The Role Of High Mobility Group Box 1 (HMGB1) In Drug Resistance Of Multiple Myeloma

BackgroundMultiple myeloma(MM)is a malignant plasma cell proliferative disease derived from B cells,which clinical manifestations including hypercalcemia,impaired renal function,anemia and osteolytic lesions.MM is the second mostly diagnosed disease among hematological malignancies after lymphoma.With the novel agents(bortezomib,lenalidomide,etc.)and autologous stem cell transplantation,the survival of MM patients has been improved significantly.But the most part of MM patients eventually suffered progression and relapse/refractory because of primary or secondary drug resistance and MM still remains incurable.Therefore,it’s necessary to explore the mechanisms of MM drug resistance and find new strategies for MM patients toovercome drug resistance and enhance drug sensitivity.High mobility group box 1(HMGB1),being as a DNA chaperone,plays an important role in DNA recombination,repair and transcription of target genes,while HMGB1 can also be secreted or released into extracellular as a damage associated molecular patterns(DAMPs).HMGB1 could participate in inflammation,cell differentiation,cell migration,angiogenesis and tumor metastasis and drug resistance through binding with its receptors.Overexpression of HMGB1 was found in a variety of tumors,and was involved in tumor invasion,metastasis and drug resistance.HMGB1 overexpression also found in hematological malignancies such as leukemia,lymphoma,et al.Chemotherapies could induce HMGB1 release from leukemia cells,while HMGB1 can protect cells from apoptosis and promote the leukemia cells drug resistance induced by autophagy.TLR4 was one of the key receptors of HMGB1 and HMGB1 could induce inflammation,angiogenesis and tumor metastasis through TLR4.Our previous study found that LPS was able to activate NF-κB and MAPK signaling pathways through interation with its receptor TLR4,so that to promote MM cell proliferation and inhibit apoptosis induced by chemotherapies.We further found that MM cells coule secret HMGB1,an endogenous ligand of TLR4.But being as the chromosome binding protein and DAMPs,the role of HMGB1 in development and drug resistance of MM remains unclear,and our research was aimed at exploring the role of HMGB1 in MM cell proliferation and drug resistance.Objective1.To clarify the expression and secretion of HMGB1 in primary MM cells and MM cell lines;2.To investigate the role of endogenous HMGB1 on the biological behavior and drug resistance of MM cells;3.To analyze the changes of HMGB1 in the cell culture supernatant after treatment with chemotherapeutic drugs,and to elucidate the role of exogenous HMGB1 on the growth and drug resistance of MM cells;4.To explore the machenism of HMGB1 in MM drug resistance in vitro and in vivo.Methods1.semi-quantitative real time-polymerase chain reaction(qRT-PCR),Western blot,flow cytometry and immunofluorescence were used to determine the mRNA and protein expression of HMGB1 and receptors(RAGE,TLR2,TLR4,TLR9)of HMGB1 in MM cell lines(MM.1S,RPMI8226,ARK,ARP-1,CAG,NCI-H929)and primary MM samples(CD 138+).ELISA was performed to analyze the level of HMGB1 in the serum of MM bone marrow or the cell culture supernatant;2.MM cells were pretreated with combination human protein HMGB1(rhHMGB1).Then,MTT,CCK8 and flow cytometry analysis was used to evalute the effect of exogenous recombinant HMGB1 on the proliferation and cell apoptosis of MM cells induced by chemotherapies;3.MM cells(RPMI8226,CAG,MM.1S and ARK)were transfected with HMGB1-knockdown lentivirus to scilencing HMGB1 expression.Then CCK8 assay and flow cytometry analysis were used to determine the proliferation and apoptosis of MM cells with or without chemotherapeutic drugs dexamethasone(Dex),adriamycin(ADM)and melphalan(Mel).Expression of apoptosis related proteins were tested by Western blot;4.Gene expression profiles(Affymetrix HTA 2.0 Array)was used to compare changes in gene expression between HMGB1-knockdown cells and the control cells and qRT-PCR and Western blot were used to verify the array results.Western bolt was performed to analyze expression of protein involved in signaling pathways after HMGB1 knockdown;5.To test the role of HMGB1 in MM in vivo,a NOD/SCID xenograft model of human myeloma using MM cells(MM.1S-HMGB1 shRNA or MM.1S-control shRNA)was established.The tumor burder was measured using calipers and immunohistochemistry analysis was used to detected protein expression in tumor tissue samples derived from the NOD-SCID mice.Results1.qRT-PCR and Western blot results showed that MM cell lines and primary MM samples both expressed high mRNA and protein levels of HMGB1.qRT-PCR and flow cytometry showed that MM cell lines and primary MM samples also expressed receptors(RAGE,TLR2,TLR4,TLR9)of HMGB1.Immunofluorescence analysis showed that CD 13 8+ plasma cells in bone marrow of MM patients expressed HMGB1.While,HMGB1 in the bone marrow serum and culture supernatant of MM cells was detected by ELISA;2.Bortezomib(Bor)and adriamycin(ADM)can significantly inhibit the proliferation of MM cells and increase the level of HMGB1 in the cell culture supernatant.CCK8 assay showed that rhHMGB1 had no significant effect on the proliferation of MM cells.Results of MTT and flow cytometry showed that rhHMGB1 could not protect MM cells from apoptosis induced by Bor,ADM and Dex;3.CCK8 and cell cycle analysis by flow cytometry showed that there was no difference in MM cells proliferation between HMGB1-knockdown group and the control group(P>0.05)and CCK8 showed that HMGB1-knockdown significantly enhanced inhibitory effect of chemotherapy with Dex in comparison with the wildtype HMGB1 control(P<0.05).Flow cytometry analysis showed that apoptosis of MM cells induced by Dex,ADM and melphalan were increased when HMGB1 expression was suppressed(P<0.05)and there was no difference in cell cycle arrest induced by Dex(P>0.05).Western blot showed that cPARP and c-caspase3 induced by Dex was significantly enhanced after interference of HMGB1;4.Gene array analysis on RPMI8226 and CAG cell lines showed that anti-apoptotic genes(Bcl-2,Bcl-xl)and MM survival related genes(DEPTOR)were decreased in knockdown cells compared to the controls.Some of the gene array results were verified by qRT-PCR(P<0.05)and Western blot;5.Western blot showed that with interference of HMGB1:the activation of mTOR pathway was upregulated,autophagy induced by starvation of MM cells was decreased,activation of p38MAPK in MM induced by Dex was increased,and the NF-κB pathway was inhibited;6.Western blot showed that the level of yH2A.X,a DNA damage response protein in MM cells was significantly upregulated with HMGB1 scilencing;7.Results in vivo of the NOD/SCID xenograft model of human myeloma showed that mice exposed to dexamethasone had a lower tumor burden in MM.1S-HMGB1 shRNA group compared with the control group(P<0.05).Immunohistochemical analysis showed that with interference of HMGB1,expression of Bcl-2,p4EBP1 were decreased;expression of pAKt(ser473)and yH2A.X were increased.After dexamethasone administration,expression of c-caspase-3 and yH2A.X in HMGB1 knockdown group were greater than the control group.Conclusions1.Primary MM cells and MM cell lines both expressed HMGB1 and its receptors and also secreted HMGB1;2.Chemotherapies could induce the release of HMGB1 to the culture supernatant of MM cell lines,but exogenous rhHMGB1 had no effect on the proliferation of MM cells and could not protect MM cells from apoptosis induced by chemotherapeutic drugs;3.Interference of HMGB1 lead to Increased Vulnerability of MM Cells to Dex and ADM and melphalan;4.Endogenous HMGB1 may induce dysregulation of the downstream signaling pathway and autophagy and regulating the expression of proliferation and apoptosis-related genes in MM cells and thus to affect the response of MM cells to chemotherapeutic drugs;5.Endogenous HMGB1 may be involved in MM cells DNA damage repair pathway to induce MM drug resistance.