A novel in vitro bioassay for screening matrix metalloproteinases activity in human cancer cell lines.

M Waheed Roomi, Shrirang P Netke, Vadim Ivanov, Matthias Rath and Aleksandra Niedzwiecki.

Proceedings of the American Association for Cancer Research 44:4559(2003)

Metastatic cancer cells secret a large amounts of matrix metalloproteinases (MMPs), which degrade ECM and basement membrane and thereby allow them to spread to distal organs. Increased activity of MMPs was associated with cancer of breast, lung, ovary, prostate and pancrease. There is compelling evidence to suggest that MMP-2 and MMP-9 play an important role in tumor invasion and metastasis. Designing new drugs to inhibit MMPs activity is therefore a priority.

However, several types of cancer cells in vitro do not express MMPs 2 & 9 in any significant amount and thereby making it difficult for an in vitro screening assay. In the present investigation we report the observation that co-culturing cancer cells with normal human dermal fibroblast (NHDF) results in an enhanced expression of MMP-2 and 9.

We suggest that this in vitro bioassy can be used to screen drugs for their ability to inhibit MMPs activity. Human skin cancer (melanoma A2058), liver cancer (Hep G2) and fibrosarcoma( HT-1080 ) cells in culture expressed MMPs-2 and 9 only weakly, the bands are faint and hardly visible on gelatinase zymography assay. Virtuall no bands were seen with breast cancer cells( MDA MB 231 and MCF –7), prostate cancer (PC-3 and LNCaP ) with colon cancer cells( HCT116).

In sharp contrast, when these cancer cells were co-cultured with NHDF, we observed a dramatic increase in MMPs expression. MMP zymogram bands were well distinct and discrete, the intensity of the bands was enhanced several folds. Both MMP-2 and MMP-9 were increased significantly in Hep G2 and HT-1080 cancer cells, and melanoma A2058 cells exhibited the most increase in MMP-2 and MMP-9. Human breast cancer cell lines MDA MB 231 and MCF-7, and colon cancer HCT116 also showed enhanced levels of MMP-2 and MMP-9 upon co-culture.

The cancer cells did not stimulate MMPs expression when they were cultured along with the condition media from NHDF, suggesting that physical contact between cancer cell and NHDF is necessary for stimulation of MMPs. In this respect, this co-culture system mimic the in vivo condition where cancer cells are often in physical contact with ECM of other neighboring cells. The maximum stimulation of MMPs occurred when cancer cells co-cultured with NHDF in 1:1 ratio. The co-culture system exhibited an excellent predictable response to both the stimulators and inhibitors of MMPs. For example, phorbol 12-myristate 13-acetate exerted stimulatory effect, while retinoic acid, epigallcatechin gallate, selenium, cycloheximide and H-7 exerted an inhibitory effect on MMPs expression.

These results demonstrate that this co-culture assay offers a better screening system for potential cancer drugs or agents that have either enhancing or inhibitory effects on MMPs.