumor cells.When cisplatin enters tumor cells,it can cause DNA damage and interfere with DNA replication.Thereby cisplatin can inhibit tumor cell proliferation and induce tumor cell apoptosis.Recent studies have shown that chemotherapeutic drugs,take cisplatin for example,have direct killing effect on tumor cells.They also affect the infiltration and function of stromal cells and immune cells in the tumor microenvironment,thereby affecting the outcome and therapeutic efficacy of the tumor.Mesenchymal stem cells or multipotent mesenchymal stromal cells(MSCs)are stem cells with multipotential differentiation potential.It exists in almost all types of tissues and participates in processes,including tissue damage repair.Studies have confirmed that MSCs are one of the important components of the tumor microenvironment.MSCs can migrate from bone marrow to tumor sites under the action of various secretory factors derived from tumor cells.And MSCs are involved in the regulation of tumor cell apoptosis,epithelial-mesenchymal transition,stem cell proliferation,angiogenesis and local immune response,thus affecting tumor development and outcome.However,there are few reports about the effects of cisplatin on MSCs.MSCs affect tumor development mainly via the secreting a variety of cytokines and chemokines,promoting tumor local immune cell infiltration and regulating immune response.Interleukin-6 is a multifunctional cytokine that regulates immune cell proliferation and differentiation.It can also recruit mononuclear-macrophages from the peripheral circulation to the tumor sites,and participate in the regulation of the development of various malignant tumors including lung cancer.After reaching the tumor site from peripheral blood,monocytes or macrophages differentiate into tumor-associated macrophages(TAMs)under the action of various factors.TAMs play a pro-tumor role by promoting tumor proliferation,invasion and metastasis,stimulating tumor angiogenesis,and leading to immunosuppression.In this paper,we systematically studied the effects of cisplatin on the expression of cytokines and chemokines in MSCs by in vitro experiments,and explored the signaling pathways involved in them,and verified the chemotaxis of MSCs to macrophages.Part I Effect of cisplatin on the expression of cytokines and chemokines in tumor mesenchymal stem cellsObjective:To study the effect of cisplatin on MSCs in vitro,to explore the effect of cisplatin on the expression of cytokines and chemokines in MSCs,and to screen out the cells/chemokines which are significantly altered.Methods:1.MSCs were isolated and purified from the bone marrow of C57/BL6 mice by adherent separation,immunomagnetic beads negative sorting and flow cytometry,followed by sorting and purification.The isolated and purified cells were identified according to the International Cell Therapy Association standards;2.MSCs were treated with cisplatin at different concentrations or for different durations,and then cell viability was measured by CCK-8 method to study the inhibitory effect of cisplatin on MSCs;3.MSCs were treated with cisplatin,and the expression changes of cytokines and chemokines after cisplatin treatment were detected by polymerase chain reaction array(PCR Array).4.The expression changes of IL-6 and other factors were further detected and verified from mRNA and secreted protein levels by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA),respectively.5.Mouse lung cancer cell Lewis lung carcinoma(LLC)cell was treated with cisplatin,and the expression of IL-6 mRNA and secreted protein were detected by qRT-PCR and ELISA,respectively;Results:1.The mouse MSCs cell line was successfully established and verified from phenotype and function.2.Cisplatin has a significant inhibitory effect on MSCs,and the inhibition is related to the concentration of cisplatin,the duration of action and the number of MSCs.With the increase of cisplatin concentration(0 ?mol/L-50 ?mol/L)or prolonged action time(6 h-72 h),the cell inhibition rate increased gradually(0%-99.83%).The number of cells seeded with MSCs before cisplatin treatment also affected the inhibition of cisplatin.As the number of cells decreased(in a 96-well plate:5000-1000 cells/well)or as the duration of action increased(24 h-72 h),the inhibition of cisplatin gradually increased(0%-99.64%).3.After cisplatin treatment of MSCs,PCR Array results show that:(1)0 h after cisplatin treatment:no cytokine or chemokine gene expression were increased in MSCs,and most of them decreased;(2)6 h after cisplatin treatment:CCL-2,CCL-20,CCL-5 and CCL-7 in the CCL family increased by 8.82,1.93,2.72 and 9.79 times,respectively.CXCL-1,CXCL-10,CXCL-16,CXCL-3 and CXCL-5 in the CXCL family were increased by 2.53,1.55,1.09,1.79,and 1.58 times,respectively.IL-2,IL-22,IL-6 and IL-7 in the IL family increased by 1.53,1.89,14.34 and 1.12 times,respectively,and the IL-6 gene was most elevated.(3)24 h after cisplatin treatment:CCL-2,CCL-20,CCL-4,CCL-5 and CCL-7 in the CCL family increased by 6.10,2.19,2.11,44.62 and 9.51 times,respectively.CXCL-1,CXCL-10,CXCL-16,and CXCL-3 in the CXCL family increased by 4.56,106.86,6.91,and 4.56 times,respectively.IL-15,IL-17f,IL-8.IL-6,IL-7 and IL-9 in the IL family increased by 2.53,24.93,2.22,21.11,7.78 and 5.77 times,respectively.4.There was no statistically significant change in IL-6 mRNA and protein expression at 3 h and 24 h after cisplatin treatment(control v.s'cisplatin treatment group:mRNA:3 h:P=0.089;24 h;P=0.073;Protein:3 h:P=0.149;24 h;P =0.149).5.The results of qRT-PCR and ELISA showed that the expression change of IL-6 mRNA and protein in MSCs after cisplatin treatment were statistically significant(P<0.001).(1)At 3,6,and 24 h,IL-6 mRNA expression increased by 1.67,2.83,and 10.15 times,respectively(3 h:P=0.218;6 h:P=0.007;24 h:P<0.001).And with the prolongation of cisplatin,IL-6 mRNA expression level increased gradually(3 hvs 6 h:P=0.050;3 hvs 24 h:P<0.001;6 hvs 24 h:P<0.001).(2)At 3,6,and 24 h,IL-6 protein secretion levels increased by 3.28,5.71,and 6.69 times(3 h:P = 0.001;6 h:P<0.001,24 h:P<0.001).And with the prolongation of cisplatin action,IL-6 secretion levels gradually increased(3 h vs 6 h:P= 0.001;3 h vs 24 h:P<0.001;6 h vs 24 h:P = 0.074).Conclusion:We successfully sorted and purified mouse MSCs and identified them from phenotype and function.After cisplatin treatment of MSCs,the expression of various cytokines and chemokines changed significantly,and the expression of IL-6 was most prominent.For mouse lung cancer LLC cells,the expression of IL-6 was not significantly changed after cisplatin treatment.These results suggest that MSCs are one of the main sources of IL-6 in the tumor microenvironment under the action of cisplatin,and its expression is significantly increased.Part II Mechanism of cisplatin regulating IL-6 expression in tumor mesenchymal stem cellsObjective:To investigate and elucidate the possible involvement of cisplatin in the regulation of IL-6 expression in MSCs,and to study the effect of MSCs-derived IL-6 expression on macrophage chemotaxis.Methods:1.Set up the following groupings according to the purpose of the experiment:(1)MSCs control group:conventionally cultured MSCs;(2)cisplatim-treated group:MSCs treated with 5 pmol/L cisplatin for 6 h;(3)p38MAPK pathway inhibitor group:MSCs were treated with 10 ?mol/L SB20358(p38MAPK pathway inhibitors);(4)IL-6 antibody group:0.03 ?g/mL IL-6 blocking antibody was added to MSCs medium;(5)Negative control group:DMEM only;(6)Positive control group:IL-6 recombinant protein at 0.25 ng/mL.2.After cisplatin treatment,the total protein of MSCs was extracted.And Western blotting was used to detect the expression of p38MAPK pathway in MSCs control group and cisplatin treatment group.3.The p38MAPK pathway was inhibited by the specific inhibitor SB20358,and the expression of IL-6 in MSCs after cisplatin treatment was detected by ELISA.4.The chemotaxis of RAW264.7 macrophages was detected by Transwell system in cisplatin-treated group MSCs and control group MSCs,respectively.5.Study the chemotaxis of MSCs-derived IL-6 on RAW264.7 macrophages by antibody blocking IL-6.Results:1.The expression of p38 and pp38 were enhanced importantly at 3,6,and 24 h after cisplatin treatment,and the ratio of pp38/p38 was significantly increased.The difference was statistically significant(MSCs control group vs cisplatin treatment group:3 h:P<0.001;6 h:P=0.019;24 h:P<0.001).The p38MAPK pathway is clearly activated.2.After inhibition of the p38MAPK pathway by specific inhibitors,the up-regulated IL-6 expression of cisplatin in MSCs was partially inhibited.The expression of IL-6 was significantly decreased compared with the control group(MSCs control group vs cisplatin-treated group:3 h:P<0.001;6 h:P<0.001;24 h:P<0.001).3.The chemotactic effect of MSCs on RAW264.7 macrophages was significantly enhanced after cisplatin treatment(cisplatin-treated groupvs MSCs control group:P<0.001).4.Chemotactic effects of cisplatin-treated MSCs on RAW264.7 macrophages were significantly down-regulated by antibodies blocking IL-6 function(cisplatin-treated vs IL-6 antibody group:P<0.001).Conclusion:The p38 MAPK pathway was significantly activated after cisplatin treatment of MSCs.The p38MAPK pathway is involved in the regulation of IL-6 expression in MSCs which can be verified by specifically inhibiting this pathway with inhibitors.The chemotaxis of MSCs to RAW264.7 macrophages was significantly enhanced after cisplatin treatment.And blocking experiments indicated that this chemotaxis was partially mediated by IL-6.