Cyclooxygenase-2 (COX-2), which catalyses a key step in conversion of arachidonic acid to prostaglandin H2, is overexpressed in human colorectal carcinoma tissue, and is associated with angiogenesis in the colorectal cancer (Williams et al. 1999). Thromboxane A2 (TXA2) is produced by thromboxane synthase (TXS), a downstream enzyme of COX-2, and is well known to induce platelet aggregation and vasoconstriction. TXA2 has been reported to be associated with endothelial migration, angiogenesis and tumor metastasis (Nie et al. 2000). Recently, we found that TXS is overexpressed in human colorectal carcinoma tissues (Sakai et al. 2003). Here we found a novel function of TXA2 in the colonic carcinoma cells.
The expression of TXS mRNA and the protein in human colonic carcinoma cell lines such as KM12-L4, HT-29, T-84, WiDr was examined by Northern blotting and Western blotting, respectively. To clarify the functional role of TXA2 in colonic carcinoma cells, the TXS protein in the cells was disrupted by using specific antisense oligonucleotide (TXS-AS). Cell proliferation assay was performed by counting the number of the cell in a 12-well plate. In each well, 1 X 105 cells were seeded and cultured for two days. All data were statistically analysed using one-way ANOVA and Tukey’s multiple comparison test.
We found that both the mRNA and protein of TXS were highly expressed in KM12-L4, HT-29, T-84 and WiDr cells. In KM12-L4 cells, the cell proliferation was significantly inhibited by the disruption of TXS protein by using TXS-AS. TXS inhibitors, such as Y-20811 (5 µM) and OKY-1581 (1 µM), also inhibited the proliferation of KM12-L4 and HT-29 cells. Furthermore, direct addition of 9, 11-epithio-11, 12-methano-TXA2 (STA2; 0.1 µM), a stable analogue of TXA2, accelerated the proliferation of KM12-L4 and HT-29 cells. After the disruption of TXS protein in KM12-L4 cells, STA2 still induced the cell proliferation.
These results indicate that TXA2 stimulates the proliferation of human colonic carcinoma cells.