Resistin Induces Multidrug Resistance In Myeloma By Inhibiting Cell Death And Upregulating ABC Transporter Expression

Objective:In recent years,the treatment of multiple myeloma has made remarkable progress because of the development of new drugs.However,myeloma remains incurable,with a high frequency of relapse and the development of drug resistance.The detailed mechanism of multidrug resistance is still not well known.A recent study revealed that bone marrow adipocytes promotes chemotherapy resistance in myeloma through adipocyte-secreted adipokines,but the mechanism underlying this effect and the specific adipokines involved are not well understood.Our study purposed to determine the role and mechanism of resistin in myeloma chemotherapy resistance.Methods:1.Human myeloma cell lines and CD138+ plasma cells from bone marrow of patients with multiple myeloma were cultured in medium containing melphalan,bortezomib,or carfilzomib,with or without resistin.Apoptosis in the cultured cells was determined by using an annexin V binding assay.2.The expression levels of apoptotic related proteins and cell survival related signaling pathway were determined by western blot analysis.Then specific inhibitors were added to cultures of myeloma cells,and apoptosis was determined by using an annexin V binding assay.3.We first investigate the impact of resistin on ABC-driven efflux of chemotherapy drugs by flow cytometry.We then examined the expression of ABCB1?ABCB3?ABCC1?ABCC3?ABCC4?ABCC5?ABCG2 using real-time PCR analysis.The protein levels of ABCG2 and ABCC5 were further determined by western blot analysis.In addition,we determined the importance of ABCG2 and ABCC5 transporters in resistin-mediated protection using si RNAs specific for human ABCG2 and ABCC5 genes.4.We used methylation-specific PCR to determine the effect of resistin in the promoter methylation of ABCG2 and ABCC5 genes.Meanwhile,real-time PCR and western blot analysis were used to examine the effect of resistin in DNA methyltransferase.5.Twenty SCID(severe combined immune deficiency)mouse were randomized into four groups.ARP-1 myeloma cells were injected into SCID mouse femurs.After 3 weeks,treatment with melphalan or resistin,singly or in combination,was begun.After treatment,mouse serum M-protein levels were measured by the human Kappa ELISA kit.The percentages of CD138+ cells from bone marrow of mice were determined by flow cytometry.Apoptosis of CD138+ cells were examined by annexin V binding assay and in situ TUNEL assay.Results:Resistin significantly reduced melphalan-induced apoptosis of human ARP-1,MM.1S,and U266 myeloma cell lines in a dose-dependent manner.Similaly,resistin significantly reduced apoptosis induced in ARP-1,MM.1S,and RPMI8226 cells by bortezomib or by carfilzomib.Resistin also reduced melphalan-induced apoptosis in the patients' s CD138+ myeloma cells.In ARP-1 and MM.1S cells,addition of resistin significantly decreased the levels of cleaved caspase-9,caspase-3,poly(ADP-ribose)polymerase(PARP),and Bax in the presence of melphalan.Meanwhile,the expression of pro-survival Bcl-2 family members Bcl-2 and Bcl-x L were significantly increased in cells treated with resistin and melphalan,compared with those in cells treated with melphalan alone.We further found that resistin increased the levels of phosphorylated I?Ba,Akt,and ERK1/2.In addition,treatment with either NF-?B inhibitor or the PI3 K inhibitor could abrogate resistin-induced protection of myeloma cells from melphalan-induced apoptosis.We found that resistin significantly increased the efflux function and m RNA levels of ABC transporters.And the upregulation of ABCG2 and ABCC5 transporters persisted in all myeloma cell lines.Knocking down ABCG2 and ABCC5 expression in ARP-1 and MM.1S cells abrogated resistin-induced protection from melphalan,bortezomib and carfilzomib-induced apoptosis.Further study revealed that resistin significantly decreased the levels of methylated ABCG2 and ABCC5 gene promoters while concomitantly increased the levels of un-methylated ABCG2 and ABCC5 promoters.In addition,resistin greatly reduced the m RNA and protein levels of DNMT1 and DNMT3 a.In vivo,we measured the serum levels of M-protein to reflect tumor burden in mice.The mice receiving both resistin and melphalan had much higher levels of serum M-protein than the mice receiving melphalan alone.Flow cytometry analysis of bone marrow cells from mice showed higher percentages of CD138+cells and lower percentages of apoptotic CD138+cells in the mice treated with both melphalan and resistin than in the mice treated with melphalan alone.Similar results were obtained from the staining of TUNEL in mouse femurs.Conclusions:1.Resistin inhibited chemotherapy-induced apoptosis of multiple myeloma cells.2.Resistin inhibited myeloma cell apoptosis though activating anti-apoptotic NF-?B and PI3K/Akt signaling pathways.3.Resistin downregulated DNMT1 and DNMT3 a expression and enhanced the expression and efflux function of ABC transporters,leading to myeloma drug resistance.4.In vivo assay further verified the effect of resistin in chemoresistance of myeloma cells.