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TNF-alpha increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes.

Katerinaki E, Evans GS, Lorigan PC, MacNeil S - Br. J. Cancer (2003)

Bottom Line: In this study, we extend these investigations asking specifically whether the TNF-alpha effect on cell invasion and migration involves activation of proteolytic enzymes.Stimulation with TNF-alpha significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity.However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-alpha were inhibited by the general protease inhibitor alpha(2) macroglobulin.

View Article: PubMed Central - PubMed

Affiliation: Section of Medicine, Division of Clinical Sciences, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK.

ABSTRACT
Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF-alpha) increases melanoma cell attachment to extracellular matrix (ECM) substrates and invasion through fibronectin. In this study, we extend these investigations asking specifically whether the TNF-alpha effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-alpha on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-alpha significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-alpha were inhibited by the general protease inhibitor alpha(2) macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-alpha on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.

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(A) The proteolytic activity of a series of trypsin concentrations was assessed by the quenched fluorescent substrate assay. Results shown are of one representative experiment. Duplicate wells were used for each trypsin concentration. (B) Inhibition of the proteolytic activity of trypsin (3.3 μg ml−1) by the addition of the general protease inhibitor α2 macroglobulin. Inhibition of 84% was observed with α2 macroglobulin at a concentration of 2 U ml−1. Results show means±s.e.m. (n=2, *P<0.05).
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fig3: (A) The proteolytic activity of a series of trypsin concentrations was assessed by the quenched fluorescent substrate assay. Results shown are of one representative experiment. Duplicate wells were used for each trypsin concentration. (B) Inhibition of the proteolytic activity of trypsin (3.3 μg ml−1) by the addition of the general protease inhibitor α2 macroglobulin. Inhibition of 84% was observed with α2 macroglobulin at a concentration of 2 U ml−1. Results show means±s.e.m. (n=2, *P<0.05).

Mentions: To confirm that the fluorescence emitted by cleavage of the BODIPY® casein substrate was representative of proteolytic enzyme activity, initial experiments used serial dilutions of trypsin (see Figure 3AFigure 3


TNF-alpha increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes.

Katerinaki E, Evans GS, Lorigan PC, MacNeil S - Br. J. Cancer (2003)

(A) The proteolytic activity of a series of trypsin concentrations was assessed by the quenched fluorescent substrate assay. Results shown are of one representative experiment. Duplicate wells were used for each trypsin concentration. (B) Inhibition of the proteolytic activity of trypsin (3.3 μg ml−1) by the addition of the general protease inhibitor α2 macroglobulin. Inhibition of 84% was observed with α2 macroglobulin at a concentration of 2 U ml−1. Results show means±s.e.m. (n=2, *P<0.05).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376936&req=5

fig3: (A) The proteolytic activity of a series of trypsin concentrations was assessed by the quenched fluorescent substrate assay. Results shown are of one representative experiment. Duplicate wells were used for each trypsin concentration. (B) Inhibition of the proteolytic activity of trypsin (3.3 μg ml−1) by the addition of the general protease inhibitor α2 macroglobulin. Inhibition of 84% was observed with α2 macroglobulin at a concentration of 2 U ml−1. Results show means±s.e.m. (n=2, *P<0.05).
Mentions: To confirm that the fluorescence emitted by cleavage of the BODIPY® casein substrate was representative of proteolytic enzyme activity, initial experiments used serial dilutions of trypsin (see Figure 3AFigure 3

Bottom Line: In this study, we extend these investigations asking specifically whether the TNF-alpha effect on cell invasion and migration involves activation of proteolytic enzymes.Stimulation with TNF-alpha significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity.However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-alpha were inhibited by the general protease inhibitor alpha(2) macroglobulin.

View Article: PubMed Central - PubMed

Affiliation: Section of Medicine, Division of Clinical Sciences, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK.

ABSTRACT
Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF-alpha) increases melanoma cell attachment to extracellular matrix (ECM) substrates and invasion through fibronectin. In this study, we extend these investigations asking specifically whether the TNF-alpha effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-alpha on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-alpha significantly increased both melanoma cell migration at 24 h (+21%) and invasion through fibronectin (+35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-alpha were inhibited by the general protease inhibitor alpha(2) macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-alpha on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.

Show MeSH
Related in: MedlinePlus