The mechanism whereby cyclooxygenase-2 and its prostaglandin (PG) products are involved in colonic carcinogenesis is not fully understood. Prostacyclin (PGI2) is a major PG with antiapoptotic activity and is produced in the gastrointestinal tract. We reported previously that a human colorectal cancer (CRC) cell line, HCA-7, produces significant levels of PGE2, PGD2, thromboxane, and PGF2α, but not PGI2. We now report that human colonic fibroblast cell lines produce significant amounts of PGI2 and that fibroblast lines derived from normal-appearing colonic mucosa of hereditary nonpolyposis CRC individuals produce 50-fold more PGI2 than normal fibroblast lines derived from individuals with nonhereditary CRC. Coculture of HCA-7 cells with hereditary nonpolyposis CRC fibroblasts, but not normal fibroblasts, markedly reduced butyrate-induced apoptosis of HCA-7 cells. This antiapoptotic effect was inhibited by the cyclooxygenase-2 inhibitor rofecoxib and was restored by the stable PGI2 analogue carbaprostacyclin. PGI2 binds either G protein-coupled cell surface PGI2 receptor or the nuclear peroxisome proliferator-activated receptor (PPAR) δ. PPAR δ likely mediates this antiapoptotic effect because HCA-7 cells express this receptor, and another PPAR δ agonist, docosahexaenoic acid, mimics the effect. We propose a novel mechanism by which stromal production of PGI2 promotes survival of colonocytes through PPAR δ activation. This mechanism may have relevance to maintenance of cells in the normal crypt and to clonal expansion of mutant colonocytes during tumorigenesis.
Genetic and epidemiological studies have implicated COX-24 in the pathogenesis of CRC. We initially reported overexpression of COX-2 in 90% of CRCs and nearly 50% of colonic adenomas (1). We subsequently identified a human CRC cell line, HCA-7, colony 29, that expresses COX-2 (2). These cells form a uniform polarizing monolayer when cultured on Transwell filters, and the epidermal growth factor receptor is found predominantly at the basolateral surface, as it is in all polarized epithelial cells. Basolateral but not apical delivery of the epidermal growth factor receptor ligand transforming growth factor α results in up-regulation of COX-2 and production of PGs that are released exclusively into the basolateral medium of polarized HCA-7 cells. These cells produce PGE2, PGD2, thromboxane, and PGF2α, but not PGI2.
In contrast, most human CRC cell lines do not express COX-2, and it now appears that, at least early in the malignant process, COX-2 is expressed chiefly in the stroma (3, 4, 5). Indeed, mounting evidence has highlighted the importance of the stromal compartment in epithelial tumors in general and intestinal tumors in particular (6, 7, 8, 9). The present studies were performed to characterize PG production by human pericryptal fibroblast cell lines and to test the hypothesis that stromal PGs affect epithelial function and contribute to colon carcinogenesis. An in vitro model system was used to simulate in vivo growth conditions whereby pericryptal fibroblast lines and HCA-7 polarized epithelial monolayers were cocultured on the opposing surfaces of Transwell filters.
Materials and Methods
All culture reagents were purchased from Hyclone (Logan, UT), and all chemicals were purchased from Sigma (St. Louis, MO), unless otherwise indicated. Rofecoxib was a generous gift from Pharmacia Upjohn (Peapack, NJ). Carbaprostacyclin, docosahexaenoic acid, and arachidonic acid were purchased from Cayman Chemical (Ann Arbor, MI).
HCA-7 cells, passage 20–35, were cultured on 12-mm Transwell filters (pore size, 0.4 μm) under previously defined conditions (2). Transepithelial electrical resistance across the Transwell filter was measured using a Millicell Electrical Resistance System (Millipore, Bedford, MA) to evaluate functional integrity of tight junctions. Experiments were conducted 7–10 days after seeding, when resistance was >400 ohms·cm2.
The isolation and propagation of LIM-PF1 and LIM-PF2 normal pericryptal fibroblasts have been described by Rockman et al. (10). In an identical manner, LIM-HPF3 pericryptal fibroblasts, as well as lines HPCF1 to HPCF6, were established from the normal-appearing colonic mucosa of seven different individuals with HNPCC by the laboratories of Whitehead and Boman, respectively. All of these fibroblast lines express α smooth muscle actin, a marker of pericryptal fibroblasts, as determined by Western blot with a mouse monoclonal antibody (Sigma-A2547). Fibroblasts were plated and grown to confluence on the bottom surface of Transwell filters, after which HCA-7 cells were plated on top of Transwell filters and maintained until they formed a confluent polarizing monolayer.
Eicosanoids were quantified by gas chromatographic/negative ion chemical ionization mass spectrometric assays using stable isotope dilution techniques as described previously (11).
Apoptosis was evaluated by TUNEL and Cytodeath M30 antibody assays (Roche Diagnostic Corporation, Indianapolis, IN). Total cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma). Cells were counted using a Zeiss Axiophot microscope, a SPOT camera (Diagnostic Instruments Inc., Sterling Heights, MI), and Metamorph software version 4.6r8 (Universal Imaging Co., Downingtown, PA). Data were evaluated by a mixed effects ANOVA method (12) and considered significant if differences between groups were P < 0.05.
We examined eicosanoid production from fibroblast cell lines established from grossly normal colonic mucosa at the distal margin of two resected nonhereditary colonic tumors (LIM-PF1 and LIM-PF2, referred to as normal fibroblasts) or from the colonic mucosa of seven individuals with HNPCC (LIM-HPF3 and HPCF1 to HPCF6, referred to as HNPCC fibroblasts). In contrast to HCA-7 cells, the two normal fibroblast lines produced readily detectable amounts of PGI2, as measured by its stable metabolite 6-keto-PGF1α (Fig. 1). HNPCC fibroblasts produced, on average, 50-fold more PGI2 than normal fibroblasts. All of these fibroblast lines generate significant amounts of PGE2, PGD2, thromboxane, and PGF2α; however, the concentrations of these eicosanoids do not differ between normal fibroblasts and HNPCC fibroblasts (data not shown).
Given the observation that HCA-7 cells do not produce PGI2 but that fibroblast cell lines do, we speculated that this eicosanoid may affect epithelial cell function. PGI2 has been shown to stimulate growth and inhibit apoptosis in other epithelial cell systems (13, 14). Because PGI2 is highly unstable, we used the stable synthetic PGI2 analogue carbaprostacyclin to study its effects on HCA-7 cells. Carbaprostacyclin added to HCA-7 cells at concentrations between 10−9 and 10−4 m had no effect on proliferation of HCA-7 cells or two gastric cancer cell lines (AGS and MKN-28) as measured by bromodeoxyuridine incorporation, tritiated thymidine incorporation, or colony formation in soft agar (data not shown).
Because it has been shown previously that PGI2 inhibits programmed cell death in hepatic epithelial cells (13), we considered whether this eicosanoid may have an antiapoptotic effect on HCA-7 cells. Butyrate, a bacterially produced short chain fatty acid found naturally at millimolar concentrations in the gut, has been reported to induce apoptosis in two CRC cell lines, HT-29 and HCT-116 (15, 16). HCA-7 cells treated with 5 mm sodium butyrate for 18 h demonstrated a more than 10-fold increase in the number of TUNEL-positive cells (control, <0.5%; butyrate treatment, >5% total nuclei). We found that polarized HCA-7 cells pretreated with carbaprostacyclin showed a significant concentration-dependent decrease in butyrate-induced apoptosis as measured by TUNEL assay (Fig. 2) or by Cytodeath M30 assay (data not shown). A similar reduction in butyrate-induced apoptosis was seen with another polarizing colon cancer cell line, HCT-8, treated with carbaprostacyclin (data not shown).
Because HNPCC fibroblasts produce significant quantities of PGI2, we hypothesized that coculturing HCA-7 cells with HNPCC fibroblasts would protect HCA-7 cells from butyrate-induced apoptosis. Cells were cultured in such a way as to simulate stromal epithelial interactions. Fibroblast lines were grown on the bottom of Transwell filters, and polarized HCA-7 epithelial cells were grown on top. Levels of apoptosis for HCA-7 cells grown in the presence of normal fibroblasts (LIM-PF1 and LIM-PF2), which produce less PGI2, were not statistically different from HCA-7 cells grown alone. However, HCA-7 cells grown in the presence of HNPCC fibroblasts (LIM-HPF3, HPCF1, and HPCF4) were significantly more resistant to butyrate-induced apoptosis than HCA-7 cells grown alone (Fig. 3).
Additional studies were undertaken to evaluate whether PGI2 is a critical mediator of HCA-7 cell resistance to butyrate-induced apoptosis. Addition of the selective COX-2 inhibitor rofecoxib reduced PGI2 production in HNPCC fibroblasts by >90% (Fig. 4,A). Administration of rofecoxib to cocultures of HNPCC fibroblasts (LIM-PF3 or HPCF2) and HCA-7 cells abrogated the antiapoptotic effect conferred by HNPCC fibroblasts on HCA-7 cells (Fig. 4 B). Addition of carbaprostacyclin to these cultures, in turn, countered the effect of the COX-2 inhibitor. Furthermore, conditioned medium from HNPCC fibroblasts did not protect HCA-7 cells against butyrate-induced apoptosis (data not shown), a finding that argues against a major protective role for the paracrine action of more stable peptide growth factors such as hepatocyte growth factor and keratinocyte growth factor, which have also been shown to protect against apoptotic stimuli (17, 18).
PGI2 is thought to induce its biological activity by binding to and activating one of two receptors, the G protein-coupled cell surface PGI2 (IP) receptor and nuclear PPAR δ (19). HCA-7 cells have been shown to express PPAR δ (20), and we were not able to detect expression of IP receptor in HCA-7 cells by Northern blot analysis (data not shown). Furthermore, we found that treating HCA-7 cells with an alternative ligand for PPAR δ, docosahexaenoic acid (21), had a virtually identical effect to that observed with carbaprostacyclin (shown in Fig. 2), reducing the number of apoptotic nuclei in a concentration-dependent manner (mean control, 6.1% versus 3.7% and 3.4% at 10−6 and 10−5 m, respectively; P < 0.05). Taken together, the similar apoptosis-attenuating effects of PGI2 and docosahexaenoic acid and lack of expression of IP receptors strongly suggest that the PGI2 effect in HCA-7 cells is mediated by PPAR δ.
We find that fibroblasts derived from seven individuals with HNPCC produce an abundance of PGI2. HNPCC patients suffer from a genetic predisposition to CRC due to germ-line mutations in DNA mismatch repair genes resulting in genomic instability in the colonic epithelium (22). Could enhanced PGI2 expression have a role in the susceptibility of HNPCC kindreds to CRC? Although there appears to be a shortened interval from adenoma to carcinoma in HNPCC individuals (1–3 years compared with 8–10 years for individuals with sporadic CRC), HNPCC individuals with CRC fare better clinically. COX-2 immunoreactivity has been reported to be reduced in the tumor epithelium of HNPCC individuals with CRC compared with individuals with sporadic CRC (23, 24). Based on our findings, it is tempting to speculate that early colonic lesions in HNPCC individuals are maintained and progress rapidly due to the nurturing influence of PGI2 produced by the adjacent stroma. As these tumors progress and lose contact with their supporting stroma, they may be more susceptible to apoptotic stimuli in the absence of local PGI2 production.
We propose a model in which overproduction of PGI2 contributes to progression of neoplastic epithelium by preventing apoptosis in the face of accumulating genetic lesions and a noxious colonic environment. Studies will now be directed toward elucidating the mechanism(s) underlying the increased PGI2 production in HNPCC-derived fibroblasts and how this antiapoptotic effect is executed. Preliminary studies reveal no evidence for microsatellite instability in these HNPCC-derived fibroblasts.5 Of interest, Sonoshita et al. (25) recently observed that COX-2 is expressed in the fibroblasts and endothelial cells of intestinal polyps of individuals with familial adenomatous polyposis.
This study provides one mechanism by which the stroma, via its production of PGI2, can regulate epithelial function, that is, by promoting epithelial survival. PGI2 would be an ideal candidate molecule for such localized signaling, given its short half-life under physiological conditions (26). The normal colonic epithelium is sheathed in a single layer of pericryptal fibroblasts. Kaye et al. (27) have described these pericryptal fibroblasts and colonocytes as forming a highly ordered unit. Whereas the epithelial cells proliferate at the crypt base and acquire more differentiated features as they migrate toward the luminal surface, the single layer of fibroblasts migrates in from the lamina propia and differentiates (5, 28). These fibroblasts then undergo very little proliferation or migration, possibly contributing local cues that promote epithelial survival, growth, and differentiation (29, 30). Future studies will address whether there are different patterns of spatial or temporal production of PGI2 by pericryptal fibroblasts along the colonic crypt in normal and diseased states.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by NIH Grants GM15431, DK48831, and CA77839 (to J. D. M.); NO1-CN-95037-44, P30 CA56036, and PO1 CA 72027-01A2 (to B. M. B.); and CA46413 and 50CA95103 Gastrointestinal Specialized Program of Research Excellence (to R. J. C.). J. D. M. is the recipient of a Burroughs Welcome Clinical Scientist Award in Translational Research.
The abbreviations used are: COX-2, cyclooxygenase-2; PGI2, prostacyclin; TUNEL, terminal deoxynucleotidyl transferase-mediated nick deoxyuridine triphosphate-biotin end labeling; CRC, colorectal cancer; PG, prostaglandin; HNPCC, hereditary nonpolyposis colorectal cancer; PPAR, peroxisome proliferator-activated receptor.
W. Grady and S. Biswas, personal communication.