The present study sought to investigate the correlation between adipose cytokines (visfatin, leptin and adiponectin) and markers of multiple myeloma bone disease, and to determine the effects and mechanism of action of adiponectin on the differentiation and maturation of osteoclasts in multiple myeloma (MM). The levels of visfatin, leptin and adiponectin were measured. Their association with the indices of myeloma tumor load and bone disease were analyzed. Reverse transcription-quantitative PcR was used to detect the expression of receptor activator of nuclear factor-κB ligand (RANKL), osteoclast associated Ig-like receptor (OScAR), tartrate-resistant acid phosphatase (TRAP) and cathepsin K genes. Flow cytometry was used to detect the expression of adiponectin receptor 1 (AdipoR1) and the phosphorylation of the mechanistic target of rapamycin kinase (mTOR) pathway-associated proteins mTOR and eukaryotic translation initiation factor 4E-binding protein (4EBP1). There were no significant correlations among leptin, visfatin and the indexes of myeloma tumor load and bone disease. Serum adiponectin levels were significantly lower in patients with newly diagnosed multiple myeloma compared with healthy volunteers (12.37±3.13 vs. 13.80±0.95; P<0.05). The number of mature osteoclasts in the adiponectin group was lower compared with in the control group. Adiponectin also inhibited the mRNA expression of the osteoclast-associated factors RANKL, OScAR, TRAP and cathepsin K. comparison between the non-adiponectin group and the adiponectin group revealed that adiponectin increased the expression of AdipoR1 on the surface of osteoclast precursor cells (26.21±4.27% vs. 29.86±6.23%; P<0.05) and reduced the expression of phosphorylated (p-)mTOR (7.89±1.00% vs. 5.91±1.26%; P<0.05) and p-4EBP1 (26.78±5.00% vs. 22.49±4.24%; P<0.05). The p-mTOR and p-4EBP1 levels in the adiponectin + MHY1485 (an mTOR signaling pathway-specific agonist) group were significantly higher compared with those in the adiponectin group. It was revealed that adiponectin may inhibit osteoclast differentiation and maturation via the mTOR pathway. In conclusion, adiponectin inhibits the differentiation and maturation of osteoclasts by increasing the expression of AdipoR1 and reducing the phosphorylation levels of mTOR and 4EBP1 in patients with MM.

The bone marrow microenvironment plays important roles in the progression of the myelodysplastic syndrome (MDS). The higher incidence of ASXL1 and TET2 gene mutations in our iron overload (IO) MDS patients suggests that IO may be involved in the pathogenesis of MDS. The effects of IO damaging bone marrow mesenchymal stromal cells (MSCs) from higher-risk MDS patients were investigated. In our study, IO decreased the quantity and weakened the abilities of proliferation and differentiation of MSCs, and it inhibited the gene expressions of VEGFA, CXCL12, and TGF-β1 in MSCs regulating hematopoiesis. The increased level of reactive oxygen species (ROS) in MSCs caused by IO might be inducing apoptosis by activating caspase3 signals and involving in MDS progression by activating β-catenin signals. The damages of MSCs caused by IO could be partially reversed by an antioxidant or an iron chelator. Furthermore, the MSCs in IO MDS/AML patients had increased levels of ROS and apoptosis, and the expressions of caspase3 and β-catenin were increased even further. In conclusion, IO affects gene stability in higher-risk MDS patients and impairs MSCs by inducing ROS-related apoptosis and activating the Wnt/β-catenin signaling pathway, which could be partially reversed by an antioxidant or an iron chelator.

Research regarding histone deacetylase (HDAC) inhibitors (HDACis) has garnered interest for the treatment of multiple myeloma (MM). In addition, the high expression of nuclear factor (NF)-κB in MM cells is considered an important factor in the occurrence and development of MM. The present study aimed to determine the short-term effects of HDACis, chidamide and valproic acid (VPA), on MM cells, their effects on NF-κB and the underlying mechanisms. The present study measured HDAC activity, and the proliferation and apoptosis of U266 and RPMI8226 MM cells following treatment with various concentrations of chidamide and VPA for 6 and 48 h. Western blotting was used to detect the expression levels of phosphorylated (p)-IκB kinase (IKK)α/β, NF-κB p65 and inhibitor of NF-κB (IκBα) in U266 and RPMI8226 cells at various time points following treatment with chidamide and VPA (0, 2, 4 and 6 h). The results revealed that chidamide and VPA had no significant effect on the HDAC activity, proliferation and apoptosis of cells at 6 h; however, cell HDAC activity and proliferation were inhibited, and apoptosis was induced at 48 h. Furthermore, the expression levels of IκBα were gradually increased over time, whereas the expression levels of NF-κB p65 gradually decreased. These findings indicated that long-term (48 h) treatment with the HDACis chidamide and VPA inhibited the proliferation and promoted the apoptosis of MM cells; however, these HDACis had little effect on cell proliferation and apoptosis in the short term (6 h). Notably, in the short term (2-6 h), hyperactivation of NF-κB was inhibited via the IκBα-NF-κB p65 pathway. These findings indicated that cell growth may be inhibited and drug susceptibility may be promoted by blocking the NF-κB pathway at an early stage, when HDACis are combined with other drugs in the treatment of MM.

Objective: As an important negative regulatory factor of immunological cells, Tim3 plays a regulating role in tumor immune microenvironment. The purpose of this study was to investigate the expression of Tim3 on MM cells and its effect on the proliferation and apoptosis of MM cells, as well as its potential mechanism. Methods: In this study, the expression of Tim3 was detected on myeloma cells (CD38 + CD138 + cells) of bone marrow by flow cytometry (FCM) from 167 patients with MM and 51 healthy donors as controls and making correlation analysis with related clinical indexes. In vitro, MM cell lines (RPMI-8226 and U266) were treated with Tim3 knockdown alone, bortezomib alone and combination of Tim3 knock-down and bortezomib, then cell proliferation, cell apoptosis and downstream signaling pathway were detected by CCK-8, FCM, RT-PCR and western blot. Results: The expression of Tim3 on myeloma cells in MM patients was significantly higher than normal control group and positively correlated with b 2 microglobulin, creatine, and plasma cells of bone marrow, negatively correlated with hemoglobin and red blood cells. In vitro, we validated the high expression of Tim3 in RPMI-8226 and U266 cell lines. After Tim3 knock-down, the cell proliferation was inhibited and cell apoptosis was induced, the relative mRNA and protein expression of Tim3 and NF-kB signal pathway (PI3K, AKT, mTOR, NF-kB) were significantly decreased. Also, the cell proliferation was inhibited, cell apoptosis was increased, the relative mRNA and protein expression of NF-kB were decreased significantly in combination group than bortezomib or Tim3 knock-down group. Conclusions: The high expression of Tim3 on MM cells is associated with progression of MM patients. Tim3 maybe regulate the proliferation of MM cells via NF-kB signal pathway. Down-regulation of Tim3 expression can inhibit proliferation and induce apoptosis of MM cells, also has an additive inhibitory effect of bortezomib on NF-kB signaling pathway, then inhibit proliferation and induce apoptosis. Therefore, Tim3 may be a potential target for the treatment of MM.

Background: Multiple myeloma (MM) is a clonal disorder characterized by the proliferation of plasma cells and their accumulation within the bone marrow (BM). The flow cytometric analysis is an essential method for the hematological diseases because of high sensitivity.Aims: This study evaluates the indication role of malignant plasmacytes (PCs) in BM detected by flow cytometry for the risk stratification of MM.Methods: Whole BM samples from 92 newly diagnosed MM patients were included in the study. We collected 10 6 cells each sample by flow cytometry. Then we analyzed the correlation between the malignant PCs in BM and the characteristics of patients.Results: In this study, patients were stratified according to different baseline characteristics and the median ratio of the malignant PCs were compared. The significant statistical differences (p < 0.05) were: Hb < 100 g/L versus ≥100 g/L; β2-microglobulin <3.5 mg/dL versus 3.5-5.5 mg/dL versus >5.5 mg/dL; LDH > 250 U/L versus LDH 250 U/L; ISS I versus ISS II versus ISS III; R-ISS I versus II versus III. The detailed data are showed in Table 2. The significant correlations were observed between the malignant PCs in BM and (Figure 1): plasma cell of biopsy, hemoglobin, β2-microglobulin, lactate dehydrogenase (LDH), creatinine. "Double hit" or "triple hit" are defined as containing any two or three of the high risk cytogenetic abnormalities (t(4;14), t(14;16), t(14;20); del17q; TP53 mutation; 1q21 gain) by mSMAR. "Double or triple hit" had independently unfavorable significance for overall survival. As expected, the malignant PCs of "double or triple hit" group is significantly higher than the group B (one high risk genetic factor) and the group A (normal cytogenetic) (p < 0.0001 and p < 0.019). Conclusion:Multiparametric flow cytometry is a highly sensitive method to identify and quantify malignant PCs. And the ratio of malignant PCs detected by MFC showed strongly correlation with the severity of the pathology of MM. Malignant PCs in BM detected by flow cytometry could be regarded as a predictor for the risk MengYue Tian and ZhaoYun Liu contributed equally to this study.