Cyclooxygenases (COX) are essential rate-limiting enzymes catalyzing the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids in cells [
11,
19]. Three isoforms of COX have been identified: COX-1 and its spliced version COX-3 constitutively expressed in tissues and COX-2, which is regulated by growth factors, cytokines or oncogenes [
20,
21]. It has been clearly shown that COX-2 has tumor-promoting properties. This enzyme has been found to be expressed in approximately 40–50% of colonic adenomas and to be significantly over-expressed in 80–90% of colorectal carcinomas [
22,
23]. To date, several mechanisms by which COX-2 contributes to carcinogenesis have been identified. They include inhibition of apoptosis, enhancement of angiogenesis and invasiveness, modulation of inflammation and immunosuppression or conversion of procarcinogens to cancerogenic factors [
24]. Finally, COX-2 up-regulation is closely linked with increased production of PGs, especially PGE
2, which supports tumor growth by induction of angiogenesis and inhibition of tumor cell apoptosis and exerts immunomodulating effects via, e.g. T-cell response inhibition [
17,
25,
26].
During tumor development and metastasis, a significant role is also attributed to nitric oxide (NO). This molecule is implicated in the regulation of tumorigenesis depending on its local concentration, tumor stage, local microenvironmental conditions or even direct interactions between tumor and stromal cells.
In our study we used single doses of each agent. However, it was intended, because we did not want to analyze the effects depending on different concentrations of inhibitors or L-arginine but the relationship between COX-2, NO and PGE2 in specific culture conditions. We wanted to show that there are changes in specific molecules concentration or whole cells reaction (motility) when other molecules concentration is limited or inhibited their activity. It was only to show that such cross-talk among analyzed molecules exists. The detailed explanation of these observations, obviously, needs further study.
The selection of the dose of each agent was performed multistage. On the basis of the literature we selected a few concentrations. Next, they were tested for their cytotoxicity because in the further experiments we wanted to show often slight and delicate changes in specific molecules amounts. We had to be sure that under any circumstances we did not influence on cells amount or their viability. Thereafter, different concentrations were analyzed on their influence on NO level on normal colon epithelial cells. After that, the most appropriate concentration of inhibitors and L-arginine adapted to our experimental model has been selected.
We had to choose either different concentrations of inhibitors and L-arginine analysis or expand cellular model. The idea of our study was to analyze the relationship among selected parameters and therefore only single doses of inhibitors and L-arginine were used. If different concentrations of these agents and different cellular model combination were analyzed then, as we suppose, the results would be difficult to explain and in consequence difficult to understand.
NO is produced from L-arginine, a semiessential amino acid which by modulating host immune functions causes variable responses against tumor growth [
29,
30]. L-arginine, in experimental settings, has been shown to exert anti-tumor effects by reducing tumor size and incidence, and retarding tumor growth and metastasis [
31]. Generally, L-arginine may reduce chemically induced colorectal carcinomas influencing host immune system functions [
32]. Supplementation with this amino acid also leads to increased NO formation by the oxidative deaminase pathway. In turn, an increased local NO level may induce cytostasis by inhibiting hyperproliferation of tumor cells or cytotoxicity in tumor cells by, e.g. formation of toxic peroxynitrite [
33]. Moreover, there is also a cross-talk between products of the NOS and COX pathways. In our study, we showed that L-arginine supplementation slightly increased NO level but had no significant influence on the amount of COX-2, and in consequence on PGE
2 production in tumor and normal cell monocultures. The interactions between NO and COX-2 and PGE
2 occur at multiple levels and therefore it is difficult to unequivocally speculate about them and give simple answers or solutions. The significant reduction of PGE
2 levels by L-arginine was found in co-cultures of tumor spheroids with colonic epithelium rather than with myofibroblasts, which may suggest an important role of NO in chemoprevention of tumor-epithelial cell interactions rather than its effect on tumor-stromal development, activity and viability. Our results are in agreement with the findings of Clancy et al. [
34] and Ghosh et al. [
35], who showed that NO inactivates COX-2 functions through inhibiting its enzymatic activity by reacting with iron in the enzyme’s heme group. Moreover, we demonstrated that L-NAME, an inhibitor of NO synthases which decreased NO level and motility of tumor cells in the experiment had a relatively weak effect on COX-2 level. These results are in agreement with the findings of other authors showing a balance and interference with each other’s synthesis between NO, COX-2 and PGE
2 [
36,
37]. These interactions are important for maintaining homeostasis in normal cells and influence invasiveness in tumors. There exist contradictory reports describing the relationships between NO and COX, showing that blockade of NOS by L-NAME may either increase or decrease COX-2 and PGE
2 synthesis [
38‐
40]. We suppose, on the basis of our study, that the NO and COX-2 relationship is closely dependent on the kind of cells that have been used, their direct interactions, the kind of inhibitor applied and the local microenvironmental conditions. These elements together influence COX-2 and PGE
2 synthesis after NO imbalance. This hypothesis is confirmed by Ohno et al. [
40], who showed that mucosal PGE
2 was decreased by SC-560, a COX-2 inhibitor, but not by L-NAME. Moreover, other authors have shown that L-NAME reduces PGE
2 generation via limitation of COX-2 expression [
41]. Therefore, L-NAME may limit the invasion and migration of tumor cells and serve as a cancer prevention factor [
10,
42]. Finally, NS398, a specific COX-2 inhibitor, decreased the enzyme and PGE
2 levels and tumor cell motility with slight effects on NO production. These results are in agreement with the findings of West et al. [
43] and Payá et al. [
44], who revealed that NS398 strongly reduced COX-2 and PGE
2 levels without affecting NO metabolites and NOS activity. We can only speculate on the mechanism by which COX-2 inhibitor also causes inhibition of the enzyme expression. It may be associated with cell cycle arrest by COX-2 inhibitor what may result in potential decrease in enzyme level. NS398, which acts by inducing a conformational change of COX-2, may also lead to unrecognizing of the protein by specific antibodies. The inhibitor may also cross-influence on molecular pathways and signal transduction connected with enzyme activity and expression. Finally there may be a feedback between COX-2 and PGE
2 levels and activity. However, these are only speculations which need experimental confirmation. Moreover, there is strong evidence indicating that specific COX-2 inhibition could down-regulate the antiapoptotic Bcl-2 protein, thus inhibiting proliferation and progression of different carcinomas including colon cancer [
14,
45,
46]. At present, there is no uniform explanation of the mechanism of the cross-talks between NO and COX-2 and PGE
2 in tumor/normal cell co-cultures. Some evidence indicates that NO produced from L-arginine contributes to COX-2 and PGE
2 activation [
47]. However, no answer has been offered to the question of the influence of the NO + O
2
−
product (ONOO
−), which is locally produced during direct tumor/normal cell interactions, on COX-2 expression. In our previous work we showed that during direct tumor/normal cell contact O
2
−
anion is overproduced [
18]. However, our data obtained so far do not allow to determine whether this is mainly free NO or its reaction product peroxynitric acid, generated through the interaction between tumor cell-derived superoxide anions and NO. In order to distinguish reaction products, additional methods should be applied. Nevertheless, NO as a short-lived and highly reactive molecule will be converted into nitrate, a stable end product of NO, and will react with free oxygen radicals forming reactive oxygen/nitrogen species. The exact kind of molecules formed needs further studies. On the other hand, a large number of experimental results indicate that NO, under certain conditions, down-regulates the prostaglandin biosynthetic pathway mainly via COX-2 inhibition [
48]. We showed that in tumor or normal cell monocultures NO had no significant influence on COX-2 and PGE
2 expression. However, in specific culture conditions when tumor cells directly interacted with normal cells, especially myofibroblasts, the addition of L-arginine resulted in a limitation of COX-2 expression; PGE
2 level was decreased when tumor cells were implanted onto normal colon epithelium. Based on these results, we may speculate that NO and COX-2 and PGE
2 cross-talks should be considered in relation to the experimental conditions and the cell culture model used.
In conclusion, we have shown the existence of cross-talks among NO, COX-2 and PGE2. This relationship is rather unidirectional where NO level influences COX-2 and PGE2 levels. Moreover, any imbalances in NO level caused by exogenous factors influence COX-2 and PGE2 amounts depending on the kind of cells, their reciprocal interactions and the local microenvironmental conditions. The knowledge of these effects may be useful in limiting colon carcinoma progression and invasion.