M.W. Roomi, N.W. Roomi, M. Rath and A. Niedzwiecki
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
Presented at:
22nd Annual Fanconi Anemia Research Fund Scientific Symposium, Minneapolis Minnesota, October 21-24, 2010.
Published in:
The Proceedings of the 22nd Annual Fanconi Anemia Research Fund Scientific Symposium, pg 95.
Abstract
Objective:
Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in FA patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have received much attention in recent years for their role in various malignances, and have been implicated in tumor invasion and metastasis. 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 immortalized fibroblasts from FA.
Materials and Methods:
Human FA immortalized fibroblast cell lines FA-A:PD220(A) and FA-D2:PD20(D2) were grown in MEM 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 1) PMA 10, 25, 50 and 100 ng/ml; TNF-alpha and IL-1 beta 0.1, 1, 10 and 25 ng/ml; or LPS 10, 25, 50 and 100 µg/ml; 2) EGCG and doxycycline (Dox), 10, 25, 50 and 100 µg/ml without and with PMA; 3) cyclohexamide, actinomycin D, retinoic acid and dexamethasone; and 4) a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract without and with PMA. After 24 h the media were removed and analyzed for MMP-2 and MMP-9 by zymography and densitometry.
Results:
Both FA cell lines expressed only one band corresponding to MMP-2. Cytokines, mitogens, inducers and inhibitors had a similar effect on MMP-2 and PMA-induced MMP-9 expression in both FA fibroblasts A and D2. PMA had a marked stimulatory dose dependent effect on MMP-9 expression and a moderate dose response effect on MMP-2. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. TNF-alpha and IL-1 beta had slight dose-response effects on MMP-2 and significant stimulatory dose-dependent effects on MMP-9 in both FA fibroblasts. EGCG and Dox, without and with PMA, down regulated the expression of MMP-2 and MMP-9 in a dose-dependent manner. In addition, actinomycin D, cyclohexamide, retinoic acid and dexamethasone also had an inhibitory effect on MMP-2. NM, without and with PMA, showed a dose-dependent decrease in MMP-2 and MMP-9 expression.
Conclusions:
The cytokines, inducers and inhibitors tested had an up and down regulatory effect on FA fibroblast A and D2 MMP-2 and -9 expression.
Translational Applicability:
Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast A and D2 MMP-2 and -9 expression, suggesting the clinical use of MMP inhibitors, especially such potent and non-toxic ones as the nutrient mixture and its component EGCG in management of FA cancers.
Comment
Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in FA patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, play a major role in in tumor invasion and metastasis. 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 immortalized fibroblasts from FA. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast A and D2 MMP-2 and -9 expressions, suggesting the clinical use of MMP inhibitors, especially such potent and non-toxic ones as the nutrient mixture and its component EGCG in management of FA cancers.
