At near confluence, the cells were washed with phosphate-buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100 ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) at 0.1-25 ng/ml; lipopolysaccharide (LPS) at 10-100 μg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100 μM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000 μg/ml; actinomycin-D and cyclohexamide at 2 and 4 μM; retinoic acid and dexamethasone at 50 μM. After 24 h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1β enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.
Running title: Fanconi anemia fibroblast MMP modulation
Keywords: matrix metalloproteinases, Fanconi anemia fibroblasts, cytokines, inducers, inhibitors