These results demonstrate, to our knowledge for the first time, that glycated serum albumin, not glucose, increases VEGF production in HMC. HMC play an important role as a source of intraperitoneal VEGF synthesis, and VEGF expression also is up-regulated in the presence of proinflammatory cytokines and thrombin. Additionally, these results confirm clinical data that the continuous exposure of the peritoneal membrane to glucose-based dialysis solutions is an important stimulus for VEGF expression. However, it is not glucose per se, but nonenzymatic glycation products like glycated albumin that up-regulate VEGF expression.

Multiple myeloma is still an incurable disease; therefore, new therapeutics are urgently needed. A771726 is the active metabolite of the immunosuppressive drug leflunomide, which is currently applied in the treatment of rheumatoid arthritis, BK virus nephropathy, and cytomegaly viremia. Here, we show that dihydroorotate dehydrogenase (DHODH) is commonly expressed in multiple myeloma cell lines and primary multiple myeloma cells. The DHODH inhibitor A771726 inhibits cell growth in common myeloma cell lines at clinically achievable concentrations in a time-and dose-dependent manner. Annexin V-FITC/ propidium iodide staining revealed induction of apoptosis of multiple myeloma cell lines and primary multiple myeloma cells. The 5-bromo-2 ¶-deoxyuridine cell proliferation assay showed that inhibition of cell growth was partly due to inhibition of multiple myeloma cell proliferation. A771726 induced G 1 cell cycle arrest via modulation of cyclin D2 and pRb expression. A771726 decreased phosphorylation of protein kinase B (Akt), p70S6K, and eukaryotic translation initiation factor 4E-binding protein-1 as shown by Western blotting experiments. Furthermore, we show that the stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on multiple myeloma cell growth is completely abrogated by A771726. In addition, synergism studies revealed synergistic and additive activity of A771726 together with the genotoxic agents melphalan, treosulfan, and doxorubicin as well as with dexamethasone and bortezomib. Taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in multiple myeloma. Considering the favorable toxicity profile and the great clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in multiple myeloma. [Mol Cancer Ther 2009;8(2):366 -75]

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The t(11;14)(q13;q32) is the most common translocation in multiple myeloma (MM), resulting in up-regulation of cyclin D1. We used a segregation fluorescence in situ hybridization (FISH)

Bendamustine induces ATM-Chk2-Cdc2-mediated G2 arrest and p53 mediated apoptosis. Inhibition of p38 MAPK augments apoptosis and abrogates G2 arrest and can be considered as a new therapeutic strategy in combination with bendamustine.

SummaryInhibition of histone deacetylase (HDAC) is a promising mechanism for novel, anti-myeloma agents. We investigated the effects of the novel HDAC inhibitor resminostat on multiple myeloma (MM) cells in vitro. Resminostat is a potent inhibitor of HDACs 1, 3 and 6 [50% inhibitory concentration (IC 50 ) = 43-72 nmol/l] representing HDAC classes I and II and induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of resminostat abrogated cell growth and strongly induced apoptosis (IC 50 = 2AE5-3 lmol/l in 3 out of 4 MM cell lines) in MM cell lines as well as primary MM cells. At 1 lmol/l, resminostat inhibited proliferation and induced G0/G1 cell cycle arrest in 3 out of 4 MM cell lines accompanied with decreased levels of cyclin D1, cdc25a, Cdk4 and pRb as well as upregulation of p21. Resminostat decreased phosphorylation of 4E-BP1 and p70S6k indicating an interference with Akt pathway signalling. Treatment with resminostat resulted in increased protein levels of Bim and Bax and decreased levels of Bcl-xL. Caspases 3, 8 and 9 were activated by resminostat. Furthermore, synergistic effects were observed for combinations of resminostat with melphalan and the proteasome inhibitors bortezomib and S-2209. In conclusion, we have identified potent anti-myeloma activity for this novel HDAC inhibitor.

Discordant bone marrow (BM) involvement in patients with a diagnosis of large-cell non-Hodgkin's lymphoma (NHL) is characterized by marrow infiltrates predominantly composed of small lymphoid cell, cytologically compatible with low-grade NHL. Although this phenomenon is well described morphologically, molecular data concerning the relationship of the two lesions are lacking. The aim of the study was to investigate the clonal relationship of discordant lymphoma manifestations by using immunoglobulin heavy chain gene (IgH), as well as bcl-2 rearrangements, as molecular markers. IgH rearrangements were amplified by PCR with consensus primers directed against framework regions 3 or 2 (FR3 and FR2), followed by automated fragment length analysis and sequencing in selected cases. Rearrangements of the bcl-2 gene were identified with primers against the major breakpoint region. Small BM infiltrates were isolated by laser capture microdissection. In addition, immunohistochemistry was performed on paraffin sections using antibodies against CD3, CD10, CD20, bcl-2, bcl-6, p53, and the Ki67 antigen. Paraffin-embedded tissues of 21 cases diagnosed as diffuse large B-cell lymphoma (DLBCL) with discordant BM involvement and no previous history of low-grade B-cell NHL were analyzed. After review of immunohistochemical stains, 5 cases were excluded either as concordant BM infiltrates by large-cell lymphoma with abundant reactive T-cells (2 cases) or as benign, reactive lymphoid infiltrates (3 cases), as confirmed by a polyclonal pattern in the IgH analysis. Of the remaining 16 cases, a common clonal origin was confirmed in 8 cases by the presence of an identical clonal IgH rearrangement or bcl-2 rearrangement. In 4 cases, identification of distinct IgH or bcl-2 rearrangements gave evidence for the presence of two clonally unrelated neoplasms. The remaining 4 cases were not evaluable for technical reasons. Morphological, phenotypical, and molecular findings were compatible with a lymphoma of germinal center origin in the majority of cases. However, in 4 cases, flow cytometric analysis of the BM infiltrates revealed a B-cell chronic lymphocytic leukemia phenotype. Two of these cases were clonally related to the DLBCL and thus represented Richter's transformation. In summary, discordant BM infiltrates in DLBCL represent a heterogeneous group of disorders, encompassing cases with a clonally related, clinically occult small-cell component, as well as cases with two clonally distinct, unrelated B-cell neoplasms presenting synchronously at different locations.

In this study, we show that adenosine monophosphate-activated protein kinase (AMPK) is expressed and activated in multiple myeloma cells. The inhibition of AMPK induced growth arrest and reduction of cell viability in the cell viability assay using the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay). Induction of apoptosis was determined by annexin-V and propidium iodide staining. The prevention of apoptosis using the pancaspase inhibitor ZVAD-fmk and caspase-3 cleavage upon incubation with the AMPK inhibitor (AMPKI) is shown. Furthermore, incubation of myeloma cells with AMPKI resulted in the downregulation of pAMPK, Mcl-1 and Bcl-xL. Coincubation of AMPKI and melphalan led to a strong additional increase of apoptosis in myeloma cells. We conclude that AMPKI has a strong antimyeloma activity in vitro and represents a new targeted strategy in the treatment of multiple myeloma.