The activity of a novel mithramycin analog is related to its binding to DNA, cellular accumulation, and inhibition of Sp1-driven gene transcription

DIG-MSK (demycarosyl-3D-β-d-digitoxosyl-mithramycin SK) is a recently isolated compound of the mithramycin family of antitumor antibiotics, which includes mithramycin A (MTA) and mithramycin SK (MSK). Here, we present evidence that the binding of DIG-MSK to DNA shares the general features of other mithramycins such as the preference for C/G-rich tracts, but there are some differences in the strength of binding and the DNA sequence preferentially recognized by DIG-MSK. We aimed at gaining further insights into the DIG-MSK mechanism of action by direct comparison with the effects of the parental MTA. Similar to MTA, MSK and DIG-MSK accumulated rapidly in A2780, IGROV1 and OVCAR3 human ovarian cancer cell lines, and DIG-MSK was a potent inhibitor of both basal and induced expression of an Sp1-driven luciferase vector. This inhibitory activity was confirmed for the endogenous Sp1 gene and a set of Sp-responsive genes, and compared to that of MTA and MSK. Furthermore, DIG-MSK was stronger than MTA as inhibitor of Sp3-driven transcription and endogenous Sp3 gene expression. Differences in the effects of MTA, MSK and DIG-MSK on gene expression may have a large influence on their biological activities. © 2014 Elsevier Ltd. All rights reserved.This work was supported by grant BFU2010-15518 from the Spanish Ministry of Science and Innovation, and the FEDER program of the European Community, and it was performed within the framework of the “Xarxa de Referencia en Biotecnologia” of the Generalitat de Catalunya. It was also supported by the Principado de Asturias Government through PCTI founds 2006-2009, 80% co-financed with the FEDER Operative Program of the Principado de Asturias 2007-2013 (project FC-11-PC-10-31) and Fondo de Investigaciones Sanitarias (Institute Carlos III) PS09/00420 and PI12/01280. A. F-G. is co-financed by the project COF-11-24 from FICYT. C.V. is recipient of a JAE-Predoc2010 fellowship (CSIC), co-financed by the European Social FundPeer Reviewe

Pleiotropic Anti-Angiogenic and Anti-Oncogenic Activities of the Novel Mithralog Demycarosyl-3D-ß-D-Digitoxosyl-Mithramycin SK (EC-8042).

Demycarosyl-3D-ß-D-digitoxosyl-mithramycin SK (DIG-MSK) is a recently isolated analogue of mithramycin A (MTA) that showed differences with MTA in the DNA binding strength and selectivity. These differences correlated with a better therapeutic index and less toxicity in animal studies. Herein, we show that DIG-MSK displays a potent anti-tumor activity against different types of cancer cell lines, ovarian tumor cells being particularly sensitive to this drug. Of relevance, DIG-MSK exerts low toxicity on fibroblasts and peripheral blood mononuclear cells, this toxicity being significantly lower than that of MTA. In correlation with its antitumor activity, DIG-MSK strongly inhibited Sp1-mediated transcription and endogenous Sp1 mRNA expression, which correlated with the inhibition of the expression of key Sp1-regulated genes involved in tumorigenesis, including VEGFA, BCL2L1 (Bcl-XL), hTERT, BRCA2, MYC and SRC in several ovarian cells. Significantly, DIG-MSK was a stronger inhibitor of VEGFA expression than MTA. Accordingly, DIG-MSK also exhibited potent anti-angiogenic activity on microvascular endothelial cells. Likewise, it significantly inhibited the gene expression of VEGFR1, VEGFR2, FGFR, PDGFB and PDGFRA and, additionally, it induced the expression of the anti-angiogenic factors angiostatin and tunstatin. These effects correlated with a pro-apoptotic effect on proliferating microvascular endothelial cells and the inhibition of the formation of endothelial capillary structures. Overall, the pleiotropic activity of DIG-MSK in inhibiting key oncogenic and angiogenic pathways, together with its low toxicity profile, highlight the therapeutic potential of this new drug

Expansion of NK cells and reduction of NKG2D expression in chronic lymphocytic leukemia. Correlation with progressive disease.

The immune system may mediate anti-tumor responses in chronic lymphocytic leukemia (CLL) which may affect disease progression and survival. In this study, we analyzed the immune characteristics of 99 consecutive previously diagnosed CLL patients and 50 healthy controls. The distribution of lymphocyte subsets at diagnosis was retrospectively analyzed. Compared with controls, leukemia patients showed an expansion of NK and CD8 T cells at diagnosis. The relative number of CD8 T cells at diagnosis was associated with time to treatment, suggesting that CD8 T cells may modify disease progression. The distribution of lymphocyte subsets was analyzed again when patients were enrolled in this study. The median time since these patients were diagnosed was 277 weeks. Compared with diagnosis, the absolute number of CD8 T cells significantly decreased in these patients, reaching similar values to healthy controls; however NK cells kept significantly elevated overtime. Nevertheless, NK cells showed an impaired expression of NKG2D receptor and a defective cytotoxic activity. This down-regulation of NKG2D expression was further enhanced in patients with advanced and progressive disease. Additionally, membrane NKG2D levels significantly decreased on CD8 T cells, but a significant increase of NKG2D+CD4+ T cells was observed in CLL patients. The cytotoxic activity of NK cells was diminished in CLL patients; however the treatments with IL-2, IL-15, IL-21 and lenalidomide were able to restore their activity. The effect of IL-2 and IL-15 was associated with the increase of NKG2D expression on immune cells, but the effect of IL-21 and lenalidomide was not due to NKG2D up-regulation. The expansion of NK cells and the reversibility of NK cell defects provide new opportunities for the immunotherapeutic intervention in CLL

MSK and DIG-MSK modulate the expression of key angiogenic genes in ovarian carcinoma cells.

<p>a) qRT-PCR analysis of the expression of several key angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared with untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p<0.05; **p<0.01; Mann-Whitney U test). b) Venn diagrams representing genes down-regulated by treatment with MSK or DIG-MSK (p<0.05). Numbers inside the intersections correspond to genes repressed by both treatments.</p

Effect of MTA and DIG-MSK on the secretion of VEGF and the surface expression of VEGFR1 and VEGFR2.

<p>a) Soluble VEGF levels were measured by ELISA in supernatants of ovarian carcinoma cells (A2780, OVCAR-3 and IGROV-1) treated with 200 nM MTA or DIG-MSK compared with untreated cells. b) The surface expression of VEGFR1 and VEGFR2 was analyzed by flow cytometry in ECs treated with 200 nM MTA or DIG-MSK relative to DMSO treated cells. Data represent the mean ± SEM of levels obtained from at least three independent experiments. (*p<0.05; Mann-Whitney U test).</p

qRT-PCR primers used and thermal profiles.

<p>qRT-PCR primers used and thermal profiles.</p

Analysis of the expression of key anti-angiogenic genes in ECs treated with MTA or DIG-MSK.

<p>qRT-PCR analysis of the expression of several key anti-angiogenic genes in the presence of 200 nM MTA or DIG-MSK compared to untreated ECs. Data represent the mean ± SEM of Ct values obtained from at least three qRT-PCR independent experiments made by duplicate in HUVEC (a) or HMEC-1 (b) endothelial cells. The relative mRNA expression was obtained by comparison of the expression profiles of untreated cells (DMSO) versus treated ones (*p<0.05; Mann-Whitney U test).</p

Effect of MTA and DIG-MSK on tube formation by human microvascular endothelial cells.

<p>a) HMEC-1 cells were seeded onto Matrigel-coated wells and incubated with DMSO or various concentrations of MTA and DIG-MSK for 48 hours. Capillary-like structures formation was captured and processed with Angiodraw Software. Angiogenic index was calculated as the number of branch points in a field. The bars represent the mean ± SEM of the angiogenic index of four independent experiments (*p<0.05; Mann-Whitney U test). b) Representative appearance of HMEC-1 tube formation. Untreated control cells and cells treated with MTA and DIG-MSK are presented.</p

Cytotoxic effect (IC₅₀) of MTA and DIG-MSK against cancer cell lines and endothelial cell lines.

<p>Ns: Non significant</p><p>Cytotoxic effect (IC₅₀) of MTA and DIG-MSK against cancer cell lines and endothelial cell lines.</p