Inhibition of experimental metastasis and cell adhesion of B16F1 melanoma cells by inhibitors of protein kinase C.
Phorbol esters which activate protein kinase C (PKC) have been shown to enhance experimental lung metastasis. Therefore, it was reasoned that inhibitors of PKC might also modulate metastasis. We have investigated this possibility using a PKC inhibitor, MDL 27,032 [4-propyl-5(4-pyridinyl)-2(3H)-oxazolone], as well as staurosporine and H-7. Treatment of B16F1 murine melanoma cells with MDL 27,032 for 24 h in culture and subsequent i.v. injection of the cells into mice resulted in greater than 90% inhibition of lung metastasis. Inhibition of metastasis was time dependent, with 90% of maximum inhibition occurring by 8 h of incubation. The 50% inhibitory concentration (IC50) for inhibition of metastasis with MDL 27,032 was 7 microM, a value similar to that for the inhibition of B16F1 membrane-associated PKC (IC50 = 13 microM) but not cytosolic PKC (IC50 = 54 microM). B16F1 cells treated with MDL 27,032 for 24 h were less adherent than untreated cells to extracellular matrix/basement membrane proteins. Adhesion to fibrinogen and collagen IV was inhibited (IC50 = 6 microM and 48 microM, respectively) by MDL 27,032, whereas adherence to laminin and fibronectin was not affected, indicating that the drug affects specific adhesion molecules. MDL 27,032-treated cells were also found to be less adherent than untreated cells to human umbilical vein endothelial cells. The phosphorylation of an 80-kDa B16F1 cell plasma membrane protein was stimulated under conditions known to stimulate PKC activity, and MDL 27,032 inhibited this phosphorylation in a dose-dependent manner. MDL 27,032 was more potent than H-7 for the inhibition of metastasis but was significantly less potent than staurosporine. These results support the hypothesis that there is a critical role for PKC-mediated phosphorylation of cell surface adhesion receptors in metastasis.
Dumont
J A
JA
Marion Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Jones
W D
WD
Bitonti
A J
AJ
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D007546:Isoquinolines / Q000494:pharmacology; D008175:Lung Neoplasms / Q000201:enzymology / Q000517:prevention & control* / Q000556:secondary*; D008546:Melanoma, Experimental / Q000201:enzymology / Q000517:prevention & control* / Q000556:secondary*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009362:Neoplasm Metastasis; D010080:Oxazoles / Q000494:pharmacology*; D010081:Oxazolone / Q000031:analogs & derivatives*; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology*; D019311:Staurosporine; D013997:Time Factors
D000470:Alkaloids; D007546:Isoquinolines; D008565:Membrane Proteins; D010080:Oxazoles; D010879:Piperazines; D011725:Pyridines; C069455:4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; D010081:Oxazolone; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D019311:Staurosporine