M. W. Roomi, T. Kalinovsky, A. Niedzwiecki, M. Rath
Dr. Rath Research Institute, Santa Clara, CA
Horizons in Cancer Research, volume 59, Nova Science Publishers, Inc., 2015
Chondrosarcoma (CS), a malignancy of cartilaginous origin, is an aggressive cancer with poor prognosis, due to both its aggressive metastatic spread and the lack of efficacy in current treatment modalities to prevent or counteract tumor progression. Chondrosarcomas are characterized by high levels of proteases, which are involved in the degradation of ECM and the basement membrane, thus allowing cancer cells to invade and metastasize to distal organs.
Numerous clinical and experimental studies have demonstrated that high levels of uPA and MMPs are associated with tumor growth, progression, metastasis and shortened survival rates in patients. We have developed natural strategies to inhibit cancer by increasing the stability and integrity of connective tissue through the synergistic effects of selected nutrients, such as lysine, proline, ascorbic acid and green tea extract (NM). This micronutrient mixture has exhibited anticancer activity in vivo and in vitro in a large variety of cancer cell lines by simultaneously affecting several key mechanisms involved in cancer. We investigated the effect of NM in chondrosarcoma cell line SW-1353 in vitro focusing on the invasive/metastatic parameters such as activities of uPA, MMPs and their tissue inhibitors (TIMPs), as well as the effects of various cytokines, mitogens, inducers and inhibitors on MMPs secretion. We also evaluated the effects of NM on cell invasion and migration. Cells were cultured in DEM and treated at confluence with NM at 0, 50, 100, 250, 500 and 1000 µg/ml. Analysis of uPA activity was carried out by fibrin zymography, MMPs by gelatinase zymography, TIMPs by reverse zymography, invasion through Matrigel and migration by scratch test. Chondrosarcoma expressed two bands corresponding to uPA subunits 1 and 2 at 55 and 33 kD, which were inhibited by NM in a dose-dependent manner with total block at 100 μg/ml in (linear trends R2=0.690 and 0.698, respectively). Gelatinase zymography showed bands corresponding to MMP-2 and -9 and strongly induced MMP-9 and MMP-9 dimer with PMA (100 ng/ml) treatment. NM inhibited their secretions in a dose-dependent manner with total block of MMP-9 dimer at 250 g/ml (linear trend R2 = 0.843), MMP-9 monomer at 500 g/ml (linear trend R2 = 0.729) and MMP-2 at 500 g/ml (linear trend R2 = 0.866). Secretion of MMP-9 dimer, which has been shown to be required for cell migration, was found to be 25% of that of MMP-9, indicating the aggressiveness of chondrosarcoma, since the MMP-9 dimer is usually present as 10-15% of the MMP-9 level. Cell migration and Matrigel invasion were inhibited by NM in a dose-dependent manner with total block at 250 and1000 g/ml, respectively. Activity of TIMPs was up regulated by NM in a dose–dependent manner. Analysis revealed a positive correlation between uPA and MMP-2 (r = 0.703) and negative correlations between uPA /MMP-2 (r = -0.901) and uPA/TIMPs (r= -0.753). A significant negative correlation was found between NM modulation of Matrigel invasion and MMP-2 secretion with chondrosarcoma SW-1353 (r= -0.942). These findings suggest the therapeutic potential of NM in treatment of chondrosarcoma.
Running Title: Nutrients inhibit chondrosarcoma progression
Key Words: Chondrosarcoma SW-1353, nutrient mixture, uPA, MMP-2, MMP-9 monomer and dimer, TIMPs, Matrigel invasion, cell migration
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