Introduction: COX-2 expression appears to be important in colorectal carcinogenesis. Prior studies implicating involvement of the Ras oncogene and growth factors on COX-2 expression were largely derived from rat small intestinal cell lines. The purpose of this study is to determine whether mouse colonocyte COX-2 levels are regulated by oncogenic Ras or TGF-b1, and whether these factors also regulate cellular invasiveness.
Methods: YAMC cells are colonocytes derived from the "Immortomouse and immortalized by the SV40 T antigen. YAMC-Ras cells were derived by transfection of YAMC cells with oncogenic Ha-Ras and are tumorigenic. Both cells were treated with TGF-b1 (5ng/ml). Lysates were collected at intervals (0,8,24h) and COX-2 level determined by western blot analysis. For PGE2 and PGI2 release, cells were treated with TGF-b1 in serum free media followed by addition of arachadonic acid (15mm). Media were collected at 0,2 and 8h and PGE2 and PGI2 release determined by using gas chromotography and mass spectrometry. The effect of TGF-b1 on cell invasiveness was examined in Boyden chambers with type I collagen-coated transwell inserts.
Results: TGF-b1 increased COX-2 expression in both cell lines, but caused a 3-fold increase in COX-2 levels in YAMC-Ras cells as compared to the YAMC cells at 8h. PGE2 and PGI2 release was increased in both cell lines by TGF-b1 but, to a larger degree in the Ras transfected cells (P<.03 PGI2 and <.006 PGE2) by 8h. Additionally, the release of these prostaglandins and the increased expression of COX-2 was inhibited with the addition of PD9805 (inhibitor of ERK/MEK activity), SB203580 (inhibitor of P38 kinase activity), AG1678 (inhibitor of receptor tyrosine kinase) and the COX-2 specific inhibitor NS-398. The number of invasive cells increased 2.4 and 1.3-fold in the YAMC-Ras and YAMC cells respectively after TGF-b1 treatment. Also, the overall number of invasive cells in the Ras vs. non-Ras cells was 4.6-fold greater.
Conclusions: Induction of COX-2 and eicosanoid release were augmented by TGF-b1 in Ras-transformed mouse colonocytes. Also, there is a correlation between this increase in COX-2 expression and increased invasiveness in these cell lines. Thus, enhanced induction of COX-2 by TGF-b1 may contribute to increased tumorigenicity and invasiveness in cells transformed by oncogenic Ras.