Induction of cyclooxygenase-2 in monocyte/macrophage by mucins secreted from colon cancer cells

Abstract

Up-regulation of cyclooxygenase-2 (COX-2) and overproduction of prostaglandins have been implicated in the initiation and/or progression of colon cancer. However, it is uncertain in which cells and how COX-2 is induced initially in the tumor microenvironment. We found that a conditioned medium of the colon cancer cell line, LS 180, contained a factor to induce COX-2 in human peripheral blood mononuclear cells. This factor was purified biochemically and revealed to be mucins. A small amount of mucins (approximately 100 ng of protein per ml) could elevate prostaglandin E2 production by monocytes. The mucins induced COX-2 mRNA and protein levels of monocytes in a dose- and time-dependent manner, indicating a COX-2-mediated pathway. We also have examined immunohistochemically the localization of COX-2 protein and mucins in human colorectal cancer tissues. It is noteworthy that COX-2-expressing macrophages were located around the region in which mucins were detectable, suggesting that COX-2 also was induced by mucins in vivo. These results suggest that mucins produced by colon cancer cells play a critical role in the initial induction of COX-2 in the tumor microenvironment.

Figure 1

PGE2 production in monocytes cocultured with LS 180 cells or cultured in the presence of conditioned medium of LS 180 cells. (A) Monocytes (1 × 10⁵ cells) (lane a) and LS 180 cells (1 × 10⁵ cells) (lane b) were cultured separately for 36 h. The same number of both cells (lane c) was cocultured for 36 h. (B) Monocytes (1 × 10⁵ cells) were cultured for 36 h in the presence of 10% FCS-MEM (lane a) and in the presence of conditioned medium of LS 180 cells treated (lane b) or not (lane c) with 5 mM phenyl α-GalNAc. Each culture supernatant was assayed for PGE2 by ELISA. The data are mean levels (n = 3) ± SD of the secreted PGE2.

Figure 2

Isolation of activating factor from conditioned medium of LS 180 cells. The conditioned medium of LS 180 cells was subjected to gel filtration on Sepharose 6B (3.0 × 105 cm) and then eluted with 25 mM sodium phosphate buffer, pH 7.5, and 0.15 M NaCl. (A) Fractions of 18.6 ml were collected, and absorbance at OD₂₈₀ was determined. One microgram of protein of each fraction was added to the culture medium of monocytes (1 × 10⁵ cells). After 24 h, secreted PGE2 was assayed by ELISA. (B) One hundred microliters of each fraction was loaded on a nylon membrane. Dot blot analysis was performed by using mAb MLS 132. (C) Purified mucin (5 μg) was subjected to SDS/PAGE with a 6% running gel, followed by silver (lane a) and periodate–Schiff (lane b) staining. Another sample was subjected to Western blotting after SDS/PAGE, and cancer-associated carbohydrate antigens were detected (lane c) as described in Materials and Methods.

Figure 3

Dose- and time-dependent PGE2 production in monocytes stimulated with mucin or BSM. (A) Monocytes (1 × 10⁵ cells) were cultured in the presence of mucin (0–2 μg of protein per ml) or BSM (0–30 μg of protein per ml) for 12 h. (B) Monocytes (1 × 10⁵ cells) were cultured in the presence of mucin (1 μg of protein per ml) or BSM (30 μg of protein per ml) for the time indicated. Each supernatant was assayed for PGE2 by ELISA.

Figure 4

Induction of COX-2 mRNA and protein in monocytes. (A) Monocytes (5 × 10⁶ cells) were cultured in the presence of mucin (1 μg of protein per ml) or BSM (30 μg of protein per ml) for the time indicated. (a) Total RNA was prepared, and COX-2 cDNA was amplified for 22 cycles and subjected to agarose gel electrophoresis as described in Materials and Methods. (b) COX-2 protein was immunoprecipitated by using anti-human COX-2 antibody and detected as described in Materials and Methods. (Ba) Monocytes (5 × 10⁶ cells) were cultured for 2 h in the presence of mucin (0–2 μg of protein per ml) or BSM (0–30 μg of protein per ml), from which total RNA was prepared. COX-2 cDNA was amplified (mucin-treated monocytes: 22 cycles; BSM-treated monocytes: 30 cycles) and analyzed as described above. (b) Monocytes (2 × 10⁷ cells) were cultured for 2 h in the presence of mucin (1 μg of protein per ml). Total RNA was prepared and Northern blot analysis was performed as described in Materials and Methods. (c) COX-2 protein was prepared from the protein fraction and detected as described above. Representative data of three experiments are shown.

Figure 5

Binding of mucins to monocytes through MSR. (A) Monocytes (1 × 10⁶ cells) were incubated with ¹²⁵I-labeled mucin at 4°C for 2 h in the presence of various inhibitors. The radioactivity bound to the cell was determined as described in Materials and Methods (duplicate experiments). (B) Binding of ¹²⁵I-labeled mucin to the MSR cDNA transfectants (1 × 10⁶ cells) was examined as described above (triplicate experiments). (C) Monocytes (1 × 10⁵ cells) were cultured for 36 h in the presence of various ligands. Each culture supernatant was assayed for PGE2 by ELISA. (D) Monocytes (5 × 10⁶ cells) were cultured for 2 h in the presence of various ligands, from which total RNA was prepared. COX-2 and β-actin cDNAs were amplified for 30 and 27 cycles, respectively, and analyzed as described above. The amounts of various ligands used are as follows: OSM and BSM, 30 μg of protein per ml; fucoidan, poly I, and poly C, 100 μg/ml; orosomucoid, 2 μg of protein per ml.

Figure 6

Immunostaining for CD 68, COX-2, and cancer-associated carbohydrate antigens in colon cancer tissues. Colon cancer tissues were immunostained by using the Vectastain avidin–biotin peroxidase complex kit as described in Materials and Methods (A and B). (A) COX-2-positive macrophages were located around the region in which carbohydrate antigens were detectable. (B) COX-2 was not expressed in macrophages without carbohydrate antigens around them. (a) CD 68; (b) COX-2; (c) Tn antigen; (d) sialyl Tn antigen; (e) control (mouse IgG); (f) control (goat IgG). (C) The same specimens as in A were double-stained with anti-CD68 and anti-COX-2 antibodies as described in Materials and Methods. Most of CD 68-positive macrophages (brownish-black deposit) were costained with anti-COX-2 antibody (blue deposit). (a) Macrophages in cancer tissues; (b) macrophages in intraluminal space of adenocarcinoma.

Figure 7

Possible role of mucins as chemical mediators in the tumor microenvironment. Mucins produced by colon cancer cells induce COX-2 in macrophages, resulting in PGE2 production. PGE2 functions as a mediator in an autocline and/or paracline fashion, leading to vascular endothelial growth factor (VEGF) production.