Mithramycin Exerts an Anti-Myeloma Effect and Displays Anti-Angiogenic Effects through Up-Regulation of Anti-Angiogenic Factors

<div><p>Mithramycin (MTM), a cytotoxic compound, is currently being investigated for its anti-angiogenic activity that seems to be mediated through an inhibition of the transcription factor SP1. In this study we evaluated its anti-myeloma effects in the syngenic 5TGM1 model <i>in vitro</i> as well as <i>in vivo</i>. <i>In vitro</i>, MTM inhibited DNA synthesis of 5TGM1 cells with an IC50 of 400 nM and induced an arrest in cell cycle progression at the G1/S transition point. Western-blot revealed an up-regulation of p53, p21 and p27 and an inhibition of c-Myc, while SP1 remained unaffected. In rat aortic ring assays, a strong anti-angiogenic effect was seen, which could be explained by a decrease of VEGF production and an up-regulation of anti-angiogenic proteins such as IP10 after MTM treatment. The administration of MTM to mice injected with 5TGM1 decreased 5TGM1 cell invasion into bone marrow and myeloma neovascularisation. These data suggest that MTM displays anti-myeloma and anti-angiogenic effects that are not mediated by an inhibition of SP1 but rather through c-Myc inhibition and p53 activation.</p></div

MTM inhibits proliferation, migration and invasion of endothelial cell lines STR4 and STR10.

<p>Proliferation and viability was assessed using an MTT assay. Wound healing assay and Boyden chamber assay were used to study migration. Cell sprouting was assessed using a spheroid sprouting assay. Cells were treated with various concentrations of MTM during 24 h and 48 h. (<b>A</b>) Increasing concentrations of MTM resulted in a minor anti-proliferative effect in STR-4 cells, while MTM inhibited STR-10 proliferation with an IC-50 of 200 nM. (<b>B</b>) MTM inhibited both STR-4 and STR-10 cell migration in the Boyden Chamber assay. (<b>C,D</b>) Wound healing assay showed that adding 200 nM MTM decreased cell repopulation by both cells lines. In the spheroid sprouting assay (<b>E, F</b>), treatment with 200 nM or 400 nM had only an effect on the STR4 cell line whereas 800 nM of MTM caused an significant decrease in vessel sprouting by both cell lines.</p

MTM induces decreased VEGF and IP10 secretion.

<p>Levels of VEGF were determined in medium conditioned by 5TGM1 cell following 24 h of MTM treatment. Levels of IP10 were determined in both conditioned medium and cell lysate. Results were obtained using ELISA assays.</p

MTM reduced the myeloma-associated neo-vascularization <i>in vitro</i> in a rat aortic ring assay.

<p><b>A</b> Representative photomicrographs (scale 25×) of aortic rings cultured for 9 days with MCDB+DMEM, VEGF, MTM 400 nM, or with conditioned medium (CM) by cancer cells treated with MTM 0 nM, 200 nM or 400 nM. <b>B</b> A grid of concentric rings is automatically drawn by making successive dilations of the aortic ring boundary. Microvessel distribution is defined as the number of microvessel intersections with this grid plotted as a function of the distance from the ring. <b>C</b> and <b>D</b> represent the mean numbers of vessels depending of the distance to the ring for control (<b>C</b>) and MTM treatment (<b>D</b>). The addition of medium conditioned by treated 5TGM1 cells to the aortic ring culture increased the development of microvessels compared to the control condition (MCDB+DMEM) (p<0.01). This angiogenic response was inhibited (p<0.001) when increased concentrations of MTM (200 nM and 400 nM) were added to 5TGM1 cells before collection of conditioned medium.</p

MTM induces a dose-dependent increase of the percentage of cells in G0/G1 phase.

<p><b>A</b> Cell cycle analysis of 5TGM1 cells treated with 0, 200 or 400 nM of MTM. Cells were treated for 24 h with 200 nM and 400 nM of MTM and DNA was stained with propidium iodide, followed by flow cytometry analysis. This treatment resulted in a dose-dependent increase of the percentage of cells in G0/G1 phase (45% and 61%, respectively) when compared to the untreated cells (37%) (p<0,01). A decreased percentage of cells in the S phase was also observed (56% versus 49% and 32% for the 200 nM and 400 nM conditions, respectively) (p<0,01). Percentages of cells in G1, S or G2 phases following MTM treatment are indicated on the graph. <b>B</b> Protein expression of c-Myc, p21, p27, p53, cyclin D/CDK4 and cyclin E/CDK2, cdk6, phospho Rb and SP1 analyzed by Western blot (duplicates, repeated 3 times). Protein loadings were normalized with β-actin. Data shown are representative of at least three independent experiments. We observed an upregulation of p53, an inhibition of c-myc, while SP-1 remained stable. Downstream, p21 and p27 were upregulated, while cyclin B, cyclin E, CDK2, CDK4, CDK6 were decreased, which finally resulted in decreased phosphorlyation of Rb.</p

MTM inhibits proliferation and induces apoptosis in 5TGM1 cells.

<p><b>A</b> Effect of MTM on 5TGM1 cell proliferation and viability. Cells were treated with various concentrations of MTM for 24 h and viability was assessed using an MTT assay. There was a dose dependent inhibition in DNA synthesis observed with an IC50 of 600 nM <b>B</b> The induction of apoptosis was determined by FACS analysis. Cells were incubated during 24 h or 48 h with MTM (0 nM, 200 nM, 400 nM and 800 nM) and annexin V/PI stainings were performed. A representative picture of FACS analysis shows no induction of apoptosis after 24 h and the presence of late apoptotic cells after 48 h of treatment.</p

MTM reduced BM colonisation by 5TGM1 (A) and splenomegaly (B).

<p>Tumour load was assessed by flow cytometry analysis of GFP cells and spleen weight was measured. Starting the day after injection (day 1), a group of mice was treated intraperitonealy twice weekly with MTM or vehicle (DMSO dissolved in PBS). At day 32, mice were sacrificed.</p

MTM decreased angiogenic response.

<p>CD31 immuno-staining on bone marrow sections of control mice (<b>A</b>) or 5TGM1 injected mice, treated with the vehicle solution (<b>B</b>) or with MTM (<b>C</b>). Original magnification 10x. <b>D</b> Quantification of microvessel density (MVD) using the hot spot technique. MVD was decreased in MTM-treated mice compared with the group treated with the vehicle solution.</p

Upregulation of miR-135b Is Involved in the Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients

<div><p>Previous studies have demonstrated that mesenchymal stem cells from multiple myeloma (MM) patients (MM-hMSCs) display a distinctive gene expression profile, an enhanced production of cytokines and an impaired osteogenic differentiation ability compared to normal donors (ND-hMSCs). However, the underlying molecular mechanisms are unclear. In the present study, we observed that MM-hMSCs exhibited an abnormal upregulation of miR-135b, showing meanwhile an impaired osteogenic differentiation and a decrease of SMAD5 expression, which is the target of miR-135b involved in osteogenesis. By gain and loss of function studies we confirmed that miR-135b negatively regulated hMSCs osteogenesis. We also found that MM cell-produced factors stimulated ND-hMSCs to upregulate the expression of miR-135b. Importantly, treatment with a miR-135b inhibitor promoted osteogenic differentiation in MM-hMSCs. Finally, we observed that MM cell-derived soluble factors could induce an upregulation of miR-135b expression in ND-hMSCs in an indirect coculture system and the miR-135b expression turned to normal level after the removal of MM cells. Collectively, we provide evidence that miR-135b is involved in the impaired osteogenic differentiation of MSCs derived from MM patients and might therefore be a promising target for controlling bone disease. </p> </div

miR-135b expression is higher in MM BM-derived hMSCs showing impaired osteogenic differentiation potential.

<p>(A) MM-hMSCs show a reduced ALP activity, a marker for osteogenic differentiation. ND-hMSCs (n=7) and MM-hMSCs (n=12) are cultured in osteogenic medium (OM) for 72 hours and the ALP activity is measured quantitatively by the alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (Sigma-Aldrich, Bornem, Belgium). ** <i>p</i><0.01 (B) miR-135b expression is significantly upregulated in MM-hMSCs compared to ND-hMSCs by quantitative real time PCR. The miR-135b expression of all hMSCs samples is normalized to the miR-135b expression of U266 MM cells. * <i>p</i><0.05 (C) miR135b expression is inversely correlated with the ALP activity in MM-hMSCs. The relative expression of miR-135b in hMSCs derived from 12 MM patients was plotted versus their ALP activity.</p