Diversity of melanoma plasma membrane proteinases: inhibition of collagenolytic and cytolytic activities by minocycline.
The tissue-destructive proteinases of B16-BL6 melanoma cells from C57BL/6 mice and subcellular fractions were examined. Cancer cell organelles were isolated following nitrogen cavitation with the use of sucrose density gradient centrifugation. Serine, cysteine, and metalloproteinases were assayed with the use of radiolabeled proteins and synthetic substrates. Tumor-induced red blood cell lysis was quantitated by measurement of the release of isotope from 59Fe-labeled red blood cells (RBC) cocultivated with melanoma cells; the RBC were from Wistar rats. Enzyme inhibitors with specificity toward different classes of proteinases were used in the above assays to categorize the enzymes responsible for substrate degradation. Results indicated that intact melanoma cells, cell organelles, and cytosol contain proteinases that can degrade collagen and gelatin and lyse normal RBC. Melanoma plasma membranes are highly enriched in collagenase, gelatinase, cysteine proteinase, plasminogen activator, and cytolytic activity. The inhibition of tumor collagenolytic, gelatinolytic, and cytolytic activities by EDTA and 1,10-phenanthroline but not by diisopropyl fluorophosphate and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates that metalloproteinases are the active enzymes in these assays. Minocycline, a synthetic tetracycline with demonstrable inhibitory activity with other mammalian collagenases, also inhibited melanoma collagenolytic and cytolytic activities.
Zucker
S
S
Lysik
R M
RM
Ramamurthy
N S
NS
Golub
L M
LM
Wieman
J M
JM
Wilkie
D P
DP
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000032:analysis*; D004912:Erythrocytes / Q000473:pathology; D005260:Female; D018093:Gelatinases; D008545:Melanoma / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors*; D008911:Minocycline / Q000494:pharmacology*; D010434:Pepsin A / Q000032:analysis; D012697:Serine Endopeptidases; D013754:Tetracyclines / Q000494:pharmacology*
D013754:Tetracyclines; D010450:Endopeptidases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase; D008911:Minocycline