Experimental Study On Effects Of Targeted Agents On Unfolded Protein Response In Multiple Myeloma Cells

Background and objectivesRecently several targeted-therapies have significantly improved the prognosis of multiple myeloma,although it remains an incurable malignancy.Bortezomib(BZ,PS-341), a proteasome inhibitor(PI) has been widely used in clinic for its highly specific cytotoxicity and relatively low side effects,and has demonstrated its efficacy.Previous reports have demonstrated that proteasome inhibition by bortezomib abrogates degradation of IκB,leading to the cytoplasmic sequestration and inhibition of the transcription factor NF-κB.Whether can NF-κB inhibition completely explain the nature of the selectivity of bortezomib for MM cells? Although constitutive NF-κB activity in MM cells has been shown to increase MM cell survival and resistance to cytotoxic agents,bortezomib was shown to have more profound effects on MM cell proliferation than a specific IκB kinase inhibitor-PS-1145 or an irreversible inhibitor of IκB phosphorylation,suggesting that there must exist other mechanisms than NF-κB inhibition by which BZ function in myeloma.Similar to their normal counterparts-normal plasma cells,MM cells also produce and secrete large amounts of immunoglobulin,which need to be properly folded into their proper tertiary structures in endoplasmic reticulum(ER).Definitely misfolded proteins, such as defective ribosome products,which are targeted for ER-associated protein degradation(ERAD),accompany the production of immunoglobulin.ERAD involves the retrograde translocation or dislocation of the misfolded proteins out of the ER and subsequent degradation by cytosolic 26S proteasomes.The inhibition of cytosolic proteasomes by PIs definitely influences ERAD,leading to the accumulation of misfolded proteins within the ER and subsequent ER stress induction and UPR activation.If the stress is severe or prolonged,UPR activation eventually leads to cell-cycle arrest and the induction of apoptosis.So MM cells are certainly very sensitive to ERAD inhibition by PIs since they produce large amounts of ER-processed proteins.Combination of PIs and chemical agents known to induce ER stress have shown obvious synergic effects on killing myeloma cells,which indirectly reflecting the association of inhibition of myeloma with UPR.How on earth PIs exert their effects on UPR in myeloma awaits further study. 2-Methoxyestradiol(2ME2),an end-metabolite of estradiol,has cytotoxic effects on various proliferating cells in vitro and its phaseⅡclinical trial is ongoing.Our previous studies have demonstrated that 2-methoxyestradiol(2ME2) at low-concentration could induce differentiation of myeloma cell lines and CD138+ primary myeloma cells from myeloma patients,and up-regulate the expression level of Xbp-1 mRNA and protein in myeloma cell lines.A functional UPR system is therefore necessary both for the differentiations of B cells into plasma cells and to ensure that only properly folded antibodies are secreted.A recent study has demonstrated that XBP-1 mediates B-cell differentiation into plasma cells via UPR pathway.The up-regulation of Xbp-1 in 2ME2-induced myeloma cell differentiation results from inhibition of Pax-5 by PRDM1, suggesting that PRDM1 probably plays important roles in UPR.Recently it was reported that PRDM1 might be target gene in UPR for UPR activation quickly followed by up-regulation of PRDM1 mRNA in B cells.PRDM1 is a member of PRDM gene family,each of which having two different transcripts with or without PR-domain.They potentially function as oncogene and tumor suppressor gene,respectively.The effects of low-dose 2ME2 on the two transcripts of PRDM1(PRDM1αwith PR-domain and PRDM1βwithout PR-domain) needs further study.Methods and results1.We detected cellular viability by MTT assay and got the IC50 concentrations in three myeloma cell lines KM3,RPMI-8226 and NCI-H929 with the treatment of BZ. Then,we respectively detected the expressions of the important UPR target gene GRP78/BiP and XBP-1(XBP-1u,XBP-1s) at different time points by both Real-time quantitative PCR assay and Western-blot assay in these myeloma cell lines treated by BZ. The results showed that different myeloma cell lines had their individual sensitivity to BZ. The IC50 concentrations were 420.3073nM,129.083nM and 70.913nM,respectively. Real-time quantitative PCR results showed the mRNA levels of chaperone BiP were upregulated time-dependently and reached the peak after 12h treatment of BZ,increasing from 0.209±0.024 to 0.350±0.015(p＜0.05),0.365±0.124 to 2.175±0.175(p＜0.05), 0.324±0.016 to 2.235±0.355(p＜0.05) in myeloma cell line KM3,RPMI8226 and NCI-H929,respectively.The BiP protein expression levels also time-dependently increased by 0.92～1.73,1.46～2.68,1.17～1.80 during 24h treatment of BZ.In contrast to the obvious decline of XBP-1s mRNA,XBP-1u-mRNA significantly increased and consequently XBP-1u/XBP-1s mRNA 24h ratios going up significantly after 4h treatment of BZ,increasing from 1.22±0.04 to 8.17±2.03(p＜0.05),5.64±2.00 to 34.23±10.03 (p＜0.05) and 3.10±0.24 to 7.56±1.76(p＜0.05) in KM3,RPMI8226 and NCI-H929, respectively.The total protein expression levels of XBP-1 had no significant differences after treatment of BZ.2.In myeloma cell line NCI-H929,RPMI8226,KM3 and LP-1,PRDM1αand PRDM1βtranscripts were detected by real-time quantitative PCR after treatment of low-dose 2ME2(0.5μmol/L) for 0h,48h and 72h.It showed that both PRDM1αand PRDM1βwere up-regulated in time-dependent manner and the PRDM1α/βratio was obviously elevated time-dependently from 1.461±0.033 to 2.663±0.381(p＜0.01),1.929±0.334 to 2.727±0.362(p＜0.05),1.471±0.012 to 4.367±0.243(p＜0.001),1.660±0.042 to 3.059±0.167(p＜0.001),positively associated with myeloma cells' differentiation degrees.Conclusions:1.Different myeloma cell lines had their individual sensitivity to PIs.The more misfolded proteins produced in cells,the high sensitivity to PIs the cells showed.The quantities of intracellular misfolded proteins are positively associated with the cellular sensitivity to PIs.2.Increased transcription and translation of ER molecular chaperones induced by UPR activation help unfolded and misfolded proteins correctly refold and assemble.BZ increases ER stress by inhibition of ERAD and activates UPR,but the expression levels of GRP78 and GRP94 only slightly increased.The results suggest MM plasma cells already express near-maximal levels of cytoprotective UPR proteins to function as secretory cells. Thus,these cells may have little additional increase in expressions of molecular chaperones following any additional stress to the ER.3.PIs inhibited the function of XBP-1 both by inhibiting production of XBP-1s with transcription activity and stabilization of XBP-1u to negatively regulate XBP-1s. Downstream target genes of XBP-1,such as chaperones,could protect cells from death induced by ER stress.PIs inhibited activation of XBP-1 and its downstream target genes, consequently blocking cytoprotection of UPR in myeloma plasma cells. 4.PIs upgraded UPR by inhibiting ERAD and increasing ER stress,and blocking activation of XBP-1 and its downstream target genes to remove their cytoprotection role, probably being PIs' another mechanism of anti-tumor effects in multiple myeloma.5.The significant increases in PRDM1α/PRDM1βtranscript ratios in low-dose 2-ME2-induced myeloma cell differentiation parallel with cell differentiation degrees,in accordance with negative-positive mechanism of PRDM family.