Reduced 15S-lipoxygenase-2 expression in esophageal cancer specimens and cells and upregulation in vitro by the cyclooxygenase-2 inhibitor, NS398 - PubMed (original) (raw)

Reduced 15S-lipoxygenase-2 expression in esophageal cancer specimens and cells and upregulation in vitro by the cyclooxygenase-2 inhibitor, NS398

Xiao-Chun Xu et al. Neoplasia. 2003 Mar-Apr.

Abstract

Alterations in arachidonic acid metabolism are involved in human carcinogenesis. Cyclooxygenase (COX) and lipoxygenase (LOX) are key enzymes in this metabolism. We analyzed the expression of 15S-lipoxygenase-2 (15-LOX-2) mRNA and protein in surgical specimens from normal (N=37) and malignant (63) esophageal tissues using in situ hybridization and immunohistochemistry (IHC), and in normal (1), premalignant (1), and malignant (5) esophageal cell lines using Northern and Western blotting. 15-LOX-2 was expressed in normal esophageal epithelial cells (EECs) at the highest levels, whereas an SV40-immortalized HET-1A line and three of five esophageal cancer cell lines failed to express it at detectable levels. 15-LOX-2 was detected in 76% (28/37) of the normal esophageal mucosae, but only in 46% (29/63) of the cancer specimens using IHC (P<.01). Transient transfection of 15-LOX-2 expression vectors into esophageal cancer cells significantly inhibited the proliferation of 15-LOX-2-negative cancer cells. The COX-2 inhibitor, NS398, induced 15-LOX-2 expression in esophageal cancer cells, which is associated with reduced cell viability. This study demonstrated that 15-LOX-2 expression is lost in esophageal cancers and that the induction of 15-LOX-2 can inhibit cancer cell proliferation. Further investigation of the effects of nonsteroidal anti-inflammatory drugs on 15-LOX-2 expression and apoptosis in esophageal cancer cells may be warranted.

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Figures

Figure 1

Analysis of 15-LOX-2 mRNA and protein using Northern (A) and Western (B) blotting. Normal esophageal cells (EECs), SV40-immortalized esophageal epithelial cell line HET-1A, and esophageal cancer cell lines TE-1, TE-3, TE-7, TE-8, and TE-12 were grown in monolayer cultures to about 90% confluence. The total RNA and protein were isolated and subjected to Northern and Western blot analyses, respectively. The experiments were repeated once with similar results.

Figure 2

Differential expression of 15-LOX-2 mRNA and protein in normal and malignant esophageal surgical specimens. 15-LOX-2 mRNA and protein were detected using ISH and IHC, respectively. Consecutive sections of formalin-fixed and paraffin-embedded human esophageal carcinomas and distant normal tissues were hybridized with 15-LOX-2 antisense digoxigenin-labeled cRNA probe, which results in purple to blue staining of the positive signal in the cytoplasm. IHC was performed by using polyclonal rabbit anti-15-LOX-2 antibody, and the AEC was the chromogen, resulting in red staining of the positive signal in the cytoplasm. (A), (C), (D), (E), (F) =200x original magnification; (B)=100x magnification.

Figure 3

Inhibition of esophageal cancer cell proliferation by the expression of 15-LOX-2. Esophageal cancer cell lines TE-1, TE-8, and TE-12 were transiently transfected with the control or 15-LOX-2 expression vectors for 12 hours and treatedwith BrdU for 8 hours. The cells were then stained with anti-BrdU antibody (see Materials and Methods) and more than 200 GFP-positive cells were counted for positive or negative BrdU staining. The percentage of inhibition of BrdU incorporation was calculated from the equation: % Inhibition =[1-(Nb/Ng)]x100, where Nb and Ng are the numbers of BrdU-positive cells in GPF-positive cells of 15-LOX-2-transfected and control cultures, respectively. The data showed that 15-LOX-2 significantly reduced BrdU incorporation in 15-LOX-2-negative esophageal cancer cells. The experiments were performed in triplicate with similar results.

Figure 4

Inhibition of BrdU incorporation by 15-LOX-2 in esophageal cancer cell lines. 15-LOX-2 expression vectors were transiently transfected into the esophageal cancer cell lines TE-1 (A), TE-8 (B), and TE-12 (C), respectively, for 12 hours and then treated with BrdU for 8 hours (see Materials and Methods). After BrdU immunostaining, more than 200 cells were counted for positive staining of GFP (green), for positive or negative BrdU (red) staining in these cells.

Figure 5

Induction of 15-LOX-2 expression after treatment with NS398 using quantitative RT-PCR. Esophageal cancer cell lines TE-1, TE-3, TE-7, TE-8, and TE-12 were grown on monolayer and treated with NS398 (100 µM) for 5 days. Afterwards, total cellular RNA was isolated and subjected to real-time RT-PCR analysis. The experiments were repeated once.

Figure 6

Cell viability assay. Esophageal cancer cell lines TE-1, TE-3, TE-7, TE-8, and TE-12 were treated with and without NS398 (50 or 100 µM) for 5 days. Culture medium was replaced once at 72 hours. On day 5, the cells were fixed with 10% trichloroacetic acid and stained with 0.4% sulforhodamine B in 1% acetic acid, and then the optical densities were read on an automated spectrophotometric plate reader at a single wavelength of 490 nm. The percentage of growth inhibition was calculated from the equation: % Control =(ODt/ODc)x100, where ODt and ODc are the values of optical densities in treated and control cultures, respectively. The experiments were repeated once.

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Reduced 15S-lipoxygenase-2 expression in esophageal cancer specimens and cells and upregulation in vitro by the cyclooxygenase-2 inhibitor, NS398 - PubMed (original) (raw)