| BackgroundMultiple myeloma a(MM)is a universally clonal B-cell neoplasm,Bortezomib(bort,Velcade)is one of the best effective treatments for MM.Although initial advantages of bort treatment of MM including higher overall response rates are promising,a number of patients develop a resistance to it over time.The main mechanism of MM drug resistance has been related to the instablity of tumor cells and the microenvironment.In MM,M?s could promote MM cells to survival,proliferate and angiogenesis,they also induce drug resistance by protecting tumor cells from chemotherapy-induced apoptosis.Further study have indicated that the protective effect of M?s in MM cells was induced by direct contact,which means the surface molecules on MOs and MM cells may exist a functional role in inducing drug resistance.B cell activating factor BAFF is expressed predominantly by stromal compartment including osteoclast,M?s,dendritic cells,and some T cells,it enhances the survival of MM cells and protects them from Dexamethasone induced apoptosis.Our previous study has found the expression of BAFF and its receptors both in M?s and MM cells.Here we aimed to characterize the role of BAFF in M?-mediated resistance of MM cells to bort,and to further understand the molecular mechanisms involved in the process.Objective1.To identify M?s induced from PBMC and to clarify the role of M?s in myeloma.2.To explore the role and mechanism of BAFF in the interaction of M?s and MM cells in vitro and vivo.Methods1.The expression of BAFF and its receptors on MM cells was detected by qRT-PCR and Flowcytometry.The expression of BAFF in primary M?s was detected by immunofluorescence.2.The morphology of M?s was observed using inverted phase contrast microscope,the expression of CD68,CD163,BAFF and its receptors was detected by Flowcytometry.3.The soluable BAFF in the supemants of MM patients' bone marrow,MM cell lines and M?s were detected using ELISA.4.CCK-8,Flowcytometry and Western blot were used for examining the proliferation and bort induced apoptosis of MM cells in the presense or absence of rhBAFF and MOs,respectively.5.Flowcytometry and Western blot were used for examining the apoptosis and protein changes of MM cells,either cocultured with M?s in the presence of BAFF neutralizing antibody,or cocultured with BAFF knockeddown M?s.6.A xenograft model of human myeloma was established with injecting ARP-1 or ARP-1/PBMC cells subcutaneously to test the role of BAFF in M?s induced Bort resistance in vivo.Results1.qRT-PCR showed that primary MM cells expressed BAFF heterogeneously,the expression of BCMA was significantly higher than that of TACI,with BAFF-R being the lowest.Similar results were observed in the six MM cell lines MM.1S,MM.1R,CAG,RPMI8226,ARP-1,and ARK.Flow cytometry showed ARP-1 and RPMI8226 expressed higher levels of BCMA and TACI,lower levels of BAFF,and virtually undetectable BAFF-R.2.We observed that the cultured M?s were adherent to the six-well plates and had a spindle-like morphology.The expression of CD68 and CD 163 of M?s from different donors was(24.6%± 0.39%)and(78.4%± 0.67%),respectively.M? had increased expression of BAFF compared with monocytes(P<0.05),and in monocyte-derived M?,BAFF had relatively high expression whereas its receptors were barely detected by flow cytometry.We also identified the expression of BAFF in CD68+ monocytes/M?s from BM aspirates of patients with MM using immunofluorescence.3.Using ELISA,median soluble BAFF levels were 1306.9 pg/ML,20.5 pg/mL,and 74.0 pg/mL in culture supernatants of BM of patients with MM,MM cell lines,and M?s,respectively.4.CCK8 showed rhBAFF could enhance ARP-1 and RPMI8226 to proliferate,200 ng/mL rhBAFF showed the highest proliferation rate.rhBAFF also made ARP-1 and RPMI8226 more resistant to bort.5.Flow cytometry results showed Bort(range 0-80 nM)had small effect in inducing apoptosis of MOs,whereas MOs co-cultured with ARP-1(bort,10nM),RPMI8226(bort,15nM)and CD 13 8+ plasma cells significantly weakened bort-induced and spontaneous apoptosis.The apoptosis rate as follows:ARP-1,50.8±2.40%vs 19.58± 2.31%(p?0.0007);RPMI8226,53.3±1.89%vs 28.74±2.04%(p=0.0004);CD138+,55± 3.20%vs 14.85 ± 2.79%(p=0.0003).6.Flow cytometry showed the BAFF-neutralizing antibody repressed M?-mediated bort resistance of ARP-1,RPMI8226 and CD 138+ plasma cells.The apoptosis rate:ARP-1,35.4 ± 1.07%vs 20.04 ±1.98%(p<0.05);RPMI8226,62.5 ± 2.89%vs 19.75±2.34%(p<0.05);CD138+cells,35.95 ±3.07%vs 21.07±1.98%(p<0.05).Also BAFF-knocked down M?s repressed M?-mediated bort resistance of ARP-1 and RPMI8226.The apoptosis rate:ARP-1,20.34± 1.07%vs 54.2±1.98%(p<0.01);RPMI8226,23.4± 2.89%vs 53.4±2.34%(p<0.01).7.Western blot showed that in ARP-1 cells co-cultured with M?s,bort-induced PARP,caspase-3 cleavage and pH2A.X had high repression,phosphorylated AKT,ERK1/2 kinase,SRC and NF-?B were activated.Neutralizing BAFF expression under co-culture conditions or ARP-1 cells co-cultured with BAFF-knocked M?s had moderate elevation of these apoptotic proteins and pH2A.X,lower levels of p.AKT,p-ERKl/2,p-SRC,and repressed the activation of NF-?B.8.Immunohistochemical analysis showed mice bearing ARP-1/PBMC had more CD68 expression compared with ARP-1 alone.Immunohistochemistry and flow cytometry showed ARP-1/M? mice contain more cPARP expression and more apoptosis cells.We also found mice bearing ARP-1/M? cells had a larger tumor volume in the presence and absence of bort,and BAFF neutralizing antibody treated ARP-1/M?mice showed small-sized volumes compared with control IgG2B-treated group with bort.Conclusions1.MM cells mainly express BCMA and TACI,M?s express BAFF,and bone marrow microenvironment is the sourse of BAFF.2.BAFF promotes MM cell to proliferate and makes MM cells more resistant to bort.3.PBMC-induced M?s were insensitive to bort and protected MM cells from bort-induced apoptosis.4.Neutralizing or blocking BAFF in MM-M?s coculture system represses the protective effect of M?s.5.BAFF is involved in MOs mediated MM bort resisitance through repressing apoptosis proteins and pH2A.X,activating AKT,ERK,SRC and NF-?B signaling. |
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