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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 under oxidative stress conditions in hypoxic and normoxic cells.(A) Galectin-3 subcellular expression was assessed by double-labelling immunofluorescence. Representative galectin-3 cell expression shows that despite galectin-3 increase under hypoxia, in the presence of catalase this was not verified. GLUT-1, included as a well-known target of hypoxic conditions, was also not increased in the presence of catalase. (B) To quantitatively evaluate galectin-3 expression under different oxidative stress conditions western blot analyses were performed. Relative intensity of the indicated protein level bands normalizes to actin were measured. In hypoxic conditions, in the presence of catalase, increased expression of galectin-3 was not observed in CMT-U27 cells. Galectin-3 expression, was kept, both in levels and quantity, similar to that seen in normoxic cells. However, in normoxia, catalase itself appears to increase the expression of galectin-3. In normoxic conditions, hydrogen peroxide treatment induced an apparent increase of a heavier molecular weight form of galectin-3 but had no additive effect on the increase of galectin-3 under hypoxia.
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pone.0134458.g002: Galectin-3 expression under oxidative stress conditions in hypoxic and normoxic cells.(A) Galectin-3 subcellular expression was assessed by double-labelling immunofluorescence. Representative galectin-3 cell expression shows that despite galectin-3 increase under hypoxia, in the presence of catalase this was not verified. GLUT-1, included as a well-known target of hypoxic conditions, was also not increased in the presence of catalase. (B) To quantitatively evaluate galectin-3 expression under different oxidative stress conditions western blot analyses were performed. Relative intensity of the indicated protein level bands normalizes to actin were measured. In hypoxic conditions, in the presence of catalase, increased expression of galectin-3 was not observed in CMT-U27 cells. Galectin-3 expression, was kept, both in levels and quantity, similar to that seen in normoxic cells. However, in normoxia, catalase itself appears to increase the expression of galectin-3. In normoxic conditions, hydrogen peroxide treatment induced an apparent increase of a heavier molecular weight form of galectin-3 but had no additive effect on the increase of galectin-3 under hypoxia.

Mentions: One of the cellular outcomes of hypoxic conditions is oxidative stress, namely the increase in reactive oxygen species (ROS) production. We hypothesized that the increase in galectin-3 expression in hypoxia could, at least to some extent be, due to oxidative stress in CMT. To test this, cells were submitted to hydrogen peroxide and/or catalase treatment, to induce oxidative stress and to detoxify, respectively. Double-labeling immunofluorescence analyses were performed to evaluate simultaneous expression of galectin-3 and GLUT-1 in normoxic and hypoxic catalase-treated CMT-U27 cells. Galectin-3 and GLUT-1 presence, as evidenced by fluorescent immunostaining, was simultaneously increased in hypoxic CMT-U27 cells when compared with normoxic controls. However, catalase-treated CMT-U27 hypoxic cells failed to show such an increase of galectin-3 and also showed a lower of an increase of GLUT-1 immunostaining (Fig 2A). Galectin-3 expression was analyzed in the CMT-U27 cell line treated with hydrogen peroxide, using Western Blot analysis (Fig 2B). An increased heavier form of galectin-3 was observed in hydrogen peroxide-treated cells kept under normoxic conditions. Next, to detoxify the culture medium, catalase was used to counteract the effect of endogenous H2O2, under otherwise hypoxic and normoxic conditions. Upon 12 hours treatment with 4μM of catalase, galectin-3 expression was overall increased in protein extracts of CMT-U27 cells under normoxia. In hypoxic conditions, upon medium detoxification, galectin-3 expression decreased when compared with hypoxic untreated CMT-U27 cells (Fig 2B), corroborating the immunofluorescence analyses.


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 under oxidative stress conditions in hypoxic and normoxic cells.(A) Galectin-3 subcellular expression was assessed by double-labelling immunofluorescence. Representative galectin-3 cell expression shows that despite galectin-3 increase under hypoxia, in the presence of catalase this was not verified. GLUT-1, included as a well-known target of hypoxic conditions, was also not increased in the presence of catalase. (B) To quantitatively evaluate galectin-3 expression under different oxidative stress conditions western blot analyses were performed. Relative intensity of the indicated protein level bands normalizes to actin were measured. In hypoxic conditions, in the presence of catalase, increased expression of galectin-3 was not observed in CMT-U27 cells. Galectin-3 expression, was kept, both in levels and quantity, similar to that seen in normoxic cells. However, in normoxia, catalase itself appears to increase the expression of galectin-3. In normoxic conditions, hydrogen peroxide treatment induced an apparent increase of a heavier molecular weight form of galectin-3 but had no additive effect on the increase of galectin-3 under hypoxia.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519331&req=5

pone.0134458.g002: Galectin-3 expression under oxidative stress conditions in hypoxic and normoxic cells.(A) Galectin-3 subcellular expression was assessed by double-labelling immunofluorescence. Representative galectin-3 cell expression shows that despite galectin-3 increase under hypoxia, in the presence of catalase this was not verified. GLUT-1, included as a well-known target of hypoxic conditions, was also not increased in the presence of catalase. (B) To quantitatively evaluate galectin-3 expression under different oxidative stress conditions western blot analyses were performed. Relative intensity of the indicated protein level bands normalizes to actin were measured. In hypoxic conditions, in the presence of catalase, increased expression of galectin-3 was not observed in CMT-U27 cells. Galectin-3 expression, was kept, both in levels and quantity, similar to that seen in normoxic cells. However, in normoxia, catalase itself appears to increase the expression of galectin-3. In normoxic conditions, hydrogen peroxide treatment induced an apparent increase of a heavier molecular weight form of galectin-3 but had no additive effect on the increase of galectin-3 under hypoxia.
Mentions: One of the cellular outcomes of hypoxic conditions is oxidative stress, namely the increase in reactive oxygen species (ROS) production. We hypothesized that the increase in galectin-3 expression in hypoxia could, at least to some extent be, due to oxidative stress in CMT. To test this, cells were submitted to hydrogen peroxide and/or catalase treatment, to induce oxidative stress and to detoxify, respectively. Double-labeling immunofluorescence analyses were performed to evaluate simultaneous expression of galectin-3 and GLUT-1 in normoxic and hypoxic catalase-treated CMT-U27 cells. Galectin-3 and GLUT-1 presence, as evidenced by fluorescent immunostaining, was simultaneously increased in hypoxic CMT-U27 cells when compared with normoxic controls. However, catalase-treated CMT-U27 hypoxic cells failed to show such an increase of galectin-3 and also showed a lower of an increase of GLUT-1 immunostaining (Fig 2A). Galectin-3 expression was analyzed in the CMT-U27 cell line treated with hydrogen peroxide, using Western Blot analysis (Fig 2B). An increased heavier form of galectin-3 was observed in hydrogen peroxide-treated cells kept under normoxic conditions. Next, to detoxify the culture medium, catalase was used to counteract the effect of endogenous H2O2, under otherwise hypoxic and normoxic conditions. Upon 12 hours treatment with 4μM of catalase, galectin-3 expression was overall increased in protein extracts of CMT-U27 cells under normoxia. In hypoxic conditions, upon medium detoxification, galectin-3 expression decreased when compared with hypoxic untreated CMT-U27 cells (Fig 2B), corroborating the immunofluorescence analyses.

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