Modulation of u-PA, MMPs and their inhibitors by a novel nutrient mixture in adult human sarcoma cell lines

M.Waheed Roomi, Tatiana Kalinovsky, Aleksandra Niedzwiecki* and Matthias Rath
Dr. Rath Research Institute, Santa Clara, CA
International Journal of Oncology 2013; 43: 39-49

Adult sarcomas are highly aggressive tumors that are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9 secretions that degrade the ECM and basement membrane, allowing cancer cells to spread to distal organs. Proteases play a key role in tumor cell invasion and metastasis by digesting the basement membrane and ECM components. Strong clinical and experimental evidence demonstrates association of elevated levels of u-PA and MMPs with cancer progression, metastasis and shortened patient survival. MMP activities are regulated by specific tissue inhibitors of metalloproteinases (TIMPs).

Our main objective was to study the effect of a nutrient mixture (NM) on activity of u-PA, MMPs and TIMPs in various human adult sarcomas.  Human fibrosarcoma (HT-1080), chondrosarcoma (SW-1353), liposarcoma (SW-872), synovial sarcoma (SW-982), and uterine leimyosarcoma (SK-UT-1) cell lines (ATCC) were cultured in their respective media and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of u-PA activity was carried out by fibrin zymography, MMPs by gelatinase zymography and TIMPs by reverse zymography. Fibrosarcoma, chondrosarcoma, liposarcoma and leimyosarcoma cancer cell lines expressed u-PA, which was inhibited by NM in a dose-dependent manner. However, no bands corresponding to u-PA were detected for synovial sarcoma cell line. On gelatinase zymography, fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with enhancement of MMP-9 with PMA (100 ng/ml) treatment. Uterine leimyosarcoma showed strong bands corresponding to inactive and active MMP-9 and a faint band corresponding to MMP-9 dimer induced with PMA treatment, but no MMP-2 band. NM inhibited their expression in a dose-dependent manner.  Activity of TIMPs was up regulated by NM in all 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. These findings suggest the therapeutic potential of NM in treatment of adult sarcomas.

Running Title: 
Modulation of adult sarcoma cancer cell line MMPs, u-PA, and TIMPs activity

Key words: 
fibrosarcoma, chondrosarcoma, liposarcoma, synovial sarcoma, uterine leimyosarcoma, 
u-PA, MMP-2 and MMP-9, TIMP-2, PMA, nutrient mixture