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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 upregulation in hypoxia depends on HIF-1α and NF-κB and is blocked by cicloheximide.A. qRT-PCR analysis of NG97ht cells treated with cycloheximide (CHX) 5 µM and 20 µM and its control (DMSO) and in cells exposed to either normoxia, CoCl2 or hypoxia in serum-deprived medium for 6 h demonstrated significant increase in gal-3 expression in cells exposed to CoCl2 and hypoxia compared to normoxia in the group treated with DMSO, but not when cells were exposed to CHX. B. The exposure to HIF-1α inhibitor 10 µm (CAS 934593-90-5) demonstrated gal-3 decreased induction by qRT-PCR in cells exposed to hypoxia, but not normoxia and CoCl2. DMSO used as control. C. The exposure to DHMEQ 5 µg/mL, an NF-κB inhibitor, decreased gal-3 induction by qRT-PCR both in CoCl2 and hypoxia compared to normoxia after 24 h in serum-deprived medium. DMSO used as control. D. Western blot analysis with the HIF-1αinhibitor demonstrated a slight decrease in gal-3 only in cells exposed to hypoxia, but not normoxia and CoCl2, compared to DMSO control (experiment performed once). E. Western blot analysis also demonstrated decreased gal-3 induction in cells treated with DHMEQ compared to DMSO in hypoxia and CoCl2 compared to normoxia after 24 h in serum-deprived medium. F. Cells in normoxia display scattered HIF-1α (green) throughout cytoplasm, while cells exposed to CoCl2 and hypoxia display HIF-1α mainly in cells nuclei (blue). G. In vivo, NF-κB demonstrates to be upregulated in pseudopalisading areas, which display increased gal-3 expression. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. **p<0,01; ***p<0.001.
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pone-0111592-g003: Gal-3 upregulation in hypoxia depends on HIF-1α and NF-κB and is blocked by cicloheximide.A. qRT-PCR analysis of NG97ht cells treated with cycloheximide (CHX) 5 µM and 20 µM and its control (DMSO) and in cells exposed to either normoxia, CoCl2 or hypoxia in serum-deprived medium for 6 h demonstrated significant increase in gal-3 expression in cells exposed to CoCl2 and hypoxia compared to normoxia in the group treated with DMSO, but not when cells were exposed to CHX. B. The exposure to HIF-1α inhibitor 10 µm (CAS 934593-90-5) demonstrated gal-3 decreased induction by qRT-PCR in cells exposed to hypoxia, but not normoxia and CoCl2. DMSO used as control. C. The exposure to DHMEQ 5 µg/mL, an NF-κB inhibitor, decreased gal-3 induction by qRT-PCR both in CoCl2 and hypoxia compared to normoxia after 24 h in serum-deprived medium. DMSO used as control. D. Western blot analysis with the HIF-1αinhibitor demonstrated a slight decrease in gal-3 only in cells exposed to hypoxia, but not normoxia and CoCl2, compared to DMSO control (experiment performed once). E. Western blot analysis also demonstrated decreased gal-3 induction in cells treated with DHMEQ compared to DMSO in hypoxia and CoCl2 compared to normoxia after 24 h in serum-deprived medium. F. Cells in normoxia display scattered HIF-1α (green) throughout cytoplasm, while cells exposed to CoCl2 and hypoxia display HIF-1α mainly in cells nuclei (blue). G. In vivo, NF-κB demonstrates to be upregulated in pseudopalisading areas, which display increased gal-3 expression. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. **p<0,01; ***p<0.001.

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 upregulation in hypoxia depends on HIF-1α and NF-κB and is blocked by cicloheximide.A. qRT-PCR analysis of NG97ht cells treated with cycloheximide (CHX) 5 µM and 20 µM and its control (DMSO) and in cells exposed to either normoxia, CoCl2 or hypoxia in serum-deprived medium for 6 h demonstrated significant increase in gal-3 expression in cells exposed to CoCl2 and hypoxia compared to normoxia in the group treated with DMSO, but not when cells were exposed to CHX. B. The exposure to HIF-1α inhibitor 10 µm (CAS 934593-90-5) demonstrated gal-3 decreased induction by qRT-PCR in cells exposed to hypoxia, but not normoxia and CoCl2. DMSO used as control. C. The exposure to DHMEQ 5 µg/mL, an NF-κB inhibitor, decreased gal-3 induction by qRT-PCR both in CoCl2 and hypoxia compared to normoxia after 24 h in serum-deprived medium. DMSO used as control. D. Western blot analysis with the HIF-1αinhibitor demonstrated a slight decrease in gal-3 only in cells exposed to hypoxia, but not normoxia and CoCl2, compared to DMSO control (experiment performed once). E. Western blot analysis also demonstrated decreased gal-3 induction in cells treated with DHMEQ compared to DMSO in hypoxia and CoCl2 compared to normoxia after 24 h in serum-deprived medium. F. Cells in normoxia display scattered HIF-1α (green) throughout cytoplasm, while cells exposed to CoCl2 and hypoxia display HIF-1α mainly in cells nuclei (blue). G. In vivo, NF-κB demonstrates to be upregulated in pseudopalisading areas, which display increased gal-3 expression. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. **p<0,01; ***p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4219723&req=5

pone-0111592-g003: Gal-3 upregulation in hypoxia depends on HIF-1α and NF-κB and is blocked by cicloheximide.A. qRT-PCR analysis of NG97ht cells treated with cycloheximide (CHX) 5 µM and 20 µM and its control (DMSO) and in cells exposed to either normoxia, CoCl2 or hypoxia in serum-deprived medium for 6 h demonstrated significant increase in gal-3 expression in cells exposed to CoCl2 and hypoxia compared to normoxia in the group treated with DMSO, but not when cells were exposed to CHX. B. The exposure to HIF-1α inhibitor 10 µm (CAS 934593-90-5) demonstrated gal-3 decreased induction by qRT-PCR in cells exposed to hypoxia, but not normoxia and CoCl2. DMSO used as control. C. The exposure to DHMEQ 5 µg/mL, an NF-κB inhibitor, decreased gal-3 induction by qRT-PCR both in CoCl2 and hypoxia compared to normoxia after 24 h in serum-deprived medium. DMSO used as control. D. Western blot analysis with the HIF-1αinhibitor demonstrated a slight decrease in gal-3 only in cells exposed to hypoxia, but not normoxia and CoCl2, compared to DMSO control (experiment performed once). E. Western blot analysis also demonstrated decreased gal-3 induction in cells treated with DHMEQ compared to DMSO in hypoxia and CoCl2 compared to normoxia after 24 h in serum-deprived medium. F. Cells in normoxia display scattered HIF-1α (green) throughout cytoplasm, while cells exposed to CoCl2 and hypoxia display HIF-1α mainly in cells nuclei (blue). G. In vivo, NF-κB demonstrates to be upregulated in pseudopalisading areas, which display increased gal-3 expression. Representative experiments performed at least in three independent assays and data are presented as mean±SEM. **p<0,01; ***p<0.001.
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