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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, COX-2 and MT1-MMP expression are increased in U87 neurospheres glioma cultures. (A) U87 glioblastoma-derived cell lines were cultured as monolayers or non-adherent neurospheres as described in the Methods section and representative phase contrast photographs were taken. (B) Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and CD133, COX-2, MT1-MMP or GAPDH immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells.
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Figure 1: CD133, COX-2 and MT1-MMP expression are increased in U87 neurospheres glioma cultures. (A) U87 glioblastoma-derived cell lines were cultured as monolayers or non-adherent neurospheres as described in the Methods section and representative phase contrast photographs were taken. (B) Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and CD133, COX-2, MT1-MMP or GAPDH immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells.

Mentions: Neurosphere-like brain CSC are thought to contribute to a sub-population of CD133(+) brain CSC [32]. Neurosphere induction in U87 cells was performed according to established protocols [33,34]. This process promoted the transition of adherent monolayer cells to non-adherent, neurosphere-like cells (Figure 1a). Immunodetection of CD133, COX-2 MT1-MMP and GAPDH was performed on the cell lysates. Neurosphere culture conditions induced CD133 and COX-2 expression in U87 cells when compared to their corresponding monolayer cultures (Figure 1b). MT1-MMP expression was also induced during neurosphere-like formation, while the house-keeping gene GAPDH remained unaffected (Figure 1b). Since MT1-MMP is known to activate proMMP-2 into MMP-2, the levels of latent proMMP-2 and active MMP-2 were also assessed in those same serum-starved monolayer and neurosphere culture conditions. We observed that proMMP-2 activation in U87 neurospheres remained unchanged. Therefore, increased MT1-MMP seems to occur independent of its capacity to induce proMMP-2 activation, suggesting that MT1-MMP may regulate alternate intracellular processes. Collectively, increased CD133, COX-2 and MT1-MMP expression characterizes neurosphere-like formation in U87 glioma cells.


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, COX-2 and MT1-MMP expression are increased in U87 neurospheres glioma cultures. (A) U87 glioblastoma-derived cell lines were cultured as monolayers or non-adherent neurospheres as described in the Methods section and representative phase contrast photographs were taken. (B) Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and CD133, COX-2, MT1-MMP or GAPDH immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: CD133, COX-2 and MT1-MMP expression are increased in U87 neurospheres glioma cultures. (A) U87 glioblastoma-derived cell lines were cultured as monolayers or non-adherent neurospheres as described in the Methods section and representative phase contrast photographs were taken. (B) Cell lysates were isolated from U87 glioblastoma-derived cells and SDS-PAGE performed (20 μg protein/well), followed by Western blotting and CD133, COX-2, MT1-MMP or GAPDH immunodetection. Gelatin zymography was also used to monitor the extent of latent proMMP-2 and active MMP-2 expression from the conditioned media of the serum-starved cells.
Mentions: Neurosphere-like brain CSC are thought to contribute to a sub-population of CD133(+) brain CSC [32]. Neurosphere induction in U87 cells was performed according to established protocols [33,34]. This process promoted the transition of adherent monolayer cells to non-adherent, neurosphere-like cells (Figure 1a). Immunodetection of CD133, COX-2 MT1-MMP and GAPDH was performed on the cell lysates. Neurosphere culture conditions induced CD133 and COX-2 expression in U87 cells when compared to their corresponding monolayer cultures (Figure 1b). MT1-MMP expression was also induced during neurosphere-like formation, while the house-keeping gene GAPDH remained unaffected (Figure 1b). Since MT1-MMP is known to activate proMMP-2 into MMP-2, the levels of latent proMMP-2 and active MMP-2 were also assessed in those same serum-starved monolayer and neurosphere culture conditions. We observed that proMMP-2 activation in U87 neurospheres remained unchanged. Therefore, increased MT1-MMP seems to occur independent of its capacity to induce proMMP-2 activation, suggesting that MT1-MMP may regulate alternate intracellular processes. Collectively, increased CD133, COX-2 and MT1-MMP expression characterizes neurosphere-like formation in U87 glioma cells.

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