Involvement of cyclooxygenase-2 in the tumor site-dependent production of parathyroid hormone-related protein in colon 26 carcinoma

Abstract

It has been shown that in the mouse colon 26 tumor model, tumors grown in the subcutis (subcutis colon 26) caused early onset of cachectic syndromes, whereas those in the liver (liver colon 26) did not. Both interleukin (IL)-6 and parathyroid hormone-related protein (PTHrP) were involved in the development of cachectic syndromes in this tumor model. However, whether expression of PTHrP and IL-6 is differently regulated in the tumor microenvironment is unclear. In the present study, culturing the colon 26 cells under different conditions in vitro revealed that IL-6 production was increased by monolayer culture under a low-glucose condition but not by spheroid culture. In contrast, PTHrP production was increased by spheroid culture but not by monolayer culture, even under a low-glucose condition. Gene expression profiling revealed that the expression of cyclooxygenase (COX)-2 was up-regulated in both subcutis colon 26 and spheroid cultures, and that COX-2 inhibitor NS-398 suppressed PTHrP production in spheroid cultures. Furthermore, administration of NS-398 decreased the PTHrP level without affecting the tumor growth in mice bearing subcutis colon 26. These results demonstrate that production of PTHrP and IL-6 largely depends on the microenvironments in which tumors are developed or metastasized and that up-regulation of COX-2 in a necrobiotic environment leads to PTHrP production, thereby causing cachectic syndromes.

Figure 1

Plasma concentrations of parathyroid hormone‐related protein (PTHrP) and interleukin (IL)‐6 in mice bearing colon 26 and amounts of transforming growth factor (TGF)‐β mRNA in subcutis colon 26 and liver colon 26. Mice were subcutaneously or intrahepatically inoculated with colon 26, and plasma levels of (a) PTHrP and (b) IL‐6 in mice bearing colon 26 were determined using radioimmuno assay (RIA) and ELISA, respectively, on days 12 and 17. Non‐tumor‐bearing mice were used as control mice (normal). (c) Quantities of TGF‐β mRNA in subcutis colon 26 and liver colon 26 on day 17 were determined using real‐time reverse transcription–polymerase chain reaction (RT‐PCR). Each group consisted of four mice, and results are shown as mean ± SEM. Day 0 represents the day when the tumors were inoculated. *Significant differences between mice without tumor (normal) and those with subcutis colon 26 or liver colon 26.

Figure 2

Production of parathyroid hormone‐related protein (PTHrP) and interleukin (IL)‐6 in the monolayer and spheroid cultures of colon 26. Monolayer cells of colon 26 were cultured in a glucose‐containing medium under a normoxic condition (normal), glucose‐free medium under a normoxic condition (low glucose), glucose‐containing medium under 1% O₂ (hypoxia), or glucose‐free medium under 1% O₂ (hypoxia, low glucose). Spheroids of colon 26 cells were cultured in the glucose‐containing medium under a normoxic condition (spheroid). After culturing spheroids for 2 days, amounts of (a) PTHrP and (b) IL‐6 in the culture media were determined using radioimmuno assay (RIA) and ELISA, respectively. Results are shown as mean ± SD from three independent experiments. Asterisks indicate significant differences between colon 26 cells cultured in monolayer under a normal condition and those cultured under the indicated conditions. HE staining of (c) spheroids, (d) subcutis colon 26 and (e) liver colon 26 resected on day 17 are shown. Arrows in panel D indicate the areas with cells undergoing necrosis.

Figure 3

Genes whose expression was markedly increased in subcutis colon 26 and spheroid cultures of colon 26. The gene expression profiles of subcutis colon 26, liver colon 26, and monolayer cultures and spheroid cultures of colon 26 were examined using GeneChip Mouse Expression Array 430A (Affymetrix). Signals that represent the levels of mRNA for each gene were compared between subcutis colon 26 and liver colon 26 and between monolayer cultures and spheroid cultures of colon 26. Monolayer cells and spheroids of colon 26 were cultured in glucose‐containing medium under a normoxic condition. Shown are those genes whose expression was more than three‐fold higher in subcutis colon 26 than in liver colon 26 and more than three‐fold higher in spheroid cultures than in monolayer cultures. Colored bars indicate the number of signals of genes in subcutis colon 26 or spheroid cultures that were higher than 1000 (yellow), between 500 and 1000 (orange), and between 100 and 500 (gray).

Figure 4

Expression of cyclooxygenase (COX)‐2 in colon 26 and effects of NS‐398 on parathyroid hormone‐related protein (PTHrP) production. (a,b) COX‐2 expression was examined in (a) subcutis colon 26 and liver colon 26 and in (b) monolayer and spheroid cultures of colon 26 using reverse transcription–polymerase chain reaction (RT‐PCR). Monolayer cells and spheroids of colon 26 were cultured in glucose‐containing medium under a normoxic condition. Total RNA was extracted from tumors from each of the following: (a) subcutis colon 26 and liver colon 26 and (b) monolayer cultures and spheroid cultures of colon 26 cells. PCR products separated on a 2% agarose gel are shown. (a) Results from two independent tumors each from subcutis colon 26 and liver colon 26 are shown. (c) Monolayer cells and spheroids of colon 26 were cultured in glucose‐containing medium under a normoxic condition in the presence of the indicated concentrations of NS‐398 for 2 days. Amounts of PTHrP in the culture media were determined using radioimmuno assay (RIA). Results are shown as mean ± SD from three independent experiments. Asterisks indicate significant differences compared to the control (spheroids without NS‐398). (d) A monolayer cells of colon 26 was cultured in glucose‐containing (normal) or glucose‐free (low glucose) medium under a normoxic condition in the presence or absence of the indicated concentrations of NS‐398 for 2 days, and the quantity of COX‐2 mRNA and interleukin (IL)‐6 in the culture media were determined using RT‐PCR and ELISA, respectively. Results are shown as mean ± SD from three independent experiments.

Figure 5

Decrease of plasma parathyroid hormone‐related protein (PTHrP) levels from NS‐398 in mice bearing subcutis colon 26. Mice were subcutaneously inoculated with colon 26 and were administrated 4 mg/kg or 40 mg/kg of NS‐398 from day 7 to day 13. Control mice received vehicle. Blood samples and tumors were collected on day 14. (a) Plasma levels of PTHrP determined using radioimmuno assay (RIA) and (b) tumor weights are shown. Each group consisted of eight mice; results are shown as mean ± SEM. Day 0 represents the day when the tumors were inoculated. Asterisks indicate significant differences between control mice and mice administered the indicated concentrations of NS‐398.

Figure 6

Induction of parathyroid hormone‐related protein (PTHrP) production by platelet‐derived growth factor (PDGF) in the monolayer cultures of colon 26 cells. (a) Mice were subcutaneously or intrahepatically inoculated with colon 26, and plasma levels on day 17 of PDGF‐BB in mice bearing colon 26 were determined using ELISA. Non‐tumor‐bearing mice were used as control mice (normal). Each group consisted of four mice, and results are shown as mean + SEM. Day 0 represents the day when the tumors were inoculated. Asterisks indicate significant differences between mice without tumor (normal) and those with subcutis colon 26 or liver colon 26. (b,c) Monolayer cells of colon 26 were cultured in glucose‐containing medium under a normoxic condition in the presence or absence of the indicated concentrations of PDGF‐BB, PDGF‐AB, or PDGF‐AA. After culturing for 2 days, amounts of (b) PTHrP and (c) interleukin (IL)‐6 in the culture media were determined using radioimmuno assay (RIA) (for PTHrP) and ELISA (for IL‐6). Results are shown as mean + SD from three independent experiments. *Significant differences between the colon 26 cells without any treatment and those treated with the indicated concentrations of PDGF‐AA, PDGF‐AB, or PDGF‐BB. (d) Monolayer cells of colon 26 were cultured in glucose‐containing medium under a normoxic condition in the presence or absence of PDGF‐BB or NS‐398. After culturing for 2 days, amounts of PTHrP in the culture media were determined using RIA. Results are shown as mean + SD from three independent experiments. Asterisks indicate significant differences between colon 26 cells cultured with 20 ng/mL PDGF‐BB and those cultured with the indicated concentrations of NS‐398 and 20 ng/mL PDGF‐BB.