The Inhibition Of Proliferation And Invasion Activity Of Melanoma By Microtubule-associated Protein 2

Malignant melanoma(also termed melanoma)is derived from melanocytes, nevus cells,high malignant degree;often involves skin,also the conjunction of skin - mueosa,choroids,leptomeningeal.In China,the incidence of melanoma is lower than in Europe and America significantly,but there is not the epidemio-logical information.The incidence of melanoma increases progressively 3%per year in America.Because that melanoma is not sensitive to therapy of medicine and radiation and operation is effective only on early stage,the incidence of mel-anoma takes the 4%of total incidence of skin malignant tumor,but the mortality takes 79%.The co -therapy including operation immunological therapy and gene therapy is recomended.The melanocytes arise from the ectodermal neural crest,exhibit certain characteristics of neural crest derivatives.For example,the morphology of mela-nocytes resemble neuron's dendritic morphology;some nevus cells that migrate into dermis morphologically resemble Schwann cells of peripheral nervous sys-tem;there are markers of neural crest derivatives in primary and metastatic mela-noma,such as,peripherin,neuron - specific enolase.The recent studies found that a neural marker,microtubule - associated proteins 2(MAP2),high - expressed in nevus,and the melanoma tumor which is in the radial growth phase(RGP)or inthe vertical growth phase(VGP),but low -expressed or absent in metastatic melanoma.MAP2,one of microtubule - associated proteins(MAPs},MAP2 and tub-lin compose microtubule(MT).MT have many physiological function,as main cytoskeletal elements,for example,involves in mitosis as spindle.MAPs consist ofα- tubulin,β- tubul,in andγ- tubulin.MAPs are a large family of proteins,and include MAP1,MAP2,MAP3,MAP4,MAP5 and MAP6.MAP2 is a group of filiform molecular structure and thermostable phos-phoproteins.It has the isoforms;MAP2a,MAP2b,MAP2c,AP2d and MAP2e. MAP2 exists in the axon and dendrite of the neuron principally.In the neuron the primary function of MAP2 is that,â‘ MAP2 has a thoroughly significant effect on the growth and the obtaining polarity of the dendrite;â‘¡Connected with the tubulin MAtE can stabilize the tubulin and increase its rigidity,in this way the tubulin can form into a long multimer;â‘¢MAP2 is the promoter in tubulin assembly.It can bundle the tubulin thus the tubulin can form a long and rigid tubulin bundle;â‘£MAP2 can intervene the interrelation between the movement protein and the tubulin,thereby it can inhibit the organelle transport regulated by the movements.We surmise that the deletion and the silencing of MAP2 may relate to the tumor progression and the tumor metastasis,and its mechanism may relate to the alteration of the function and the character of microtubule,which caused by the deletion and the silencing of MAP2.Up -regulating the MAP2 expression in the metastatic melanoma may inhibit the tumorous proliferation and metastasis by in-fecting the microtubule.So to study the effect and the mechanism of MAP2 in vivo or in vitro,to comprehend their role in the melanoma progression is-signifi-cant for designing the new treatment strategy of melanoma.Materials and methodsâ… .Materials1.Cells and ceil culture;Human embryo kidney cell line HEK- 293 cells (provided by Yusen professor,Chinese military medical academy,Beijing), mouse-invasive melanoma ceil line B16C29 cell(provided by Dr.Vijaysaradhi Set.aluri,Wake Forest University,America},mouse invasive melanoma cell lines B16(purchased from Dalian medical university,Dalian),human metastatic mel-anoma cell line WM451Lu cells(purchased from Beijing medical university, Beijing).HEK293 cells were cultivated in High Glucose medium supplemented with 10%Bovine Calf Serum DMEM,under a atmosphere of 5%CO₂ at 37℃. mouse invasive melanoma cell lines B16C29,mouse invasive melanoma cell lines B16 and Human metastatic melanoma cells WM451Lu were cultivated in the medium supplemented with 10%Bovine Calf Serum RPMI- 1640 medium, under a atmosphere of 5%CO₂ at 37℃.2.Agents;The primary antibody;mouse anti - MAP2,mouse anti - Green Fluorescent Protein(GFP)monoclonal antibody {America,Sigma},Secondary Antibody;mouse anti - FITC monoclonal antibody(Denmark,DaKo),Texas red -conjugated sheep anti mouse polyclonal antibody(Britain,Amersham Life Sci-ence),phycoerythrin - conjugated rabbit anti - mouse polyclonal antibody(A-merica,Sigma);PI - ANNEXIN - V apoptosis kit(America,Becton Dickin-son);Boyden chamber and Martrigel(America,Becton Dickinson);Bovine Calf Serum,DMEM and RPMI- 1640 medium;Hang Zhou Sijiqing Biological Engineering Material Co.,Ltd..Ad- MAP2 vectors were gifts from Dr Vijay-saradhi Setaluri of Wake Forest University,America.3.Animals;6～8 weeks- old Balb/c female nude mice were bred in(with body weight of 18～20 g),semi - barrier system at a constant temperature of 25 -27℃and at a humidity of 45%-50%.â…¡.MethodsA Preparation and.detection of adenovirus vectors1.Generation of Ad -MAP2;The HEK -293 ceils were used to replicate a great deal of adenovirus.2.Ad - MAP2 were purified by CsC1 ultracentrifugation.3.The virus titer was detected with Plaque Assay.4.The transfection efficiency was detected.B Experiments in vitro1.The study of the inhibition on proliferation of melanoma ceils by MAP2.1.1 The ceils proliferation was-detected with MTT test.1.2 The cell growth curve was detected.2.The study of that MAP2 induce melanoma cells apoptosis and arrests Cell -cycle in vitro. 2.1When the Ad - MAP2 had been transfected into the melanoma ceils, the apoptosis circumstance was detected with Flow Cytometry(PI-ANNEXIN -V staining),2.2.When the Ad - MAP2 had been transfected,the DNA in the melano-ma cells was quantitatively analyzed with Flow Cyometry.Then combining with Modfit analysis software the distribution of each Cycle- Phase was calculated.3.The study about the effect on the intracellular microtubule character and the morphology of melanoma cell after MAP2 transfectin in vitro.3.1 The morphologic changes of the Ad - MAP2 - transfected cells were observed under a light microscope.3.2 The morphologic change of the transfected cells and the intracellular microtubule character change were observed under a electron microscope.4.The study of the inhibition of the tumor invasion by MAP2 in vitro.By Boyden chamber and the Matrigel,cell invasiveness was observedC Experiments in vivo1.The set up of the melanoma nude mouse model.2.The expression of MAP2 was detected by immuno - histochemistry.3.The study of the inhibition effect of tumor by MAP2 in vivo.4.The study of the feature of MT in melanoma cells in vivo. nificantly with No obvious change was found in negative control group.3.Influence of MAP2 on the microtubule character in mouse invasive mela-noma ceilline B16C29 in vitro.;6days after MAtE - transfected,the cell bodies extended out long and thin dendrites,and abundant microtubules under a trans-mission electron microscope。Compare with the control group,the microtubules became longer,thicker and more microtubules -bundles extended along the cell dendrites.4.The MAP2 induce mouse invasive melanoma cell line B16C29,B16 to apoptosis in vitro;6days after Ad - MAP2 transfected,cells appeared apopt0sis detected with Flow Cytometry(PI- ANNEXIN -V staining}.The total apopto-sis proportion of B16C29 ceils and B16 ceils in the transfected group were 21. 34%,and 17.06%,respectively,higher than that of the negative control group signiticantly.5.The influence of MAP2 on the cell cycle in mouse invasive melanoma cell line B16C29,human metastatic melanoma ceil line WM451Lu;Mter MAP2 -transfection,the proportion of the G0/G1 phase and G2/M phase cells of the B16C29 cells,the WM451Lu ceils induced,the G2/M phase ceils reduced sig, nificantly.The proportion of S phase ceils increased,and there was significant difference compare with the control group.6.The influence of MAP2 on the cell invasiveness in vitro;MAP2 has sig-nificant inhibition to the cell invasiveness in Boyden chamber invasive model, Compared with Ad -GFP control group.7.MAP2 has the inhibition effect on the tumor proliferation in vivo.;The nude mice melanoma models were set up using the mouse invasive melanoma cell,B16C29 and the human invasive melanoma cells,WM451Lu.;From the 6th day after infected,the nude mice subcutaneous tumors which were injected with Ad- MAP2 grew more slowly compare with the negative control Ad-GFP group,and there was significant difference.8.Influence of MAP2 on the microtubule character in melanoma ceil line B16C29 in vivo;the nude mouse melanoma model,which set up by mouse inva-sire melanoma cell,B16C29 and the human invasive melanoma cells, WM451Lu,the microtubules longer,thicker and more microtubules - bundles under a transmission electron microscope in MAP2 -transfected group,compare with the control.Conclusions1.MAP2 has the inhibition effect on the growth of the mouse invasive mela-noma cell line B16C29 and the human metastatic melanoma cell line WM451Lu in vitro.2.Up - regulaion of MAP2 can arrest the mouse invasive melanoma cell line B16C29,human metastatic melanoma cell line WM451Lu from G2/M phase,induce mouse invasive melanoma cell line B16C29,B16 cell apoptosis.3.The over - expression of MAP2 in vitro can make melanoma cell den-drite longer,thiner,extend and recover the morphology of melanocytes.The mi-crotubules in the dendrite,longer,thicker,more microtuble -bundles.The change of microtubule is the reason of morphology change of melanoma cells.4.The over - expression of MAP2 in vitro can inhibit the invasion of mela-noma.It's mechanism may relate to the change of cell.morphology and microtu-bule.5.The over expression of MAP2 in vivo can inhibit the proliferation of melanoma,the microtubules in the dendrite,longer,thicker,more microtuble -bundles,that proves the results in vitro.