Nitric Oxide (NO) and Cyclooxygenase-2 (COX-2) Cross-Talk in Co-Cultures of Tumor Spheroids with Normal Cells

Abstract

Cyclooxygenases (COX), prostaglandin E(2) (PGE(2)) and nitric oxide (NO) are believed to be some of the most important factors related to colon cancer growth and metastasis. In this study, we aimed to investigate the associations between COX-2, PGE(2) and NO in co-cultures of human colon cancer spheroids obtained from different tumor grades with normal human colonic epithelium and myofibroblast monolayers. L-arginine (2 mM), a substrate for nitric oxide synthases (NOS), decreased COX-2 and PGE(2) levels, while N( G )-nitro-L-arginine methyl ester (L-NAME) (2 mM), a NOS inhibitor, had no influence on COX-2 and PGE(2) levels but limited tumor cell motility. NS398 (75 μM), a selective COX-2 inhibitor, had no significant influence on NO level but decreased motility of tumor cells. COX-2, PGE(2) and NO levels depended on the tumor grade of the cells, being the highest in Duke's stage III colon carcinoma. Summing up, we showed that addition of L-arginine at doses which did not stimulate NO level caused a significant decrease in COX-2 and PGE(2) amounts in co-cultures of colon tumor spheroids with normal epithelial cells and myofibroblasts. Any imbalances in NO level caused by exogenous factors influence COX-2 and PGE(2) 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.

Fig. 1

The effect of L-arginine (2 mM), L-NAME (2 mM) and NS398 (75 μM) on the migration capacity of colon tumor HT29 cells. a wounded monolayer, b tumor cell migration after 24 h in control culture, c tumor cell migration after 24-h incubation with L-arginine, d tumor cell migration after 24-h incubation with L-NAME and e tumor cell migration after 24-h incubation with NS398. Bar = 500 μm

Fig. 2

a–c Nitric oxide (NO) production in co-cultures of colon carcinoma cell spheroids HT29 (a), LS180 (b) and SW948 (c) with normal colon epithelial cells (841CoTr) and myofibroblasts (18Co) during 24 h of incubation with L-arginine (2 mM), L-NAME (2 mM), and NS398 (75 μM). Exposure of cells to L-arginine non-significantly enhanced NO production, while L-NAME significantly inhibited NO secretion as compared to an appropriate sample control. NS398 had no significant influence on NO production. ^* p ≤ 0.05—a co-culture of tumor/normal cells compared to an appropriate monoculture of normal cells. # p ≤ 0.05—a culture of tumor and/or normal cells after treatment compared to an appropriate non-treated culture

Fig. 3

Western blot analysis of COX-2 after 24 h of incubation with L-arginine, L-NAME or NS398 in HT29 colon carcinoma cells, myofibroblasts and their co-culture. The increase in band density indicates an increase in protein levels. L-arginine limited COX-2 expression and in tumor cells co-cultured with myofibroblasts the amino acid significantly decreased the enzyme level as compared to a non-treated co-culture. L-NAME non-significantly decreased COX-2 expression as compared to a non-treated co-culture control. NS398 caused a significant inhibition of COX-2 expression

Fig. 4

a–c Semiquantitative results of a densitometric analysis of the bands of COX-2 protein detected by immunoblotting performed on material obtained from co-cultures of tumor cell spheroids HT29 (a), LS180 (b) and SW948 (c) with normal cells. Only co-cultures of tumor spheroids with colonic epithelium and myofibroblasts were analyzed. Note the lack of COX-2 expression in normal colonic epithelial cells. ^* p < 0.05—co-cultures treated with L-arginine, L-NAME or NS398 compared to an untreated co-culture

Fig. 5

a–c PGE₂ production in co-cultures of colon carcinoma cell spheroids HT29 (a), LS180 (b) and SW948 (c) with normal colon epithelial cells and myofibroblasts during 24 h of incubation with L-arginine, L-NAME or NS398. ELISA test. Tumor cells produced significantly higher PGE₂ than normal cells. Myofibroblasts expressed significantly higher PGE₂ than colonic epithelium. L-arginine limited PGE₂ production in tumor cells, myofibroblasts and their co-cultures. L-NAME had no significant influence on PGE₂ level, which remained unchanged or slightly decreased in tumor cells, normal cells and their co-cultures as compared to untreated controls. NS398, inhibited PGE₂ production in the tested cells. ^* p ≤ 0.05—a co-culture of tumor/normal cells compared to an appropriate monoculture of normal cells. # p ≤ 0.05—a culture of tumor and/or normal cells after treatment compared to an appropriate non-treated culture

Fig. 6

Schematic graph showing reciprocal relations among nitric oxide (NO), nitric oxide synthases (NOS), cyclooxygenase-2 (COX-2), and prostaglandin E₂ (PGE₂)