C6 Glioma Paracrine S100B Induces Malignant Transformation Of Mesenchymal Stem Cells And Its Possible Mechanism

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C6 glioma-conditioned medium induces malignant transformation of mesenchymal stem cellsObjective:To identification the possible changes of C6 glioma-conditioned medium induces bone marrow-derived mesenchymal stem cells,and the underlying mechanism.Methods:Four weeks old male SD rats,weight range from 40 g to 50 g were sacrificed and obtained primary bone marrow-derived mesenchymal stem cells.Primary cells were labeled with mesenchymal stem cells and hemopoietic system markers to test the BMSCs ratio.C6 glioma-conditioned medium was collected daily and mixed with fresh complete culture medium(1:1).The second passage of BMSCs(P2)was divided into 2 groups.One was replaced GCM every 24 h.Another group was treated with normal culture medium as control.Cell morphology,cell cycle,proliferation,migration,invasion,senescence and the in vivo tumor genesis were analyzed after GCM culture completed.The RT-PCR,immunofluorescence staining and Western blot were followed to detect mRNA and protein levels of Akt,STAT3,NF-kB,C-myc,Cyclin D1,Bcl-xl,Bad.To evaluate the tumorigenicity,both CM and GCM treated BMSCs were injected into nude mice,after 4 weeks of injection the animals were sacrificed,tumor tissue was embedded in OCT,frozen,sectioned and stained with hematoxylin-eosin(HE).Ki-67,Des,Vim,Nes,GFAP ?S100B were analyzed to identification tumor source.Results:1.Flow cytometric and immunofluorescence staining analysis of specific mesenchymal stem cells surface markers showed that the primary cultured MSCs were positive for CD73,CD90 and CD105,but negative for CD34 and CD45.2.Normal BMSCs are spindle like fibroblast cells,but GCM cultured BMSCs exhibited more rounded cells,but also changed cellular shape,increased the nucleus/cytoplasm ratio,inhibited cell contact inhibition ability and grew in layers and clusters which have altered to tumor cell morphology.3.Cell cycle demonstrated that GCM significantly increased G2+S phase cell ratio(25.48%)than that of the control group(14.36%).4.Cell viability assay showed that GCM induced an increased proliferation rate of BMSCs compared with normal CM from day 3 to day 4(P<0.05)and day 5 to day 7(P<0.01).5.The invasion and migration ability of cells was assessed by transwell-based assay.The results revealed that both invasion(P<0.01)and migration(P<0.05)ability of BMSCs were significantly increased by GCM.6.Notably,almost half of control BMSCs(53.3 ± 19.2%)showed ?-gal staining positively at P6.In contrast,GCM cultured BMSCs were detected few staining positivity(8.1 ± 6.8%),that seen to GCM cultured BMSCs escaped from senescence like tumor cells.7.The mRNA expression levels of Aktl,STAT3,C-myc,IL-6 and GFAP were significantly increased in GCM cultured BMSCs compare to normal group.8.Immunofluorescent staining demonstrated that GCM significantly increased Akt1,p-Akt1 and p-STAT3 in cellular nucleus,not the STAT3.9.Western blot analysis showed an up-regulation of Akt1,p-Akt1,STAT3,p-STAT3,Cyclin D1,Bcl-xl,Bad and p-Bad(P<0.01).10.The histological analysis reveals the tumors were heterogeneous such as atypical round cells,enlarged nuclei and decreased karyoplasms which arranged in solid clusters and high vascularity.The control animals injected with normal BMSCs did not reveal any differences or malignant tissue other than dermis and musculus.Conclusions:1.C6 conditioned medium induced BMSCs inhibited cell contact inhibition ability,increased proliferation rate and G2+S phase cell ratio,escaped from senescence,up-regulated invasion and migration ability and tumorigenesis.2.Some paracrine cytokine in GCM may responsible for mRNA and protein level up-regulation of Akt,STAT3,CyclinD1,Bcl-xl and Bad.Chapter ? The possible mechanism of RAGE/S100B pathway in malignant transformation proceedingObjective:To investigate the underlying mechanism of RAGE/S100B pathway in GCM induced BMSCs malignant transformation.Methods:Primary rat bone marrow-derived mesenchymal stem cells were prepared.The concentration of EGF,IGF-1,PDGF-BB and S100B in normal CM of BMSCs and C6 CM were detected by ELISA assay.In order to reveal the link between S100B and GCM induced malignant transformation,RAGE and S100B protein was tested with immunofluorescence staining and Western blot among the different cells.The rats were intracerebroventricularly injected by CM and GCM treated BMSCs respectively,after that HE staining was followed to reveal BMSCs growing condition.Double immunofluorescence staining was used to detect the S100B and GFAP expression status around the injection site in rat brain.FPS-ZM1,a specific RAGE inhibitor was used to verify the S100B/RAGE pathway.Drug toxicity was analyzed to ensure the suitable FPS concentration by CCK-8 assay.The total RNA of BMSCs in the seven groups was collected,and Aktl,STAT3,RAGE,S100B,Bcl-xl and Bad mRNA expression were detected by real-time PCR.Meanwhile,the protein levels of Akt1,p-Akt1,STAT3,p-STAT3,RAGE,S100B,Bcl-xl,Bad and p-Bad were detected by Western blot.Results:1.The ELISA assay showed that the S100B concentration in golima condition medium(30.35 pg/ml)was much higher than BMSCs cultured medium(12.65 pg/ml).2.Both immunofluorescence staining and Western blot revealed an up-regulation of inner S100B and RAGE protein in BMSCs.3.The rat intracerebroventricularly injection model demonstrated that GCM induced BMSCs around injection site exhibited a aggressive proliferation and infiltration behavior compared to CM cultured BMSCs.4.The immunofluorescence staining of brain slices showed that S100B positive cells in rat brain surrounded BMSCs;this in vivo environment of BMSCs was similar to C6 micro environment in vitro.5.Drug toxicity assay revealed the suitable concentration of RAGE inhibitor FPS-ZM1 was 50 ?M.6.Cell proliferation test showed that like GCM,S100B increased BMSCs proliferation rate significantly,RAGE inhibitor blocked GCM and S100B increased cell proliferation rate.7.The mRNA expression of RAGE,S100B,Akt1,STAT3,Bcl-xl and Bad were detected by real-time PCR,GCM and S100B culture up-regulated RAGE and inner S100B,RAGE inhibitor blocked GCM and S100B induced RAGE and inner S100B expression.Furthermore,GCM and S100B culture increased Akt1 and STAT3 mRNA expression,RAGE inhibitor reversed GCM and S100B induced Akt1 up-regulation but only attenuated STAT3 expression.Bcl-xl and Bad also increased by GCM and S100B treatment,and FPS-ZM1 suppressed GCM and S100B induced Bad activation,not Bcl-xl.8.Western blot analysis revealed GCM and S100B increased BMSCs RAGE and intracellular S100B protein,RAGE inhibitor could blocked this augmentation.Furthermore,the protein levels of Akt1,p-Akt1,STAT3 ?p-STAT3 were activitaed by GCM and S100B,and S100B induced protein activitation reversed by RAGE inhibitor treatment,but RAGE inhibitor only attenuated these activitation.The expression of Bcl-xl,Bad and p-Bad protein was increased by GCM and extracellular S100B,FPS-ZM1 significantly reduced Bad and p-Bad up-regulation,not GCM induced Bcl-xl up-regulation.Conclusions:1.C6 glioma paracrine high concentration of S100B protein in its microenvironment and up-regulated RAGE.2.S100B increased the proliferation rate of BMSCs,and activating Akt and STAT3 pathway,up-regulated Bcl-xl and Bad.3.Specific RAGE inhibitor FPS-ZM1 blocked S100B induced Akt and STAT3 activation.