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Effect of monensin on secretion of t-PA from melanoma (Bowes)
1Department of Physiology, Kinki University, School of Medicine, Osakasayama, Japan.
Abstract:
The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of 3H-mannose- and 35S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of 3H-mannose-related glycoprotein to 35S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation.
Insights
Monensin treatment inhibits tissue-type plasminogen activator (t-PA) secretion from melanoma cells by causing abnormal glycosylation. This leads to intracellular t-PA accumulation and altered sugar chain modifications.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Melanoma cell secretion of tissue-type plasminogen activator (t-PA) is crucial for tumor progression.
- Understanding the regulatory mechanisms of t-PA secretion is vital for developing targeted therapies.
Purpose of the Study:
- To investigate the effect of monensin on t-PA secretion from Bowes melanoma cells.
- To elucidate the role of glycosylation in monensin-induced alterations of t-PA secretion.
Main Methods:
- Monensin treatment of melanoma cells.
- Measurement of secreted and intracellular t-PA levels.
- Electrophoretic enzymography and ultracentrifugation for t-PA localization.
- Metabolic labeling with 3H-mannose and 35S-methionine.
- Enzymatic analysis of t-PA sugar chains using endoglycosidase H and N-glycanase.
Main Results:
- Monensin inhibited t-PA secretion in a dose- and time-dependent manner.
- Intracellular accumulation of t-PA was observed following monensin treatment.
- Monensin treatment altered t-PA glycosylation, leading to the presence of high-mannose type sugar chains in secreted t-PA.
Conclusions:
- Monensin disrupts normal t-PA secretion by inducing abnormal glycosylation.
- Altered glycosylation of t-PA impacts its secretion pathway from melanoma cells.
- These findings provide insights into the post-translational modification of t-PA and its secretion regulation.