Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential

Abstract

Recent epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in individuals who take nonsteroidal antiinflammatory drugs on a regular basis compared with those not taking these agents. One property shared by all of these drugs is their ability to inhibit cyclooxygenase (COX), a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. Human colon cancer cells (Caco-2) were permanently transfected with a COX-2 expression vector or the identical vector lacking the COX-2 insert. The Caco-2 cells, which constitutively expressed COX-2, acquired increased invasiveness compared with the parental Caco-2 cells or the vector transfected control cells. Biochemical changes associated with this phenotypic change included activation of metalloproteinase-2 and increased RNA levels for the membrane-type metalloproteinase. Increased invasiveness and prostaglandin production were reversed by treatment with sulindac sulfide, a known COX inhibitor. These studies demonstrate that constitutive expression of COX-2 can lead to phenotypic changes that alter the metastatic potential of colorectal cancer cells.

Figure 5

Dose-dependent sulindac sulfide inhibition of PGE₂ production and cell invasion. Either control or COX-2-expressing Caco-2 cells were treated with sulindac sulfide at the indicated concentrations for 24 hr prior to harvest. In A, PGE₂ concentrations were determined and the results plotted on the y axis. The cells were then evaluated in the invasion chamber assay described. The number of cells that invaded through the Matrigel and lodged onto the filter were counted and the results plotted on the y axis in B. Results are expressed as number of cells per high power field (×400). Five separate fields were counted per filter and the experiment was repeated four separate times.

Figure 1

COX-2 expression levels in permanently transfected Caco-2 cells. (Upper) Immunoblotting results from protein extracts (50 μg) taken from control and COX-2-expressing Caco-2 cells. (Lower) Northern blotting results from RNA samples (20 μg total RNA) taken from both control and COX-2-expressing Caco-2 cells. The agarose gel from which this Northern transfer was stained with ethidium bromide to ensure equal loading, and the filter was also probed with an 18 S RNA probe to verify equal loading (data not shown).

Figure 2

PGE₂ production from control and COX-2-expressing Caco-2 cells. Medium was taken from either the control or COX-2-expressing Caco-2 cells and used for ELISA assay for PGE₂ levels. Samples were measured in triplicate, and this experiment was repeated three separate times.

Figure 3

Matrix MMP-2 and MT-MMP-1 expression in Caco-2 cells. (Top) Gelatin zymography of proteins secreted into the cell culture medium. Lane 3 demonstrates the results following treatment with sulindac sulfide (SS; 25 μM) for 24 hr. A 68- and 62-kDa band are seen in the COX-2-expressing Caco-2 cells; whereas, only the inactive 68-kDa band is seen in the control Caco-2 cells. (Middle) Immunoblotting results with an anti-MMP-2 antibody in an identical experiment to that shown in the Top panel. This result confirms the gelatin zymography data shown in the Top panel. (Bottom) Northern blotting results which demonstrates a 2- to 3-fold increase in MT-MMP RNA in the COX-2-expressing Caco-2 cells which is inhibited in lane 3 by treatment of the cells with 25 μM sulindac sulfide for 6 hr. RNA loading controls were carried out as described for the Northern blot in Fig. 1 (data not shown).

Figure 4

Invasiveness and colony formation of Caco-2 cells. (A) Results of the invasion chamber assays quantitating the number of cells that migrate through the Matrigel layer to the filter placed on the bottom of the upper chamber. Cells were counted at a magnification of ×400. (B) Morphology of the colonies formed by control and COX-2-expressing Caco-2 cells. Cells were plated in Matrigel and incubated for 2 weeks prior to photography. (B1) Results for the control Caco-2 cells. (B2) Results for the COX-2-expressing Caco-2 cells. (×200.)