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Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages.

Machado CM, Andrade LN, Teixeira VR, Costa FF, Melo CM, dos Santos SN, Nonogaki S, Liu FT, Bernardes ES, Camargo AA, Chammas R - Cancer Med (2014)

Bottom Line: Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM.TGFβ1 induced secretion of VEGF only in WT-BMDM.Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Oncologia Experimental-LIM24, Departamento de Radiologia e Oncologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil; Depto. de Radiologia e Oncologia, Centro de Investigação Translacional em Oncologia, Instituto do Câncer do Estado de São Paulo, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil; Laboratório de Investigação Médica Radioisotopos-LIM/43, Departamento de Radiologia e Oncologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

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Methylation status of galectin-3 promoter region in melan-A and Tm1 cells. (A) Sequence of promoter, first exon and first intron (from −137 upstream to +377 downstream) of the galectin-3 gene (GenBank, # L08649). The analysis of these regions using CpGplot indicated that these regions lie within a putative CpG island. CpG dinucleotides methylated are represented as black circles whereas unmethylated CpG dinucleotides are represented in open circles. (B) Western blotting to galectin-3 in melan-A, Tm1 and transfected cells with a plasmid containing human cDNA for galectin-3 or the empty vector (Tm1G3 and Tm1N3) showing the presence of the murine galectin-3 in melan-A (molecular weight, ∼30 kDa), and the human galectin-3 in Tm1G3 (molecular weight, ∼25 kDa). Each lane corresponded to 20 μg from total protein cell extracts. (C) Representative graph of tumor volume after Tm1-galectin-3-nonexpressing cells implantation in C57black/6 Lgals3+/+ (WT) or Lgal3s3−/− (KO) mice. After 14 days post inoculation a palpable mass was detected. (D) After Tm1G3 injections in WT mice (WTG3) or KO mice (KOG3) a palpable mass was detected after 11 days post inoculation. The dashed line represents the cutoff tumoral volume to compare inocula of Tm1 and its transfected cells. The results in each graph corresponded to mean ± SEM and test used was two-way ANOVA with post test comparing all pair of columns. WT, wild type; KO, knockout; ANOVA, analyses of variance.
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fig01: Methylation status of galectin-3 promoter region in melan-A and Tm1 cells. (A) Sequence of promoter, first exon and first intron (from −137 upstream to +377 downstream) of the galectin-3 gene (GenBank, # L08649). The analysis of these regions using CpGplot indicated that these regions lie within a putative CpG island. CpG dinucleotides methylated are represented as black circles whereas unmethylated CpG dinucleotides are represented in open circles. (B) Western blotting to galectin-3 in melan-A, Tm1 and transfected cells with a plasmid containing human cDNA for galectin-3 or the empty vector (Tm1G3 and Tm1N3) showing the presence of the murine galectin-3 in melan-A (molecular weight, ∼30 kDa), and the human galectin-3 in Tm1G3 (molecular weight, ∼25 kDa). Each lane corresponded to 20 μg from total protein cell extracts. (C) Representative graph of tumor volume after Tm1-galectin-3-nonexpressing cells implantation in C57black/6 Lgals3+/+ (WT) or Lgal3s3−/− (KO) mice. After 14 days post inoculation a palpable mass was detected. (D) After Tm1G3 injections in WT mice (WTG3) or KO mice (KOG3) a palpable mass was detected after 11 days post inoculation. The dashed line represents the cutoff tumoral volume to compare inocula of Tm1 and its transfected cells. The results in each graph corresponded to mean ± SEM and test used was two-way ANOVA with post test comparing all pair of columns. WT, wild type; KO, knockout; ANOVA, analyses of variance.

Mentions: We evaluated the protein levels of galectin-3 in Tm1 cells, as compared to Melan-A and observed a total absence of this lectin in Tm1 cells, suggesting that galectin-3 expression was downregulated upon malignant transformation. It is well known that aberrant methylation of CpG dinucleotides is responsible for gene silencing 28 and, based on that, we first performed in silico analyses to identify putative CpG islands in the promoter region of the galectin-3 murine gene (GenBank sequence number L08649.1). Analysis of these sequences using CpG plot (EMBL) showed 33 CpG dinucleotides in the 5′ upstream region of the galectin-3 gene, including the first exon and first intron (Fig. 1A). The high content of CpG dinucleotides around regulatory regions of the galectin-3 gene suggests a possible role for DNA methylation in its control. To evaluate DNA methylation status of melan-A and Tm1 cell lines, we performed bisulfite genomic sequencing of a genomic region comprising the 33 CpG dinucleotides present in the 5′ upstream region of the galectin-3 gene. Sequencing of bisulfite-converted DNA revealed that all CpG dinucleotides were indeed methylated in the tumorigenic cell line and unmethylated in melan-A (Fig. 1A). Moreover, these regions had three extra CpG dinucleotides as compared to the actual galectin-3 sequence of Sv129 mice deposited in the GenBank.


Galectin-3 disruption impaired tumoral angiogenesis by reducing VEGF secretion from TGFβ1-induced macrophages.

Machado CM, Andrade LN, Teixeira VR, Costa FF, Melo CM, dos Santos SN, Nonogaki S, Liu FT, Bernardes ES, Camargo AA, Chammas R - Cancer Med (2014)

Methylation status of galectin-3 promoter region in melan-A and Tm1 cells. (A) Sequence of promoter, first exon and first intron (from −137 upstream to +377 downstream) of the galectin-3 gene (GenBank, # L08649). The analysis of these regions using CpGplot indicated that these regions lie within a putative CpG island. CpG dinucleotides methylated are represented as black circles whereas unmethylated CpG dinucleotides are represented in open circles. (B) Western blotting to galectin-3 in melan-A, Tm1 and transfected cells with a plasmid containing human cDNA for galectin-3 or the empty vector (Tm1G3 and Tm1N3) showing the presence of the murine galectin-3 in melan-A (molecular weight, ∼30 kDa), and the human galectin-3 in Tm1G3 (molecular weight, ∼25 kDa). Each lane corresponded to 20 μg from total protein cell extracts. (C) Representative graph of tumor volume after Tm1-galectin-3-nonexpressing cells implantation in C57black/6 Lgals3+/+ (WT) or Lgal3s3−/− (KO) mice. After 14 days post inoculation a palpable mass was detected. (D) After Tm1G3 injections in WT mice (WTG3) or KO mice (KOG3) a palpable mass was detected after 11 days post inoculation. The dashed line represents the cutoff tumoral volume to compare inocula of Tm1 and its transfected cells. The results in each graph corresponded to mean ± SEM and test used was two-way ANOVA with post test comparing all pair of columns. WT, wild type; KO, knockout; ANOVA, analyses of variance.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3987071&req=5

fig01: Methylation status of galectin-3 promoter region in melan-A and Tm1 cells. (A) Sequence of promoter, first exon and first intron (from −137 upstream to +377 downstream) of the galectin-3 gene (GenBank, # L08649). The analysis of these regions using CpGplot indicated that these regions lie within a putative CpG island. CpG dinucleotides methylated are represented as black circles whereas unmethylated CpG dinucleotides are represented in open circles. (B) Western blotting to galectin-3 in melan-A, Tm1 and transfected cells with a plasmid containing human cDNA for galectin-3 or the empty vector (Tm1G3 and Tm1N3) showing the presence of the murine galectin-3 in melan-A (molecular weight, ∼30 kDa), and the human galectin-3 in Tm1G3 (molecular weight, ∼25 kDa). Each lane corresponded to 20 μg from total protein cell extracts. (C) Representative graph of tumor volume after Tm1-galectin-3-nonexpressing cells implantation in C57black/6 Lgals3+/+ (WT) or Lgal3s3−/− (KO) mice. After 14 days post inoculation a palpable mass was detected. (D) After Tm1G3 injections in WT mice (WTG3) or KO mice (KOG3) a palpable mass was detected after 11 days post inoculation. The dashed line represents the cutoff tumoral volume to compare inocula of Tm1 and its transfected cells. The results in each graph corresponded to mean ± SEM and test used was two-way ANOVA with post test comparing all pair of columns. WT, wild type; KO, knockout; ANOVA, analyses of variance.
Mentions: We evaluated the protein levels of galectin-3 in Tm1 cells, as compared to Melan-A and observed a total absence of this lectin in Tm1 cells, suggesting that galectin-3 expression was downregulated upon malignant transformation. It is well known that aberrant methylation of CpG dinucleotides is responsible for gene silencing 28 and, based on that, we first performed in silico analyses to identify putative CpG islands in the promoter region of the galectin-3 murine gene (GenBank sequence number L08649.1). Analysis of these sequences using CpG plot (EMBL) showed 33 CpG dinucleotides in the 5′ upstream region of the galectin-3 gene, including the first exon and first intron (Fig. 1A). The high content of CpG dinucleotides around regulatory regions of the galectin-3 gene suggests a possible role for DNA methylation in its control. To evaluate DNA methylation status of melan-A and Tm1 cell lines, we performed bisulfite genomic sequencing of a genomic region comprising the 33 CpG dinucleotides present in the 5′ upstream region of the galectin-3 gene. Sequencing of bisulfite-converted DNA revealed that all CpG dinucleotides were indeed methylated in the tumorigenic cell line and unmethylated in melan-A (Fig. 1A). Moreover, these regions had three extra CpG dinucleotides as compared to the actual galectin-3 sequence of Sv129 mice deposited in the GenBank.

Bottom Line: Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM.TGFβ1 induced secretion of VEGF only in WT-BMDM.Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Oncologia Experimental-LIM24, Departamento de Radiologia e Oncologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil; Depto. de Radiologia e Oncologia, Centro de Investigação Translacional em Oncologia, Instituto do Câncer do Estado de São Paulo, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil; Laboratório de Investigação Médica Radioisotopos-LIM/43, Departamento de Radiologia e Oncologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil.

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Related in: MedlinePlus