Abstract

Nonsteroidal antiinf lammatory drugs reduce the risk of colon cancer, possibly via cyclooxygenase (COX) inhibition. The growth factor-inducible COX-2, which is overexpressed in neoplastic colonic tissue, is an attractive target to mediate this effect. Herein we have exploited the ability of a human colon cancer cell line, HCA-7 Colony 29, to polarize when cultured on Transwell (Costar) filters to study COX-2 production and the vectorial release of prostaglandins (PGs). Administration of type ␣ transforming growth factor to the basolateral compartment, in which the epidermal growth factor receptor (EGFR) resides, results in a marked induction of COX-2 immunoreactivity at the base of the cells and the unexpected appearance of COX-2 in the nucleus. The increase in COX-2 protein is associated with a dose-and time-dependent increase in PG levels in the basolateral, but not apical, medium. Amphiregulin is the most abundantly expressed EGFR ligand in these cells, and the protein is present at the basolateral surface. EGFR blockade reduces baseline COX-2 immunoreactivity, PG levels, and mitogenesis in a concentration-dependent manner. Two specific COX-2 inhibitors, SC-58125 and NS 398, also, in a dose-dependent manner, attenuate baseline and type ␣ transforming growth factor-stimulated mitogenesis, although PG levels are decreased >90% at all concentrations of inhibitor tested. These findings show that activation of the EGFR stimulates COX-2 production and its translocation to the nucleus, vectorial release of PGs, and mitogenesis in polarized HCA-7 Colony 29 cells.In the gastrointestinal tract, prostaglandins (PGs) mediate important functions, including motility, vascular tone, angiogenesis, mucosal protection, and immune responsiveness (1). Inasmuch as epithelial cells are capable of PG synthesis, it is feasible that PGs synthesized in the gastrointestinal epithelium regulate these functions by paracrine pathways in response to luminal or serosal stimuli. Although data exist in support of vectorial release of PGs in the isolated rat colon (2), as well as in other tissues and polarized kidney-derived cells (3-5), regulatory mechanisms have not been defined more precisely.Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid (AA) to PGs and other eicosanoids. Two isoforms of the enzyme have been characterized. COX-1 in most cells is expressed constitutively, and a second inducible form known as COX-2 has been identified (refs. 6-8; for review see ref. 9). Recent observations indicate that many colonic polyps and cancers overexpress COX-2 (10-12) and that inhibition of this enzyme by nonsteroidal antiinflammatory drugs decreases the risk of colonic neoplasia (13-20), emphasizing the importance of defining potential autocrine and paracrine pathways for regulation of gastrointestinal epithelial growth by COX. Signaling through the epidermal growth factor receptor (EGFR) induces COX-2 expression, and unregulated overexpression of COX-2 results in a tumorigenic phenotype in the rat intestinal e...

BACKGROUND: Human colorectal cancer is caused by mutations and is thought to be maintained by a population of cancer stem cells. Further phenotypic changes occurring at the invasive edge suggest that colon cancer cells are also regulated by their microenvironment. Type I collagen, a promoter of the malignant phenotype in pancreatic carcinoma cells, is highly expressed at the invasive front of human colorectal cancer. METHODS: This study investigates the role of type I collagen in specifying the colorectal cancer cell phenotype. The effect of type I collagen on morphology, localisation of cell -cell adhesion proteins, differentiation and stem cell-like characteristics was examined in a panel of human colorectal carcinoma cell lines. RESULTS: Human colorectal carcinoma cells grown on type I collagen in serum-free medium show an epithelial -mesenchymal-like transition (EMT-like), assuming a more flattened less cohesive morphology. Type I collagen downregulates E-cadherin and b-catenin at cell -cell junctions. Furthermore, type I collagen inhibits differentiation, increases clonogenicity and promotes expression of stem cell markers CD133 and Bmi1. Type I collagen effects were partially abrogated by a function-blocking antibody to a2 integrin. CONCLUSION: Together, these results indicate that type I collagen promotes expression of a stem cell-like phenotype in human colorectal cancer cells likely through a2b1 integrin.

Human colorectal epithelium is composed mainly of columnar, mucous and endocrine cells; origin of these cell lineages from a multi-potential stem cell at the base of the crypt (the Unitarian hypothesis) has been proposed but not yet demonstrated. Gut endocrine cells have variously been considered of neural crest or endodermal origin, but conclusive evidence, particularly in humans, is lacking. It has been shown that in mouse gastrointestinal tract, a single progenitor cell gives rise to both columnar and mucous cells, but it has yet to be demonstrated that such a progenitor cell can also give rise to endocrine cells. Here, a single human rectal adenocarcinoma cell has been shown to differentiate into columnar, mucous and endocrine cells; therefore all epithelial lineages are of clonal origin. Additionally, these results show that human colorectal enteroendocrine cells, at least in neoplastic epithelium, have an endodermal origin.

1 Three stable epithelial cell lines (HCA-7, HCA-7-Col 1 and HCA-7-Col 3) all derived from the same human adenocarcinoma have been cultured on collagen-coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to a variety of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. 2 Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA-7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4°C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo-oxygenase. 3 Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA-7-Col 1 monolayers while responses were virtually absent in HCA-7-Col 3. A similar profile was seen with carbachol except that responses of HCA-7 and HCA-7-Col I monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. 4 The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. 5 Treatment with forskolin increased SCC in HCA-7 and HCA-7-Col 1 monolayers with little effect in HCA-7-Col 3. Nevertheless cyclic AMP levels were elevated most in HCA-7-Col 3 and least in HCA-7-Col I monolayers, in reciprocal relationship to the functional response. 6 A23 187 increased SCC when applied to HCA-7 and HCA-7-Col 3 monolayers with little effect on HCA-7-Col 1. 7 The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA-7-Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.