Cyclooxygenase (COX)-2-derived prostaglandins (PGs) are thought to contribute to tumor growth and resistance to radiation therapy. COX-2 protein expression is increased in many tumors including those of the breast. COX-2-derived PGs have been shown to protect cells from radiation damage. This study evaluated the role of COX-2-derived PG in radiation treatment by using the NMF11.2 mammary tumor cell line originally obtained from HER-2/neu mice that overexpress HER-2/neu. We determined whether the effects of the COX-2 inhibitor SC236 on cell growth, radiation-induced PGE2 production and COX expression, cell cycle redistribution, and vascular endothelial growth factor (VEGF) were acting through COX-2-dependent mechanisms. The NMF11.2 cells expressed both COX-1 and COX-2 protein and mRNA. The radiation treatment alone led to a dose-dependent increase in the levels of COX-2 mRNA and COX-2 protein, which was associated with an increase in the production of PGE2 and prostacyclin (PGI2). Treating NMF11.2 cells with high concentrations (20 μm) of SC236 for 48 h reduced the radiation-induced increase in COX-2 activity and also decreased cell growth. SC236 (20 μm) increased the accumulation of the cells in the radiosensitive G2-M phase of the cell cycle. However, a low concentration (5 μm) of SC236 was adequate to reduce COX-2 activity. The lower concentration of SC236 (5 μm) also decreased cell growth after a longer incubation period (96 h) and, in combination with a 2 or 5 Gy dose, led to an accumulation of cells in G2-M phase. Restoring PG to control values in cells treated with 5 μm SC236 prevented the growth inhibition and G2-M cell cycle arrest. Radiation treatment of NMF11.2 cells also increased VEGF protein expression and VEGF secretion in a dose-dependent manner, which was blocked in those cells pretreated with 20 μm SC236 but not in those pretreated with 5 μm SC236. These findings indicate that the COX-2 inhibitor SC236 reduced cell growth and arrested cells in the G2-M phase of the cell cycle by mechanisms that are both dependent and independent of PG production while its effects on VEGF appear to be independent of COX-2.

Breast cancer is the most common type of cancer and is the second leading cause of death in women in the United States. Although successful treatments are available to treat estrogen receptor (ER)-positive breast cancer, strategies to treat ER-negative breast cancer are limited and associated with undesirable side effects. This tumor phenotype is found in 25–30% of human breast cancers (1). These tumors occur more often in premenopausal women and they do not respond well to traditional therapies.

Evidence is growing to support the use of the nonsteroidal anti-inflammatory (NSAIDs) drugs for the chemoprevention of many types of cancer. A recent meta-analysis also suggests that NSAIDs may protect against breast cancer (2). These drugs inhibit cyclooxygenase (COX), the enzyme controlling the conversion of arachidonic acid into prostaglandins (PGs). Two isoforms of COX exist: COX-1 is thought to be the constituent isoform involved in homeostasis of normal cell functioning and COX-2 can be induced by cytokines, growth factors, and tumor promoters. COX-2 is up-regulated in many types of human cancer including those of the breast and is thought to contribute to tumor growth and metastasis (3–11). In support of this view, overexpression of COX-2 in a transgenic mouse model was sufficient to induce mammary tumors with increased concentrations of PGE2, PGD2, and PGF2α (12). Increased expression of COX-2 is associated with elevated concentrations of PGE2 in human breast cancers (13, 14). Breast tumors that have ER-negative status and amplification of HER-2/neu tend to overexpress COX-2 (15, 16). Recently, increased amounts of COX-2 protein were found in HER-2/neu-positive breast tumors (17). Moreover, COX-2 protein was induced after transfecting colon cancer cells with HER-2/neu (18). Recent studies have shown that select inhibitors of COX-2 retard the growth of established mammary tumors that express COX-2 (19–21). Thus, suppressing COX-2 may be an important target in the treatment of breast cancer.

There is a growing interest in the potential use of select COX-2 inhibitors in combination with chemotherapy or radiation therapy. Earlier studies have demonstrated that tumors were more responsive to radiation when pretreated with the NSAID, indomethacin, prior to radiation treatment (22, 23). More recently, Milas et al. (24) demonstrated that select COX-2 inhibitors improved the radiation response in mice bearing a new fibrosarcoma and Kishi et al. (25) confirmed this finding. Peterson et al. (26) demonstrated in vitro that COX-2 inhibitors increased the radiosensitivity of human glioma cells that express COX-2. Another study reported that the radiosensitivity of the COX-2 inhibitors was found only in cells that expressed COX-2 (27). Raju et al. (28) observed that the radioenhancing effect of the COX-2 inhibitors in murine sarcoma cells could be attributed to an accumulation of cells in the radiosensitive G2-M phase of the cell cycle. The COX products, PGE2 and PGI2, which are elevated in many types of tumors, have been shown to protect cancer cells from radiation damage (22). PGE2 and PGI2 support the growth of tumors by stimulating angiogenesis through up-regulation of proangiogenic factors such as vascular endothelial growth factor (VEGF), which serve to increase the supply of oxygen and nutrients to the tumor (20, 29–32). Because previous studies have used very high concentrations of COX-2 inhibitors, above what would be sufficient to reduce PG synthesis, we do not know whether PGs have a role in the radiosensitizing effect of the COX-2 inhibitors. The aim of this study was to evaluate the role of COX-2-derived PGs after radiation treatment by determining the effects of the COX-2 inhibitor SC236 on cell growth, radiation-induced increases in PGE2 production and COX expression, VEGF expression, and cell cycle redistribution at doses that are relevant clinically. We used the NMF11.2 cell line derived from mammary tumor cells from HER-2/neu mice. Mammary tumors from HER-2/neu mice are a model of ER-negative breast cancer; the HER-2/neu mice develop mammary tumors spontaneously after a long period of growth and maturation and express COX-2.

Cell Culture

The mouse mammary tumor virus HER-2/neu cell line NMF11.2 (kindly provided by Sandra J. Gendler, Mayo Clinic, Scottsdale, AZ) was derived from mammary gland tumors of HER-2/neu mice. Cells were cultured in improved MEM zinc option medium without phenol red (Life Technologies, Inc., Grand Island, NY; catalog 10373-017). The medium was supplemented with 5% fetal bovine serum, which had been pretreated with charcoal and dextran (Hyclone Laboratories, Logan, UT), and 1% Glutamax-1 supplement solution (Life Technologies) as an additional source of glutamine. Cells were maintained at 37°C in an atmosphere of 5% CO2. To remove the adherent cells from the flask for passaging or counting, cells were washed with HBSS without calcium or magnesium, incubated with a small volume of 0.25% trypsin-EDTA solution (Sigma Chemical Co., St. Louis, MO) for 5–10 min, and washed with culture medium by centrifugation.

Cell Viability

Cell viability was determined using trypan blue exclusion or a cell growth and viability assay (Intergen Co., Purchase, NY). Cells (2.3 × 106 cells/flask) were plated and incubated for 96 h with different concentrations of SC236. Then, cells were trypsinized and washed with culture medium by centrifugation and the cell pellet was resuspended in 0.5 or 1 ml of culture medium. The cell suspension was placed in a trypan blue solution and an aliquot was counted using a hemocytometer. Cell number was determined as the average of three counts.

Cells were plated on a 96-well plate at a concentration of 9.4 × 103 cells/well in 100 μl. Twenty-four hours later, each well was incubated for 24, 48, or 72 h with various concentrations of SC236 dissolved in DMSO (<0.1%). At the end of the experiment, 20 μl ProCheck reagent was added to each well and the plates were placed in the incubator for 4 h. After incubation, the absorbance of the supernatant was determined at 475 nm. This assay is based on the reduction of a water-soluble tetrazolium salt, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt, by cellular enzymes in viable cells. The absorbance of the reduced product is proportional to the cell number. To relate cell number to absorbance, various numbers of cells were added to the wells of a 96-well plate. After allowing the cells to attach, ProCheck reagent (20 μl) was added to each well. The plate was incubated, and subsequently, the absorbance in the wells was determined. A graph of absorbance versus cell number was constructed to serve as a standard curve.

Cell Cycle Analysis

Cells (2.5 × 105) were preincubated with 5 or 20 μm SC236 overnight followed by γ irradiation using an X-ray machine (PanTak, Bradford, CT) at doses of 0, 2, 5, and 10 Gy and maintained in culture for an additional 48 h. Attached and floating cells were harvested, fixed in 70% ethanol, digested with RNase A, stained with propidium iodide, and analyzed by fluorescence-activated cell sorting using a Beckman Coulter XL-MCL flow cytometer with System II software.

Western Blot Analysis of COX Enzymes

The protein expression of COX-2 was measured in the cells treated with and without SC236 and irradiated. At the termination of the experiment, the cell monolayer was placed in the freezer overnight. The next day, 5 ml of 0.2% SDS solution containing 5% protease inhibitor (Sigma Chemical; catalog P8340) were placed in the flask, and after a short incubation, the cells were removed by scraping. The cell suspension was sonicated, incubated on ice for 30 min, and centrifuged at 100,000 × g for 30 min. Protein extracts from cells were electrophoresed at constant voltage (100 V) on a 7.5% SDS-PAGE under reducing conditions and transferred to nitrocellulose paper. The blots were incubated overnight with PBS containing 0.1% Tween 20 and 5% powered milk (blocking solution) and then incubated with either goat anti-COX-1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-COX-2, or rabbit anti-VEGF antibodies. The membranes were washed six times with blocking solution, incubated with either mouse anti-goat or mouse monoclonal anti-rabbit IgG conjugated to alkaline phosphatase (Sigma-Aldrich, St. Louis, MO), and washed six times with blocking solution. Bands were visualized by chemiluminescence (CDP Star; NEN Life Science Products, Boston, MA).

RNA Isolation

Total RNA was extracted from cells using the acid guanidium-phenol-chloroform extraction method described by Chomczynski and Sacchi (33).

Reverse Transcription-PCR

An aliquot (50 ng) of total RNA was reverse transcribed to cDNA using oligo and Sensiscript Reverse Transcriptase Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer's instruction. The COX-1 cDNA was amplified using forward primer (5′-AGGAGATGGCTGCTGAGTTGG-3) corresponding to 1496–1516 and reverse primer (5′-CTCAGAGCTCAGTGGAGCGTC-3) corresponding to 1846–1826 of the mouse COX-1 gene (Genbank accession no. NM008969). The COX-2 cDNA was amplified using forward primer (5′-ACACACTCTATCACTGGCACC-3′) corresponding to 1227–1247 and reverse primer (5′-AGCAGGCAGGGTACAGTTCC-3′) corresponding to 1605–1585 of the mouse COX-2 gene (Genbank accession no. NM011198). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment was amplified using forward primer (5′-CACCACCATGGAGAAGGCTG-3′) corresponding to 330–349 and reverse primer (5′-ATGATGTTCTGGGCTGCCCC-3′) corresponding to 644–625 of the rat GAPDH gene (Genbank accession no. NM017008). Amplification was provided using HotStarTaq Master Mix Kit (Qiagen) according to the manufacturer's instructions. Amplified PCR products were analyzed by electrophoresis using 2% agarose gel. Gels were analyzed using Quantity One Bio-Rad System (Bio-Rad Laboratories, Hercules, CA). Intensity of the COX-1 and COX-2 cDNA bands was normalized to the GAPDH cDNA band.

Production of PGI2 and PGE2

PGE2 and PGI2 were measured by RIA using polyclonal rabbit to PGE2 or polyclonal rabbit to 6-keto PGF (34, 35). To assess PGE2 and PGI2 production in the presence of excess arachidonic acid, arachidonic acid (10 μm) was added to the medium 48 h after radiation and the cells were incubated for an additional 20 min; then, the medium was harvested by centrifugation at 5000 × g at 4°C and assayed.

Production of VEGF

NMF11.2 cells (2.3 × 106 cells/flask) were plated, allowed to attach overnight, pretreated with or without 20 μm SC236 for 24 h, irradiating with 0, 2, 5, and 10 Gy, and incubating for an additional 48 h. The medium was removed, centrifuged at 5000 × g for 15 min, and analyzed for VEGF by ELISA according to the manufacturer's instructions (R&D Systems, Inc., Minneapolis, MN). Data were normalized to cell number in each flask.

Statistical Analysis

Data are mean values of at least three replicate experiments and expressed as means ± SD. PG concentrations, cell growth, and cell cycle data were analyzed by analysis of variance. P < 0.05 is considered statistically significant.

SC236 Inhibits Radiation-Induced COX-2 Activity in NMF11.2 Cells

NMF11.2 cells produce PGE2 and PGI2. We evaluated the ability of a low and high dose of SC236 to block the radiation-induced increase in COX-2 activity. To determine the low dose concentration, COX activity was measured by incubating cells with 1, 3, or 5 μm SC236 for 72 h and measuring the concentration of PGE2 and PGI2 produced in the presence of excess arachidonic acid (data not shown). We observed that the 5 μm dose inhibited production of PGE2 below the level of detection by the RIA.

The effect of radiation on COX-2 activity in NMF11.2 cells was evaluated by preincubating the cells with 5 or 20 μm SC236 for 24 h, irradiated with 0, 2, 5, and 10 Gy, and incubated for an additional 48 h. Then, the medium was removed and fresh medium with 10 μm arachidonic acid was added to the cells and they were incubated for an additional 20 min. The production of PGE2 and PGI2 was increased after 2, 5, and 10 Gy in comparison with nonirradiated cells. Both concentrations of SC236 (5 and 20 μm) inhibited the radiation-induced increase in COX activity (Table 1).

Table 1.

Effect of SC236 on radiation-induced increase in COX activity

Treatment groupPGE2 (ng/106 cells)PGI2 (ng/106 cells)
−SC236   
 0 Gy 1.95 ± 0.047 0.38 ± 0.033 
 2 Gy 2.13 ± 0. 071 0.69 ± 0.022 
 5 Gy 2.70 ± 0.146 0.92 ± 0.044 
 10 Gy 3.11 ± 0.131 1.32 ± 0.027 
+5 μm SC236   
 0 Gy <0.100 0.139 ± 0.013* 
 2 Gy <0.100 0.122 ± 0.013* 
 5 Gy <0.1001 0.146 ± 0.004* 
 10 Gy <0.100 0.206 ± 0.038* 
+20 μm SC236   
 0 Gy <0.100 <0.100 
 2 Gy <0.100 <0.100 
 5 Gy <0.100 <0.100 
 10 Gy <0.100 <0.100 
Treatment groupPGE2 (ng/106 cells)PGI2 (ng/106 cells)
−SC236   
 0 Gy 1.95 ± 0.047 0.38 ± 0.033 
 2 Gy 2.13 ± 0. 071 0.69 ± 0.022 
 5 Gy 2.70 ± 0.146 0.92 ± 0.044 
 10 Gy 3.11 ± 0.131 1.32 ± 0.027 
+5 μm SC236   
 0 Gy <0.100 0.139 ± 0.013* 
 2 Gy <0.100 0.122 ± 0.013* 
 5 Gy <0.1001 0.146 ± 0.004* 
 10 Gy <0.100 0.206 ± 0.038* 
+20 μm SC236   
 0 Gy <0.100 <0.100 
 2 Gy <0.100 <0.100 
 5 Gy <0.100 <0.100 
 10 Gy <0.100 <0.100 

Note: NMF11.2 cells were pretreated with 5 or 20 μm SC236 for 24 h, irradiated with 0, 2, 5, and 10 Gy, and incubated for an additional 48 h. Total COX-2 activity was measured by adding 10 μm arachidonic acid to the media and incubating for an additional 20 min. Then, the medium was harvested and assayed for PGE2 and PGI2 by RIA. <0.100 was below the limit of accurate detection. Means ± SD for n = 4.

*

P < 0.05, SC236-treated cells are statistically different from corresponding group.

COX-2 Protein and COX-2 mRNA Levels Are Increased after Radiation

Western blot analysis showed that NMF11.2 cells express COX-1 and COX-2 protein. COX-1 and COX-2 mRNA were detected in NMF11.2 cells by reverse transcription-PCR (Fig. 1). Irradiating the cells did not alter the expression of COX-1 in the cells. However, the radiation-induced increase in PGE2 and PGI2 was associated with a dose response increase in COX-2 protein measured by Western blot and COX-2 mRNA was analyzed by reverse transcription-PCR. We also observed that SC236 alone increased the expression of COX-2 protein and COX-2 mRNA levels, although COX-2 activity was decreased (Table 1).

Figure 1.

Radiation increased the levels of COX-2 protein and COX-2 mRNA. A, COX-1 protein and COX-1 mRNA. B, COX-2 protein and COX-2 mRNA. NMF11.2 cells were incubated with or without 20 μm SC236 for 24 h before irradiation with 0, 2, 5, and 10 Gy. Cells were then incubated for an additional 48 h. Total RNA from NMF11.2 cells was subject to RT-PCR. Amplification products were electrophoresed in a 2% agarose gel. Intensity of each band was normalized to GAPDH cDNA band. Western blots were performed by using a rabbit anti-COX-2 antibody.

Figure 1.

Radiation increased the levels of COX-2 protein and COX-2 mRNA. A, COX-1 protein and COX-1 mRNA. B, COX-2 protein and COX-2 mRNA. NMF11.2 cells were incubated with or without 20 μm SC236 for 24 h before irradiation with 0, 2, 5, and 10 Gy. Cells were then incubated for an additional 48 h. Total RNA from NMF11.2 cells was subject to RT-PCR. Amplification products were electrophoresed in a 2% agarose gel. Intensity of each band was normalized to GAPDH cDNA band. Western blots were performed by using a rabbit anti-COX-2 antibody.

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The COX-2 Inhibitor SC236 Decreases Cell Growth in NMF11.2 Cells

NMF11.2 cells were incubated with several concentrations of SC236 (0–40 μm) for 0, 24, 48, and 72 h. Analysis of cell growth using the ProCheck cell viability assay showed that SC236 produced a concentration-dependent and time-dependent inhibition of cell growth. SC236 significantly decreased the number of viable cells at 10, 20, and 40 μm concentrations after 48 and 72 h of incubation in comparison with untreated cells (Fig. 2).

Figure 2.

The COX-2 inhibitor SC236 inhibited cell growth and viability in a dose-dependent and time-dependent manner. NMF11.2 cells were treated with different concentrations of the SC236 for 24, 48, or 72 h. Cell growth and viability were quantified by the ProCheck cell viability assay. Points, mean of three experiments; bars, SD. *, P < 0.05, statistically different from untreated cells.

Figure 2.

The COX-2 inhibitor SC236 inhibited cell growth and viability in a dose-dependent and time-dependent manner. NMF11.2 cells were treated with different concentrations of the SC236 for 24, 48, or 72 h. Cell growth and viability were quantified by the ProCheck cell viability assay. Points, mean of three experiments; bars, SD. *, P < 0.05, statistically different from untreated cells.

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To determine whether SC236 also had a COX-2-dependent effect on cell growth, we conducted a separate experiment in which NMF11.2 cells were incubated for 96 h with several concentrations of SC236 (0–40 μm). Figure 3 shows that 5 μm SC236, which was a sufficient concentration to inhibit COX activity, significantly decreased cell growth after the longer incubation time. To confirm that this effect was due to the decrease in PG, we added PGE2 to the incubation medium at a concentration similar to that produced by the control cells. We chose to add only PGE2 because the NMF11.2 cells produced more PGE2 than prostacyclin (PGI2). Cells were incubated for 96 h; every 48 h, the medium was removed and fresh medium was added. The addition of PGE2 to the medium prevented the SC236-induced decrease in cell growth (Table 2). In contrast, addition of PGE2 to the medium of cells treated with 20 μm SC236 did not abrogate the growth inhibitory effects of SC236 (data not shown).

Figure 3.

The COX-2 inhibitor SC236 reduced viability of NMF11.2 cells. NMF11.2 cells were treated with different concentrations of SC236 for 96 h. Cell viability was determined by trypan blue exclusion. Columns, mean of three separate experiments; bars, SD. *, P < 0.05, statistically different from untreated cells.

Figure 3.

The COX-2 inhibitor SC236 reduced viability of NMF11.2 cells. NMF11.2 cells were treated with different concentrations of SC236 for 96 h. Cell viability was determined by trypan blue exclusion. Columns, mean of three separate experiments; bars, SD. *, P < 0.05, statistically different from untreated cells.

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Table 2.

PGE2 abolishes the growth inhibitory effect of SC236

TreatmentTotal number of cells (1 × 106)
Control 18.3 ± 0.84 
SC236 (5 μm12.1 ± 0.54* 
SC236 (5 μm) + PGE2 19.1 ± 0.92* 
TreatmentTotal number of cells (1 × 106)
Control 18.3 ± 0.84 
SC236 (5 μm12.1 ± 0.54* 
SC236 (5 μm) + PGE2 19.1 ± 0.92* 

Note: NMF11.2 cells were treated with 5 μm SC236 or 5 μm SC236 + 2 ng/ml PGE2 for 96 h. The medium was changed every 48 h and medium with fresh compounds was added. Cell viability was determined by trypan blue exclusion. Means ± SD of triplicates of three separate experiments.

*

P < 0.05, SC236-treated cells are statistically different from the controls.

SC236 Alters Cell Cycle Progression of NMF11.2 Cells

To evaluate whether the growth inhibitory effects of SC236 on NMF11.2 cells were correlated with changes in cell cycle progression, we pretreated the cells for 24 h with and without 20 μm SC236 prior to radiation and incubated them for an additional 48 h after radiation. The cells were then stained with propidium iodide and analyzed by flow cytometry. NMF11.2 cells treated with radiation alone showed an increase in the G2-M phase (Table 3). Pretreating the cells for 24 h with SC236 prior to radiation further increased the percentage of cells in the G2-M phase (Table 3).

Table 3.

Effect of SC236 on cell cycle phase distribution of NMF11.2 cells

TreatmentG0/G1 (%)S (%)G2-M (%)Apoptosis (%)
0 Gy 45.6 ± 2.2 25.2 ± 1.7 23.6 ± 1.4 0.80 ± 0.06 
2 Gy 43.9 ± 2.6 22.4 ± 1.4 26.0 ± 1.5 0.99 ± 0.08 
5 Gy 38.8 ± 2.0 17.5 ± 1.6 35.2 ± 1.9 1.51 ± 0.19 
10 Gy 27.4 ± 1.5 15.0 ± 1.1 48.6 ± 2.1 1.50 ± 0.12 
0 Gy + SC236 42.8 ± 2.6 22.1 ± 1.3 33.9 ± 1.9* 0.89 ± 0.04 
2 Gy + SC236 39.9 ± 1.9 17.8 ± 1.2 36.3 ± 1.2* 1.08 ± 0.10 
5 Gy + SC236 26.8 ± 1.6* 11.2 ± 0.6 58.6 ± 2.5* 1.69 ± 0.08 
>10 Gy + SC236 22.1 ± 1.5 10.6 ± 0.8 61.7 ± 2.7* 2.52 ± 0.59 
TreatmentG0/G1 (%)S (%)G2-M (%)Apoptosis (%)
0 Gy 45.6 ± 2.2 25.2 ± 1.7 23.6 ± 1.4 0.80 ± 0.06 
2 Gy 43.9 ± 2.6 22.4 ± 1.4 26.0 ± 1.5 0.99 ± 0.08 
5 Gy 38.8 ± 2.0 17.5 ± 1.6 35.2 ± 1.9 1.51 ± 0.19 
10 Gy 27.4 ± 1.5 15.0 ± 1.1 48.6 ± 2.1 1.50 ± 0.12 
0 Gy + SC236 42.8 ± 2.6 22.1 ± 1.3 33.9 ± 1.9* 0.89 ± 0.04 
2 Gy + SC236 39.9 ± 1.9 17.8 ± 1.2 36.3 ± 1.2* 1.08 ± 0.10 
5 Gy + SC236 26.8 ± 1.6* 11.2 ± 0.6 58.6 ± 2.5* 1.69 ± 0.08 
>10 Gy + SC236 22.1 ± 1.5 10.6 ± 0.8 61.7 ± 2.7* 2.52 ± 0.59 

Note: NMF11.2 cells were incubated for 24 h with vehicle or 20 μm SC236, irradiated with 0, 2, 5, and 10 Gy, and cultured for 48 h. Cells were harvested and stained with propidium iodide. DNA content was analyzed by flow cytometry as described in Materials and Methods. Means ± SD of three independent experiments.

*

P < 0.05, SC236-treated cells are statistically different from corresponding group not treated with SC236.

We also determined whether SC236 could alter the cell cycle in a COX-2-dependent manner by using a lower concentration of SC236. NMF11.2 cells were preincubated with 5 μm SC236 for 96 h prior to radiation. The medium was changed every 48 h. Then, cells were irradiated and incubated for an additional 48 h. Our findings show that pretreating the cells for 96 h with a low concentration of SC236, similar to the concentration found to be adequate for inhibition of PGE2 production in NMF11.2 cells, resulted in accumulation of cells in G2-M after radiation (Table 4). To confirm the role of COX-2-derived PGE2, we added PGE2, in the concentration produced by the control cells, to see if we could reverse the effects of SC236. Adding back PGE2 prevented the SC236-induced increase in G2-M at 2 and 5 Gy but not at 10 Gy.

Table 4.

COX-2-dependent effects of SC236 on cell cycle phase distribution of NMF11.2 cells

TreatmentG0/G1 (%)S (%)G2-M (%)Apoptosis (%)
0 Gy 48.2 ± 1.4 22.5 ± 1.9 23.1 ± 1.7 0.68 ± 0.07 
2 Gy 45.3 ± 1.8 20.4 ± 1.6 27.8 ± 1.5 0.76 ±0.06 
5 Gy 39.1 ± 1.2 15.7 ± 1.5 37.4 ± 1.1 1.85 ± 0.35 
10 Gy 30.4 ± 1.7 13.2 ± 1.4 47.2 ± 1.6 1.65 ± 0.91 
0 Gy + SC236 38.3 ± 1.5* 23.1 ± 1.5 32.5 ± 2.1* 0.76 ± 0.08 
2 Gy + SC236 36.5 ± 1.9* 18.4 ± 0.9 37.1 ± 2.3* 1.24 ± 0.36 
5 Gy + SC236 28.4 ± 1.8* 12.6 ± 1.6 51.6 ± 2.1* 1.45 ± 0.66 
10 Gy + SC236 32.6 ± 2.0 12.7 ± 1.4 50.6 ± 2.4 1.11 ± 0.57 
0 Gy + SC236 + PGE2 47.8 ± 1.8 24.1 ± 1.3 22.4 ± 1.6 0.70 ± 0.09 
2 Gy + SC236 + PGE2 48.1 ± 1.6 22.3 ± 1.7 24.8 ± 0.8 0.76 ± 0.05 
5 Gy + SC236 + PGE2 29.0 ± 1.5 15.4 ± 0.9 47.3 ± 2.2 1.96 ± 0.09 
10 Gy + SC236 + PGE2 29.6 ± 1.6 12.3 ± 0.7 49.7 ± 2.1 1.88 ± 0.15 
TreatmentG0/G1 (%)S (%)G2-M (%)Apoptosis (%)
0 Gy 48.2 ± 1.4 22.5 ± 1.9 23.1 ± 1.7 0.68 ± 0.07 
2 Gy 45.3 ± 1.8 20.4 ± 1.6 27.8 ± 1.5 0.76 ±0.06 
5 Gy 39.1 ± 1.2 15.7 ± 1.5 37.4 ± 1.1 1.85 ± 0.35 
10 Gy 30.4 ± 1.7 13.2 ± 1.4 47.2 ± 1.6 1.65 ± 0.91 
0 Gy + SC236 38.3 ± 1.5* 23.1 ± 1.5 32.5 ± 2.1* 0.76 ± 0.08 
2 Gy + SC236 36.5 ± 1.9* 18.4 ± 0.9 37.1 ± 2.3* 1.24 ± 0.36 
5 Gy + SC236 28.4 ± 1.8* 12.6 ± 1.6 51.6 ± 2.1* 1.45 ± 0.66 
10 Gy + SC236 32.6 ± 2.0 12.7 ± 1.4 50.6 ± 2.4 1.11 ± 0.57 
0 Gy + SC236 + PGE2 47.8 ± 1.8 24.1 ± 1.3 22.4 ± 1.6 0.70 ± 0.09 
2 Gy + SC236 + PGE2 48.1 ± 1.6 22.3 ± 1.7 24.8 ± 0.8 0.76 ± 0.05 
5 Gy + SC236 + PGE2 29.0 ± 1.5 15.4 ± 0.9 47.3 ± 2.2 1.96 ± 0.09 
10 Gy + SC236 + PGE2 29.6 ± 1.6 12.3 ± 0.7 49.7 ± 2.1 1.88 ± 0.15 

Note: NMF11.2 cells were incubated for 96 h with vehicle, 5 μm SC236, or 5 μm SC236 + 2 ng/ml of PGE2, irradiated with 0, 2, 5, and 10 Gy, and cultured for 48 h. Cells were harvested and stained with propidium iodide. DNA content was analyzed by flow cytometry as described in Materials and Methods. Means ± SD for three independent experiments.

*

P < 0.05, SC236-treated cells are statistically different from corresponding group not treated with SC236.

P < 0.05, SC236- and PGE2-treated cells are statistically different from corresponding group treated with SC236 alone.

SC236 Inhibits Radiation-Induced Increase in VEGF Protein

Because COX-2 inhibitors have been shown to inhibit angiogenesis (20, 36), we evaluated whether SC236 would alter the protein expression of VEGF, one of the most potent angiogenic growth factors. We observed that radiation increased the protein expression of VEGF while pretreating the cells for 24 h with 20 μm SC236 prior to radiation blocked the up-regulation of VEGF (Fig. 4). The lower dose of SC236 (5 μm), which was adequate to reduce PG production, did not have an effect on the radiation-induced increase in VEGF expression (data not shown).

Figure 4.

SC236 inhibited the radiation-induced increase in VEGF protein. NMF11.2 cells were incubated with or without 20 μm SC236 for 24 h before irradiation with 0, 2, 5, and 10 Gy. Cells were then incubated for an additional 48 h. Western blots were performed by using a rabbit anti-VEGF antibody.

Figure 4.

SC236 inhibited the radiation-induced increase in VEGF protein. NMF11.2 cells were incubated with or without 20 μm SC236 for 24 h before irradiation with 0, 2, 5, and 10 Gy. Cells were then incubated for an additional 48 h. Western blots were performed by using a rabbit anti-VEGF antibody.

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SC236 Inhibits Radiation-Induced Increase in VEGF Secretion

Radiation also increased the secretion of VEGF in the media, which was measured by an ELISA and treating the cells with 20 μm SC236 blocked the radiation-induced increase in VEGF secretion (Table 5).

Table 5.

VEGF concentration in the culture media from NMF11.2 cells treated with SC236 alone, radiation alone, or combination of SC236 and radiation

Treatment groupVEGF (pg/106 cells)
−SC236  
 0 Gy 631 ± 40 
 2 Gy 753 ± 17 
 5 Gy 968 ± 18 
 10 Gy 990 ± 17 
+20 μm SC236  
 0 Gy 667 ± 23 
 2 Gy 602 ± 24* 
 5 Gy 513 ± 24* 
 10 Gy 499 ± 25* 
Treatment groupVEGF (pg/106 cells)
−SC236  
 0 Gy 631 ± 40 
 2 Gy 753 ± 17 
 5 Gy 968 ± 18 
 10 Gy 990 ± 17 
+20 μm SC236  
 0 Gy 667 ± 23 
 2 Gy 602 ± 24* 
 5 Gy 513 ± 24* 
 10 Gy 499 ± 25* 

Note: NMF11.2 cells were incubated with or without 20 μm SC236 for 24 h before irradiation with 0, 2, 5, and 10 Gy. Culture media was collected after 48 h of incubation and VEGF concentrations in the media were determined by ELISA. Results were normalized to cell number in each flask. Means ± SE for n = 6.

*

P < 0.05, statistically different from untreated cells.

In this report, we have demonstrated that the antitumor effect of the COX-2 inhibitor SC236 can be explained by both COX-2-dependent and COX-2-independent mechanisms in HER-2/neu-positive mammary tumor cells. We observed that mammary tumor cells from HER-2/neu mice express COX-2 protein and radiation treatment increased COX-2 expression and COX-2 activity in a dose-dependent manner. After 48 h of incubation, 5 μm SC236 was adequate to inhibit COX activity but not proliferation, whereas 20 μm SC236 reduced both COX-2 activity and cell growth, which suggests that the higher dose was acting through COX-2-independent mechanisms. A longer incubation time (96 h) was required for the lower dose of SC236 (5 μm) to inhibit cell proliferation. To test further for a COX-2-dependent mechanism, we added PGE2 to the medium with SC236 at a concentration observed in control cells. Cell growth was restored, which provides additional evidence that the SC236-induced decrease in cell growth was dependent on inhibition of PGs. A recent study has also documented COX-2-dependent and COX-2-independent effects in oral cancer cells (37). These findings suggest that the inhibition of cell growth by SC236 in NMF11.2 cells can occur by mechanisms that are dependent and independent of PG production. A similar conclusion may also be made with regard to colon cancer cells (38). There is evidence to support that COX-2 inhibitors act by COX-2-dependent and COX-2-independent mechanisms. COX-2 inhibitors have been shown to inhibit proliferation in cells that do not express COX-2 (39–42). COX-2 derivatives that do not have COX activity have also been found to inhibit proliferation and induce apoptosis (42). In contrast, there is evidence to suggest that the antitumor effects of COX-2 inhibitors are dependent on inhibition of COX-2 activity. Treating mice carrying the APCΔ716 gene with a select COX-2 inhibitor reduced the number of polyps in a dose-dependent manner (43). Furthermore, deleting the COX-2 gene in mice carrying a mutation in the FAP gene at codon 716 (FAPΔ716) produced significantly fewer intestinal polyps than FAPΔ716 controls with COX-2 (39). Our laboratory and another have also demonstrated that the COX-2 inhibitor celecoxib reduced the incidence of mammary tumors from HER-2/neu mice that express COX-2 (44, 45).

The mechanisms for the radiosensitizing effect of the COX-2 inhibitors are still unclear and appear to involve multiple mechanisms (46). COX-2 inhibitors have been shown to decrease cell proliferation (47, 48), alter progression of the cell cycle (23), induce apoptosis (40–42), and regulate angiogenesis (49, 50). In this study, we show that irradiating NMF11.2 mammary tumors cells increased COX-2 activity and the expression of COX-2 protein and COX-2 mRNA. Another laboratory has also observed increased production of PGs and COX-2 protein in a prostate cancer cell line following radiation (51). Addition of SC236 to NMF11.2 cells reduced the radiation-induced increase in COX-2 activity but did not alter COX-2 protein or COX-2 mRNA; this finding is consistent to what has been observed in vivo (25). However, SC236 treatment alone increased COX-2 protein and mRNA levels in NMF11.2, which may reflect alterations in the transcription, post-transcription, post-translation, or stability of COX-2. The concentration of PG may regulate synthesis of COX through a negative feedback mechanism; thus, the decrease in COX activity by the COX-2 inhibitor may trigger the production of COX-2. NSAIDs have been shown to stimulate the production of COX-2 mRNA and protein in chick embryo fibroblasts (52), the macrophage cell line J774 (53), and fetal hepatocytes (54).

The increased production of PGE2 and PGI2 is thought to protect cancer cells from radiation damage (19), stimulate angiogenic growth factors (30), and promote angiogenesis (20, 29, 31, 32). The COX-2 inhibitors have been shown to inhibit neovascularization resulting in reduced tumor growth (20, 36). Zweifel et al. (55) further demonstrated that the reduction in COX-2-derived PGs accounted for the antitumor effects of the COX-2 inhibitor celecoxib. However, the mechanisms for this effect are not clear. Ionizing radiation increased VEGF production in several human cell lines (56). However, to our knowledge, this is the first report showing that radiation induced VEGF protein expression in mammary cancer cells and VEGF secretion from the cells that can be blocked by pretreatment with a COX-2 inhibitor. Our findings suggest that the COX-2-derived PGs may not be involved in the regulation of VEGF as pretreating the cells with the lower concentration of SC236 (5 μm), which was sufficient to lower PGs, was not effective in blocking the increase in VEGF induced by radiation. An early study showed that nonspecific COX inhibitors might enhance the radioresponse of tumor cells by producing an accumulation of cells in the radiosensitive G2-M phase, which is considered to be most sensitive phase to radiation (23). A more recent report showed that SC236 radiosensitized murine sarcoma cells to radiation by inducing a block of the cells in the G2-M phase of the cell cycle (28). This mechanism also appears to be involved in the radioenhancement of SC236 of NMF11.2 mammary tumor cells. Our results demonstrate that SC236 arrests NMF11.2 cells in the G2-M phase prior to radiation and that PGs have a role in this effect.

In summary, SC236 reduced cell growth and increased the accumulation of NMF11.2 mammary tumor cells in the radiosensitive G2-M phase of the cell cycle by mechanisms that are both dependent and independent of PG synthesis. We also showed that SC236 inhibited the induction of VEGF protein expression by radiation, which may be a mechanism for the SC236-induced inhibition of angiogenesis observed in radiated tumors from mice bearing the fibrosarcoma (24, 25). The antiangiogenic effects of SC236 on VEGF may be attributed to other cellular processes independent of PG production, although PG-dependent effects cannot be excluded. Our findings show the improved radioresponse observed with SC236 involves mechanisms that are dependent and independent of COX-2. Further investigation is needed as to which cellular responses are COX-2 dependent in the radiosensitizing effect of the COX-2 inhibitors.

Grant support: U.S. Army Medical Research Material Command grant DAMD17-00-1-0299 (S. Lanza-Jacoby), National Institute of Cancer Research grant CA89784, and Commonwealth of Pennsylvania Tobacco Settlement Act (R. Burd and A. Dicker).

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.

We thank Sandra Gendler for providing the NMF11.2 cell line from mammary tumors from HER-2/neu mice.

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