Low-molecular-weight membrane component inhibits the metastatic phenotype of B16-F10 melanoma

Treatment of the metastatic melanoma cell lines B16-F1 and B16-F10 with 1.5-2 per cent butanol elevates their experimental metastatic potential, whereas reconstitution of butanol-extracted B16 cells with crude butanol extracts decreases the number of experimentally induced lung foci. We partially purified the biologically active components from crude butanol extracts of B16-F1 by high-performance liquid chromatography and isoelectric focusing, and found the inhibitory activity (i) was in the low-molecular-weight (5-10 kDa) fraction of the chromatogram, (ii) had an isoelectric pH between 5.6 and 5.8, (iii) was distinct from a thiol-protease activity eluted from the isoelectric focusing bed at pH 4.9-5.3 and (iv) was not itself an inhibitor of serine or thiol proteases. Incubation of butanol-extracted B16-F10 cells with known inhibitors of serine, acid and thiol protease inhibitors had no effect on the experimental metastatic phenotype. Although the apparent molecular weight was low, the inhibitor(s) tended to aggregate after focusing, probably owing to the presence of carrier ampholines. Using two-dimensional gel electrophoresis, we observed slight differences between intact and butanol-extracted cells, most of them in the low-molecular-weight region. These results suggest that butanol treatment may reversibly release certain inhibitors of cell surface enzymes other than proteases, which might be involved in invasion and metastasis.