The activating mutations of ras
occur in ~30% of all human tumors. In squamous carcinomas, these mutations appear at early or intermediate stages of neoplasia. In colon cancers, ras
gene mutations were found in 50% of adenocarcinomas and larger adenomas 28
. To initiate studies on the involvement of the cyclooxygenases in transformation and malignancy, we developed cells that lack COX-1, COX-2, or both, and studied ras
-induced malignant transformation. We found that transformation by SV40 alone downregulated the expressions of both cyclooxygenases. However, addition of the activated ras
oncogene induced malignant transformation accompanied by an increased expression of either or both cyclooxygenase enzymes. Although most of the clones exhibit increased expression of either or both cyclooxygenases, some do not express either of the genes at increased levels. This result by itself suggests that the expression of either cyclooxygenase does not have a direct implication on mechanisms of transformation. Furthermore, our transformation experiments conducted in the cyclooxygenase-null cells provide still stronger evidence that transformation can occur in the absence of either of the cyclooxygenases, and that cyclooxygenase overexpression is not necessary to induce neoplastic transformation.
Nevertheless, there is abundant evidence suggesting that high levels of cyclooxygenase activity do play a critical role in some malignancies. Recent reports show that forced overexpression of cyclooxygenase enzymes increases metastatic potential, invasiveness, and angiogenesis of tumor cells both in vitro and in vivo 293031
. Moreover, carcinogenesis is a chronic, sequential, and progressive process that usually involves accumulation of mutations of protooncogenes and tumor suppressor genes in later steps. Mechanisms involving cyclooxygenases in mutagenesis have been suggested 3233
. COX-2 overexpression also leads to resistance to apoptosis and increases in the cellular life-span 1011
; these phenotypic changes may result in the additional development of mutations leading to more malignant behavior. Elevated cyclooxygenase expression is also known to increase malondialdehyde generation, providing another mechanism to foster higher cellular mutation rates 34
. So while activation of ras
by itself is not usually sufficient for malignancy, the elevations in cyclooxygenases induced by the activation of ras
could well predispose the cells to mutations in p53
or other genes that participate in the later steps towards high-grade malignancy. It is also noteworthy that in addition to COX-2, COX-1 expression is often induced by the activated ras
oncogene, suggesting that both cyclooxygenases may serve some overlapping roles in tumorigenesis. Further studies are required to clarify the functional differences and relative importance of the two isozymes in cell malignancy and in the different stages of carcinogenesis.
NSAIDs such as sulindac and indomethacin have been shown to be beneficial for the prevention and in some cases for the treatment of certain cancers, although the underlying mechanisms are still unknown. Inhibition of cellular proliferation and/or induction of apoptosis are reported to be the mechanisms that cause the regression of tumors in vivo 35
. The antiproliferative and apoptotic effects of NSAIDs have been documented in a variety of cells and cell lines, including colon cancer cells, breast cancer cells, and fibroblasts 2223
. Considerable recent data suggest that some of the NSAIDs actions may not be entirely explained by inhibition of cyclooxygenases. First, some NSAID-related compounds, which are not cyclooxygenase inhibitors, can also induce antineoplastic changes in both the cell cycle and cellular apoptotic responses 3637
. Second, the antineoplastic or anticarcinogenic activity of NSAIDs may not coincide with the inhibition of PG production in some tumor cells and animals 38
. Finally, Hanif et al. 39
found that sulindac and piroxicam inhibit the growth of and induce apoptosis in HCT-15 cells, which were shown to produce neither COX-1, COX-2, nor PGs. Elder et al. 40
also showed that NS-398 induced apoptosis in S/KS cells, which lack COX-2 protein. In contrast, Murphy et al. 41
showed that the growth of the cells expressing COX-2 was not suppressed by COX-2–selective inhibitors. In this study, we further illustrate that a selective knockout of cyclooxygenase activity does not change the ability of NSAIDs to inhibit transformation or to induce apoptosis. These results confirm that inhibition of cyclooxygenase is not the only mechanism responsible for the anticarcinogenic and antineoplastic activity of NSAIDs.
NSAID actions that are independent of cyclooxygenase have been reviewed by Abramson and Weissmann 42
. New evidence suggests that many of the traditional NSAIDs have targets other than cyclooxygenases. A recent report by Herrmann et al. 24
demonstrated that sulindac sulfide inhibits the ras
signaling apparatus, potentially via an eicosanoid-independent pathway. Lehmann et al. 43
demonstrate that many NSAIDs are ligands of peroxisome proliferation–activated receptors (PPARs) α and γ. Chan et al. 25
demonstrated that the elevation of the PG precursor AA could be involved in the induction of apoptotic response by the NSAIDs (i.e., sulindac and indomethacin) through production of ceramide. However, due to the higher concentration of NSAIDs used in their study, it is not clear whether the induction of AA is a result of cyclooxygenase enzyme inhibition or direct production of AA through activation of phospholipases. NSAIDs are a large group of structurally diverse compounds; therefore, differences in the actions of individual NSAIDs are also expected. For example, aspirin inhibits the activation of the transcription factor NF-κB, whereas indomethacin does not 44
. Recently COX-2–specific inhibitors were demonstrated to have potent antiproliferative effects and to induce apoptosis in cultured cells 204546
. Whether or not actions exerted by these drugs are mediated through inhibition of COX-2 needs additional investigation.
We have also observed in our studies that there are differences between cell lines in their sensitivity to NSAIDs that are independent of cyclooxygenase expression (). Thus, it seems likely that the differences in the sensitivity of tumor cell lines to NSAIDs as reported in the literature may also be independent of cyclooxygenase expression, particularly when NSAIDs are used at high concentrations.
Finally, the effects of NSAIDs on apoptosis, cell growth, and DNA synthesis studied here are those often cited to account for the antitumor activity of this class of compounds, yet in accord with the results of others, our data show that NSAID inhibition of the cyclooxygenases does not account for the antiproliferative and apoptotic actions seen in transformed embryonic fibroblasts. Our results further demonstrate that although cellular transformation by ras and SV40 usually leads to a dramatic increase in the expression of COX-1 and COX-2 when present, neither these inductions nor the presence of either of the enzymes is required for neoplastic transformation per se. These results suggest that the involvement of cyclooxygenase expression in neoplastic growth may occur at steps beyond those involved in transformation.