The Effect Of Bone Morphogenetic Protein9on The Cross-talk Between Breast Cancer Cells MDA-MB-231and Mesenchymal Stem Cells HS-5

Objective: To investigate the effect of bone morphogenetic protein9(BMP9) on the cross-talk between breast cancer cells MDA-MB-231andbone marrow mesenchymal stem cells HS-5and its molecular mechanism.Methods: The co-culture system composed of breast cancer cellsMDA-MB-231and bone marrow mesenchymal stem cells HS-5intranswell chamber was exposed to the conditioned media which wereproduced in HCT116cells infected by AdBMP9and AdGFP recombinatedadenovirus respectively.MDA-MB-231+HS-5,MDA-MB-231+HS-5+GFPand MDA-MB-231+HS-5+BMP9were respectively considered as blankgroup,control group and experimental group.For MDA-MB-231cells of the coculture system,Cell wounding assayand transwell invasion assay were carried out to detect the changes ininvasion and migration.The metastases-related genes(IL-6,IL-8,RANKL,IL-11,RANK,PTH-rp,MMP2,C-myc,CXCR4,MMP9) were detected by Real time PCR.Western blot were carried out to observe the changes in theproteins(IL-6,PTH-rp,MMP9)and in the metastases-related signalingpathways proteins(p-P38,P38,p-ERK1/2,ERK1/2,p-JNK1/2, p-AKT, AKT,pGSK-3β,GSK-3β,β-catenin).For HS-5cells of the coculture system,the changes in the activity andexpression of alkaline phosphatase (ALP),calcium deposition andproliferation were observed by chemiluminescence assay,cytochemicalstaining,Alizarin Red S staining and MTT assay respectively. RT-PCR andWestern blot were carried out to detect the mRNA and protein expressionsof osteocalcin(OCN)、 osteopontin(OPN)、 receptor activator of nuclearfactor κB ligand(RANKL) and osteoprotegerin(OPG).The genes(IL-6,IL-8and RANKL)，influencing the signaling pathways MAPK and AKT，weredetected by Real time PCR.The RANKL concentration in the coculture sustem was detected byELISA assay.After6ng/ml OPG antibody was added into the coculturesystem containing BMP9,the changes in invasion and migration ofMDA-MB-231and in the activity of AKT signaling pathways wereinvestigated.MDA-MB-231+HS-5+BMP9,MDA-MB-231+HS-5+BMP9+IgG and MDA-MB-231+HS-5+BMP9+OPG anbody were respectivelyconsidered as blank group,control group and experimental group.Results: The conditioned medium contained BMP9protein afterHCT116cells were infected AdBMP9adenovirus vector.When the coculture system were added into BMP9conditioned medium,forMDA-MB-231cells in experimental group,comparing with controlgroup,the healing rate was signifcantly decreased from (78.3±3.2)%to(28.4±0.7)%(P<0.05);the number of invading cells which moved across thematrix barrier was decreased from (408.7±17.2) to (169.0±12.5)(P<0.05);the mRNA expressions of IL-6,PTH-rp and MMP9weredecreased by43%,38%and51%respectively (P<0.05),the mRNAexpressions of IL-8,C-myc and CXCR4were increased by113%,97%and164%respectively (P<0.05),while the mRNA expressions of IL-11andRANK didn’t make significant changes;the protein expressions ofIL-6,PTH-rp and MMP9were decreased;the protein expression of p-P38was decreased at30min and1h respectively;the protein expression ofp-ERK1/2was decreased at1h;the protein expression of p-JNK1/2wasdecreased at6h;the protein expression of p-AKT was decreased at15min,30min and1h respectively;the protein expression of PGSK-3β wasdecreased at12h,while the protein expression of β-catenin was increased at6h.For HS-5cells in experimental group,compared with control group,theALP activity was increased in a time-dependent manner and reached thepeak at the ninth day,and the cytochemical staining further confirmed that;the calcium deposition was increased;the mRNA and protein expressions ofOCN and OPN was increased;proliferation was elevated;the mRNA expressions of RANKL was decreased by54%(P<0.05),while the mRNAexpressions of IL-8and IL-6were increased by39%and212%respectively (P<0.05);the mRNA and protein expressions of OPG/β-actinwere increased from (0.048±0.008,0.126±0.020) to(0.335±0.042,0.332±0.055) respectively(P<0.05),while the mRNA andprotein expressions of RANKL/β-actin were decreased from(0.070±0.008,0.099±0.034) to (0.036±0.004,0.055±0.006) respectively(P<0.05).RANKL concentration in experimental group was246±22pg/ml whichwas lower than that in control group.After6ng/ml OPG antibody wasadded into the coculture system containing BMP9, Compared with thecontrol group,the AKT signaling pathway in the experimental grouprecovered, the number of invading MDA-MB-231cells which movedacross the matrix barrier were decreased,while the healing rate ofMDA-MB-231cells didn’t make significant changes.Conclusions:BMP9can regulate the cross talk betweenMDA-MB-231cells and HS-5cells in the coculture system.BMP9reducethe RANKL secretion of HS-5cells to inhibit the invasion ofMDA-MB-231through suppressing it’s AKT signaling pathway activity,meanwhile BMP9can promote osteogenesis and proliferation of HS-5cellsin the coculture system.