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A MT1-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.

Annabi B, Laflamme C, Sina A, Lachambre MP, Béliveau R - J Neuroinflammation (2009)

Bottom Line: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration.MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function.COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire d'Oncologie Moléculaire, Département de Chimie, Centre de Recherche BIOMED, Université du Québec à Montréal, Quebec, Canada. annabi.borhane@uqam.ca

ABSTRACT

Background: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration. Screening of a large panel of glioma samples increasing histological grade demonstrated frequencies of CD133(+) cells which correlated with high expression of cyclooxygenase (COX)-2 and of membrane type-1 matrix metalloproteinase (MT1-MMP).

Methods: We used qRT-PCR and immunoblotting to examine the molecular interplay between MT1-MMP and COX-2 gene and protein expression in parental, CD133(+), and neurospheres U87 glioma cell cultures.

Results: We found that CD133, COX-2 and MT1-MMP expression were enhanced when glioma cells were cultured in neurosphere conditions. A CD133(+)-enriched U87 glioma cell population, isolated from parental U87 cells with magnetic cell sorting technology, also grew as neurospheres and showed enhanced COX-2 expression. MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function. COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

Conclusion: We provide evidence for enhanced COX-2 expression in CD133(+) glioma cells, and direct cell-based evidence of NF-kappaB-mediated COX-2 regulation by MT1-MMP. The biological significance of such checkpoint control may account for COX-2-dependent mechanisms of inflammatory balance responsible of therapy resistance phenotype of cancer stem cells.

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CD133-sorted U87 glioma cells grow as neurospheres and express high levels of COX-2. (A) U87 CD133(+) cells were isolated from the parental U87 cells as described in the Methods section using MACS technology. Evaluation of CD133 cell surface expression was then performed by flow cytometry on parental U87 and CD133(+) sorted cells. (B) U87 CD133(+) cells were put back into culture and show a typical neurosphere phenotype, unlike their parental counterpart. (C) Total RNA was extracted from parental U87 and CD133(+) U87 cells and gene expression levels were assessed by qRT-PCR for CD133 (white bars), β-actin (black bars), MT1-MMP (lined bars) and COX-2 (grey bars).
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Figure 3: CD133-sorted U87 glioma cells grow as neurospheres and express high levels of COX-2. (A) U87 CD133(+) cells were isolated from the parental U87 cells as described in the Methods section using MACS technology. Evaluation of CD133 cell surface expression was then performed by flow cytometry on parental U87 and CD133(+) sorted cells. (B) U87 CD133(+) cells were put back into culture and show a typical neurosphere phenotype, unlike their parental counterpart. (C) Total RNA was extracted from parental U87 and CD133(+) U87 cells and gene expression levels were assessed by qRT-PCR for CD133 (white bars), β-actin (black bars), MT1-MMP (lined bars) and COX-2 (grey bars).

Mentions: In order to evaluate the potential contribution of the CD133(+) cell subpopulation to the MT1-MMP/COX-2 signalling axis, we used magnetic cell sorting (MACS) technology to isolate CD133(+) cells from the parental U87 glioma cell population. We found that the CD133(+) U87 cell population represented ~0.15% of the total parental U87 glioma cells (Figure 3a, left panel). Sorting of the CD133(+) cells was then performed and we evaluated the cells as being ~27% CD133 positive (Figure 3a, right panel). The isolated subpopulation, with an enrichment of ~180-fold for CD133(+) U87 cells, was put into culture. Cell morphologies of the parental and CD133(+) U87 glioma cells were compared and we observed that the CD133(+) cells formed spontaneous neurospheres (Figure 3b), a characteristic of brain CSC in agreement with previous reports [33,35]. Total RNA was isolated from both parental and CD133(+) glioma cells in order to assess gene expression levels of CD133, COX-2, and β-Actin. We found that CD133 gene expression was increased by ~6-fold in the sorted CD133(+) U87 glioma cells (Figure 3c), in agreement with the increased CD133 cell surface expression (Figure 3a). Moreover, MT1-MMP and COX-2 gene expression were also increased by ~4-fold in CD133(+) U87 cells (Figure 3c).


A MT1-MMP/NF-kappaB signaling axis as a checkpoint controller of COX-2 expression in CD133+ U87 glioblastoma cells.

Annabi B, Laflamme C, Sina A, Lachambre MP, Béliveau R - J Neuroinflammation (2009)

CD133-sorted U87 glioma cells grow as neurospheres and express high levels of COX-2. (A) U87 CD133(+) cells were isolated from the parental U87 cells as described in the Methods section using MACS technology. Evaluation of CD133 cell surface expression was then performed by flow cytometry on parental U87 and CD133(+) sorted cells. (B) U87 CD133(+) cells were put back into culture and show a typical neurosphere phenotype, unlike their parental counterpart. (C) Total RNA was extracted from parental U87 and CD133(+) U87 cells and gene expression levels were assessed by qRT-PCR for CD133 (white bars), β-actin (black bars), MT1-MMP (lined bars) and COX-2 (grey bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2655289&req=5

Figure 3: CD133-sorted U87 glioma cells grow as neurospheres and express high levels of COX-2. (A) U87 CD133(+) cells were isolated from the parental U87 cells as described in the Methods section using MACS technology. Evaluation of CD133 cell surface expression was then performed by flow cytometry on parental U87 and CD133(+) sorted cells. (B) U87 CD133(+) cells were put back into culture and show a typical neurosphere phenotype, unlike their parental counterpart. (C) Total RNA was extracted from parental U87 and CD133(+) U87 cells and gene expression levels were assessed by qRT-PCR for CD133 (white bars), β-actin (black bars), MT1-MMP (lined bars) and COX-2 (grey bars).
Mentions: In order to evaluate the potential contribution of the CD133(+) cell subpopulation to the MT1-MMP/COX-2 signalling axis, we used magnetic cell sorting (MACS) technology to isolate CD133(+) cells from the parental U87 glioma cell population. We found that the CD133(+) U87 cell population represented ~0.15% of the total parental U87 glioma cells (Figure 3a, left panel). Sorting of the CD133(+) cells was then performed and we evaluated the cells as being ~27% CD133 positive (Figure 3a, right panel). The isolated subpopulation, with an enrichment of ~180-fold for CD133(+) U87 cells, was put into culture. Cell morphologies of the parental and CD133(+) U87 glioma cells were compared and we observed that the CD133(+) cells formed spontaneous neurospheres (Figure 3b), a characteristic of brain CSC in agreement with previous reports [33,35]. Total RNA was isolated from both parental and CD133(+) glioma cells in order to assess gene expression levels of CD133, COX-2, and β-Actin. We found that CD133 gene expression was increased by ~6-fold in the sorted CD133(+) U87 glioma cells (Figure 3c), in agreement with the increased CD133 cell surface expression (Figure 3a). Moreover, MT1-MMP and COX-2 gene expression were also increased by ~4-fold in CD133(+) U87 cells (Figure 3c).

Bottom Line: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration.MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function.COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratoire d'Oncologie Moléculaire, Département de Chimie, Centre de Recherche BIOMED, Université du Québec à Montréal, Quebec, Canada. annabi.borhane@uqam.ca

ABSTRACT

Background: The CD133(+) stem cell population in recurrent gliomas is associated with clinical features such as therapy resistance, blood-brain barrier disruption and, hence, tumor infiltration. Screening of a large panel of glioma samples increasing histological grade demonstrated frequencies of CD133(+) cells which correlated with high expression of cyclooxygenase (COX)-2 and of membrane type-1 matrix metalloproteinase (MT1-MMP).

Methods: We used qRT-PCR and immunoblotting to examine the molecular interplay between MT1-MMP and COX-2 gene and protein expression in parental, CD133(+), and neurospheres U87 glioma cell cultures.

Results: We found that CD133, COX-2 and MT1-MMP expression were enhanced when glioma cells were cultured in neurosphere conditions. A CD133(+)-enriched U87 glioma cell population, isolated from parental U87 cells with magnetic cell sorting technology, also grew as neurospheres and showed enhanced COX-2 expression. MT1-MMP gene silencing antagonized COX-2 expression in neurospheres, while overexpression of recombinant MT1-MMP directly triggered COX-2 expression in U87 cells independent from MT1-MMP's catalytic function. COX-2 induction by MT1-MMP was also validated in wild-type and in NF-kappaB p65-/- mutant mouse embryonic fibroblasts, but was abrogated in NF-kappaB 1 (p50-/-) mutant cells.

Conclusion: We provide evidence for enhanced COX-2 expression in CD133(+) glioma cells, and direct cell-based evidence of NF-kappaB-mediated COX-2 regulation by MT1-MMP. The biological significance of such checkpoint control may account for COX-2-dependent mechanisms of inflammatory balance responsible of therapy resistance phenotype of cancer stem cells.

Show MeSH
Related in: MedlinePlus