Effect And Mechanism Of ?-elemene On Recruitment Of Macrophages By Lung Cancer Cells Induced By Radiation And Hypoxia

Tumor associated macrophages are a group of macrophages infiltrating in the tumor area.They are among the most abundant normal cells in the tumor microenvironment.Researchers found that number of tumor-associated macrophage increased in radiotherapy or hypoxia and they often present tumor-promoting phenotype.They promote tumor survival,angiogenesis and inhibit immune response.It makes tumor resistant to radiotherapy.Therefore,inhibiting macrophage infiltration is beneficial for tumor treatment and makes tumor more sensitive to radiotherapy.?-elemene is extracted from the Chinese medicine herb Curcuma Wenyujin,it is confirmed as a radiosensitizer in recent years.It sensitized the lung cancer cell to radiotherapy by enhancing DNA damage and inhibiting DNA repair,or by increasing apoptosis in radiation.Whether it has an effect on the infiltration of macrophages in the tumor,there is no research yet.Radiation induced reactive oxygen species(ROS),activated Prx-1 and nuclear factor-kappa B(NF-?B)pathway.Hypoxia is a main feature of solid tumors,and radiation will destroy the vascular structure of the tumor,aggravating the hypoxia.Hypoxia promotes the expression of hypoxia inducible factor-1?(HIF-1?),it regulate the transcription of many genes to make cells to adapt the hypoxic environment.Whether the Prx-1,NF-?B and HIF-1? regulate macrophages infiltration needs to be further study.This study observed the effects of radiotherapy and hypoxia on macrophageinfiltration,the effect of ?-elemene on macrophage infiltration and the mechanism of lung cancer cells recruitment of macrophage.The aim of this study was to reveal the reason of macrophage infiltration in radiotherapy and hypoxia,so as to facilitate targeted treatment.At the same time,it further investigate the radiosensitization mechanism of ?-elemene,and provide more information for its application in clinical.Objective:1.To study the recruitment effect of lung cancer cells on macrophages and the effect of ?-elemene on this process in radiation and hypoxia.2.To study the expression of Prx-1,NF-?B and HIF-1? in lung cancer cells in radiation and hypoxia,the role of these factors in the recruitment of macrophages,and explore the relationship between these factors.3.To study the effects of radiation and ?-elemene on the growth of transplanted tumor and the infiltration of macrophages in mice,validate the experimental results in vitro.Methods:Part one: The recruitment effect of lung cancer cells on macrophages and the effect of ?-elemene on this process in radiation and hypoxia.1.Growth inhibition in Lewis cells after exposure to different concentrations of?-elemene for 24 h was determined by MTT,calculated IC50,IC20 and IC10.2.Experimental groupings: Lewis cells were divided into(1)Control: received DMEM medium;(2)?-elemene: treated with 45 ?g/ml ?-elemene for 24h;(3)Irradiation: irradiated with 4Gy X-irradiation;(4)?-elemene and Irradiation: after incubation with ?-elemene for 24 h,the cells were irradiated with 4Gy X-irradiation;(5)Hypoxia: received DMEM medium and hypoxia culture;(6)Irradiation and Hypoxia:irradiated with 4Gy X-irradiation,hypoxia culture 24h;(7)?-elemene and Hypoxia:after incubation with ?-elemene for 24 h,then hypoxia culture 24h;(8)?-Elemene,Irradiation and Hypoxia: after incubation with ?-elemene for 24 h,the cells were irradiated with 4Gy X-irradiation,then hypoxia culture 24 h.The supernatant(conditioned medium,CM)of cells was collected.The recruitment of RAW264.7 wasdetected by Transwell assay.3.q RT-PCR and ELISA were used to detect the expression of m RNA and protein of chemokines in Lewis cells and CM.4.Added chemokine neutralizing antibody into CM collected from Control,Irradiation,Hypoxia,Irradiation and Hypoxia groups,Transwell assay was used to detect the recruitment of RAW264.7.5.After culturing RAW264.7 cells with CM,the protein and m RNA expression of macrophage phenotype marker gene was detected by Western Blot and q RT-PCR.Part two: The expression of Prx-1,NF-?B and HIF-1? of lung cancer cells in radiation and hypoxia,the role of these factors in the recruitment of macrophages and the relationship between these factors.1.Western Blot,q RT-PCR and immunofluorescence were used to detect the expression of Prx-1,NF-?B and HIF-1? protein and m RNA expression in Lewis cells in radiation and hypoxia.2.Lewis cells were transfected with Prx-1,NF-?B and HIF-1? si RNA.CM were collected and ELISA was used to detect the chemokine expression in radiation and hypoxia.3.Western Blot detected the expression of other two genes.4.Lewis cells were divided into(1)Control(2)?-elemene(3)Irradiation(4)?-elemene and Irradiation(5)Hypoxia(6)Irradiation and Hypoxia(7)?-elemene and Hypoxia(8)?-elemene,Irradiation and Hypoxia;The protein expression of Prx-1,NF-?B and HIF-1? in Lewis cells were detected by Western Blot.Part three: Effects of radiotherapy and ?-elemene on the growth of transplanted tumor and the infiltration of macrophages in mice.1.C57BL/6 mouse transplanted tumor models were established by subcutaneous inject of cell suspension and randomly divided into four groups(n=6): control group(Na Cl),45 mg/kg ?-elemene group,radiotherapy group(Na Cl + radiotherapy),45 mg /kg ?-elemene + radiotherapy group2.Measure the volume of transplanted tumor volume every other day and draw thetumor growth curve.3.After treatment,weigh the transplanted tumor and calculate the tumor inhibition rate 14 days later.4.Immunohistochemical was used to detect macrophage infiltration and expression of chemokine MCP-1.Results:Part one:1.The IC10,IC20 and IC50 of Lewis cells were 73?g/ml,83?g/ml and 103?g/ml.2.Compared with the control group,the chemotaxis of macrophages of irradiation group,hypoxia group and irradiation+hypoxia group increased(P<0.05).After adding?-elemene,the increased chemotaxis is suppressed(P<0.05).3.The m RNA expression of MCP1 in Lewis cells was increased in the irradiation group,hypoxia group and hypoxia+irradiation group(P<0.05),and they were all inhibited after the addition of ?-elemene(P<0.05).The MCP-1 secretion in lung cancer cells in irradiation group,hypoxia group and hypoxia group were also increased(P<0.05)and they were all inhibited after the addition of ?-elemene(P<0.05).4.The chemotaxis of macrophages of irradiation group,hypoxia group and irradiation+hypoxia group were decreased after treating with MCP-1 neutralizing antibody(P<0.05).5.The protein expression of ARG1 and the m RNA expression of IL-10?MRC1were increased in RAW264.7 cells after culturing with CM of irradiation group and irradiation+hypoxia group(P<0.05).Part two:1.The protein and m RNA expression of Prx-1,NF-?B/p65 and HIF-1? in radiation and hypoxia: 1)the protein and m RNA expression of Prx-1 are increased in irradiation group and irradiation+hypoxia group(P<0.05);the protein and m RNA expression of Prx-1 are not changed in hypoxia group(P>0.05);2)the protein and m RNA expression of HIF-1? and the nucleus p65 expression are increased in the irradiation group,hypoxia group and irradiation+hypoxia group(P<0.05).Suggesting that radiationregulates the expression of Prx-1,HIF-1? and nucleus p65,whereas hypoxia does not regulate Prx-1 expression and regulates the expression of nucleus p65 and HIF-1? only.2.The secretion of MCP-1 of Lewis cells when they were transfected with Prx-1,NF-?B/p65,HIF-1? si RNA: 1)Compared with the untransfected Prx-1 si RNA group,the levels of MCP-1 in the irradiation group and the irradiation+hypoxia group were significantly inhibited(P<0.05),but the MCP-1 in the hypoxia group were not affected(P>0.05),suggesting that inhibition of Prx-1 expression affects radiation-induced MCP-1 secretion,but does not affects hypoxia-induced increasing of MCP-1;2)When cells were transfected with NF-?B/p65 si RNA,MCP-1 secretion in the irradiation group,hypoxia group and irradiation+hypoxia group were no longer increased,compared with the untransfected group(P<0.05),indicating that inhibition of NF-?B/p65 expression affects radiation and hypoxia-induced MCP-1 secretion;3)When cells were transfected with HIF-1? si RNA,the secretion of MCP-1 in irradiation group,hypoxia group and irradiation+hypoxia group was inhibited(P<0.05).It suggested inhibition of HIF-1? expression affects both radiation and hypoxia-induced MCP-1 secretion.3.The expression of other two genes when Lewis cells were transfected with Prx-1,NF-?B/p65,HIF-1? si RNA,respectively: 1)Inhibited Prx-1 expression,total HIF-1?and nucleus p65 protein expression did not increase in irradiation group and irradiation+hypoxia group was inhibited(P<0.05),suggesting that Prx-1 regulated the expression of HIF-1? and NF-?B/p65;2)After transfecting with NF-?B/p65 si RNA,the expression of HIF-1? was inhibited in the irradiation group,hypoxia group and irradiation+hypoxia group(P<0.05).The expression of Prx-1 in irradiation group and irradiation+hypoxia was not affected(P>0.05),suggesting NF-?B/p65 regulates the expression of HIF-1? in radiation and hypoxia,but does not regulate Prx-1 expression;3)After transfecting with HIF-1? si RNA,the expression of p65 nucleus protein in irradiation group,hypoxia group and irradiation+hypoxia group were not affected(P>0.05),and the expression of Prx-1 in irradiation group and irradiation+hypoxia group was not affected(P>0.05),suggesting that HIF-1? does not regulate NF-?B/p65 and Prx-1 expression in radiation and hypoxia.4.TLR4 is an important factor that mediates the activation of NF-?B pathway.Therefore,Lewis cells were transfected with TLR4 si RNA,though expression of Prx-1was increased(P<0.05)in irradiation group,but expression of nucleus p65 was inhibited in irradiation group(P <0.05),suggesting that Prx-1 activities NF-?B depend on TLR4.5.?-elemene inhibit the expression of Prx-1,nucleus p65 in irradiation group and irradiation+hypoxia group(P<0.05);inhibit the expression of HIF-1? in irradiation group,hypoxia group and irradiation+hypoxia group(P<0.05);But it has no effect on the expression of nucleus p65 in the hypoxic group(P>0.05).Part three:1.The mice transplanted tumor model was successfully established,and the growth curve of each group was obtained according to tumor volume.On the 7th day,the tumor volume of each treatment group was smaller than that of the control group(P <0.05).On the 15 th day,the tumor volume of ?-elemene combined with radiotherapy group was significantly smaller than that of other groups(P<0.05).EF=1.65,suggesting that?-elemene has a radiosensitizing effect.2.The tumor weight of radiotherapy group and ?-elemene group was lower than that of control group(P<0.05),the tumor weight of ?-elemene combined with radiotherapy group is lower than that of radiotherapy group and ?-elemene group(P<0.05),and the tumor inhibition rates of radiotherapy group,?-elemene group,?-elemene combined with radiotherapy group are 46.01%,36.81% and 72.39%respectively.3.Compared with the control group,the macrophage infiltration of the radiotherapy group was increased(P<0.05),and the infiltration of the macrophages in the ?-elemene combined with radiotherapy group was less than that of the radiotherapy group(P<0.05).4.The expression of MCP-1 in the radiotherapy group was significantly higher than that of the control group(P<0.05),and MCP-1 expression in ?-elemene combinedwith radiotherapy group was lower than that of radiotherapy group(P<0.05).Conclusion:Part one:1.Radiation and hypoxia promote lung cancer cell recruitment of macrophages,and radiation can also promotes macrophages transform to M2 phenotype.2.Radiation and hypoxia promote the secretion of MCP-1 in lung cancer cells.3.Radiation and hypoxia promote lung cancer cells recruitment of macrophages depend on MCP-1.4.?-elemene inhibits radiation and hypoxia induced recruitment of macrophages.5.?-elemene inhibits radiation and hypoxia induced MCP-1 secretion in lung cancer cells.Part two:1.Radiation induces the expression of Prx-1,activates NF-?B and promotes the expression of HIF-1?.Hypoxia has no effect on the expression of Prx-1,but it can activates NF-?B and promotes HIF-1? expression.2.Radiation-induced MCP-1 expression depends on Prx-1,NF-?B,HIF-1?;hypoxia-induced MCP-1 expression does not depend on Prx-1 but dependent on NF-?B and HIF-1?.3.Radiation activated the Prx-1/NF-?B/HIF-1? pathway,hypoxia activated the NF-?B/HIF-1? pathway.4.Radiation activates NF-?B depends on TLR4.5.?-elemene inhibits the expression of Prx-1,the activation of NF-?B and the expression of HIF-1? in radiation;inhibits the expression of HIF-1? in hypoxia.Part three:1.Radiotherapy and ?-elemene can inhibit the growth of transplanted tumor in mice,the inhibit effect of radiotherapy combined with ?-elemene is more obvious,?-elemene has radiosensitizing effect.2.Radiotherapy promotes the recruitment of macrophages in transplanted tumor,and ?-elemene inhibit the recruitment.3.Radiotherapy induces expression of MCP-1 in transplanted tumor,and?-elemene inhibited the expression of MCP-1.