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
Proceedings of the American Association for Cancer Research 44:4559(2003)
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
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.
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
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
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
are often in physical contact with ECM of other neighboring cells.
The maximum stimulation of MMPs occurred when cancer cells co-cultured
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
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.