4’，5，7三羟基异黄酮抑制骨髓瘤基质细胞bcl-2，bcl-xl，CyclinD1，ICAM-1和IL-6基因表达

目的:研究4’,5,7三羟基异黄酮(Genistein)对人多发骨髓瘤(MM)基质细胞增殖和bcl-2,bcl-xl,CyclinD1,ICAM-1和IL-6基因表达的影响。方法:体外培养人多发骨髓瘤基质细胞,依据浓度梯度分别给予Genistein,用噻唑蓝染色法(MTT)观察Genistein对骨髓瘤基质细胞增殖的影响;10,20,40 mg.L-1Genistein与溶剂对照组分别作用骨髓瘤基质细胞48 h后,半定量RT-PCR法检测骨髓瘤基质细胞bcl-2,bcl-xl,CyclinD1,ICAM-1和IL-6基因表达。结果:Genistein作用骨髓瘤基质细胞48 h,随浓度增加,细胞增殖逐渐下降;10,20,40 mg.L-1Genistein处理组bcl-2,bcl-xl,CyclinD1,ICAM-1,IL-6基因mRNA的表达呈浓度依赖性下降,与溶剂对照组比较,差异有统计学意义(P<0.05,P<0.01)。结论:Genistein可能通过下调骨髓瘤基质细胞bcl-2,bcl-xl,CyclinD1,ICAM-1和IL-6基因mRNA的表达,抑制骨髓瘤基质细胞的生长。     

Genistein down‐regulates the constitutive activation of nuclear factor‐κB of bone marrow stromal cells in multiple myeloma,leading to suppression of gene expression and proliferation

Bone marrow stromal cells (BMSCs) play a central role in human multiple myeloma(MM) cell survival and proliferation. We explored the possibility of using BMSCs as a target for MM treatment using genistein, a chemopreventive agent with little or no toxicity in humans. NF‐κB was constitutively active in BMSCs and genistein down‐regulated NF‐κB in BMSCs as measured by an electrophoretic mobility gel shift assay. BMSCs also showed constitutively active Akt phosphorylation that was suppressed by genistein. Genistein also down‐regulated the expression of NF‐κB‐regulated gene products, including bcl‐2, bcl‐xl, Cyclin D1, IL‐6, and ICAM‐1, which was associated with the suppression of BMSC proliferation. MM cells can survive if co‐cultured with BMSCs, suggesting that the bone marrow microenvironment is important. However, genistein‐treated BMSCs provide little support to MM cells. Overall, our results indicate that genistein down‐regulates NF‐κB and phospho‐Akt of BMSCs in human myeloma, leading to the suppression of gene expression of bcl‐2, bcl‐xl, Cyclin D1, IL‐6, and ICAM‐1 suppression proliferation, thus providing a potential new target for the treatment of MM. Drug Dev Res 69:219–225, 2008. © 2008 Wiley‐Liss, Inc.     

BAFF activates Erk1/2 promoting cell proliferation and survival by Ca-CaMKII-dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca²⁺-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca²⁺-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca²⁺ may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.     

Evaluation of the multipotent character of human adipose tissue-derived stem cells isolated by Ficoll gradient centrifugation and red blood cell lysis treatment

In the present study, the multipotent potential of two differential isolated human adipose-derived stem cell (hADSC) populations was evaluated. More specifically, hADSC isolated by means of classical Ficoll (F) gradient centrifugation were compared to hADSC isolated by means of red blood cell (RBC) lysis treatment and subsequent cultivation as 3D spheres.No significant difference in the genotypic expression of the multipotent markers Oct-4, Sox-2, Nanog, Klf-4 and cMyc could be observed between both isolation methods. Upon adipogenic and osteogenic differentiation, both hADSC populations showed lipid droplet accumulation and mineral deposition, respectively. Although, a more pronounced mineral deposition was observed in hADSC-RBC, suggesting a higher osteogenic potential. Upon exposure to keratinogenic media, both hADSC populations expressed the keratinocyte markers filaggrin and involucrin, evidencing a successful keratinogenic differentiation. Yet, no differences in expression were observed between the distinctive isolation procedures. Finally, upon exposure to neurogenic differentiation media, a significant difference in marker expression was observed. Indeed, hADSC-RBC only expressed vimentin and nestin, whereas hADSC-F expressed vimentin, nestin, NF-200, MBP and TH, suggesting a higher neurogenic potential.In summary, our data suggest that the choice of the most efficient isolation procedure of hADSC depends on the differentiated cell type ultimately required.