Article Figures & Data
Figures
- Fig. 1.
COX-2 is overexpressed in respiratory papillomas. A, immunohistochemical staining of formalin-fixed, paraffin-embedded normal laryngeal and papilloma tissues. Tissue sections were stained with goat polyclonal anti–COX-2 or with PBS (control), detected using 3,3′-diaminobenzidine and lightly counterstained with hematoxylin. Note abundant COX-2 in the cytoplasm of papilloma tissues. Arrows, location of the basement membrane. Bar, 24 μm. B, representative Western blot showing expression of COX-2 levels in three papillomas and two samples of normal laryngeal tissue. A squamous carcinoma of the head and neck served as a positive control. Equal amounts of protein (40 μg/lane) were separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, incubated with goat polyclonal anti–COX-2 antibody followed by antigoat IgG-horseradish peroxidase, and detected by chemiluminescense. Membranes were then stripped and incubated with goat polyclonal anti–β-actin. Columns, mean for COX-2 expression, analyzed by densitometry, normalized to actin, and expressed as relative level of COX-2 in six papillomas compared with seven normal tissues; bars, SD (*P < 0.01). C, analysis of COX-2 mRNA levels in papilloma and normal laryngeal tissues. COX-2 mRNA was quantified by real-time reverse transcription-PCR assays with specific COX-2 primers and expressed relative to mRNA for ribosomal phosphoprotein PO (36B4). Columns, means of three normal tissues and three papilloma tissues from different patients; bars, SD. COX-2 mRNA was elevated nearly 7-fold in papillomas compared with normal laryngeal tissue (*P < 0.01). D, amounts of PGE2 produced by papilloma and normal cells. Cell culture medium from 105 normal laryngeal cells or papilloma cells was analyzed for PGE2 levels by enzyme immunoassay. Papilloma cells produced more PGE2 than normal laryngeal cells, consistent with differences in amounts of COX-2. Columns, mean from three separate experiments; bars, SD (*P < 0.01).
- Fig. 2.
Levels of COX-2 are higher in papilloma versus normal laryngeal cells under basal conditions and following EGF treatment. Cells were cultured in serum-free medium without growth factors for 24 hours before being stimulated with KGM containing either 10 ng/mL EGF (A) or 1 ng EGF + 5 μg/mL insulin (B) for 24 hours. Levels of COX-2 were determined by Western blot analysis. Representative results are shown. For quantitation of Western blots, COX-2 expression was normalized to actin levels in each lane and expressed relative to levels in normal cells. A, left columns, means for two normals; right columns, means for three papillomas; bars, SD. B, columns, means for four normals and four papillomas; bars, SD (*P < 0.05).
- Fig. 3.
COX-2 expression is regulated by EGFR→PI3K signaling in papilloma cells. A, normal laryngeal cells and papilloma cells in serum-free KGM were starved of growth factors for 24 hours and then stimulated for 1 hour with 10 ng/mL EGF in the presence or absence of 25 μmol/L LY294002. Cells were lysed and immunoblot analysis was done using the indicated antibodies. Results are representative of three experiments. B, cells were pretreated with 1 μmol/L PD153035, 50 μmol/L PD98059, 25 μmol/L LY294002, or vehicle (control); stimulated with 10 ng/mL EGF for 5 hours in the presence of inhibitors; and analyzed by immunoblot. Results shown are from two normal cell cultures and three papilloma cultures (*P < 0.01). C, cells were pretreated with inhibitors as in (B), stimulated with 1 ng/mL EGF plus 5 μg/mL insulin, and analyzed at 48 hours. Results shown are representative of experiments with three normal laryngeal cell cultures and five papilloma cell cultures (*P < 0.01). D, dose-dependent reduction in COX-2 expression in papilloma cells treated with PD153035 or LY294002. Cells were pretreated with inhibitors at the indicated concentrations, stimulated with 1 ng/mL EGF and 5 μg/mL insulin, and analyzed at 48 hours. Results are representative of three experiments. E, cells were treated as described in (C). Subsequently, amounts of PGE2 in the culture medium were determined by enzyme immunoassay. Production of PGE2 was significantly reduced in papilloma cells treated with 1 μmol/L PD153035 or 25 μmol/L LY294002 compared with control (*P < 0.01).
- Fig. 4.
The activities of COX-2 and EGFR are determinants of COX-2 expression. Papilloma cells were incubated for 48 hours with vehicle (control), 500 nmol/L PGE2, 3 μmol/L celecoxib, 3 μmol/L celecoxib plus 500 nmol/L PGE2, 1 μmol/L PD153035, or 500 nmol/L PGE2 plus 1 μmol/L PD153035. Representative Western blots from three separate experiments are shown. Treatment with PGE2 induced COX-2 whereas celecoxib reduced COX-2 protein levels. The suppressive effect of celecoxib was reversed by cotreatment with PGE2. The EGFR tyrosine kinase inhibitor PD153035 suppressed COX-2 expression. The induction of COX-2 by PGE2 was suppressed by cotreatment with PD153035. COX-2 expression was normalized to total protein in each lane and expressed relative to control cells. Columns, mean of three separate experiments; bars, SD (*P < 0.01 compared with control).
- Fig. 5.
Effects of PGE2 and celecoxib on cell proliferation and apoptosis. A, assay of cell growth measuring metabolic reduction of tetrazolium as a surrogate measure of cell number. Treatment of papilloma cells for 48 hours with PGE2 stimulated cell growth (*P < 0.01). B, celecoxib caused dose-dependent inhibition of proliferation of papilloma cells. Cells were treated with 2.5, 5.0, or 7.5 μmol/L celecoxib or vehicle (control) for 24 hours, with BrdUrd added for the last 18 hours. Reduction in the relative fraction of cells incorporating BrdUrd into nuclear DNA was indicative of inhibition of proliferation (*P < 0.05). The proliferating fraction in the control cells was ∼20%. C, apoptosis assay, measured by nucleosome release into the cytoplasm, showing a dose-dependent increase in apoptosis after 24 hours treatment of papilloma cells with 2.5, 5.0, or 7.5 μmol/L celecoxib compared with control cells treated with vehicle (*P < 0.05). Columns, means from three separate experiments for (A) and two each for (B) and (C); bars, SD.
Volume 11, Issue 17
