Human RCC 786-0 ATCC) was grown in RPMI medium supplemented with 10% FBS, penicillin, and streptomycin in 24-well tissue plates. At near confluence, the cells were washed with PBS; the serum free medium was incubated with various inducers: phorbol ester (PMA; tumor necrosis factor alpha (TNFα); interleukin 1-beta ( IL-1ß); and lipopolysaccharides (LPS). Cells were also incubated with inhibitors: EGCG; doxycycline; a nutrient mixture with and without PMA; retinoic acid; dexamethasone; H-7; actinomcin D; and cyclohexamide. After 24 hours, the medium was removed and analyzed for MMP-2 and MMP-9 by gelatinase zymography.
RCC 786-0 secreted two bands, a major band corresponding to MMP-2 and a faint band corresponding to MMP-9. PMA and TNF-α, with increased concentration, increased MMP-9 secretion, while IL-1ß and LPS did not significantly modify MMP-9 activity. MMP-2 secretion was not affected by any of the inducers. All the inhibitors tested without and with PMA showed a dose-dependent decrease in both MMP-2 and -9 expression.
In conclusion, this study has shown that exposure of the highly metastatic renal cell carcinoma cell line 786-0 to different growth factors increases secretion of MMP-9 but not MMP-2; MMP-2 and-9 activity was decreased by exposure to inhibitors. Further studies are in progress to confirm the role of MMP-9 on Matrigel invasion using PMA, cytokines and LPS.