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Galectin-3 up-regulation in hypoxic and nutrient deprived microenvironments promotes cell survival.

Ikemori RY, Machado CM, Furuzawa KM, Nonogaki S, Osinaga E, Umezawa K, de Carvalho MA, Verinaud L, Chammas R - PLoS ONE (2014)

Bottom Line: In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction.Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death.Similar results were also found in a human GBM cell line, T98G.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Medicina da Universidade de São Paulo, Instituto do Câncer do Estado de São Paulo, São Paulo, SP, Brazil.

ABSTRACT
Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

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NG97ht cells demonstrate increased cell death only when exposed to oxygen and nutrient deprivation, which is accompanied by the increase of reactive oxygen species and caspase-7 activation.A. Experiments performed in complete medium did not demonstrate differences in cell death rates in cells exposed to hypoxia compared to normoxia, however, cells in CoCl2 demonstrated cell death after 48 h of exposure. B. In serum-deprived medium, longer periods of exposure (>24 h) significant cell death rates were observed in CoCl2 and hypoxia compared to normoxia. C. ROS detection by DHE also demonstrated the increase of these molecules in cells exposed to hypoxia and CoCl2 regardless serum concentrations and in normoxia in nutrient deprivation compared to normoxia in complete medium. D. NG97ht cells exposed to DHMEQ in serum-deprived medium presented increased cell death in cells exposed to normoxia and hypoxia, but not CoCl2. E. Caspase 7 analysis showed increased active caspase-7 in cells exposed do CoCl2 and hypoxia, both in complete or serum-deprived medium. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; *** p<0.001.
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pone-0111592-g004: NG97ht cells demonstrate increased cell death only when exposed to oxygen and nutrient deprivation, which is accompanied by the increase of reactive oxygen species and caspase-7 activation.A. Experiments performed in complete medium did not demonstrate differences in cell death rates in cells exposed to hypoxia compared to normoxia, however, cells in CoCl2 demonstrated cell death after 48 h of exposure. B. In serum-deprived medium, longer periods of exposure (>24 h) significant cell death rates were observed in CoCl2 and hypoxia compared to normoxia. C. ROS detection by DHE also demonstrated the increase of these molecules in cells exposed to hypoxia and CoCl2 regardless serum concentrations and in normoxia in nutrient deprivation compared to normoxia in complete medium. D. NG97ht cells exposed to DHMEQ in serum-deprived medium presented increased cell death in cells exposed to normoxia and hypoxia, but not CoCl2. E. Caspase 7 analysis showed increased active caspase-7 in cells exposed do CoCl2 and hypoxia, both in complete or serum-deprived medium. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; *** p<0.001.

Mentions: To evaluate the cell death rates at different experimental conditions, cell death assays with propidium iodide staining were performed. In complete medium, cell death rates were the same in both normoxic and hypoxic conditions (Fig. 4A), however, when cells were exposed to oxygen and serum deprivation, increased cell death rates were observed after 48–72 h (Fig. 4B). Regarding cells exposed to CoCl2, we could observe increased cytotoxicity, regardless serum concentration, in time points >24 h (Fig. 4A/B). The detection of reactive oxygen species (ROS) (Fig. 4C) demonstrated ROS induction in cells exposed to CoCl2 and hypoxia both in complete or deprived medium.


Galectin-3 up-regulation in hypoxic and nutrient deprived microenvironments promotes cell survival.

Ikemori RY, Machado CM, Furuzawa KM, Nonogaki S, Osinaga E, Umezawa K, de Carvalho MA, Verinaud L, Chammas R - PLoS ONE (2014)

NG97ht cells demonstrate increased cell death only when exposed to oxygen and nutrient deprivation, which is accompanied by the increase of reactive oxygen species and caspase-7 activation.A. Experiments performed in complete medium did not demonstrate differences in cell death rates in cells exposed to hypoxia compared to normoxia, however, cells in CoCl2 demonstrated cell death after 48 h of exposure. B. In serum-deprived medium, longer periods of exposure (>24 h) significant cell death rates were observed in CoCl2 and hypoxia compared to normoxia. C. ROS detection by DHE also demonstrated the increase of these molecules in cells exposed to hypoxia and CoCl2 regardless serum concentrations and in normoxia in nutrient deprivation compared to normoxia in complete medium. D. NG97ht cells exposed to DHMEQ in serum-deprived medium presented increased cell death in cells exposed to normoxia and hypoxia, but not CoCl2. E. Caspase 7 analysis showed increased active caspase-7 in cells exposed do CoCl2 and hypoxia, both in complete or serum-deprived medium. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; *** p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219723&req=5

pone-0111592-g004: NG97ht cells demonstrate increased cell death only when exposed to oxygen and nutrient deprivation, which is accompanied by the increase of reactive oxygen species and caspase-7 activation.A. Experiments performed in complete medium did not demonstrate differences in cell death rates in cells exposed to hypoxia compared to normoxia, however, cells in CoCl2 demonstrated cell death after 48 h of exposure. B. In serum-deprived medium, longer periods of exposure (>24 h) significant cell death rates were observed in CoCl2 and hypoxia compared to normoxia. C. ROS detection by DHE also demonstrated the increase of these molecules in cells exposed to hypoxia and CoCl2 regardless serum concentrations and in normoxia in nutrient deprivation compared to normoxia in complete medium. D. NG97ht cells exposed to DHMEQ in serum-deprived medium presented increased cell death in cells exposed to normoxia and hypoxia, but not CoCl2. E. Caspase 7 analysis showed increased active caspase-7 in cells exposed do CoCl2 and hypoxia, both in complete or serum-deprived medium. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; *** p<0.001.
Mentions: To evaluate the cell death rates at different experimental conditions, cell death assays with propidium iodide staining were performed. In complete medium, cell death rates were the same in both normoxic and hypoxic conditions (Fig. 4A), however, when cells were exposed to oxygen and serum deprivation, increased cell death rates were observed after 48–72 h (Fig. 4B). Regarding cells exposed to CoCl2, we could observe increased cytotoxicity, regardless serum concentration, in time points >24 h (Fig. 4A/B). The detection of reactive oxygen species (ROS) (Fig. 4C) demonstrated ROS induction in cells exposed to CoCl2 and hypoxia both in complete or deprived medium.

Bottom Line: In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction.Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death.Similar results were also found in a human GBM cell line, T98G.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Medicina da Universidade de São Paulo, Instituto do Câncer do Estado de São Paulo, São Paulo, SP, Brazil.

ABSTRACT
Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7-2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions.

Show MeSH
Related in: MedlinePlus