Effect of Tropolone, Azulene and Azulenequinone Derivatives on Prostaglandin E₂ Production by Activated Macrophage-like Cells

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) (Invitrogen Corp, Carlsbad, CA, USA); fetal bovine serum (FBS) (Gemini Bio-Products, Woodland, CA, USA), LPS from Escherichia coli (Serotype 0111:B4) (Sigma Chemical Ind., St. Louis, MO, USA) and tropolone (T-3) (Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan).

Synthesis of tropolone, azulene and azulenequinone derivatives. T-21 (8), A-9 (9), A-11 (9), AQ-8 (10, 11) and AQ-13 (10, 12, 13) were prepared, according to the reports in the references cited. Cell culture. The RAW264.7 cells, established from the peritoneal fluid of BALB/c mice and showing the phenotypic characteristics of monocytes and macrophages (14), were subcultured in DMEM supplemented with 10% heat-inactivated FBS under a humidified 5% CO₂ atmosphere and incubated with various concentrations of test compounds in the presence or absence of LPS (100 ng/ml).

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Figure 1.

Structure of compounds used in this study.

Assay for cytotoxic activity. The cytotoxic activity of the azulenes was determined by the MTT method, and expressed as absorbance at 540 nm of the MTT-stained cells. The 50% cytotoxic concentration (CC₅₀) was determined from the dose-response curve (1-3).

Assay for NO concentration. Near confluent RAW264.7 cells were incubated for 24 hours with each test sample in phenol red-free DMEM supplemented with 10% FBS, and the NO production was quantified by Greiss reagent, using the standard curve of NO ^-₂. To eliminate the interaction between the sample and the Greiss reagent, the NO concentration in the culture medium without cells was also measured, and subtracted from the value obtained with the cells. The concentration that inhibited the LPS-stimulated NO production by 50% (50% inhibitory concentration: IC₅₀) was determined from the dose-response curve (1-3). The efficacy of inhibition of NO production was evaluated by the selectivity index (SI), which was calculated by the following equation: SI=CC₅₀ / IC₅₀.

Measurement of PGE₂ production. RAW264.7 cells were subcultured in 24-well plates and incubated with various concentrations of test compounds in the presence of LPS (100 ng/ml). The culture supernatant was collected by centrifugation and the PGE₂ concentration was determined by EIA kit (Cayman Chemical Co, Ann Arbor, MI, USA).

Western blotting. The cell pellets were lysed with 100 μl of lysis buffer (10 mM Tris-HCl [pH 7.6], 1% Triton® X-100, 150 mM NaCl, 5 mM EDTA-2Na and 2 mM phenylmethylsulfonyl fluoride (PMSF) for 10 minutes in ice water, and then incubated for 50 minutes at 4°C with RT-5 ROTATOR (Titec, Saitama, Japan). The cell lysates were centrifuged at 16,000 ×g for 20 minutes at 4°C to remove the insoluble materials and the supernatant was collected. The protein concentrations of the supernatant were measured by Protein Assay Kit (Bio Rad, Hercules, CA, USA). An equal amount of the protein from the cell lysates (10 μg) was mixed with 2x sodium dodecyl sulfate (SDS)-sample buffer (0.1 M Tris-HCl [pH 6.8], 20% glycerol, 4% SDS, 0.01% bromphenol blue, 1.2% 2-mercaptoethanol), boiled for 10 minutes, applied to SDS-8% polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skim milk in phosphate-buffered saline (PBS(-)) plus 0.05% Tween 20 for 90 minutes and incubated for 90 minutes at room temperature with anti-PLA₂, anti-COX-2 or anti-iNOS antibody (1:1,000; Santa Cruz Biotechnology, Delaware, CA, USA) or anti-actin antibody (1:2,000-4,000; Sigma) and then incubated with horseradish peroxidase-conjugated anti-mouse or goat IgG for 90 minutes at room temperature. The immunoblots were developed with a Western Lightning™ Chemiluminescence Reagent Plus (Perkin Elmer Life Sciences, Boston, MA, USA) and analyzed on a Macintosh (Power Macintosh 7600/120) computer using the public domain NIH Image program (National Technical Information Service, Springfield, Virginia, USA, part number PB95-500195GEI).

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Figure 2.

Effect of six selected compounds on PGE₂ production by LPS-activated RAW264.7 cells. RAW264.7 cells were incubated for 24 hours without (control), or with the indicated concentrations of tropolone (T-3), 2,4-dibromo-7-methoxytropone (T-21), diethyl 2-chloroazulene-1,3-dicarboxylate (A-9), 1,3-difluoroazulene (A-11), 3-morpholino-1,5-azulenequinone (AQ-8), or 3,7-dibromo-1,5-azulenequinone (AQ-13), and the extracellular concentration of PGE₂ was then measured.