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Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis.

de Oliveira JT, Ribeiro C, Barros R, Gomes C, de Matos AJ, Reis CA, Rutteman GR, Gärtner F - PLoS ONE (2015)

Bottom Line: Increased galectin-3 expression was confirmed at the mRNA level.Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions.In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology (IPATIMUP), University of Porto, Porto, Portugal; Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Faculty of Veterinary Medicine of the Lusophone University of Humanities and Technologies, Lisbon, Portugal.

ABSTRACT
The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness.

No MeSH data available.


Related in: MedlinePlus

Galectin-3 expression in CMT-U27 cell line under hypoxic conditions.Galectin-3 protein expression was assessed by western blot and FACS using an anti-galectin-3 antibody (M3/38) (A) Western Blot analyses show increased expression levels of galectin-3 (p<0.001), HIF1-α and GLUT-1 in protein extracts from malignant CMT-U27 cell line after 6 hours 0.1% oxygen exposure. Relative intensity of the indicated protein level bands were normalized to actin. (B) FACS analysis of galectin-3 expression on CMT-U27 cells. CMT-U27 cells exposed to hypoxia and controls were stained with M3/38 anti-galectin-3 antibody. Galectin-3 expression was measured by florescence intensity of each cell, representative cells correspond statistically to median fluorescence of all cells. Galectin-3 was increased in CMT-U27 cells under hypoxia (1.238x105± mean fluorescence intensity) when compared with cells kept in normoxia (8.56x104± mean fluorescence intensity).
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pone.0134458.g001: Galectin-3 expression in CMT-U27 cell line under hypoxic conditions.Galectin-3 protein expression was assessed by western blot and FACS using an anti-galectin-3 antibody (M3/38) (A) Western Blot analyses show increased expression levels of galectin-3 (p<0.001), HIF1-α and GLUT-1 in protein extracts from malignant CMT-U27 cell line after 6 hours 0.1% oxygen exposure. Relative intensity of the indicated protein level bands were normalized to actin. (B) FACS analysis of galectin-3 expression on CMT-U27 cells. CMT-U27 cells exposed to hypoxia and controls were stained with M3/38 anti-galectin-3 antibody. Galectin-3 expression was measured by florescence intensity of each cell, representative cells correspond statistically to median fluorescence of all cells. Galectin-3 was increased in CMT-U27 cells under hypoxia (1.238x105± mean fluorescence intensity) when compared with cells kept in normoxia (8.56x104± mean fluorescence intensity).

Mentions: Up-regulation of galectin-3 under hypoxia has been described in a non-neoplastic context [20]. In order to assess a possible regulation of galectin-3 by hypoxia in neoplastic settings, we used a highly metastatic canine mammary cancer cell line, CMT-U27. CMT-U27 cells were cultured for 12 hours in hypoxia (0.1% O2) or normoxia (21% O2) as a control condition. HIF-1α and GLUT-1 protein expression were also determined by Western Blot as hypoxia readouts [34, 35]. Galectin-3 expression was significantly increased (p<0.001) in protein extracts from CMT-U27 cells exposed to hypoxia for 12 hours (paralleled by HIF-1α and GLUT-1 increased expression) when compared with normoxic controls (Fig 1A). In accordance, FACS analysis showed increased galectin-3 expression in CMT-U27 cells exposed to 0.1% oxygen (1.239x105±83.67 mean fluorescence intensity) when compared with cells kept in normoxia (8.25x104±65.12 mean fluorescence intensity) (Fig 1B). Galectin-3 expression was also increased in protein extracts of hypoxic CMT-U27 cells after 12 hours but not after 24 hours of 0.1% oxygen exposure (S1 Fig). Despite galectin-3 may be secreted to the extracellular space in an energy-independent manner [36], our present data seem not to support any additional differences in the secretion of the lectin at different points of hypoxia exposure (S2 Fig). Likewise, no differences were found in the expression of galectin-3 when CMT-U27 cells were treated with a proteasome inhibitor, either in normoxic or in hypoxic conditions (S3 Fig).


Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis.

de Oliveira JT, Ribeiro C, Barros R, Gomes C, de Matos AJ, Reis CA, Rutteman GR, Gärtner F - PLoS ONE (2015)

Galectin-3 expression in CMT-U27 cell line under hypoxic conditions.Galectin-3 protein expression was assessed by western blot and FACS using an anti-galectin-3 antibody (M3/38) (A) Western Blot analyses show increased expression levels of galectin-3 (p<0.001), HIF1-α and GLUT-1 in protein extracts from malignant CMT-U27 cell line after 6 hours 0.1% oxygen exposure. Relative intensity of the indicated protein level bands were normalized to actin. (B) FACS analysis of galectin-3 expression on CMT-U27 cells. CMT-U27 cells exposed to hypoxia and controls were stained with M3/38 anti-galectin-3 antibody. Galectin-3 expression was measured by florescence intensity of each cell, representative cells correspond statistically to median fluorescence of all cells. Galectin-3 was increased in CMT-U27 cells under hypoxia (1.238x105± mean fluorescence intensity) when compared with cells kept in normoxia (8.56x104± mean fluorescence intensity).
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Related In: Results  -  Collection

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pone.0134458.g001: Galectin-3 expression in CMT-U27 cell line under hypoxic conditions.Galectin-3 protein expression was assessed by western blot and FACS using an anti-galectin-3 antibody (M3/38) (A) Western Blot analyses show increased expression levels of galectin-3 (p<0.001), HIF1-α and GLUT-1 in protein extracts from malignant CMT-U27 cell line after 6 hours 0.1% oxygen exposure. Relative intensity of the indicated protein level bands were normalized to actin. (B) FACS analysis of galectin-3 expression on CMT-U27 cells. CMT-U27 cells exposed to hypoxia and controls were stained with M3/38 anti-galectin-3 antibody. Galectin-3 expression was measured by florescence intensity of each cell, representative cells correspond statistically to median fluorescence of all cells. Galectin-3 was increased in CMT-U27 cells under hypoxia (1.238x105± mean fluorescence intensity) when compared with cells kept in normoxia (8.56x104± mean fluorescence intensity).
Mentions: Up-regulation of galectin-3 under hypoxia has been described in a non-neoplastic context [20]. In order to assess a possible regulation of galectin-3 by hypoxia in neoplastic settings, we used a highly metastatic canine mammary cancer cell line, CMT-U27. CMT-U27 cells were cultured for 12 hours in hypoxia (0.1% O2) or normoxia (21% O2) as a control condition. HIF-1α and GLUT-1 protein expression were also determined by Western Blot as hypoxia readouts [34, 35]. Galectin-3 expression was significantly increased (p<0.001) in protein extracts from CMT-U27 cells exposed to hypoxia for 12 hours (paralleled by HIF-1α and GLUT-1 increased expression) when compared with normoxic controls (Fig 1A). In accordance, FACS analysis showed increased galectin-3 expression in CMT-U27 cells exposed to 0.1% oxygen (1.239x105±83.67 mean fluorescence intensity) when compared with cells kept in normoxia (8.25x104±65.12 mean fluorescence intensity) (Fig 1B). Galectin-3 expression was also increased in protein extracts of hypoxic CMT-U27 cells after 12 hours but not after 24 hours of 0.1% oxygen exposure (S1 Fig). Despite galectin-3 may be secreted to the extracellular space in an energy-independent manner [36], our present data seem not to support any additional differences in the secretion of the lectin at different points of hypoxia exposure (S2 Fig). Likewise, no differences were found in the expression of galectin-3 when CMT-U27 cells were treated with a proteasome inhibitor, either in normoxic or in hypoxic conditions (S3 Fig).

Bottom Line: Increased galectin-3 expression was confirmed at the mRNA level.Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions.In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; Institute of Molecular Pathology and Immunology (IPATIMUP), University of Porto, Porto, Portugal; Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), University of Porto, Porto, Portugal; Faculty of Veterinary Medicine of the Lusophone University of Humanities and Technologies, Lisbon, Portugal.

ABSTRACT
The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness.

No MeSH data available.


Related in: MedlinePlus