Cancer Res Treat.  2006 Jun;38(3):152-158.

Inhibition of Phorbol Ester-induced Mouse Skin Tumor Promotion and COX-2 Expression by Celecoxib: C/EBP as a Potential Molecular Target

• Chun KS
• Kundu JK
• Park KK
• Chung WY
• Surh YJ

Abstract

PURPOSE: Inflammation acts as a driving force for the development of cancer. Multiple lines of evidence suggest that nonsteroidal anti-inflammatory drugs, especially those that specifically target cyclooxygenase-2 (COX-2), are effective in preventing certain cancers. The present study was aimed at investigating the antitumor promoting potential of celecoxib in chemically induced mouse skin tumorigenesis, as well as elucidating the underlying molecular mechanisms.
MATERIALS AND METHODS
To study the antitumor promoting effects of celecoxib, we used the classical two-stage mouse skin tumorigenesis model that involves initiation with a single application of 7,12-dimethylbenz[alpha]anthracene (DMBA) followed by promotion with repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). The effects of celecoxib on the expression of COX-2, vascular endothelial growth factor (VEGF), p65 and the different isoforms of CCAAT/enhancer binding protein (C/EBP) were examined by performing Western blot analysis. Electrophoretic mobility gel shift assay was used to examine the effects of celecoxib on the TPA-induced DNA binding activities of various transcription factors.
RESULTS
Our study revealed that topical application of celecoxib (10 micromol) significantly reduced the multiplicity of papillomas in DMBA-initiated and TPA-promoted mouse skin. Pretreatment with celecoxib also diminished the expression of COX-2 and VEGF in the mouse skin papillomas. Pretreatment with celecoxib attenuated DNA binding of transcription factor (C/EBP) in the TPA-stimulated mouse skin. Moreover, celecoxib suppressed the TPA-induced nuclear expression of C/EBPdelta, but not C/EBPbeta, in mouse skin in vivo.
CONCLUSION
Our study demonstrates the inhibitory effects of celecoxib on mouse skin tumor promotion, which was associated with a decreased expression of COX-2 and VEGF, as well as inhibition of C/EBP activation.

Keyword

Celecoxib; Chemoprevention; Mouse skin carcinogenesis; Cyclooxygenase-2; VEGF; NF-kappaB; C/EBP

MeSH Terms

Animals
Blotting, Western
Carcinogenesis
Carrier Proteins
Chemoprevention
Cyclooxygenase 2
DNA
Inflammation
Mice*
NF-kappa B
Papilloma
Protein Isoforms
Skin*
Transcription Factors
Vascular Endothelial Growth Factor A
Celecoxib
Carrier Proteins
Cyclooxygenase 2
DNA
NF-kappa B
Protein Isoforms
Transcription Factors
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Celecoxib inhibits the promotion of mouse skin tumor. male ICR mice were treated with 0µmol (○) 1µmol (▼) or 10µmol (▽) µmol of celecoxib dissolved in 0.2 ml acetone or they were treated with the solvent alone 30 min prior to each topical application of TPA (10 nmol/0.2 ml acetone) after initiation with DMBA (0.2 µmol in 0.2 ml acetone). The control animals (●) were treated with acetone in lieu of TPA. Each treatment group consisted of 25 mice. (A) Comparison of the average numbers of papillomas per mouse in the different treatment groups. The multiplicity of papillomas in the celecoxib (10µmol) pretreated group is significantly lower than that observed in the group treated with DMBA plus TPA only (p<0.05). (B) The percent incidence of papillomas in the different treatment groups.

• 

  Fig. 2 Celecoxib diminishes the expression of COX-2 and VEGF in mouse skin papillomas. Whole tissue extracts or tumor extracts that were prepared from both control and treated animals were subjected to Western blot analysis for checking the levels of COX-1, COX-2 (A) and VEGF (B). Quantification of the COX-2 and VEGF immunoblotting was performed via an image densitometer, and the relative band intensity was normalized to that of actin; this was followed by statistical analysis (C). *p<0.001 (Control vs. DMBA plus TPA-treated group); †p<0.01 (DMBA plus TPA-treated group vs DMBA plus celecoxib plus TPA- treated group).

• 

  Fig. 3 Effects of celecoxib on TPA-induced NF-κB activation in mouse skin. The shaven backs of female ICR mice were treated with celecoxib (1 or 10µmol) 30 min prior to topical application of 10 nmol TPA. The animals were sacrificed 1 h after the TPA treatment, and the epidermal nuclear extracts were prepared as described in the Materials and Methods section. (A) The nuclear extracts (10µg) were incubated with radio-labeled NF-κB oligonucleotide, and NF-κB DNA binding assay was performed by EMSA. Lane 1, probe only; Lane 2, acetone control; Lane 3, TPA treatment only; Lane 4, celecoxib (1µmol) plus TPA; Lane 5, celecoxib (10µmol) plus TPA; Lane 6, celecoxib (10µmol) alone. (B) The nuclear protein (50µg) was separated by 10% SDS-polyacrylamide gel, and immunoblotting was performed by using a specific primary antibody to detect p65 protein.

• 

  Fig. 4 Celecoxib suppresses TPA-induced DNA binding of C/EBP in mouse skin. Animals were treated as described in Fig. 3. (A) The nuclear extracts (10µg) were incubated with radio-labeled C/EBP oligonucleotide, and the DNA binding assay was done by EMSA. Lane 1, probe only; Lane 2, acetone control; Lane 3, TPA treatment only; Lane 4, celecoxib (1µmol) plus TPA; Lane 5, celecoxib (10µmol) plus TPA; Lane 6, lane 2 plus excess cold probe. (B) The nuclear proteins (50µg) were analyzed for the expression of C/EBPβ and C/EBPδ.

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