Study On The Molecular Mechanism Of Acute Inflammation Influence On Proliferation And Angiogenesis In Melanoma

As early as 1863,Virchow first postulated that cancer originates at sites ofchronic inflammation,in part based on his hypothesis that some classes of irritantsthat cause inflammation also enhance cell proliferation.In the past decades,scientistshave made considerable progress in the research on the relationship between cancerand inflammation.This process of inflammation involves the classic model ofinflammatory response,including the formation of granulation tissue,leukocyteinfiltration,angiogenesis factor,and cytokines network.All of those are very similarto tumor growth and progression,but since there are two different consequences,theformer is limited and controlled;the latter is unlimited and irregular.In this study,amouse tumor model undergoes manufactured surgical wound,building a model ofacute inflammation,to evaluate the relationship between acute inflammation orwound healing and the process of tumor growth.HE staining was performed tocompare the microvessel density (MVD) among these groups.Immunhistochemistrywas employed to detect the expression of VEGF,metalloproteinases-2 (MMP-2) andMMP-9.Gelatin zymography was used to examine the levels of MMP-2 andMMP-9 activity.In vitro experiments,the role of TGF-β/IFN-γin the inflammationwas evaluated.This study provides a better understanding of the relationship betweentumor and inflammation,and contributes to a deeper understanding of tumor cells andattacks on immunity.The main contents are as following:1.The Construct of melanoma mouse model of acute inflammationMelonoma,metastasis occurs in the early phase by blood,is selected toconstructed the model.When the volum of melanoma is up to about 500 mm~3,B16F10 melanoma cells suspension was used to establish different types ofmelanoma - acute inflammation model.Group A:Melanoma- a simple model of trauma,Group B:Melanoma-therepeated debridement model,Group C:Melanoma - traumatic suppurative bacteriamodel,Group D:Melanoma - the trauma model of glacial acetic acid,E:controlgroup--imple tumor.Information of tumor from daily observation was used to mapthe curves of growth to compare the different effects of inflammation in the tumor model.The experimental results show that:In this experiment,the tumor volume of thecontrol group and experimental group focused on among 450-550 mm~3 before theestablishment of acute inflammation model.After treantments with the repeatedimposition of the wound debridement,Staphylococcus aureus and glacial acetic acidinterference,different trends of the tumor growth of emerged.Tumor growth ofGroup A is similar to that of Group E,tumor growth rate slowed down in otherexperimental groups,the volume is less than the control group gradually.On the fifthday of modeling,tumor volume of Group B,C and D focused on between 350-450mm~3,which suggest that the acute inflammation had no effect on tumor growth untilto a certain intensity and duration.When reaches a certain intensity and duration,inflammation can inhibit tumor growth.All differences of three groups had statistical significancein in inhibition of tumorgrowth (P＜0.05),t here was no difference between them (P＞0.05) .All three waysof modeling can achieve the effect of sustained inflammatory stimulus,but thewounds and other conditions vary:Mice in Group B has clean wound,cruste and exudative are not clear,thetransition of tissue from the old to the new,suitable for higher sterile experiments.But it needs daily debridement and it's complicated to operate.Mice in Group B havepus covered eggplant,modeling method is simple,but there is risk of suffering fromsepsis,Staphylococcus aureus infection and as a result of strong demands for keepingseparate;Wound of mice in Group D is cleaner than that of Group C;Composition ofwound in Group D is more homogeneous than that of Group B.But acetic acid hasthe capability to cause corrosion and is strict with the experimental site.In the processof modeling,it is easy to damage large and medium-sized blood vessels and othersensitive parts.Also there are less interfere effect on B models.Based on the aboveexperimental results,this study selected melanoma - the repeating debridement as ouranimal experimental model of the next step.2.The effect of acute inflammation on angiogenesis in melanoma.Tumor angiogenesis is a complex process of the interaction between tumor cells,vascular endothelial cells with their micro-environment.A variety of active substances can regulate tumor angiogenesis.A variety of cytokines resulting from theprocess of inflammation may also control the activation,migration,proliferation,remodeling and apoptosis of endothelial cells,which play a critical role in promotingtumor angiogenesis.According to the first part of the experimental results,we selectmelanoma - the continuing debridement as our animal experimental model.In orderto investigate the effect of acute inflammation on angiogenesis in melanoma,HEstaining was performed to compare MVD among these groups andimmunhistochemistry was employed to detect the expression of VEGF.Mice will bedivided into two groups:The control group:tumor without wound;the experimentgroup:tumor with wound on the opposite side of bodies.After modeling,tumorvolume of the control group and the experiment group were measured on alternatedays and calculated according to the method reported in the literature.Tumor growthcurve were mapped.On the 7th day of modeling,the animals were sacrificed,andthen tumor tissues were isolated,formalin fixed for paraffin block preparation.The experimental results show that:1.The experimental model present two phasein the process of tumor-inflammation interaction:shown as inhibition phase (before7d) and inhibition-missing phase (after 11 d).2.The MVD in the in two groups weresignificantly different in inhibition phase:Control＞Experiment (P＜0.05) 3.Thepositive expression ratio of VEGF in the experiment group statistically differentsfrom the control group in inhibition phase (P＜0.05);Model established in this study was used to observe the direct and indirect impactof inflammation on tumor tissue through the simulation of the pathophysiology ofacute inflammatory process,with particular attention to effect of acute inflammationon angiogenesis in melanoma.The effect characterized by inhibition,including theinhibition of tumor growth and angiogenesis.3.The effect of acute inflammation on expression of MMPsStudy found that cancer cells in tumor tissue are not simply the sum.There isclose collaboration between tumor cells and stromal cells.These cells includefibroblasts,inflammatory cells such as macrophages and endothelial cells.These cellsrely on extracellular matrix (ECM) and link with each other constitute thecomponents of tumor stroma.Stromal tumor components have an important impact on the growth of tumor cells themselves and migration.Matrix metalloproteinasefamily participates in the degradation and remodeling of ECM in the tumor and itsmicro-environment.Acute inflammation plays an important role in tumor growth,invasion and metastasis,as well as wound healing and the formation of granulationtissue.MMP-2 and MMP-9 is two key members of MMPs,which closely effecttumor microcirculation and wound healing.Study in this part is to investigate the effect of acute inflammation on expressionof MMP-2 and MMP-9.The expression of MMP-9 and MMP-2 were examined byimmunohistochemistry staining and Gelatin Zymography.Evaluate the effect of acuteinflammation on the expression of MMPs under the condition of the existence ofacute inflammation and tumors at the same time.The experimental results show that:immunohistochemical staining was used todetect positive cell count,in 7d,MMP-2 expression was 2-fold down-regulationcompared to the control group,same as MMP-9.both statistically significantdifference (P = 0.000);but at lld,MMP-2 has missing statistically significantdifference,MMP-9 also restored.Gelatin zymography analysis showed that theactivity of MMP-2 and MMP-9 were down-regulated compared with the controlgroup within the tumor tissue,both statistically significant (P＜0.05) .At 11 d thisdifference were whole missing.The results suggest that the expression and theactivity of MMP-2 and MMP-9 are both subject to the effects of acute inflammation.Acute inflammation may effect expression and activity of MMP-2 and MMP-9 bysome way.This effect is also shown to the early inhibition phase and loss of inhibitionin late-phase,with the second part of the experimental results anastomosis.This isidentical with the result in the second part.4.A pilot study of IFN-γand TGF-βon melanoma cell proliferation,migrationand invasionThe process of acute inflammation may involve two stages:the initial stage ofrelease of inflammatory factors and the late stage of repairing granulation tissue.As aresult of continued wound debridement in this experimental,it mainly served as theinitial stage of release of inflammatory factors.This stage involved a wide range ofinflammatory cytokines and infiltration of inflammatory cell.Local areas of inflammation characterized by inflammatory cell infiltration,mainly myeloid cells,and the effect on the tumor may be caused by the release of inflammatory factors,directly or indirectly.In order to further confirm the effect on tumor cell proliferation,migration andinvasion capacity caused by the local release of inflammatory factors,serum from thesecond part was used to analysis and screening cytokine.We found that dynamicchanges of IFN-γand TGF-βhave certain significance.So we choose B16F10 celllines in vitro cell culture in our experiment.Effects on on the B16F10 cellproliferation,migration and invasion capacity caused by cytokines IFN-γandTGF-βweer observed.The SRB show that the survival rate of cells in TGF-βgroup ishigher than that of control group,with statistically significant differences (P＜0.05) ;but the survival rate of cells in IFN-γgroup is lower than that of control group,withstatistically significant differences (P＜0.05) ;The survival rate of cells in experimentgroup is lower than that of control group when IFN-γand TGF-βwere administeredat the same time.Cell morphology varied with the changes of cell survival result fromdifferent administration of cytokines.All of that suggest that IFN-γinhibited cellsproliferation,migration,and invasion,and TGF-βcan confront the role of IFN-γ,sothat tumor cells can restore proliferation,migration,as well as the ability to resumeinvasion and the activity of MMPs.Growth curve of IFN-γinjection group (injectionthrough mouse tail vein) showed inhibition in early phase and loss of inhibition inlate-phase.Gelatin zymography analysis showed that IFN-γcan reduce the activity ofMMP-2 and MMP-9.On the other hand,TGF-βconfronted IFN-γto recover theactivity of MMPs,and increased the activity of MMP-2 and MMP-9.The experiment revealed that the tumor cells can secrete TGF-βand IFN-γ,whichexplains the possible mechanisms of inhibition in early phase and loss of inhibition inlate-phase.This suggested that the balance of IFN-γ/TGF-βplay an important role inthe interaction between acute inflammation and tumor.In conclusion,we established animal models of acute inflammation.Immunohistochemistry,gelatin zymography and in vitro cell culture techniques werecarried out to detect the effect of inflammation on tumor volume,microvessel density,expression of VEGF-a,MMP-2 and MMP-9 ,activity of MMP-2 and MMP-9 and role of balance of IFN-γ/TGF-βin tumor.The main conclusions are as follows:1.The Construct of melanoma mouse model of acute inflammation.This studyselected melanoma - the repeated debridement as our animal experimental model ofthe next step.2.The effect of acute inflammation on angiogenesis in melanoma.Theinhibitory effect not only results from the direct stimulation of inflammatory factors,but also may from the release of certain inflammatory cytokines into the blood.3.The effect of acute inflammation on expression of MMPs.The results suggestthat the expression and the activity of MMP-2 and MMP-9 are both subject to theeffects of acute inflammation.4.A pilot study of IFN-γand TGF-βon melanoma cell proliferation,migrationand invasion.The experiment revealed that the tumor cells can secrete TGF-βandIFN-γ,which explains the possible mechanisms of inhibition in early phase and lossof inhibition in late-phase.This suggested that the balance of IFN-γ/TGF-βplay animportant role in the interaction between acute inflammation and tumor.