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Mithramycin exerts an anti-myeloma effect and displays anti-angiogenic effects through up-regulation of anti-angiogenic factors.

Otjacques E, Binsfeld M, Rocks N, Blacher S, Vanderkerken K, Noel A, Beguin Y, Cataldo D, Caers J - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA-Research), University of Liège, Liège, Belgium.

ABSTRACT
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 in vitro as well as in vivo. In vitro, 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.

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MTM induces a dose-dependent increase of the percentage of cells in G0/G1 phase.A 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 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.
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pone-0062818-g002: MTM induces a dose-dependent increase of the percentage of cells in G0/G1 phase.A 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 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.

Mentions: To determine whether MTM affects cell cycle progression in 5TGM1 cells, cells were treated for 24 h with 200 nM and 400 nM of MTM and the DNA was stained with propidium iodide, followed by flow cytometry analysis. As shown in Figure 2A, treatment of cells with 200 nM and 400 nM of MTM resulted in a dose-dependent increase of the percentage of cells in G0/G1 phase (45% and 61%, respectively) when compared to the non-treated cells (37%) (p<0,01). A decreased percentage of cell in the S phase was also observed (56. % versus 49% and 32% for the 200 nM or 400 nM conditions, respectively) (p<0,01). Knowing that MTM acts through inhibiting SP1 or c-Myc, we investigated protein expression of these two transcription factors by Western blot. These experiments showed a reduction of c-Myc protein levels, while SP1 levels were unaffected (Figure 2B). Since p53 signalling cascade is known to play a role in the cell cycle arrest in response to cellular stress like DNA damage [26], p53 production was investigated using Western blot. Following MTM treatment, a distinct increase in p53 protein expression was observed (Figure 2B). Moreover, to understand the molecular mechanisms underlying the observed MTM-induced inhibition of cell growth [27], the expression of several intracellular regulators of the cell cycle was assessed by Western blot analysis (Figure 2B). For this, 5TGM1 cells were treated for 24 h with 200 nM and 400 nM of MTM. MTM exposure resulted in a significant reduction of the expression of cell cycle regulatory proteins, cyclins D1, E, CDK2, CDK4, CDK6 and c-Myc, particularly with 400 nM treatment. Knowing that CDK4, CDK6 and also Cyclin E, through activation of CDK2, are responsible for phosphorylation of pRb, the effect of MTM on the expression of pRb was investigated. MTM treatment caused a decrease of pRb phosphorylation in 5TGM1 cells. Because p21 and p27 mediate the p53-dependent G1 phase arrest in response to a variety of stress stimuli, their protein expression was evaluated by Western blot, showing an increase in their protein expression (Figure 2B). In summary, cell cycle arrest at the transition of G1 to S phase seems mediated by a decreased expression of cell cycle inducers (such as cyclins and cyclin-dependent kinases) but also by an increased expression of inhibitors such as p21 and p27 (which are under control of p53).


Mithramycin exerts an anti-myeloma effect and displays anti-angiogenic effects through up-regulation of anti-angiogenic factors.

Otjacques E, Binsfeld M, Rocks N, Blacher S, Vanderkerken K, Noel A, Beguin Y, Cataldo D, Caers J - PLoS ONE (2013)

MTM induces a dose-dependent increase of the percentage of cells in G0/G1 phase.A 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 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.
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Related In: Results  -  Collection

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pone-0062818-g002: MTM induces a dose-dependent increase of the percentage of cells in G0/G1 phase.A 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 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.
Mentions: To determine whether MTM affects cell cycle progression in 5TGM1 cells, cells were treated for 24 h with 200 nM and 400 nM of MTM and the DNA was stained with propidium iodide, followed by flow cytometry analysis. As shown in Figure 2A, treatment of cells with 200 nM and 400 nM of MTM resulted in a dose-dependent increase of the percentage of cells in G0/G1 phase (45% and 61%, respectively) when compared to the non-treated cells (37%) (p<0,01). A decreased percentage of cell in the S phase was also observed (56. % versus 49% and 32% for the 200 nM or 400 nM conditions, respectively) (p<0,01). Knowing that MTM acts through inhibiting SP1 or c-Myc, we investigated protein expression of these two transcription factors by Western blot. These experiments showed a reduction of c-Myc protein levels, while SP1 levels were unaffected (Figure 2B). Since p53 signalling cascade is known to play a role in the cell cycle arrest in response to cellular stress like DNA damage [26], p53 production was investigated using Western blot. Following MTM treatment, a distinct increase in p53 protein expression was observed (Figure 2B). Moreover, to understand the molecular mechanisms underlying the observed MTM-induced inhibition of cell growth [27], the expression of several intracellular regulators of the cell cycle was assessed by Western blot analysis (Figure 2B). For this, 5TGM1 cells were treated for 24 h with 200 nM and 400 nM of MTM. MTM exposure resulted in a significant reduction of the expression of cell cycle regulatory proteins, cyclins D1, E, CDK2, CDK4, CDK6 and c-Myc, particularly with 400 nM treatment. Knowing that CDK4, CDK6 and also Cyclin E, through activation of CDK2, are responsible for phosphorylation of pRb, the effect of MTM on the expression of pRb was investigated. MTM treatment caused a decrease of pRb phosphorylation in 5TGM1 cells. Because p21 and p27 mediate the p53-dependent G1 phase arrest in response to a variety of stress stimuli, their protein expression was evaluated by Western blot, showing an increase in their protein expression (Figure 2B). In summary, cell cycle arrest at the transition of G1 to S phase seems mediated by a decreased expression of cell cycle inducers (such as cyclins and cyclin-dependent kinases) but also by an increased expression of inhibitors such as p21 and p27 (which are under control of p53).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hematology, Groupe Interdisciplinaire de Génoprotéomique Appliquée (GIGA-Research), University of Liège, Liège, Belgium.

ABSTRACT
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 in vitro as well as in vivo. In vitro, 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.

Show MeSH
Related in: MedlinePlus