Modulation of MMP-2 and -9 Secretion by Cytokines, Inducers and Inhibitors in Human Melanoma A-2058 Cells


Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in regulation of MMP-2 and -9 in human melanoma A-2058 cells. Human A-2058 cells were grown in DMEM supplemented with 15% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS and incubated in serum-free media with phorbol 12-myristate 13-acetate (PMA) at 10, 25, 50 and 100 ng/ml; TNF-α and IL-1β at 0.1, 1, 10 and 25 ng/ml; LPS at 10, 25, 50 and 100 µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10, 25, 50 and 100 µM without and with PMA; a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA at 10, 50, 100, 500 and 1,000 µg/ml; actinomycin D and cyclohexamide at 2 and 4 µM; retinoic acid and dexamethasone at 50 µM. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry. Melanoma A-2058 demonstrated strong expression of MMP-2 and slight expression of MMP-9. PMA at 100 ng/ml showed no effect on MMP-2 secretion but potently upregulated MMP-9 secretion to 400% that of control. TNF-α showed no significant overall effect on expression of MMP-2 but potent dose-dependent increased MMP-9 secretion with 200% of control at 25 ng/ml. IL-1β showed no significant effect on MMP-2 or MMP-9 secretion by A-2058 cells, except at 25 ng/ml where MMP-2 level was reduced by ~40% and MMP-9 secretion ~50%. LPS treatment showed no significant effect on MMP-2 secretion and enhanced MMP-9 secretion up to 25 µg/ml followed by decreased level. EGCG, NM and doxycycline, without and with PMA, downregulated the expression of MMP-2 and MMP-9 in a dose-dependent manner.Actinomycin D, cyclohexamide and retinoic acid had inhibitory effects on MMP-2, while dexamethasone showed slight stimulatory effect on MMP-2 secretion. Our results showed that select cytokines, mitogens and inhibitors modulated A-2058 MMP-2 and MMP-9 expression. They suggest the clinical potential of MMP inhibitors, especially the non-toxic ones, such as the nutrient mixture and its component EGCG in management of melanoma.

Running title: 
Melanoma MMP modulation

Key words: 
matrix metalloproteinases, melanoma A-2058, cytokines, inducers, inhibitors