Scientific Presentations

Scientific Presentations: Cancer

Downregulation of Urokinase Plasminogen Activator (U-PA), Matrix Metalloproteinases (MMPS) and Upregulation of Their Inhibitors (TIMPS) by a Novel Nutrient Mixture in Human Prostate Cancer Cell Lines PC-3 and DU-145

M.W. Roomi, N.W. Roomi, M. Rath and A. Niedzwiecki
Dr. Rath Research Institute, Cancer Division, Santa Clara, CA, USA

Presented at: 
100th Annual Meeting of the American Association for Cancer Research, Denver, Colorado, April 18-22, 2009.

Published in: 
Proceedings of the 100th Annual Meeting of the AACR, Abstract #1127,p 270.

Abstract

Introduction: 
Prostate cancer is the most frequently diagnosed cancer among men in the U.S. Cancer turns deadly when it invades and metastasizes to other parts of the body. Proteases play a key role in tumor cell invasion and metastasis due to their ability to digest basement membrane and ECM components. Two families of proteases: serine (u-PA) and MMPs-2 and-9 are necessary for invasive and metastatic potential. U-PA and MMPs are expressed in many tumor cell lines. Strong clinical and experimental evidence shows that elevated levels of u-PA and MMPs are associated with tumor growth, cancer progression, metastasis and shortened survival in patients. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs).

Objective:
Biological agents that prevent ECM degradation have been shown to be a promising therapeutic approach to cancer. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract showed anticancer activity against a number of cancer cell lines. We investigated the effect of NM on the activity of u-PA, MMPs and their inhibitor TIPMs on human prostate cancer cell lines PC-3 and DU-145.

Materials and Methods: 
Human prostate cancer cell lines PC-3 and DU-145 (ATCC) were grown in MEM media with 10% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with NM at 0, 50, 100, 250, 500 and 1000 μg/ml in triplicate at each concentration. U-PA activity was carried out by fibrin zymography on 10% SDS-PAGE gels containing fibrinogen and plasminogen, MMPs by gelatinase zymography and TIMPs were analyzed by reverse zymography.

Results: 
U-PA activity was detected in both PC-3 and DU-145 prostate cancer cell lines, showing two bands corresponding to molecular weights 35 and 33 kD. NM inhibited their expression in a dose response manner, with significant reduction in u-PA activity at 250 μg/ml  NM. PC-3 on gelatinase zymography showed two bands for MMP-2 and MMP-9. The activity of MMP-2 and MMP-9 was significantly inhibited at 250 μg/ml NM with total inhibition at 500 μg/ml. Interestingly DU-145 did not exhibit MMP bands. Activity of TIMPs was upregulated in both cancer cell lines PC-3 and DU-145 in a dose-dependent manner. Minimum activity was expressed at 50 μg/ml and maximum at 1000 μg/ml. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between  u-PA/MMPs and TIMPS.

Conclusion: 
Based on these studies it is tempting to suggest that NM could potentially be developed as a new anticancer agent that inhibits u-PA and MMPs and increases TIMPs.

Comments

Proteases play a key role in tumor cell invasion and metastasis due to their ability to digest basement membrane and ECM components. Two families of proteases: serine (u-PA) and MMPs-2 and-9 are necessary for invasive and metastatic potential and clinical studies have shown that elevated levels of u-PA and MMPs are associated with tumor growth, cancer progression, metastasis and shortened survival in patients. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs). We studied the effect of a nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract (NM) on the activity of u-PA, MMPs and their inhibitor TIPMs on human prostate cancer cell lines PC-3 and DU-145. U-PA activity was detected in both PC-3 and DU-145 prostate cancer cell lines; NM inhibited their expression in a dose response manner, with significant reduction in u-PA activity at 250 μg/ml  NM. PC-3 expression of MMP-2 and MMP-9 was significantly inhibited at 250 μg/ml NM with total inhibition at 500 μg/ml. Interestingly DU-145 did not exhibit MMP bands. Activity of TIMPs was upregulated in both cancer cell lines in a dose-dependent manner. Analysis revealed a positive correlation between u-PA and MMPs and a negative correlation between u-PA/MMPs and TIMPS. The results of these studies are significant since they suggest that NM could potentially be developed as a new anticancer agent that inhibits u-PA and MMPs and increases TIMPs.



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