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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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Gal-3 is upregulated in hypoxia in NG97ht cells.Analysis of gal-3 by qRT-PCR and western blot in NG97ht cells exposed to normoxia, CoCl2 and hypoxia in complete or serum-deprived medium. A/B. qRT-PCR demonstrated gal-3 mRNA upregulation after 24 h of exposure either in complete (A) or serum-deprived medium (B) in cells exposed to CoCl2 and hypoxia compared to normoxia. β-actin was used as normalizer. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; ***p<0.001. C. Gal-3 protein accumulated after 48 h, in CoCl2 and hypoxia both in complete and serum-deprived medium. Two electrophoretic forms of gal-3, 28.8 kDa and 29.7 kDa, were observed. D. NG97ht cell protein extracts derived from cells exposed to normoxia, CoCl2 and hypoxia in deprived medium for 48 h were submitted to treatment with acid phosphatase (AP) 100 µg/mL and analyzed by two-dimensional gel electrophoresis. Five main forms (1 to 5) were detected in the NG97ht cell line with their isoelectric point (IP) ranging from 8.00 to 9.33 and molecular sizes from 27.5 and 29.7 kDa. Spots 4 and 5 display each two forms of same IP, but different molecular sizes. E. The quantification between isoforms 4–5 (basic) and 2–3 (acidic) demonstrated increase in the first mainly in hypoxia when compared to normoxia. F. In the treatment of the protein extracts with AP, there was a decrease in isoforms 2 and 3 compared to control samples mainly in cells exposed to normoxia. Isoforms 4–5 did not demonstrate changes between normoxia, CoCl2 and hypoxia. G. U87MG protein extracts derived from cells exposed to normoxia and hypoxia in deprived medium for 48 h also demonstrated differences in two-dimensional gel electrophoresis, showing five forms (1 to 5) with IP ranging from 7 to 9 and molecular sizes from 28.3 to 24.5 kDa. Treatment of protein extracts with AP resulted in the IP modification in gal-3 in normoxia.
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pone-0111592-g002: Gal-3 is upregulated in hypoxia in NG97ht cells.Analysis of gal-3 by qRT-PCR and western blot in NG97ht cells exposed to normoxia, CoCl2 and hypoxia in complete or serum-deprived medium. A/B. qRT-PCR demonstrated gal-3 mRNA upregulation after 24 h of exposure either in complete (A) or serum-deprived medium (B) in cells exposed to CoCl2 and hypoxia compared to normoxia. β-actin was used as normalizer. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; ***p<0.001. C. Gal-3 protein accumulated after 48 h, in CoCl2 and hypoxia both in complete and serum-deprived medium. Two electrophoretic forms of gal-3, 28.8 kDa and 29.7 kDa, were observed. D. NG97ht cell protein extracts derived from cells exposed to normoxia, CoCl2 and hypoxia in deprived medium for 48 h were submitted to treatment with acid phosphatase (AP) 100 µg/mL and analyzed by two-dimensional gel electrophoresis. Five main forms (1 to 5) were detected in the NG97ht cell line with their isoelectric point (IP) ranging from 8.00 to 9.33 and molecular sizes from 27.5 and 29.7 kDa. Spots 4 and 5 display each two forms of same IP, but different molecular sizes. E. The quantification between isoforms 4–5 (basic) and 2–3 (acidic) demonstrated increase in the first mainly in hypoxia when compared to normoxia. F. In the treatment of the protein extracts with AP, there was a decrease in isoforms 2 and 3 compared to control samples mainly in cells exposed to normoxia. Isoforms 4–5 did not demonstrate changes between normoxia, CoCl2 and hypoxia. G. U87MG protein extracts derived from cells exposed to normoxia and hypoxia in deprived medium for 48 h also demonstrated differences in two-dimensional gel electrophoresis, showing five forms (1 to 5) with IP ranging from 7 to 9 and molecular sizes from 28.3 to 24.5 kDa. Treatment of protein extracts with AP resulted in the IP modification in gal-3 in normoxia.

Mentions: It was observed gal-3 mRNA upregulation after 24–48 h either in complete or serum-deprived medium (Fig. 2A/B) in the NG97ht cell line. This hybrid cell line only expresses the murine gal-3 from its genome, since it lacks the human gal-3 gene. Protein analysis demonstrated accumulation of gal-3 in cells exposed to either CoCl2 or hypoxia after 48 h compared to normoxia (Fig. 3C). Two electrophoretic forms of gal-3, ranging from 28.8 kDa to 29.7 kDa, were observed in the experiments, with accumulation of the higher molecular weight form of gal-3.


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)

Gal-3 is upregulated in hypoxia in NG97ht cells.Analysis of gal-3 by qRT-PCR and western blot in NG97ht cells exposed to normoxia, CoCl2 and hypoxia in complete or serum-deprived medium. A/B. qRT-PCR demonstrated gal-3 mRNA upregulation after 24 h of exposure either in complete (A) or serum-deprived medium (B) in cells exposed to CoCl2 and hypoxia compared to normoxia. β-actin was used as normalizer. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; ***p<0.001. C. Gal-3 protein accumulated after 48 h, in CoCl2 and hypoxia both in complete and serum-deprived medium. Two electrophoretic forms of gal-3, 28.8 kDa and 29.7 kDa, were observed. D. NG97ht cell protein extracts derived from cells exposed to normoxia, CoCl2 and hypoxia in deprived medium for 48 h were submitted to treatment with acid phosphatase (AP) 100 µg/mL and analyzed by two-dimensional gel electrophoresis. Five main forms (1 to 5) were detected in the NG97ht cell line with their isoelectric point (IP) ranging from 8.00 to 9.33 and molecular sizes from 27.5 and 29.7 kDa. Spots 4 and 5 display each two forms of same IP, but different molecular sizes. E. The quantification between isoforms 4–5 (basic) and 2–3 (acidic) demonstrated increase in the first mainly in hypoxia when compared to normoxia. F. In the treatment of the protein extracts with AP, there was a decrease in isoforms 2 and 3 compared to control samples mainly in cells exposed to normoxia. Isoforms 4–5 did not demonstrate changes between normoxia, CoCl2 and hypoxia. G. U87MG protein extracts derived from cells exposed to normoxia and hypoxia in deprived medium for 48 h also demonstrated differences in two-dimensional gel electrophoresis, showing five forms (1 to 5) with IP ranging from 7 to 9 and molecular sizes from 28.3 to 24.5 kDa. Treatment of protein extracts with AP resulted in the IP modification in gal-3 in normoxia.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4219723&req=5

pone-0111592-g002: Gal-3 is upregulated in hypoxia in NG97ht cells.Analysis of gal-3 by qRT-PCR and western blot in NG97ht cells exposed to normoxia, CoCl2 and hypoxia in complete or serum-deprived medium. A/B. qRT-PCR demonstrated gal-3 mRNA upregulation after 24 h of exposure either in complete (A) or serum-deprived medium (B) in cells exposed to CoCl2 and hypoxia compared to normoxia. β-actin was used as normalizer. Representative experiment performed at least in three independent assays and data are presented as mean±SEM. *p<0.05; **p<0.01; ***p<0.001. C. Gal-3 protein accumulated after 48 h, in CoCl2 and hypoxia both in complete and serum-deprived medium. Two electrophoretic forms of gal-3, 28.8 kDa and 29.7 kDa, were observed. D. NG97ht cell protein extracts derived from cells exposed to normoxia, CoCl2 and hypoxia in deprived medium for 48 h were submitted to treatment with acid phosphatase (AP) 100 µg/mL and analyzed by two-dimensional gel electrophoresis. Five main forms (1 to 5) were detected in the NG97ht cell line with their isoelectric point (IP) ranging from 8.00 to 9.33 and molecular sizes from 27.5 and 29.7 kDa. Spots 4 and 5 display each two forms of same IP, but different molecular sizes. E. The quantification between isoforms 4–5 (basic) and 2–3 (acidic) demonstrated increase in the first mainly in hypoxia when compared to normoxia. F. In the treatment of the protein extracts with AP, there was a decrease in isoforms 2 and 3 compared to control samples mainly in cells exposed to normoxia. Isoforms 4–5 did not demonstrate changes between normoxia, CoCl2 and hypoxia. G. U87MG protein extracts derived from cells exposed to normoxia and hypoxia in deprived medium for 48 h also demonstrated differences in two-dimensional gel electrophoresis, showing five forms (1 to 5) with IP ranging from 7 to 9 and molecular sizes from 28.3 to 24.5 kDa. Treatment of protein extracts with AP resulted in the IP modification in gal-3 in normoxia.
Mentions: It was observed gal-3 mRNA upregulation after 24–48 h either in complete or serum-deprived medium (Fig. 2A/B) in the NG97ht cell line. This hybrid cell line only expresses the murine gal-3 from its genome, since it lacks the human gal-3 gene. Protein analysis demonstrated accumulation of gal-3 in cells exposed to either CoCl2 or hypoxia after 48 h compared to normoxia (Fig. 3C). Two electrophoretic forms of gal-3, ranging from 28.8 kDa to 29.7 kDa, were observed in the experiments, with accumulation of the higher molecular weight form of gal-3.

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